To see the other types of publications on this topic, follow the link: Fungi – Biotechnology.
Dissertations / Theses on the topic 'Fungi – Biotechnology'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'Fungi – Biotechnology.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Wordon, Brett Arthur. "The use of fluorescent flow cytometry to evaluate the inactivation of Saccharomyces cerevisiae by sequential application of ultrsound (20kHz) and heat." Thesis, Cape Peninsula University of Technology, 2009. http://hdl.handle.net/20.500.11838/828.
Full textAbstract:
Thesis (MTech (Food Technology)--Cape Peninsula University of Technology, 2009<br>The primary aim of this study was to establish the effects of both cavitation, (20 KHZ), and
heat (55°C or 60•C) on Saccharomyces cerevisiae GC210 (MATa lys2) suspended in
physiological saline. Fluorescent flow cytometry was used to determine the effects of moist
heat and acoustic cavitation on S. cerevisiae cells. Results from this study could be used as
a guide for use by the food industry for the combined use of heat and sonication to disinfect
various solutions contaminated with S. cerevisiae.
2
Brohée, Sylvain. "Etude bioinformatique du réseau d'interactions entre protéines de transport ches les Fungi." Doctoral thesis, Universite Libre de Bruxelles, 2008. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210432.
Full textAbstract:
Les protéines associées aux membranes sont d'une importance cruciale pour la cellule. Cependant, en raison d'une plus grande difficulté de manipulation, les données biochimiques les concernant sont très lacunaires, notamment au point de vue de la formation de complexes entre ces protéines.<p><p>L'objectif global de notre travail consiste à combler ces lacunes et à préciser les interactions entre protéines membranaires chez la levure Saccharomyces cerevisiae et plus précisément, entre les transporteurs. Nous avons commencé notre travail par l'étude d'un jeu de données d'interactions à grande échelle entre toutes les perméases détectées par une méthode de double hybride spécialement adaptée aux protéines insolubles (split ubiquitin). Premièrement, la qualité des données a été estimée en étudiant le comportement global des données et des témoins négatifs et positifs. Les données ont ensuite été standardisées et filtrées de façon à ne conserver que les plus significatives. Ces interactions ont ensuite été étudiées en les modélisant dans un réseau d'interactions que nous avons étudié par des techniques issues de la théorie des graphes. Après une évaluation systématique de différentes méthodes de clustering, nous avons notamment recherché au sein du réseau des groupes de protéines densément interconnectées et de fonctions similaires qui correspondraient éventuellement à des complexes protéiques. Les résultats révélés par l'étude du réseau expérimental se sont révélés assez décevants. En effet, même si nous avons pu retrouver certaines interactions déjà décrites, un bon nombre des interactions filtrées semblait n'avoir aucune réalité biologique et nous n'avons pu retrouver que très peu de modules de protéines de fonction semblable hautement inter-connectées. Parmi ceux-ci, il est apparu que les transporteurs d'acides aminés semblaient interagir entre eux.<p><p>L'approche expérimentale n'ayant eu que peu de succès, nous l'avons contournée en utilisant des méthodes de génomique comparative d'inférence d'interactions fonctionnelles. Dans un premier temps, malgré une évaluation rigoureuse, l'étude des profils phylogénétiques (la prédiction d'interactions fonctionnelles en étudiant la corrééélation des profils de présence - absence des gènes dans un ensemble de génomes), n'a produit que des résultats mitigés car les perméases semblent très peu conservées dès lors que l'on considère d'autres organismes que les \<br>Doctorat en Sciences<br>info:eu-repo/semantics/nonPublished
3
Suleman, Essa. "The role of pacC in Aspergillus flavus." Thesis, Nelson Mandela Metropolitan University, 2007. http://hdl.handle.net/10948/612.
Full textAbstract:
Many microorganisms, and in particular fungi, are able to grow over a wide pH range. Thus, these microorganisms must possess some regulatory mechanism or system that senses the environmental pH signal and ensures that gene expression of certain molecules is tailored to the pH of the environment (Penalva and Arst, 2002). In Aspergillus species and several other fungi, pH regulation is mediated by seven genes viz. palA, palB, palC, palF, palH, palI and the global pH regulatory gene, pacC (MacAbe et al, 1996; Negrete-Urtasun, 1999; Denison, 2000). The activated form of the PacC protein activates genes that are required at alkaline pH, e.g. genes coding for alkaline phosphatases, and represses certain genes that are functional at acidic pH, e.g. genes encoding acid phosphatases (Negrete-Urtasun, 1999). PacC (and its homologues) also positively regulates genes involved in penicillin biosynthesis, e.g. the isopenicillin N synthase gene, ipnA, in A. nidulans (Penalva and Arst, 2002). It has also been hypothesised that pacC may negatively regulate aflatoxin biosynthesis, a carcinogenic secondary metabolite in several species of Aspergillus (Keller et al, 1997). To elucidate the role of pacC a novel method of post-transcriptional gene silencing known as RNA interference was utilized. This method involved the cloning of a partial pacC gene fragment first in the forward and then the reverse orientations in a fungal expression cassette to create an RNA interference (RNAi) vector. The unique structure of this vector would allow the cloned fragments to be expressed and the resulting RNA to immediately form a double stranded stem-loop structure or short hairpin RNA (shRNA; McDonald et al, 2005). The formation of this shRNA, in turn, would be responsible for activating the endogenous RNA degradation complexes that would lead to mRNA degradation and subsequent gene silencing (Liu et al, 2003; Kadotoni et al, 2003; McDonald et al, 2005). The results presented here have shown that confirmed pacC RNAi mutants produced aflatoxins irrespective of environmental pH (i.e. the mutants produce aflatoxins under acidic and alkaline conditions). Thus, pacC is essential for pH regulation of aflatoxin production in A. flavus. There are numerous other biological (e.g. presence of oxylipins, lipooxygenases) and non-biological factors (pH, carbon source etc.) which affect maize colonisation and aflatoxin production by A. flavus (Burrow et al, 1996; Wilson et al, 2001; Calvo et al; 2002; Tsitsigiannis et al, 2006). However, all the genetic mechanisms involved have as yet not been identified. It has been shown by Caracuel et al (2003) that pacC acts as a negative virulence regulator in plants and these workers have hypothesised that PacC prevents expression of genes that are important for infection and virulence of the pathogen. Therefore the physiological effects that pacC silencing had on the growth, conidiation and pathogenicity of A. flavus mutants were also investigated. The results of this study showed that pacC does not play a significant role in primary growth and development but does affect conidial production. SEM results showed that mutants have many “open ended” phialides and poorly developed conidiophores. This would suggest that pacC activation of conidial production genes is also required. Furthermore, pacC RNAi silencing severely impaired the ability of the A. flavus mutants to infect and cause damage on maize. The results obtained here are similar to that of pacC null mutants in A. nidulans, C. albicans and F. oxysporum which also exhibited low pathogenicity (Davis et al, 2000; Fonzi, W.A, 2002; Caracuel et al, 2003; Bignell et al, 2005 and Cornet et al, 2005). This study indicates that pathogenicity of A. flavus on maize is directly related to the structural integrity of conidia, which in turn is greatly influenced by PacC. This gene is a global transcriptional regulator and may either repress or activate one or many genes in each of the above pathways (Penalva and Arst, 2002). Studies on the genetic mechanisms of pacC regulation on these pathways are needed to elucidate the mechanisms of activation or repression of these genes.
4
Speers, Robert Alexander. "Rheological and colloidal properties of commercial brewing yeast suspensions." Thesis, University of British Columbia, 1991. http://hdl.handle.net/2429/31517.
Full textAbstract:
A three part study was carried out to examine rheological, colloidal and floe microstructural aspects of industrial brewing yeast strains. Following a review of the literature, the rheological properties of four yeast strains (two flocculent ale and lager types and their non-flocculent variants) were examined. In related colloidal studies, orthokinetic flocculation rates of these strains as well as their cell surface charge were determined. Floc microstructure was characterized using both light and scanning electron microscopy. In a summary chapter, the cell floc model (a modification of Hunter's elastic floc model) was used to the explain the rheological and colloidal behaviour of brewing yeast suspensions.
Flow behaviour studies of the commercial yeast suspensions suspended in a calcium-containing sodium acetate buffer revealed that yeast flocculent characteristics had an important influence on their suspension flow behaviour. As cell concentrations increased, suspension flow properties become increasingly non-Newtonian and could be described by the Casson model at low rates of shear and the Bingham model at shear rates above 100 s⁻¹. The cell floc model was proposed to explain the Bingham flow behaviour of these csuspensions. The Bingham yield stress in these suspensions was believed to be a function of the orthokinetic capture coefficient, cell volume and the energy to break up doublet cells. Increasing temperature tended to lower the Bingham yield stress in lager strains and increase the yield stress in ale strains. A semi-empirical explanation for the viscosity increase of deflocculated cell suspensions and the estimation of pseudo-capture coefficients was presented.
Furthermore, studies of the flow behaviour of yeast strains suspended in decarbonated ale and lager beer revealed that: 1) suspensions of flocculent strains show
higher yield stress values than their non-flocculent variants, 2) ale strain suspensions tended to have higher yield values than the lager strains and 3) yeast dispersed in beer had higher yield stress values than when suspended in buffered calcium suspensions. This last observation was believed to reflect the influence of ethanol on the cell binding process which has important implications for future measurements of yeast flocculation.
Colloidal studies revealed for the first time, that the orthokinetic rate of flocculation of brewing yeast cells could be modelled by a first order equation, as predicted by fundamental colloid theory. While subject to considerable variation, measured rate constants led to the calculation of orthokinetic capture coefficients. Yeast cell zeta potential values generally agreed with literature data but could not be employed in the DLVO model of colloid flocculation to explain measured orthokinetic capture coefficient values. Examination of the cell zeta potential data indicated that the data had non-normal distributions.
SEM examination of the four industrial yeast strains suggested that a number of distinct structures mediated cell-to-cell interaction and that intra-strain differences occurred. These findings, along with the observation of non-normal surface charge distributions, indicated that these industrially pure strains had undergone substantial variation. Treatment of the flocculent cells with pronase tended to reduce cell-to-cell contacts.
In the summary chapter the cell floe model was employed to describe the rheological behaviour of the yeast suspensions. Estimation of the force needed to separate doublet yeast cells were made using critical shear rate data (i.e., the point at which Bingham flow begins). This estimate was similar to that reported for single antibody bonds and may be due to the presence of lectin-like structures on the yeast cell wall.<br>Land and Food Systems, Faculty of<br>Graduate
5
Campos, Katherine Helen de Sa. "Identification and characterization of components that overcome secretion limitations of the yeast Pichia pastoris." Scholarly Commons, 2013. https://scholarlycommons.pacific.edu/uop_etds/844.
Full textAbstract:
The methylotrophic yeast. Pichia pastoris, is a powerful, adaptable, and inexpensive recombinant expression system commonly used to secrete heterologous protein. Although P. pastoris is a popular host organism, secretion inefficiency continues to be a major hurdle in its ability to produce high levels of foreign protein. Optimization of cis- and trans-acting factors has greatly enhanced the secretory capabilities of P. pastoris, however protein-specific engineering of a host organism is costly and not always effective. P. pastoris' secretion inefficiency is commonly due to trans-acting factors. Strains of S. cerevisiae have been engineered, through random genomic mutation, that are capable of overcoming these /ram-acting factors to secrete high levels of foreign protein. The Lin-Cereghino laboratory at University of the Pacific has developed a screen to identify mutations in P. pastoris capable of circumventing secretion obstacles. The P. pastoris genome was randomly disrupted through restriction enzyme-mediated integration of an antibiotic resistance marker. Supersecretion mutants were identified by their ability to secrete β-galactosidase, a reporter enzyme not natively secreted by P. pastoris. Sixteen β-galactosidase secretion (bgs) mutants were initially isolated by the Lin-Cereghino lab. This research focused on characterizing one of the resultant bgs mutants, ///. Initial sequencing and alignment studies identified the predicted LI1p sequence to be homologous to S. cerevisiae protein kinase C (PKC). Considering the role of PKC in the Cell Wall Integrity pathway of S. cerevisiae. the cell wall and secretory organelles of III were closely examined using transmission electron microscopy. Additionally, a qualitative alkaline phosphatase assay was used to evaluate the cell wall integrity of ///. Finally, the secretory phenotype of 111 was examined using a group of structurally and functionally diverse reporter proteins. In characterizing the bgs mutant, III, this research contributes to an understanding of cellular components that limit protein secretion in the yeast, P. pastoris.
6
Govender, Patrick. "Industrial yeast strains engineered for controlled flocculation." Thesis, Stellenbosch : University of Stellenbosch, 2009. http://hdl.handle.net/10019.1/1450.
Full textAbstract:
Thesis (PhD (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2009.<br>In many industrial fermentation processes, Saccharomyces cerevisiae yeast should ideally meet two partially conflicting demands. During fermentation a high suspended yeast count is of paramount importance to maintain a rapid fermentation rate, whilst efficient flocculation should ideally be initiated only on completion of the primary alcoholic fermentation, so as to enhance product clarification and recovery. Most commercial wine yeast strains are non-flocculent, probably because this trait was counter-selected to avoid fermentation problems. In this study, we assessed molecular strategies to optimise the flocculation behaviour of non-flocculent laboratory and wine yeast strains. For this purpose, the chromosomal copies of three dominant flocculation genes, FLO1, FLO5 and FLO11, of a non-flocculent S. cerevisiae laboratory strain (FY23) and two commercial wine yeast strains (BM45 and VIN13) were placed under the transcriptional control of the stationary phase-inducible promoters of the S. cerevisiae ADH2 or HSP30 genes.
Under standard laboratory media and culture conditions, all six promoter-gene combinations resulted in specific flocculation behaviours in terms of timing and intensity. The data show that the strategy resulted in the expected and stable expression patterns of these genes in both laboratory and industrial wine yeast strains. Most importantly, the data confirm that inducible expression of the native FLO1 and FLO5 open reading frames, albeit to varying degrees, are responsible for a quantifiable cell-cell adhesion phenotype that can be characterized as a Flo1 flocculation phenotype. On the other hand, we found that inducible expression of the native FLO11 ORF under these conditions resulted in flor/biofilm formation and invasive growth phenotypes. However, the specific impact of the expression of individual dominant FLO genes with regard to characteristics such as flocculation efficiency, cell wall hydrophobicity, biofilm formation and substrate adhesion properties showed significant differences between the commercial strains as well as between commercial and laboratory strains. These adhesion phenotype differences may at least in part be attributed to wine yeast FLO gene open reading frames containing significantly smaller intragenic repeat regions than laboratory strains.
The data show that the ADH2 regulatory sequences employed in this study were unsuitable for the purpose of driving FLO gene expression under wine-making conditions. However, HSP30p-based FLO1 and FLO5 wine yeast transformants displayed similar flocculent phenotypes under both synthetic and authentic red wine-making conditions, and the intensities of these phenotypes were closely aligned to those observed under nutrient-rich YEPD conditions. The fermentation activities of
HSP30p-based transgenic yeast strains were indistinguishable from that of their parental host wine yeast strains. The chemical composition of wines obtained using transgenic yeast strains were similar to those produced by parental strains. The BM45-derived HSP30p-FLO5 transformant in particular was capable of generating compacted or ‘caked’ lees fractions, thereby providing a distinct separation of the fermented wine product and lees fractions. Furthermore, in this study we report a novel FLO11 induced flocculation phenotype that seems to exclusively develop under authentic red wine-making conditions. This strong FLO11 flocculation phenotype was not wine yeast strain dependant, possessed both Ca2+-dependant and Ca2+-independent flocculation characteristics and was insensitive to inhibition by both glucose and mannose. A distinct advantage of this unique FLO11 phenotype was highlighted in its ability to dramatically promote faster lees settling rates. Moreover, wines produced by HSP30p-FLO11 wine yeast transformants were significantly less turbid than those produced by their wild type parental strains. The benefit of this attractive property is it facilitates simpler and faster recovery of wines and also promotes greater volume recovery of the wine product.
7
Mocke, Bernard A. "The breeding of yeast strains for novel oenological outcomes." Thesis, Link to the online version, 2005. http://hdl.handle.net/10019/1117.
Full text
8
Demirtas, Umut. "Fungi Mediated Enantioselective Biohydrogenation Of Benzils To Benzoins." Master's thesis, METU, 2008. http://etd.lib.metu.edu.tr/upload/2/12609232/index.pdf.
Full textAbstract:
Benzoin is an important a-hydroxy ketone which can be used as chiral intermediate
for the synthesis of several drugs. In this study, it was aimed to synthesize this
compound by high stereoslectivity and yield by the use of fungal bioconversions. For
this purpose, whole cells of four different Fusarium spp. (F. anguoides, F. roseum, F.
solanii, F.bulbigenum) were used for reduction of readily available achiral compound
benzil. The reaction conditions were optimized as glucose peptone broth consisting
of 30g/L glucose and 10 g/L peptone, inoculum size as 20 mg/L and substrate
concentration as 200 mg/L. A complete set of derivatives substituted with electron
donating and electron withdrawing groups of the benzils were also reduced to the
corresponding benzoin derivatives with the same optimized condition with up to 98%
ee.
9
Rizzi, John. "Production of emulsifier by Torulopsis petrophilum." Thesis, McGill University, 1987. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=64014.
Full text
10
Pather, Simisha. "Marine biotechnology : evaluation and development of methods for the discovery of natural products from fungi." Thesis, Rhodes University, 2005. http://hdl.handle.net/10962/d1007652.
Full textAbstract:
One of the major impediments in the development of marine natural products is the provision of biologically active natural products in sufficient quantity for complete pharmacological evaluation, clinical trials and eventual commercial production. Marine microorganisms show great promise in providing a renewable source of biologically active natural products. The main aim of this study was to develop and evaluate methods for the isolation, identification and cultivation of marine fungi from the South African marine environment for the production of biologically active secondary metabolites. Twenty-four species of fungi were isolated from marine algae collected from the intertidal zone near Port Alfred, South Africa. The fungi were cultivated in small-scale under static and agitated conditions and their crude intra- and extracellular organic extracts were screened by ¹H NMR and a series of bioassays. Using this as a basis, one isolate was selected for further study. By analyses of the lTS1 region of the ribosomal DNA, the fungal isolate was identified as a marine-derived isolate of Eurotium rubrum (Aspergillus ruber). Although E. rubrum has been isolated from the marine environment, no investigations have been undertaken to determine the adaptation of these isolates to the marine environment. In order to optimise productivity, creativity and incubation time, the fungus was cultivated in small-scale using a variety of carbon (glucose, fructose, lactose, sucrose, marmitol and maltose) and nitrogen sources (ammonium tartrate, urea, peptone and yeast extract). An HPLC-DAD method was developed to assess the metabolic creativity and productivity under different fermentation conditions. Distinctive variations in the range and yield of metabolites produced as well as morphology and growth time were observed. The crude extracts from all fermentations were combined and six known compounds were isolated by reversed-phase chromatography and their structures elucidated by spectroscopic techniques. The known compounds were fIavoglaucin, aspergin, isodihydroauroglaucin, isotetrahydroauroglaucin, neoechinuline A and physcion. Neoechinuline A, isodihydroauroglaucin and isotetrahydroauroglaucin showed activity against oesophageal and cervical cancer cell lines.
11
Sekhohola, Lerato M. "Evaluation of Fungcoal as a bioprocess technology for self-cladding of waste coal dumps." Thesis, Rhodes University, 2016. http://hdl.handle.net/10962/d1019992.
Full textAbstract:
Low-grade coal, a poor source of energy, has long been regarded as waste material by the coal mining industry. Biological degradation of this coal material by ligninolytic fungal strains presents a viable strategy towards eliminating this unusable fossil fuel. To this end, a novel and patented bioprocess termed Fungcoal was developed. Fungcoal is a biological process utilised in the in situ treatment of waste coal and is based on the mutualistic relationship between the fungus Neosartorya fischeri and the graminaceous species Cynodon dactylon. The process facilitates the rapid conversion of waste coal into soil-like material that stimulates establishment of vegetation for eventual coal dump rehabilitation. While a number of in vitro studies have identified various fungal strains as efficient coal degraders, the mechanisms involved in the Fungcoal-stimulated degradation process have not been fully elucidated. Furthermore, implementation of Fungcoal at both pilot and commercial scale has not been achieved. Thus the objective of this work was to investigate Fungcoal as a bioprocess via examining the role of coal degrading fungi (CDF) and grasses as biocatalysts in coal biodegradation and for the self-cladding of waste coal dumps. Initially, waste coal degradation by N. fischeri, strain ECCN 84, was investigated, specifically focusing on the mechanisms underpinning the process. In vitro studies showed the addition of waste coal induced active fungal colonisation resulting in increased fungal biomass. Increased extracellular laccase (LAC) activity, occuring concomitantly with an increase in hyphal peroxisome proliferation, was also observed in the coal supplied fungal cultures. Analysis of the colonised waste coal revealed a time dependent reduction in the percentage weight of elemental carbon coupled with an increase in elemental oxygen. The results supported metabolism and degradation of waste coal by N. fischeri strain ECCN 84 and involvement of fungal extracellular laccase. The contribution of C. dactylon, a C4 grass species to in situ biodegradation of waste coal in the presence of coal degrading and mycorrhizal fungi (MF) was also investigated. Enhanced degradation of the waste coal into a humic soil-like material was observed within the rhizosphere. Analysis of the resultant substrate revealed an increased concentration of highly oxidised humic-like substances (HS). Fungi remained viable in the rhizosphere up to 47 weeks post-inoculation and cultivation of C. dactylon, indicating the resultant humic substance-rich rhizosphere provided an environment conducive for microbial proliferation and activity. Furthermore, humic substance enrichment of waste coal substrates supported germination and seedling emergence of several agronomic species including Zea mays (corn), Phaseolus vulgaris (bean), Pisum sativum (pea), and Spinacia oleracea (spinach). Use of various cladding materials to support coal biodegradation, by fungus-grass mutualism and rehabilitation of waste dumps was evaluated at commercial scale. While substantial physico-chemical changes were not evident in the absence of cladding or where waste coal was used as cladding material, successful establishment of grass cover and diversity was achieved within three hydrological cycles on dumps cladded with weathered coal. Work presented in this thesis successfully demonstrates the degradation of waste coal by N. fischeri. The biodegradation process included enhanced extracellular LAC activity coupled with increased 3 waste coal oxidation. Increased HS concentration of waste coal substrate supported germination and early seedling establishment of several agronomic species. At commercial scale a co-substrate in the form of carbon-rich weathered coal was essential to support fungus-grass mutualism and Fungcoal-induced rehabilitation. These findings support the developed Fungcoal concept and the underpinning rationale that the phyto-biodegradation of waste coal indeed depends on the mutualistic interactions between grass root exudates and the ligninolytic and mycorrhizal fungi. Taken together, these findings provide practical evidence of the contribution of fungi and grasses as mutualists in the biodegradation of waste coal and sustainable rehabilitation of waste coal dumps
12
Belewa, Xoliswa Vuyokazi. "The antifungal activity of an aqueous Tulbaghia violacea plant extract against Aspergillus flavus." Thesis, Nelson Mandela Metropolitan University, 2015. http://hdl.handle.net/10948/5858.
Full textAbstract:
Phytochemical analysis of both HEA1 and the crude plant extract showed the presence of phenolics, tannins and saponins. Saponins were the predominant secondary metabolites and were mostly abundant in the plant extract and to a lesser extent in the active compound. Steroidal saponins, tannins and phenolics were also detected in the plant extract, but only the phenolics were detected in the active compound. The results of the phytochemical analysis showed that those compounds that were not present in the active compound could be removed from the crude extract during the TLC purification process. Investigation on the mechanism of action of the crude plant extract on the sterol production by A. flavus showed that the plant extract affected ergosterol biosynthesis by causing an accumulation of oxidosqualene in the ergosterol biosynthetic pathway resulting in a decline in ergosterol production. An oscillatory response in lanosterol production was observed in the presence of the plant extract, which may be an adaptation mechanism of A. flavus to unfavourable conditions and compensation for the loss of enzyme activity which may have occurred as a result of the accumulation of oxidosqualene. The antifungal activity of the plant extract on ergosterol production by A. flavus may also be due to saponins which target the cell membrane and ergosterol production in fungi. The effect of the plant extract on the fungal cell wall of A. flavus also showed that the plant extract caused a decline in β-(1, 3) glucan production by inhibiting β-glucan synthase. The plant extract also affected the chitin synthesis pathway of A. flavus, by causing a decline in chitin production, which was due to the inhibition of chitin synthase. Investigation of chitinase production using 4MU substrates showed that the plant extract caused an accumulation of chitobioses, by activating chitobiosidases and endochitinases. A decline in N-acetylglucosaminidase activity in the presence of the plant extract was observed and this prevented the formation of N-acetylglucosamine. The accumulation of chitobiosidase and endochitinase may be as a result of autolysis that may be triggered by A. flavus as a survival mechanism in the presence of the plant extract and as a compensatory mechanism for the loss of β-glucans and chitin. The antifungal effect of the plant extract on various components of the cell wall of A. flavus, makes T. violacea aqueous plant extract an ideal chemotherapeutic agent against both human and plant pathogens of Aspergillus. The broad spectrum of antifungal activity of T. violacea against A. flavus also eliminates any chances of the fungus developing resistance towards it and would make it a candidate for use as a potential antifungal agent. Further identification and possible chemical synthesis is needed to shed light on the safety and efficacy of the active compound for further development as a chemotherapeutic agent.
13
Voigt, Paul George. "Bioethanol production from waste paper through fungal biotechnology." Thesis, Rhodes University, 2010. http://hdl.handle.net/10962/d1013447.
Full textAbstract:
Bioethanol is likely to be a large contributor to the fuel sector of industry in the near future. Current research trends are geared towards utilizing food crops as substrate for bioethanol fermentation; however, this is the source of much controversy. Utilizing food crops for fuel purposes is anticipated to cause massive food shortages worldwide. Cellulose is the most abundant renewable resource on earth and is subject to a wide array of scientific study in order to utilize the glucose contained within it. Waste paper has a high degree of cellulose associated with it, which makes it an ideal target for cellulose biotechnology with the ultimate end goal of bioethanol production. This study focussed on producing the necessary enzymes to hydrolyse the cellulose found in waste paper and using the sugars produced to produce ethanol. The effects of various printing inks had on the production of sugars and the total envirorunental impact of the effluents produced during the production line were also examined. It was found that the fungus Trichoderma longibrachiatum DSM 769 grown in Mandel's medium with waste newspaper as the sole carbon source at 28 °C for 6 days produced extracellular cellulase enzymes with an activity of 0.203 ± 0.009 FPU.ml⁻¹, significantly higher activity as compared to other paper sources. This extracellular cellulase was used to hydrolyse waste newspaper and office paper, with office paper yielding the highest degree of sugar production with an end concentration of 5.80 ± 0.19 g/1 at 40 °C. Analysis by HPLC showed that although glucose was the major product at 4.35 ± 0.12 g/1, cellobiose was also produced in appreciable amounts (1.97 ± 0.71 g/1). The sugar solution was used as a substrate for Saccharomyces cerevisiae DSM 1333 and ethanol was produced at a level of 1.79 ± 0.26 g/1, the presence of which was confirmed by a 600 MHz NMR spectrum. It was found that cellobiose was not fermented by this strain of S. cerevisiae. Certain components of inks (the PAHs phenanthrene and naphthalene) were found to have a slight inhibitory effect (approximately 15% decrease) on the cellulase enzymes at very high concentrations (approximately 600 μg/1 in aqueous medium), while anthracene had no effect. Whole newsprint ink was shown not to sorb glucose. The environmental analysis of the effluents produced showed that in order for the effluents to be discharged into an aqueous ecosystem they would have to be diluted up to 200 times. They were also shown to have the potential to cause severe machinery damage if reused without proper treatment.
14
Kriel, Johan Hendrik. "Development of synthetic signal sequences for heterologous protein secretion from Saccharomyces cerevisiae." Thesis, Stellenbosch : Stellenbosch University, 2003. http://hdl.handle.net/10019.1/53364.
Full textAbstract:
Thesis (MSc)--Stellenbosch University, 2003.<br>ENGLISH ABSTRACT: Protein secretion and intracellular transport are highly regulated processes and
involve the interplay of a multitude of proteins. A unique collection of thermosensitive
secretory mutants allowed scientists to demonstrate that the secretory pathway of the
yeast Saccharomyces cerevisiae is very similar to that of the higher eukaryotes. All
proteins commence their journey in the endoplasmic reticulum, where they undergo
amino-linked core glycosyl modification. After passage through the Golgi apparatus,
where the remodelling of the glycosyl chains is completed, proteins are transported to
their final destinations, which are either the cell surface, periplasmic space or the
vacuole.
Proteins destined for secretion are usually synthesised with a transient
amino-terminal secretion leader of varying length and hydrophobicity, which plays a
crucial role in the targeting and translocation of their protein cargo. Considerable
effort has been made to elucidate the molecular mechanisms involved in these
processes, especially due to their relevance in a rapidly expanding biotech industry.
The advantages of S. cerevisiae as a host for the expression of recombinant
proteins are well documented. Unfortunately, S. cerevisiae is also subject to a
number of drawbacks, with a relative low product yield being one of the major
disadvantages.
Bearing this in mind, different secretion leaders were compared with the aim of
improving the secretion of the LKA 1 and LKA2 a-amylase enzymes from the
S. cerevisiae secretion system. The yeast Lipomyces kononenkoae is well known for
its ability to degrade raw starch and an improved secretion of its amylase enzymes
from S. cerevisiae paves the way for a potential one-step starch utilisation process.
Three sets of constructs were prepared containing the LKA 1 and LKA2 genes
separately under secretory direction of either their native secretion leader, the
S. cerevisiae mating pheromone a-factor (MFa1) secretion leader, or the MFa1
secretion leader containing a synthetic C-terminal spacer peptide (EEGEPK). The
inclusion of a spacer peptide in the latter set of constructs ensured improved Kex2p
proteolytic processing of the leader/protein fusion. Strains expressing the amylase
genes under their native secretion leaders resulted in the highest saccharolytic
activity in the culture medium. In contrast to this, strains utilising the synthetic
secretion leader produced the highest fermentation yield, but had a lower than
expected extracellular activity. We hypothesise that the native amylase leaders may
function as intramolecular chaperones in the folding and processing of their
passenger proteins, thereby increasing processing efficiency and concomitant
enzyme activity.<br>AFRIKAANSE OPSOMMING: Proteïensekresie en intrasellulêre transport is hoogs gereguleerde prosesse en
betrek die onderlinge wisselwerking van 'n verskeidenheid proteïene. 'n Unieke
versameling van temperatuur-sensitiewe sekresiemutante het wetenskaplikes in staat
gestelom die ooreenkoms tussen die sekresiepad van die gis
Saccharomyces cerevisiae en dié van komplekser eukariote aan te toon. Alle
proteïene begin hul reis in die endoplasmiese retikulum, waartydens hulle ook
amino-gekoppelde kernglikosielveranderings ondergaan. Nadat die proteïene deur
die Golgi-apparaat beweeg het, waar die laaste veranderings aan die
glikosielkettings plaasvind, word hulle na hul finale bestemmings, waaronder die
seloppervlak, die periplasmiese ruimte of die vakuool, vervoer.
Proteïene wat vir sekresie bestem is, word gewoonlik met 'n tydelike,
amino-eindpuntsekresiesein, wat 'n kritiese rol in die teiken en translokasie van hul
proteïenvrag speel, gesintetiseer. Heelwat pogings is in hierdie studie aangewend
om die molekulêre meganismes betrokke by hierdie prosesse te ontrafel, veral as
gevolg van hul toepaslikheid in 'n vinnig groeiende biotegnologiebedryf.
Die voordele van S. cerevisiae as 'n gasheer vir die uitdruk van rekombinante
proteïene is alombekend. S. cerevisiae het egter ook verskeie nadele, waaronder die
relatiewe lae produkopbrengs die belangrikste is.
Teen hierdie agtergrond, is verskillende sekresieseine met mekaar vergelyk met
die doelom die sekresie van die LKA 1 en LKA2 a-amilasegene vanuit die
S. cerevisiae-uitdrukkingsisteem te verbeter. Die gis Lipomyces kononenkoae is
bekend vir sy vermoeë om rou stysel af te breek en 'n verbeterde sekresie van sy
amilasegene vanuit S. cerevisiae baan die weg vir 'n moontlike een-stap
styselgebruiksproses. Drie stelle konstrukte is gemaak wat die LKA 1- en LKA2- gene
onafhanklik onder sekresiebeheer van onderskeidelik hul inheemse sekresiesein, die
S. cerevisiae paringsferomoonsekresiesein (MFa1) of die MFa1-sekresiesein met 'n
sintetiese koppelingspeptied aan die C-eindpunt (EEGEPK), plaas. Die insluiting van
'n koppelingspeptied in die laasgenoemde stel konstrukte verseker verbeterde Kex2p
proteolitiese prosessering van die sein/proteïenfusie. Rasse wat die amilasegene
onder beheer van hul inheemse sekresieseine uitdruk, het die beste saccharolitiese
aktiwiteit in die kultuurmedia getoon. In teenstelling hiermee, het rasse wat van die
sintetiese sekresiesein gebruik maak, die beste fermentasie-opbrengs getoon, maar
met 'n laer as verwagte ekstrasellulêre aktiwiteit. Ons vermoed dat die inheemse
amilaseseine as intramolekulêre begeleiers optree in die vou en prosessering van hul
proteïenpassasiers, wat lei tot verbeterde prosessering en ensiemaktiwiteit.
15
Brady, Dean. "Bioaccumulation of metal cations by yeast and yeast cell components." Thesis, Rhodes University, 1993. http://hdl.handle.net/10962/d1004107.
Full textAbstract:
The aim of the project was to determine whether a by-product of industrial fermentations, Saccharomyces cerevisiae, could be utilized to bioaccumulate heavy metal cations and to partially define the mechanism of accumulation. S. cerevisiae cells were found to be capable of accumulating Cu²⁺in a manner that was proportional to the external Cu²⁺ concentration and inversely proportional to the concentration of biomass. The accumulation process was only minimally affected by temperature variations between 5 and 40°C or high ambient concentrations of sodium chloride. The accumulation process was however considerably affected by variations in pH, bioaccumulation being most efficient at pH 5 - 9 but becoming rapidly less so at either extreme of pH. Selection for copper resistant or tolerant yeast diminished the yeast's capacity for Cu²⁺ accumulation. For this and other reasons the development of heavy metal tolerance in yeasts was deemed to be generally counterproductive to heavy metal bioaccumulation. The yeast biomass was also capable of accumulating other heavy metal cations such as c0²⁺ or Cd²⁺. The yeast biomass could be harvested after bioaccumulation by tangential filtration methods, or alternatively could be packed into hollow fibre microfilter membrane cartridges and used as a fixed-bed bioaccumulator. By immobilizing the yeast in polyacrylamide gel and packing this material into columns, cu²⁺, C0²⁺ or Cd²⁺ could be removed from influent aqueous solutions yielding effluents with no detectable heavy metal, until breakthrough point was reached. This capacity was hypothesized to be a function of numerous "theoretical plates of equilibrium" within the column. The immobilized biomass could be eluted with EDTA and recycled for further bioaccumulation processes with minor loss of bioaccumulation capacity. Yeast cells were fractionated to permit identification of the major cell fractions and molecular components responsible for metal binding. Isolation of the yeast cell walls permitted investigation of their role in heavy metal accumulation. Although the amino groups of chitosan and proteins, the carboxyl groups of proteins, and the phosphate groups of phosphomannans were found to be efficient groups for the accumulation of copper, the less effective hydroxyl groups of the carbohydrate polymers (glucans and mannans) had a similar overall capacity for copper accumulation owing to their predominance in the yeast cell wall. The outer (protein-mannan) layer of the yeast cell wall was found to be a better Cu²⁺ chelator than the inner (chitinglucan) layer. It appeared that the physical condition of the cell wall may be more important than the individual macromolecular components of the cell wall in metal accumulation. It was apparent that the cell wall was the major, if not the sole contributor to heavy metal accumulation at low ambient heavy metal concentrations. At higher ambient metal concentrations the cytosol and vacuole become involved in bioaccumulation. Copper and other metals caused rapid loss of 70% of the intracellular potassium, implying permeation of the plasma membrane. This was followed by a slower "leakage" of magnesium from the vacuole which paralleled Cu²⁺ accumulation, suggesting that it may represent some form of ion-exchange. An intracellular copper chelating agent of approximately 2 kDalton molecular mass was isolated from copper tolerant yeast. This chelator was not a metallothionein and bound relatively low molar equivalents of copper compared to those reported for metallothionein. Treatment of the biomass with hot alkali yielded two biosorbents, one soluble (which could be used as a heavy metal flocculent), and an insoluble biosorbent which could be formed into a granular product to be used in fixed-bed biosorption columns. The granular biosorbent could accumulate a wide range of heavy metal cations in a semispecific manner and could be stored in a dehydrated form indefinitely, and rehydrated when required. Bioaccumulation by live algae was investigated as an alternative to yeast based processes. Various strains of algae, of which Scenedesmus and Selenastrum were the most effective, were found to be capable of accumulating heavy metals such as Cu²⁺, Pb²⁺ and Cr³⁺.
16
Smit, Annel. "Maltotriose transport in yeast." Thesis, Stellenbosch : Stellenbosch University, 2007. http://hdl.handle.net/10019.1/21760.
Full textAbstract:
Dissertation (PhD)--University of Stellenbosch, 2007.<br>ENGLISH ABSTRACT: The conversion of sugar into ethanol and carbon dioxide is a process that has been
intertwined with human culture and long as civilized man has existed. This fermentation
process has been dominated by the micro-organism Saccharomyces cerevisiae and from
providing ancient seafaring explorers of a non perishable beverage to equipping bakers
with a raising agent to turn flour into bread; this organism with its fermentative potential,
has formed an essential part of most societies.
In more recent times, many industries still rely on this basic principle. The
complexities and efficiencies of the conversion of sugar into its various fermentative byproducts
have been studied and optimised extensively to meet the specific demands of
industries. Depending on the raw material used as starting point, the major beneficiaries of
the useful characteristics have been alcoholic beverage producers (wine, beer, and
whiskey amongst others), bakers (bread leavening) and biofuel producers.
One of the obstacles in fermentation optimisation is the sugar consumption
preferences displayed by the organism used. S. cerevisiae can consume a wide variety of
sugars. Depending on the complexities of its structures, it shows a preference for the
simpler saccharides. The fermentation of certain more complex sugars is delayed and runs
the risk of being left residually after fermentation. Many of the crops utilised in
fermentation-based products contain large amounts of starch. During the starch
degradation process many different forms of sugars are made available for fermentation.
Improved fermentation of starch and its dextrin products would benefit the brewing,
whiskey, and biofuel industries. Most strains of Saccharomyces ferment glucose and
maltose, and partially ferment maltotriose, but are unable to utilise the larger dextrin
products of starch. This utilisation pattern is partly attributed to the ability of yeast cells to
transport the aforementioned mono-, di- and trisaccharides into the cytosol. The
inefficiency of maltotriose transport has been identified as the main cause for residual
maltotriose. The maltotriose transporting efficiency also varies between different
Saccharomyces strains.
By advancing the understanding of maltotriose transport in yeast, efforts can be
made to minimise incomplete fermentation. This aim can be reached by investigating the
existing transporters in the yeast cell membrane that show affinity for maltotriose. This
study focuses on optimising maltotriose transport through the comparison of the alpha
glucoside transporter obtained from different strains of Saccharomyces. Through specific
genetic manipulations the areas important for maltotriose transport could be identified and
characterised.
This study offers prospects for the development of yeast strains with improved maltose
and maltotriose uptake capabilities that, in turn, could increase the overall fermentation
efficiencies in the beer, whiskey, and biofuel industries.<br>AFRIKAANSE OPSOMMING: Die transformasie van suiker na etanol en koolstof dioksied is so oud soos die beskawing self, en dit is van die vroegste tye af onlosmaaklik met die mens se kultuur verbind. Hierdie fermentasie-proses word gedomineer deur die Saccharomyces cerevisiae mikroorganisme. Hierdie organisme het antieke seevaarders voorsien van ‘n nie-bederfbare drankie en van ouds af aan bakkers ‘n rysmiddel verskaf waarmee meel in brood verander kon word. As gevolg van hierdie fermenteringspotensiaal het hierdie organisme ‘n onmisbare rol in meeste beskawings gespeel. Baie industrieë is steeds op hierdie basiese beginsel gebou. Die kompleksiteite en effektiwiteit van die transformasie van suiker na sy verskeie gefermeenteerde neweprodukte is breedvoerig bestudeer en geoptimiseer om aan die spesifieke behoeftes van verskeie industrieë te voeldoen. Afhangend van die grondstowwe wat as beginpunt gebruik is, is die primêre begunstigdes van die fermentasie proses die alkoholiese drankprodusente (onder andere die wyn-, bier- en whiskey produsente), bakkers en biobrandstofprodusente. Die suikerverbruik-voorkeur van die organisme wat die fermentering fasiliteer is een van die struikelblokke in die optimisering van die proses. S. cerevisiae kan ‘n wye spektrum van suikers verbruik maar dit toon ‘n voorkeur vir die eenvoudiger suikers. Die fermentasie van sekere van die meer komplekse suikers is vertraag en loop die risiko om agtergelaat te word na fermentasie. Vele van die gewasse wat in die gefermenteerde produkte gebruik word bevat groot hoeveelhede stysel. Vele soorte suikers word gedurende die afbreek van die stysel beskikbaar gestel vir fermentasie. Die brouers-, whiskey- en biobrandstof industrieë sal almal voordeel trek uit die verbeterde fermentasie van stysel en sy gepaardgaande dekstrin produkte. Meeste Saccharomyces gisrasse fermenteer glucose en maltose; maltotriose word gedeeltelik gefermenteer, maar die meer komplekse dekstrien produkte gevind in stysel word nie gefermenteer nie. Hierdie verbruikerspatroon kan gedeeltelik toegeskryf word aan die vermoë van gisselle om die bogenoemde mono-, di- and trisaccharides in die sitosol op te neem. Die oneffektiwiteit van maltotriose transport is identifiseer as die hoofoorsaak van post-fermentatiewe, oortollige maltotriose. Die effektiwiteit van maltotriose transport verskil ook tussen verskillende Saccharomyces rasse. Pogings om onvolledige fermentasie te veminder kan bevorder word deur die kennis rondom maltotriose transport in gis uit te bou. Hierdie oogmerk kan bereik word deur die bestaande transporters in die gissel se membraan wat ‘n affiniteit vir maltotriose toon te ondersoek. Hierdie studie fokus op die optimisering van maltotriose transport deur die vergelyking van die alpha glucoside transporter (AGT1) wat van verskillende Saccharomyces rasse afkomstig is. Die areas wat relevant is tot maltotriose transport kon deur spesifieke genetiese manipulasies identifiseer en gekarakteriseer word. Hierdie studie bevorder die vooruitsig op die ontwikkeling van gisrasse met verbeterde
maltose en maltotriose transport vermoëns wat op sy beurt weer kan aanleiding gee tot die
verbeterde fermentasie effektiwiteit in die bier, whiskey en biobrandstof industrieë.
17
Trollope, Kim. "Investigation of resveratrol production by genetically engineered Saccharomyces cerevisiae strains." Thesis, Stellenbosch : University of Stellenbosch, 2006. http://hdl.handle.net/10019.1/2230.
Full textAbstract:
Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2006.<br>Resveratrol is a phytoalexin that is produced in the leaves and skins of grape berries in response to biotic and abiotic factors. Substitution and polymerisation of resveratrol units produce an array of compounds which form part of the active disease defence mechanism in grapevine.
Wine is one of the major sources of resveratrol in the human diet. Resveratrol is one of the phenolic compounds present in wine that mediates protective effects on human health. It has been shown to prevent the development of cardiovascular disease, cancer and pathogenesis related to inflammation.
Red wines contain higher levels of resveratrol than white wines owing to extended maceration times during fermentation on the skins. During white wine vinification skin contact is limited as skins are removed prior to fermentation. Thus, the extraction of resveratrol into white wines is minimal. The principal focus of our research is the development of a wine yeast strain capable of resveratrol production during grape must fermentation. It is proposed that red and white wines produced with such a resveratrol-producing yeast will contain elevated levels of resveratrol, and that added health benefits may be derived from their consumption.
Initial work done in our laboratory established that expressing multiple copies of the genes encoding coenzyme A ligase (4CL216) and resveratrol synthase (vst1) in laboratory yeast enabled the yeast to produce resveratrol, conditional to the supplementation of the growth medium with p-coumaric acid. This study focused on the optimisation of resveratrol production in Saccharomyces cerevisiae. It involved the integration and constitutive expression of 4CL216 from hybrid poplar and vst1 from grapevine. Integration and expression of these genes in three laboratory strains was confirmed by Southern and Northern blot analyses.
The evaluation of resveratrol production by yeast required the initial optimisation of the analytical techniques. We optimised the method for sample preparation from the intracellular fraction of yeast and devised a procedure for the assay of the extracellular fractions. The LCMSMS method was further developed to encompass detection and quantification of other compounds related to resveratrol production in yeast.
Comparison of resveratrol production in three different yeast genetic backgrounds indicated that the onset of production and the resveratrol yield is yeast strain dependent. Precursor feeding studies indicated that p-coumaric acid availability was a factor limiting maximal resveratrol production. Early indications were obtained that endogenously-produced resveratrol may have an impact on yeast viability during extended culture periods.
This study has broadened our understanding of the resveratrol production dynamics in S. cerevisiae and provided important indications as to where further optimisation would be beneficial in order to optimally engineer a wine yeast for maximal resveratrol production.
18
Armstrong, Gareth Owen. "The production of resveratrol by wine yeast." Thesis, Stellenbosch : Stellenbosch University, 2001. http://hdl.handle.net/10019.1/52557.
Full textAbstract:
Thesis (MSc)--Stellenbosch University, 2001.<br>ENGLISH ABSTRACT: Grapevine is constantly under attack from a wide variety of pathogens including viruses,
bacteria and fungi. In order to ensure survival, the grapevine has developed a vast array of
defense mechanisms to combat invading organisms. A key element of this disease
resistance is the production of phytoalexins, of which resveratrol is the primary component.
The synthesis of resveratrol, together with other structural and biochemical defense
mechanisms equips the plant to combat a number of pathogens resulting in the production
of healthy grapes for the vinification of top quality wine. As part of the active disease
response resveratrol is synthesised de novo in the berry skin at the site of infection, on
recognition of the pathogen. Here it is able to limit the damage caused by the pathogen as
well as preventing it from spreading. This gives the plant the opportunity to initiate its
systemic acquired resistance thereby protecting the rest of the plant and preventing
secondary infections.
The fermentation of red wine on the grape skins allows for the extraction of resveratrol
from the skin into the wine. Red wines therefore have a significantly higher concentration
of resveratrol than white varieties, which contain little or no resveratrol at all. It is for this
reason that the moderate consumption of wine, in particular red wine, is synonymous with
a healthy lifestyle. The antioxidant and anti-inflammatory activities of resveratrol are
important contributors to the cardiovascular benefits derived from the consumption of red
wine. It now seems, however, that significant cardiovascular protection is derived from the
synergistic action of resveratrol, the polyphenols and the alcohol in wine.
With the wholesomeness of any food or beverage being of extreme importance, the
aim of this project was to manipulate wine yeast to produce resveratrol during
fermentation. This required the introduction of an entire metabolic pathway, by integrating
plant genes into the yeast. Resveratrol synthase utilises three malonyl-CoA and one pcoumaroyl-
CoA molecules to produce one molecule of resveratrol, Saccharomyces
cerevisiae produces malonyl-CoA but no p-coumaroyl-CoA. Therefore, the following genes
were obtained to enable yeast to produce p-coumaroyl-CoA: PAL, encoding phenylalanine
ammonia-lyase to convert phenylalanine into cinnamic acid; C4H, encoding cinnamate-4-
hydroxlyase to convert cinnamic acid into p-coumaric acid; and 4CL9 or 4CL216 encoding
CoA-ligases to convert the p-coumaric acid into p-coumaroyl-CoA. To attain high-level
expression, the genes were subcloned under the control of the phosphoglycerate kinase
gene (PGK1) promoter and terminator. Due to integration problems with these expression
cassettes and the fact that the yeast was able to consume p-coumaric acid, the 4CL9,
4CL216 and Vst1 (encoding resveratrol synthase) genes were subcloned under the control
of the alcohol dehydrogenase (ADH2) and PGK1 promoters into episomal plasmids,
respectively. A laboratory yeast strain containing both the Vst1 and 4CL9, or the Vst1 and
4CL216 genes was evaluated for its ability to utilise p-coumaric acid and produce
resveratrol. Northem analysis confirmed that the Vst1, 4CL9 and 4CL216 genes were transcribed and over-expressed compared to the control strain. The transformants
expressing the CoA-ligase genes utilised the p-coumaric acid faster than the control,
although it was not possible to determine whether p-coumaroyl-CoA was produced. No
resveratrol was produced under the assay conditions used. The results indicated that the
yeast is unable to produce active resveratrol synthase, which is required to catalyse the
final reaction in the production of resveratrol. Posttranslational modification, such as overglycosylation
and disulphide formation, of the heterologous protein in yeast has been
indicated as the possible reason for the lack of enzyme activity. This introduces an exciting
area of research for the development of biotechnological tools with the ability to increase
the production of active heterologous proteins in yeast.<br>AFRIKAANSE OPSOMMING: Wingerde word voortdurend deur 'n groot verskeidenheid patogene, insluitende virusse,
bakteriee en swamme, aangeval. Ten einde oorlewing te verseker, het die wingerdstok In
wye reeks verdedigingsmeganismes ontwikkel om weerstand te bied teen indringerorganismes.
'n Belangrike faktor in hierdie weerstand teen siektes is die produksie van
fitoaleksiene, waarvan resveratrol die hoofkomponent is. Oeur die sintese van resveratrol,
asook ander strukturele en biochemiese verdedigingsmeganismes, word die plant
toegerus om weerstand te kan bied teen In hele aantal patogene ten einde gesonde
druiwe te produseer wat gebruik kan word vir die vinifikasie van topgehalte wyn. As deel
van die aktiewe reaksie teen siektes, word resveratrol de novo in die dop van die korrel by
die plek van infeksie gesintetiseer sodra 'n patogeen herken word. Hier kan dit die skade
deur die patogeen veroorsaak, beperk en verhoed dat dit versprei. Oit gee aan die plant
die geleentheid om sy sistemies-verworwe weerstand te inisieer, en daardeur die res van
die plant te beskerm, sowel as sekondere infeksies te verhoed.
Die fermentasie van rooiwyn op die druifdoppe maak voorsiening vir die ekstraksie van
resveratrol uit die dop na die wyn. Die konsentrasie van resveratrol in rooiwyn is dus
beduidend hoer as in die wit varietelte, wat geen of baie min resveratrol bevat. Oit is dan
juis die rede waarom die matige inname van wyn, veral rooi wyn, gesien word as In
integrale deel van 'n gesonde leefwyse. Resveratrol se aktiwiteit as antioksidant en antiinflammatoriese
middel lewer In belangrike bydrae tot die kardiovaskulere voordele wat
verkry word uit die inname van rooiwyn. Oit blyk egter nou dat die beduidende
kardiovaskulere beskerming gesetel is in die sinergistiese werking van resve ratro I, die
polifenole en die alkohol in wyn.
Aangesien die heilsaamheid van enige voedsel of drank van die uiterste belang is,
was dit die doel van hierdie projek om wyngis te manipuleer ten einde tydens die
fermentasieproses resveratrol te produseer. Hiervoor moes 'n volledige metaboliese pad
daargestel word deur plantgene in die gis te inkorporeer. Resveratrol-sintase maak
gebruik van drie maloniel-KoA-molekules en een p-kumarotel-Kos-molekule om een
molekule resveratrol te produseer. Saccharomyces cerevisiae produseer maloniel-KoA,
maar nie p-kumaroiel-Kcs, nie. Oie volgende gene is dus aangewend om die gis in staat
te stel om p-kumarolel-Koe, te produseer: PAL, wat fenielalanien-ammoniak-liase
enkodeer om fenielalanien om te sit na kaneelsuur; C4H, wat sinnamaat-4-hidroksliase
enkodeer om kaneelsuur om te sit na p-kumaarsuur; en 4CL9 of 4CL216 wat KoA-ligases
enkodeer om p-kumaarsuur om te sit na p-kumarolel-Kos, Om hoevlak-uitdrukking te
verkry, is die gene gesubkloneer onder beheer van die fosfogliseraat-kinase-geen(PGK1)-
promotor en -terminator. As gevolg van integrasieprobleme met hierdie
uitdrukkingskassette en die feit dat die gis die p-kumaarsuur kon verteer, is die 4CL9-,
4CL216- en Vst1- (wat resveratrol-sintase enkodeer) gene na episomale plasmiede
gesubkloneer onder beheer van die alkohol-dehidrogenase(ADH2)- en PGK1-promotors onderskeidelik. 'n Laboratorium-gisstam wat 6f beide die Vst1-geen en die 4CL9-geen, 6f
die Vst1-geen en die 4CL216-geen bevat het, is geevalueer vir die verrnoe om pkumaarsuur
te benut en resveratrol te produseer. Noordelike klad analises het bevestig
dat die Vst1-, 4CL9- en 4CL216-gene getranskribeer en ooruitgedruk was in vergelyking
met die kontrole-stam. Die transformante wat die KoA-ligases uitgedruk het, het die pkumaarsuur
vinniger benut as wat die kontrole dit gedoen het, alhoewel dit nie moontlik
was om vas te stel of o-kurnarotel-Kos, geproduseer is nie. Met die essai-kondisies wat
gebruik is, is geen resveratroI geproduseer nie. Die resultate het daarop gedui dat die gis
nie daartoe in staat is om aktiewe resveratrol-sintase, wat nodig is vir die katalise van die
finale reaksie in die produksie van resveratrol, te produseer nie. Naomsettingsmodifikasies
van die heteroloe protelen in die gis, soos oor-glikosilasie en
disulfiedvorming, is aangewys as die moontlike rede vir die gebrek aan ensiemaktiwiteit.
Dit stel In opwindende veld vir verdere navorsing voor, naamlik die ontwikkeling van
biotegnologiese middele met die vermoe om die produksie van aktiewe heteroloe
protelene in gis te verhoog.
19
Leukes, W. "Development and characterisation of a membrane gradostat bioreactor for the bioremediation of aromatic pollutants using white rot fungi." Thesis, Rhodes University, 1999. http://hdl.handle.net/10962/d1004092.
Full textAbstract:
Bioremediation of aromatic pollutants using the ligninolytic enzymes of the white rot fungi has been thoroughly researched and has been shown to have considerable potential for industrial application. However, little success in scale-up and industrialisation of this technology has been attained due to problems associated with the continuous production of the pollutant-degrading enzymes using conventional bioreactor systems. The low productivities reported result from the incompatibility of conventional submerged culture reactor techniques with the physiological requirements of these fungi which have evolved on a solid-air interface, viz. wood. The enzymes are also produced only during the stationary phase of growth and can therefore be regarded as secondary metabolites. This study reports the conceptualisation, characterisation and evaluation of a novel bioreactor system as a solution to the continuous production of idiophasic pollutant degrading enzymes by the white rot fungus Phanerochaete chlysosporium. The reactor concept evolved from observation of these fungi in their native state, i. e. the metabolism of lignocellulosic material and involves the immobilisation of the organism onto a capillary ultrafiltration membrane. Nutrient gradients established across the biofilm, an inherent characteristic of fixed bed perfusion reactors, are exploited to provide both nutrient rich and nutrient poor zones across the biofilm. This allows growth or primary metabolism in the nutrient rich zone, pushing older biomass into the nutrient poor zone where secondary metabolism is induced by nutrient starvation. In effect, this represents a transformation of the events of a batch culture from a temporal to a spatial domain, allowing continuous production of secondary metabolites over time. Direct contact of the outer part of the biofilm with an air stream simulated the solid-air interface of the native state of the fungus. In order to facilitate the practical application of the membrane gradostat reactor (MGR) concept, conventional capillary membranes and membrane bioreactor modules were first evaluated. These were found to be unsuitable for application of the MGR concept. However, critical analysis of the shortcomings of the conventional systems resulted in the formulation of a set of design criteria for the development of a suitable membrane and module. These design criteria were satisfied by the development of a novel capillary membrane for membrane bioreactors, as well as a transverse flow membrane module, which is a novel approach in membrane bioreactor configuration. For the physiological characterisation of the MGR concept, a single fibre bioreactor unit was designed, which allowed destructive sampling of the biofilm for analysis. Using this system, it was shown that distinct morphological zones could be observed radially across the mature biofilm obtained through MGR operation. That these morphotypes do represent the temporal events of a typical batch culture in a spatial domain was confirmed by following the morphological changes occurring during batch culture of the immobilised fungus where the onset of primary and secondary metabolic conditions were manipulated through control of the nutrient supply. The different morphotypes were correlated to distinct growth phases by comparison of the morphology to the secretion of known enzymatic markers for secondary metabolism, viz. succinate dehydrogenase and cytochrome C oxidoreductase. Detailed structure-function analysis of the biofilm using transmission electron microscopy and adapted enzyme cytochemical staining techniques showed that the biofilm appeared to operate as a co-ordinated unit, with primary and secondary metabolism apparently linked in one thallus through nutrient translocation. This study provided new insights into the physiology of P. chrysosp,o rium and a detailed descriptive model was formulated which correlates well to existing models of wood degradation by the white rot fungi (WRF). Evaluation of the process on a laboratory scale using a novel transverse flow membrane bioreactor showed that a volumetric productivity of 1916 U.L.⁻¹day⁻¹ for manganese peroxidase, one of the pollutant degrading enzymes, could be attained, corresponding to a final concentration of 2 361 U.L.⁻¹ This may be compared to the best reported system (Moreira el at. 1997), where a volumetric productivity of 202 U.L.⁻¹day⁻¹was achieved with a final concentration of 250 U.L.⁻¹ However, MGR productivity is yet to be subjected to rigorous optimisation studies. The process could be operated continuously for 60 days. However, peak productivity could not be maintained for long periods. This was found to be due to physical phenomena relating to the fluid dynamics of the system which caused fluid flow maldistribution, which would have to be resolved through engineering analysis. In evaluation of the MGR concept for aromatic pollutant removal, in this case ρ- cresol, from growth medium, good performance was also achieved. The VmaxKm calculated by linear regression for the MGR was 0.8 (R² = 0.93), which compared favourably to that reported by Lewandowski et al. (1990), who obtained a Vmax/Km of 0.34 for a packed bed reactor treating chlorophenol. It was concluded that the MGR showed suitable potential to warrant further development, and that the descriptive characterisation of the biofilm physiology provided a sufficient basis for process analysis once engineering aspects ofthe system could be resolved.
20
Bulkan, Gülru. "Valorization Of Whole Stillage With Filamentous Fungi Cultivation Using Membrane Bioreactors." Thesis, Högskolan i Borås, Akademin för textil, teknik och ekonomi, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:hb:diva-26252.
Full textAbstract:
A significant by-product of bioethanol plants is whole stillage, commonly used to produce animal feed due to its nutritious value, has a potential to be used to produce various value-added products while eliminating a costly process step is an alternative approach. In this study, production and separation of additional ethanol, fungal biomass and enzyme were successfully achieved with the cultivation in membrane bioreactors in batch process condition. Process optimization studies regarding fermentation and filtration conditions were carried out. Up to 10.4 g/l ethanol per litre of used whole stillage can be produced in simultaneous saccharification and fermentation (SSF) condition without any pH adjustment and additional pretreatment step. Also, 50% diluted whole stillage provided 87% higher ethanol production comparing to non-diluted medium. Moreover, 71 % higher biomass production was obtained with the filtrate of 50% diluted whole stillage comparing to 25% diluted one. Considering the achieved results, a two-stage cultivation using SHF (Separate Hydrolysis and Fermentation) strategy in membrane bioreactors for separation of ethanol, lignin-rich stream, protein-rich fungal biomass and enzymes was proposed. The present thesis showed that the integration of filamentous fungi with membrane bioreactors can increase the range of products that can be produced from whole stillage.
21
Beckhouse, Anthony Gordon Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "The transcriptional and physiological alterations in brewers yeast when shifted from anaerobic to aerobic growth conditions." Awarded by:University of New South Wales. School of Biotechnology and Biomolecular Sciences, 2006. http://handle.unsw.edu.au/1959.4/24201.
Full textAbstract:
Yeast are exposed to many physical and chemical stresses when used in large-scale industrial fermentations, particularly the initial stages in which yeast are shifted from anaerobic storage to aerated wort. This work investigated the transcriptional and physiological responses of yeast that had been shifted from anaerobic to aerobic growth conditions. Microarray technology was employed to determine the transcriptional changes that occurred in the first hour of a pilot-plant fermentation compared to the 23rd hour. It was found that over 100 genes were up-regulated initially including genes involved in the synthesis of the essential membrane sterol ergosterol and genes for the protection of cells against oxidative stress. It was also determined that cells which accumulate ergosterol precursors in the absence of ergosterol were more sensitive to exogenous oxidative stresses, indicating a role for ergosterol in oxidative stress tolerance. Aeration of anaerobically grown cells did not affect their growth kinetics or viability. However, anaerobically grown cells were hypersensitive to exogenous oxidative stress compared to their aerobic counterparts. Anaerobic cells that underwent a short period of aeration prior to treatment with hydrogen peroxide generated a tolerance to the oxidant, indicating that the period of aeration produced an adaptive-like response. Microarray analysis of the cells during the period of aeration showed that representative genes from the oxidative stress response family were up-regulated rapidly and it was determined that the response was controlled by the Yap1p and Skn7p transcription factors. Deletion of the transcription factor genes indicated that they were responsible for the creation of tolerance to oxidant. Target gene products of the two transcription factors (Gpx2p, Gsh1p and Trx2p) were shown to be induced during the shift to aeration; however, the glutathione redox balance did not seem to be affected as the cells were shifted from highly reduced to oxidising environments. Unexpectedly, it was discovered that genes involved in the synthesis of amino acids were up-regulated during anaerobic growth and stringently downregulated upon aeration of cells. The transcriptional activator of those genes (Gcn4p) was essential for growth in anaerobic media which included amino acid supplementation.
22
Giraldo, Marielle Aleixo [UNESP]. "Purificação e caracterização bioquímica da invertase extracelular produzida pelo fungo filamentoso Aspergillus terreus." Universidade Estadual Paulista (UNESP), 2011. http://hdl.handle.net/11449/87963.
Full textAbstract:
Made available in DSpace on 2014-06-11T19:23:04Z (GMT). No. of bitstreams: 0
Previous issue date: 2011-05-09Bitstream added on 2014-06-13T20:10:32Z : No. of bitstreams: 1
giraldo_ma_me_araiq.pdf: 658373 bytes, checksum: 20a8019dced41467e4d92b76d704ce76 (MD5)<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)<br>Os microrganismos, de modo especial os fungos filamentosos, possuem papel fundamental na decomposição de matéria orgânica, sendo interessantes como modelos para realização de diferentes estudos biológicos. Além disso, são de fácil manejo e as condições de cultivo podem ser facilmente adaptadas em laboratório. Outra vantagem é, geralmente, o baixo custo e o fácil acesso aos nutrientes necessários para o crescimento dos mesmos. Entre os microrganismos, os fungos filamentosos têm se destacado na obtenção de enzimas de interesse biotecnológico, como é o caso das invertases, as quais podem ser empregadas nas indústrias de alimentos e bebidas. As invertases (EC 3.2.1.26) são hidrolases que podem ser encontradas em uma grande variedade de organismos, realizando a hidrólise da ligação - D-frutofuranosídica, agindo sobre a sacarose, gerando como produtos D-glicose e D-frutose, em quantidades equimolares. Essa mistura é conhecida como açúcar invertido e é geralmente utilizada pelas indústrias de alimentos. Desta maneira, o objetivo deste trabalho foi estudar a produção de invertase extracelular pelo fungo filamentoso Aspergillus terreus, em fermentação submersa e em fermentação em substrato sólido, bem como purificar e caracterizar a enzima bioquimicamente. Entre os diversos meios de cultura testados em FSbm, a maior produção invertásica foi obtida em meio M5 mantido em agitação orbital (100 rpm) a uma temperatura de 30ºC, por um período de 48 horas de incubação. Já na FSS, o melhor período de incubação foi de 72 horas, também a 30ºC. Entre todas as fontes de carbono testadas, a maior produção invertásica foi obtida utilizandose farinha de centeio para a FSbm e soja moída para FSS. A enzima iv extracelular obtida em FSbm foi purificada 139 vezes com uma recuperação de 11%. A invertase extracelular...<br>The microorganisms, especially filamentous fungi, have a fundamental role in the decomposition of organic matter, and they are interesting as models for carrying out different biological studies. Moreover, they are easy to handle, and their growing conditions can be easily adapted in the laboratory. Another advantage is, generally, the low cost and easy access to the nutrients needed for their growth. Among the microorganisms, filamentous fungi have been essential in obtaining enzymes of biotechnological interest, such as invertase, which may be employed in the food and beverage industries. The invertases (EC 3.2.1.26) are hydrolases that can be found in a great variety of organisms, performing the hydrolysis of the -D fructofuranosidic bond acting on sucrose, generating products such as D-glucose and D-fructose, in equimolar amounts. This mixture is known as inverted sugar and it is commonly used by food industries. This way, the aim of this work was to study the production of extracellular invertase by the filamentous fungus Aspergillus terreus in submerged fermentation and solid state fermentation, as well as its purification and biochemical characterization. Among the various media tested in FSbm, the highest yield of invertase was obtained by using M5 medium under orbital agitation (100 rpm) at 30°C for 48 hours of incubation. In the FSS, the best incubation period was 72 hours, also at 30°C. Among all carbon sources tested, the highest invertase production was obtained using rye flour for FSbm and soybean meal for FSS. The extracellular enzyme obtained from FSbm was purified 139 fold with 11% recovery. The extracellular invertase of A. terreus is a heterodimer of native vi molecular mass of 74.67 kDa consisting of two subunits, one of 46.77 kDa and another of 26.92 kDa determined... (Complete abstract click electronic access below)
23
Ndlovu, Thulile. "Mannoprotein production and wine haze reduction by wine yeast strains." Thesis, Stellenbosch : Stellenbosch University, 2012. http://hdl.handle.net/10019.1/71938.
Full textAbstract:
Thesis (PhD)--Stellenbosch University, 2012.<br>ENGLISH ABSTRACT: Wine protein haze formation is a major challenge for wine makers, and several wine clarifying agents such as bentonite are used in the industry to protect wine from this occurrence. However, clarifying agents may have an undesirable impact on wine quality. Yeast mannoproteins have been shown to possess haze-protective properties, while also positively impacting on the sensorial properties of the product. However, while such mannoproteins are released into the wine during the wine making process, the amounts are low and therefore of limited oenological significance. However, and although commercial wine yeast strains display significant genotypic and phenotypic diversity, no broader assessment of haze protective activity and of mannoproteins release by different wine yeast strains has been undertaken.
In this study, several yeast strains were screened for their impact on wine haze formation in Chardonnay must and in a grape juice model system. The data show that strains of the species Saccharomyces paradoxus possess better haze protective properties than the common Saccharomyces cerevisiae wine yeast strains. Differences in the nature of the proteins released by these two species were investigated, and indicated that several mannoproteins were released at significantly higher levels by S. paradoxus, and that some of these proteins might indeed contribute to the haze-protective activity. A further exploration of yeast cell wall properties indicated that the cell walls of haze-protective S. paradoxus strains contained higher levels of chitin than non-haze protective strains. Grape chitinases are likely to be primarily responsible for wine haze formation, and the data clearly demonstrate that these enzymes are able to bind to the yeast cell walls, and that strains with higher amounts of chitin in the cell wall will bind more chitinases. This finding suggests that the haze-protective nature of the strains is at least in part linked to the chitin levels of the strains.
Furthermore, the impact of some genetic modifications in two wine strains (namely S. cerevisiae VIN13 and S. paradoxus RO88) suggests that several proteins contribute to wine haze protection. However, none of the mannoprotein-encoding flocculation genes, FLO1, FLO5, and FLO11 showed any impact on this property.
Further studies are required to assess the full impact of the S. paradoxus strains on haze protection. In particular, the possible use of such strains as starter cultures or the use of S. paradoxus yeast hulls as clarifying agent needs to be further explored.<br>AFRIKAANSE OPSOMMING: Wyn proteïen-waas vorming is 'n groot uitdaging vir wynmakers en verskeie wyn verhelderings agente soos bentoniet word in die wynbedryf gebruik om wyn te beskerm teen die vorming van waas. Hierdie verheldering agente het egter 'n ongewenste impak op wynkwaliteit. Gis mannoproteïene is uitgewys as proteïene met moontlike waas-beskermende eienskappe wat ook 'n positiewe uitwerking op die sensoriese eienskappe van die produk het. Al word hierdie mannoproteïene egter vrygestel in die wyn tydens die wynmaak proses, is die hoeveelhede oor die algemeen te laag om van wynkundige belang te wees. Verder, ten spyte van die beduidende genotipiese en fenotipiese diversiteit van kommersiële wyngisrasse is daar nog geen breër assessering van die waas beskermende aktiwiteit van mannoproteïene, vrygestel deur verskillende rasse, tot dusver onderneem nie.
In hierdie studie is verskeie gisrasse gekeur vir hul impak op wyn waas-vorming in Chardonnay mos en ook in 'n model druiwesap. Die data wys dat rasse van die spesie Saccharomyces paradoxus besit beter waas beskermende eienskappe as die algemene Saccharomyces cerevisiae wyngisrasse. Verskille in die aard van die proteïene wat vrygestel is deur hierdie twee spesies is ondersoek, en dit is aangedui aangedui dat verskeie mannoproteins vrygestel aan aansienlik hoër vlakke deur S. Paradoxus. Dit is ook aangedui dat sommige van hierdie proteïene wel bydra tot die waas-beskermende aktiwiteit.
'n Verdere verkenning van gis selwand eienskappe het aangedui dat die selwande van waas-beskermende rasse van S. paradoxus hoër vlakke chitien as nie-waas beskermende stamme bevat. Druiwe chitinases is waarskynlik hoofsaaklik verantwoordelik vir wyn waas vorming, en die data toon duidelik dat hierdie ensieme in staat is om te bind aan die gis selwande, en dat die stamme met hoër vlakke chitien in die selwand meer chitinases sal bind. Hierdie bevinding dui daarop dat die waas-beskermende aard van die stamme ten minste gedeeltelik gekoppel is aan die chitien vlakke van die stamme. Die impak van sekere genetiese modifikasies in twee verskillende gisrasse, naamlik die S. cerevisiae ras VIN13 en die S. paradoxus ras RO88, dui verder daarop dat verskeie proteïene dra by tot die beskerming teen wyn waas. Geeneen van die mannoprotein-koderende flokkulasie gene, FLO1, FLO5 en FLO11 het egter 'n impak op hierdie eienskap nie.
Verdere studies is nodig om die volle impak van die S. paradoxus rasse op waas beskerming te assesseer. In die besonder, die moontlike gebruik van sulke rasse as 'n inkolasie kultuur of die gebruik van S. paradoxus gis doppe as verheldering agent moet verder ondersoek word.
24
Nguyen, Jack. "Development of a high throughput reporter system using β-Galactosidase in the yeast : Pichia Pastoris". Scholarly Commons, 2005. https://scholarlycommons.pacific.edu/uop_etds/623.
Full textAbstract:
Pichia pastoris is a methylotrophic yeast gaining acclamation for its capabili ties in heterologous protein expression. In contrast to other host organisms such as bacteria or mammalian cells, P. pastoris offers many advantages over its counterparts. For example, P. pastoris is cost-effective in that it can grow to high cell densities on simple media. The optional use of a constitutive (GAP) or inducible (A OXI) promoter and the ab ility to perfo1m post-translational protein modifications are additional qualities that make for a powerful heterologous expression system. This study focuses on harnessing the benefits described to develop a high-throughput reporter system for the screening of potential super-secreting mutant strains of P. pastoris. Plasmid constructs were engineered with the lacZ reporter gene, which encodes for the β-galactosidase protein, and fused to the S. cerevisiae MATa signal sequence. Expression plasmids were successfully transformed in P. pastoris strain yGS 115 followed by induction. Western blot analyses confirm the expression of β-galactosidase and colorimetric activity assays further validate enzymatic function. A mutant library containing cis- and/or trans-acting mutations was created by treating P. pas loris clones harboring the β-galactosidase expression plasmid with ultraviolet (UV) radiation. A colorimetric plate assay was combined with a replica-plating system that enabled the screening of thousands of potential super-secreting mutant colonies in a high-throughput format. This study sheds light onto our current understanding of secretion in yeast and further contributes to developing P. pastoris into a valuable heterologous protein expression system.
25
Forde, Kohler Lois J. "The effects of ophiostoma piliferm on wood pulp : investigation." Thesis, Georgia Institute of Technology, 1995. http://hdl.handle.net/1853/5982.
Full text
26
Strauss, Colin Earl, and University of Lethbridge Faculty of Arts and Science. "Development of Pichia pastoris as a ruminal escape vehicle." Thesis, Lethbridge, Alta. : University of Lethbridge, Faculty of Arts and Science, 2000, 2000. http://hdl.handle.net/10133/148.
Full textAbstract:
The yeast expression system Pichia pastoris was investigated as an encapsulation technology capable of serving as a rumen escape vehicle. Cellularly encapsulated protein is protected from the ruminal environment so long as the cell membrane, which surrounds and isolates the intracellular protein is physically intact. Intracellular expression of Green Fluorescent Protein (GFP) allows for the monitoring of cellular integrity as necessary for the protection of encapsulated protein from ruminal proteases. Upon cellular lysis GFP is exposed to extracellular proteases which result in both the proteolytic degradation of the protein-based GFP chromophore and its associated fluorescence. Visualization of rumen fluid under epifluorescent microscopy revealed a high level of background autofluorescence owing to the fluorescent plant particles, microbes, and fluorescent compounds therein. Visualization of GFP in rumen fluid can be optimized through GFP variant selection, filter set design, and light source selection based on bulb emission spectra. Incubation of intracellular GFP expressing P. pastoris in batch culture ruminal in vitro simulations demonstrated that 93%, 97%, and 25% of the P. pastoris inoculum maintained cellular integrity in clarified rumen fluid, bacterial fraction of rumen fluid, and whole rumen fluid, respectively, when incubated over 36 to 48 h. Continuous fermentation in vitro rumen simulations (Rusitec) demonstrated a P. pastoris escape rate of 19% when added daily to fully adapted Rusitec vessels having a dilution rate of 0.75d-1. Abomasal in vitro simulations demonstrated that 84% of the P. pastoris inoculum was lysed within 12 h, as necessary for the release of encapsulated protein. P.pastoris may be an effective post-fuminal delivery vehicle, provided that similar results are obtained in vivo.<br>xiv, 120 leaves : ill. ; 28 cm.
27
Souza, Helenires Queiroz de. "Detecção de antimicrobianos e enzimas de basidiomycetes da Amazônia, Brasil." Universidade Federal do Amazonas, 2006. http://tede.ufam.edu.br/handle/tede/3133.
Full textAbstract:
Made available in DSpace on 2015-04-20T12:31:52Z (GMT). No. of bitstreams: 1
Helenires Quiroz de Souza.pdf: 4253901 bytes, checksum: 8968bf2a9d9d6e181ad4528e0b805f1b (MD5)
Previous issue date: 2006-05-31<br>The Class Basidimycetes is formed by those fungi called mushroom ande ar-of-wood, beyond
others groups less known. Due to the importance of this group of fungi, the biotechnological
potential was investigated in this study. The mushroom were collected in forest areas of
campus of University of Amazonas (UFAM), Bosque da Ciência and campus V8 (INPA),
Reserva de Campina (INPA), located in BR 174, km 45, Manaus, Amazonas and Urucu
(Coari/AM). After the isolation micelial, the fungi were evaluated as regards the growth at
280C, for 30 days in liquid cultures GPY and BD to determine the antimicrobial activity. The
samples were filtered and the mycelium was dry to determine the biomass. The culture
filtrates had been tested against Ralstonia solanacearum, agent of bacterial wilt of several
vegetal species, Corynespora sp., Colletotrichum sp., Escherichia coli, Staphylococcus
aureus, Bacillus cereus e Salmonella anatum. For an enzyme production study, the fungus
had been cultivated in liquid culture and tested in solid medium for proteases, amylases,
cellulases, pectinases and phenoloxidases. In another stage, the production of amylases and
proteases in different nutritives sources was studied. Sixty samples of Basidiomycetes were
collected from February to June 2003. The families Agaricaceae, Auriculariaceae,
Cantharellaceae, Ganodermataceae, Hygrophoraceae, Polyporaceae, Russulaceae, Stereaceae,
Tremellaceae e Tricholomataceae had been identified. The Reserva de Campina was the one
that presented greater diversity of fungi. The results demonstrated inhibitory effect of culture
filtrates against bacterial strains tested. The species E. coli, S. anatum and B. cereus, beyond
inhibition halos, had presented halos of grown stimulus. It was not effect against the fungi
Corynespora sp. and Colletotrichum sp.. The culture filtrate Trametes sp. presented greater
activity of inhibition. The medium BD favored greater production of dry biomass. The
production of protease an amylase was detected for all the isolated ones, four produced
amylases, five produced phenoloxidases and one produced pectinase. For production of
amylases, fungi cultivated in medium added of wheat bran had gotten biggest halos. For
proteases, the biggest halos had been observed for the fungi grown in way with fish flour.<br>A classe Basidiomycetes é formada por aqueles fungos chamados de cogumelos e orelhas-depau,
além de outros grupos menos conhecidos. Devido à importância deste grupo de fungos, o
presente trabalho teve como objetivo investigar o seu potencial biotecnológico quanto à
produção de antimicrobianos e enzimas. Os basidiomas dos fungos foram coletados em áreas
de floresta do campus da UFAM, do Bosque da Ciência, Campus/V8 e Reserva de Campina
do INPA, localizada na BR 174, km 45, Manaus/AM e Urucu em Coari/AM. Após o
isolamento micelial, os fungos foram avaliados quanto ao crescimento radial em meio sólido.
Para verificar a produção de antimicrobianos, os fungos foram cultivados em meio líquido BD
e GPY durante 30 dias, em repouso a 28oC. Após esse período, o micélio foi seco para
determinar a biomassa e os filtrados das culturas foram testados contra Ralstonia
solanacearum, agente da murcha de várias espécies vegetais, Corynespora sp.,
Colletotrichum sp., Escherichia coli, Staphylococcus aureus, Bacillus cereus e Salmonella
anatum. Quanto à produção de enzimas, os fungos foram cultivados em meio líquido e
testados em meio sólido para proteases, amilases, celulases, pectinases e fenoloxidases. Em
outra etapa, foi estudada a produção de amilases e proteases em diferentes fontes nutricionais.
Foram coletados 60 fungos, de fevereiro a junho de 2003, e identificadas as famílias
Agaricaceae, Auriculariaceae, Cantharellaceae, Ganodermataceae, Hygrophoraceae,
Polyporaceae, Russulaceae, Stereaceae, Tremellaceae e Tricholomataceae. A Reserva de
Campina foi a que apresentou maior diversidade de fungos. Todas as 18 amostras de filtrados
de cultura dos Basidiomycetes, utilizadas nos bioensaios, apresentaram efeito inibitório contra
um ou mais microrganismos-teste. As espécies E. coli, S. anatum e B. cereus, além de halos
de inibição, apresentaram halos de estímulo de crescimento. Não foi observado efeito
inibitório contra os fungos Corynespora sp. e Colletotrichum sp. O filtrado do fungo
Trametes sp., cultivado em meio GPY, foi o que apresentou maior atividade de inibição. O
meio de cultura BD favoreceu maior produção de biomassa seca. Dos dez fungos estudados,
foi detectada a produção de proteases e celulases por todos os isolados, quatro produziram
amilases, cinco fenoloxidases e um pectinase. Para a produção de amilases, os fungos
cultivados em meio acrescido de farelo de trigo apresentaram os maiores halos. Para a
produção de proteases, os maiores halos foram observados para os fungos crescidos em meio
com farinha de peixe.
28
Russell, Ingrid Margaret. "The development of an immobilised-enzyme bioprobe for the detection of phenolic pollutants in water." Thesis, Rhodes University, 1999. http://hdl.handle.net/10962/d1006211.
Full textAbstract:
The possibility of developing an immobilised-enzyme bioprobe, based on mushroom polyphenol oxidase, for the purely biological detection and quantification of phenolic pollutants in water was investigated. Polyphenol oxidase catalyses the bioconversion of many phenolic compounds into quinone-related coloured products. Thus, in an immobilised form, the enzyme serves as a visible indicator of the presence and concentration of phenolic pollutants in water. The objective of this research was to develop a portable, disposable bioprobe incorporating polyphenol oxidase for this purpose. The intensity of the colour changes produced by the enzyme on reaction with p-cresol, p-chlorophenol and phenol was found to increase proportionally with increasing concentrations of these substrates in solution. Immobilisation of the enzyme on various supports did not appear to significantly affect the catalytic activity of the enzyme. The enzyme was immobilised by adsorption and cross-linking on polyethersulphone, nitrocellulose and nylon membranes with the production of various colour ranges on reaction with the phenolic substrates. The most successful immobilisation of the enzyme, in terms of quantity and distribution of enzyme immobilised and colour production, was obtained with the enzyme immobilised by adsorption on nylon membranes in the presence of 3-methyl-2-benzothiazolinone hydrazone (MBTH). The enzyme, immobilised using this method, produced ranges of maroon colours in phenolic solutions and orange colours in cresylic solutions. The colour intensities produced were found to increase proportionally with increasing substrate concentration after 5 minutes exposure to the substrates. The bioprobe had a broad substrate specificity and was sensitive to substrate concentrations down to 0.05 mg/L. The enzyme activity of the bioprobe was not significantly affected in a pH range from 4 to 10 and in a temperature range from 5-25⁰C. The bioprobe activity was not affected by various concentrations of salt and metal ions and the bioprobe was able to detect and semi-quantify phenolic substrates in industrial effluent samples. These features of the bioprobe indicate that the commercialisation of such a bioprobe is feasible and this technology has been patented (Patent No. SA 97/0227).<br>KMBT_363<br>Adobe Acrobat 9.54 Paper Capture Plug-in
29
Dyantyi, S. D. (Simphiwe David). "Fungal pretreatment of unextracted and pressurized hot water extracted Eucalyptus Grandis wood chips." Thesis, Stellenbosch : Stellenbosch University, 2007. http://hdl.handle.net/10019.1/21655.
Full textAbstract:
Thesis (MScFor)--University of Stellenbosch, 2007.<br>ENGLISH ABSTRACT: Unextracted (control) and PHWe Eucalyptus grandis wood chips were pulped at 15% active
alkali (AA) and 1% antraquinone (AQ). Another batch of wood chips were then inoculated
with fungal co-cultures of Aspergillus flavipes and Pycnoporus sanguineus. FCCi wood chips
were incubated for four weeks; one PHWe inoculated experimental treatment was incubated
for three weeks. The full pulping cycle (160 min) was used to digest the experimental
treatments with the exception of one lot of PHWe wood chips that were pulped for 150
minutes. A further experimental treatment of PHWe wood chips was cooked at a reduced AA
charge of 14% and 1% AQ. Analysis of variance (ANOVA) of the data from all the
experimental treatments was conducted and the differences within the experimental treatments
were determined using Statistica (v7, 1984–2006). The F-value (Fischer distribution) and the
p-value as well as a non-parametric test known as the Mann-Whitney procedure was tested at
the 95% confidence limit. For a further enhancement of the 95% confidence limit the screened
yield data was tested by the Bootstrap method. Scanning electron micrographs clearly
demonstrated the changed structure and appearance of the chip cross-sectional area after the
different pretreatments.
Although the mean average results of all the screened pulp yields showed no significant
statistical difference (p> 0.05), differences in screened yield of up to 2.5% were obtained. All
the weighted means of the rejects showed a significant difference (p < 0.05). Other pulp
properties like shive content, chemical consumption, Kappa number, handsheet brightness and
strength tests showed mixed results i.e. rejected or accepted the hypothesis (p> or =or < 0.05).
The hypothesis that the combined PHWE and FCCI of wood chips would further increase the
pulp yield had to be rejected. It is however anticipated that the combination of PHWE with
successive co-culture fungal pretreatment would be very beneficial in obtaining higher pulp
yields for fully bleached chemical pulp. Further research would be required to test this
assumption. This investigation confirmed the expected beneficial effects of combined PHWE
and FCCI pretreatments of wood chips on the strength properties. In addition the combined
treatment also improved the initial bonding strength potential of the unbeaten fibres.<br>AFRIKAANSE OPSOMMING: Onbehandelde en met onder druk, warm water uitgeloogde Eucalyptus grandis houtspaanders
is respektiefwelik met 15% aktiewe alkali (AA) en 1% antrakinoon (AQ) verpulp. Hierdie is
dan met swamkokulture van Aspergillus flavipes en Pycnoporus sanguineus inokuleer en
respektiewelik vir drie en vier weke inkubeer. Onder druk uitgeloogde houtspaanders is ook
vir 150 minute verpulp by 15% AA 1% AQ en by ‘n verminderde AA van 14%.
Pulpevaluasies is uitgevoer op alle eksperimentele behandelinge. Alle onder druk uitgeloogde
en met swamkokultuur inokuleerde houtspaanders het ‘n laer pulpopbrengs, uitskot,
skilferinhoud, Kappanommer en ‘n hoër RAA en helderheid opgelewer in vergelyking met die
vars houtspaanders. Die vars en warm water uitgeloogde houtspaanders het soortgelyke
pulpopbrengs opgelewer.
‘n Variansieanalise (ANOVA) van die data van alle eskperimentele behandelings is uitgevoer
gebruikmakende van Statistica (V7, 1984 – 2006). Die F-waarde (Fischer-verspreiding) an die
p-waarde so wel as ‘n parametriese toets (Mann-Whitney prosedure) is getoets by ‘n 95%
betroubaarheidsgrens. Vir ‘n verdere verhoging van die 95% betroubaarheidsgrens van die
pulpopbrengs, is die beskikbare data weer getoets met die Bootstrap-metode.
Alle gemiddelde pulpopbrengswaardes het geen beduidende statistiese verkil opgelewer nie
(p>0.05), alhoewel verskille van tot 2.5% in pulpopbrengs verkry is. Alle gemiddelde
uitskotwaardes het ‘n beduidende verskil getoon (p<0.05). Die ander pulpeienskappe soos
skilferinhoud, verbruik aan chemikalieë, Kappagetal, handvel helderheid en sterktewaardes
het gemengde resultate opgelewer maw verwerping of aanvaarding van die hipotese p> or =or
< 0.05. Die hipotese dat die gekombineerde PHWE en FCCI van die houtspaanders die
pulpopbrengs verder sou verhoog moes verwerp word. Daar word egter verwag dat die
kombinasie van PHWE met opeenvolgende swamkokultuur behandeling baie voordelig sou
wees op die pulpopbrengs van ‘n ten volle gebleikte chemiese pulp. Verdere navorsing is
nodig om hierdie veronderstelling te toets. Die ondersoek het die verwagte woordelige effek
van die gekombineerde PHWE en FCCI voorbehandelings van die houtspaanders op die
papierstrkte-eienskappe bevestig. Bo en behalve dit, het die gekombineerde behandeling ook
die aavanklikte bindsterkte potensiaal van die ongeklopte vessels verbeter.
30
Grimme, Eva. "Mycofumigation with Muscodor albus effects on Verticillium wilt and black dot root rot of potato, effects on Glomus intraradices and ectomycorrhizal fungi, and M. albus proliferation in soil /." Thesis, Montana State University, 2008. http://etd.lib.montana.edu/etd/2008/grimme/GrimmeE1208.pdf.
Full textAbstract:
Muscodor albus Worapong, Strobel & Hess, isolate CZ-620 (MA) is an endophytic fungus that produces volatile organic compounds (VOCs) and non-volatile antimicrobial compounds. The use of these VOCs to inhibit or kill a wide range of microorganisms is termed mycofumigation. This dissertation focuses on parameters of MA mycofumigation including: production and bioactivity of previously un-described water-soluble antimicrobial compounds produced by MA; distribution of antimicrobial compounds from a MA point source in three soil types as measured by effects on Verticillium dahliae and Colletotrichum coccodes; control of V. dahliae and C. coccodes on potato; the ability of MA to colonize soil; and the effects of mycofumigation on ectomycorrhizal fungi (EMF) in vitro and on the colonization of onion roots by the arbuscular mycorrhizal (AM) fungus Glomus intraradices. The bioactivity of water-soluble compounds produced in potato dextrose broth was significantly increased as measured in growth reduction of C. coccodes, V. dahliae, and Rhizoctonia solani. No reduction was observed for Aphanomyces cochlioides and Pythium ultimum. Antimicrobial compounds from a MA colonized barley point source reduced V. dahliae and C. coccodes populations in soils by 60-100% at distances up to 9 cm from the inoculation source depending on soil type. Mortality rate ranging from 70-100% was observed within a 3 cm radius from the inoculation source. In both field and greenhouse trials, MA colonized barley formulation reduced Verticillium wilt and black dot root rot severity and reduced populations of both pathogens in potato tissue as measured by real-time quantitative PCR and serial dilution. Planting directly into mycofumigated soil previously infested with V. dahliae or C. coccodes resulted in equal control of the pathogens when compared to a one-week mycofumigation period prior to planting. After six weeks of incubation MA did not colonize sterile soil further than 0.5 cm away from a MA inoculation point. In vitro experiments showed that most of the tested EMF were inhibited in the presence of MA VOCs, but were able to resume growth when removed from VOCs. Incorporating MA into soil had no negative but supportive effect on onion root colonization by the AM fungus G. intraradices.
31
La, Grange-Nel Karin. "Characterisation and improvement of whiskey yeast." Thesis, Stellenbosch : Stellenbosch University, 2003. http://hdl.handle.net/10019.1/53327.
Full textAbstract:
Thesis (MSc)--Stellenbosch University, 2003.<br>ENGLISH ABSTRACT: Scotch whiskey is of two main types, namely Scotch malt whiskey, made from malted
barley alone, or Scotch grain whiskey, made from cereals, such as wheat or maize,
together with malted barley. In both processes, the enzymes from the barley are
responsible for starch conversion and should always be derived entirely from the
malted barley. No exogenous enzymes are allowed to be added to any mashing.
The enzymes involved in the conversion process to fermentable sugars, are the aand
p-amylases, limit dextrinase and p-glucosidase.
Maize, on the other hand, contains no enzyme activity, therefore enzymes need
to be added when producing whiskey from maize alone. In other whiskey-producing
countries where maize is freely available and cheaper than barley, the use of
exogenous enzymes are allowed in the mashing process and is crucial for the
formation of fermentable sugars from complex carbohydrates. The cost of the
enzymes, however, can push the production cost of whiskey to higher levels.
Saccharomyces cerevisiae does not have any amylolytic activity, but is an
excellent fermenter and produces favourable organoleptic notes, which makes it very
suitable for producing potable spirit. Efforts have been made to genetically improve
industrial strains, relying on classical genetic techniques followed by the selection of
broad traits, such as ethanol tolerance, absence of off-flavours and
carbohydrate/starch utilisation. No strain has thus far been selected for total starch
degradation during the fermentation of whiskey mash.
Over the last decade, considerable progress has been made in the development
of genetically improved strains for the distilling, wine, brewing and baking industries.
The expression of heterologous genes introduced a new dimension in approaches to
the genetic improvement of industrial strains. It would therefore be cost-effective to
use a yeast strain that can produce active and sufficient enzymes to ferment raw
starch efficiently to alcohol without lowering the quality of the end product. No such
strain has been developed to date, but the continuous improvement of starch-utilising
strains has made this goal more achievable.
Two a-amylase genes, namely LKA 1 and LKA2, were previously isolated from
Lipomyces kanonenkoae. In this study, we selected 4 strains on the basis of criteria
that are important for whiskey-specific strains. The selected strains were
transformed with LKA 1, as well as with a combination of LKA 1 and LKA2 genes. The
wine yeast VIN13 was included in the transformation of LKA1 and LKA2 because of
its rapid fermentation rate. The genes were integrated into the genomes of the yeast
strains and were stable after many generations. Assays showed that a significant
increase in enzyme activity was induced in the whiskey strains, compared to the
untransformed strains. The strains also showed good fermentation ability in whiskey
fermentations, although optimum alcohol production was still not achieved.<br>AFRIKAANSE OPSOMMING: Skotse whiskey bestaan uit 2 tipes, nl. mout whiskey, gemaak slegs van mout d.w.s.
gars wat die mout proses ondergaan het, en graan whiskey wat gemaak word van
gewasse soos mielies of koring, waarby mout gevoeg word. Die ensieme afkomstig
van die mout is verantwoordelik vir die omsetting van stysel na fermenteerbare
suikers en geen eksogene ensieme mag by die gars- of graanmengsel gevoeg word
nie. Die ensieme wat betrokke is by die omsetting van stysel, is die a- en ~-
arnitases, limiet dekstrinase en ~-glukosidase.
Mielies bevat geen ensiemaktiwiteit nie, dus moet ensieme by die proses gevoeg
word indien slegs mielies vir die vervaardiging van whiskey gebruik word. In whiskey
produserende lande waar mielies vryelik beskikbaar is en goedkoper is as gars, word
eksogene ensieme by die graanmengsel gevoeg vir die vrystelling van
fermenteerbare suikers vanaf komplekse koolhidrate. Die hoë koste van die ensieme
kan egter die produksiekoste van whiskey verhoog.
Saccharomyces cerevisiae besit geen amilolitiese aktiwiteit nie, maar is 'n
uitstekende fermenteerder en produseer gewensde organoleptiese geure. Om
hierdie redes is S. cerevisiae baie geskik vir die produksie van drinkbare etanol.
Navorsingspogings om industriële rasse geneties m.b.v. klassieke genetiese
metodes te verbeter, kom wydverspreid in die literatuur voor. Dit sluit in die seleksie
van rasse met 'n verskeidenheid van eienskappe soos etanol toleransie, die
afwesigheid van afgeur produksie en koolhidraat/stysel benutting. Geen ras is egter
tot op hede geselekteer vir totale stysel afbraak gedurende fermentasie nie.
Groot vordering is gedurende die laaste dekade gemaak in die ontwikkeling van
genetiese verbeterde rasse vir die wyn- stokery- en brouers industrieë. Die uitdruk
van heterogene gene in gisrasse gee 'n nuwe dimensie aan die genetiese
verbetering van industriële rasse. Die gebruik van 'n gisras wat aktiewe en
genoegsame ensieme produseer om rou stysel te fermenteer, sonder om die kwalitiet
van die eindproduk nadelig te beïnvloed, kan die produksiekoste van whiskey
aansienlik verminder. Geen gisras met hierdie eienskap is tot op hede ontwikkel nie,
maar die voortdurende verbetering van rasse om stysel af te breek maak hierdie doel
meer bereikbaar.
Twee a-amilase gene, nl. LKA 1 en LKA2 is voorheen uit Lipomyces
kononenkoae geïsoleer. In hierdie studie is 4 gisrasse geselekteer op grond van die
kriteria wat nodig is vir whiskey giste. Die geselekteerde rasse is getransformeer met
LKA 1 sowel as 'n kombinasie van LKA 1 en LKA2 gene. Die wyngis VIN13 is
ingesluit by die transformasie met die LKA1 en LKA2 gene, omrede VIN13 bekend is
as 'n vinnige fermenteerder. Die gene is geïntegreer in die genoom van die
verskillende gisrasse en is stabiel na vele generasies. Die getransformeerde rasse
het 'n betekenisvolle verhoging in ensiemaktiwiteit teenoor die nie-getransformeerde rasse getoon. AI die transformante het ook goeie fermentasie vermoë getoon in
whiskey fermentasie proewe. Optimum alkoholproduksie is egter nie verkry nie.
32
Wilhelmi, Brendan Shane. "The removal and recovery of toxic and valuable metals from aqueous solutions by the yeast Saccharomyces cerevisiae." Thesis, Rhodes University, 1998. http://hdl.handle.net/10962/d1004062.
Full textAbstract:
This project considered the use of the yeast Saccharomyces cerevisiae as a biosorbent for the removal and recovery of a range of metals from contaminated waters. S. cerevisiae, as a biosorbent, has the potential to provide a cost effective, selective and highly efficient purification system. Initial studies focused on metal accumulation by an immobilized baker's S. cerevisiae biosorbent. The parameters affecting metal uptake were investigated, these included metal concentration, time and solution pH. Metal uptake was rapid. Gold and cobalt reached saturation within 5 min of contact with the biosorbent in batch reactors. Copper, zinc, nickel, cadmium and chromium reached saturation within 30 min of contact. Metal accumulation was pH dependent and was generally unaffected at a solution pH ≥ 4, and was substantially decreased at pH ≤ 2. The exception was gold which was preferentially accumulated at a solution pH of 2. The immobilized baker's yeast accumulated metals with maximum binding capacities in the order of gold > cadmium > cobalt > zinc > copper > chromium > nickel. A rapid method to assess metal recovery was developed. Bioaccumulated metal was efficiently recovered using dilute mineral acids. Copper recovery of ≥ 80 % was achieved by decreasing the solution pH of the reaction mixture to 2 with the addition of nominal quantities of HCl, H₂SO₄ or RNO₃. Adsorption-desorption over 8 cycles had no apparent adverse effect on metal uptake or recovery in batch reactors. Transmission electron microscopy showed no evidence of damage to cells used in copper adsorption-desorption investigations. Biosorption columns were investigated as bioreactors due to their application potential. The metals investigated were effectively removed from solution. At a saturation threshold, metal uptake declined rapidly. Most metals investigated were desorbed from the columns by eluting with 0.1 M HCl. Initially recoveries of copper, cobalt and cadmium were as high as 100%. Desorbed copper, zinc, cadmium, nickel and cobalt were concentrated in 10 to 15 ml of eluent, representing up to a 40 fold decrease in solution volume. Cadmium, nickel and zinc uptake increased with the second application to the columns. Initial accumulation of gold and chromium was 42.2 μmol/g and 28.6 μmol/g, however, due to the low recoveries of these two metals, a second application was not investigated. Copper was applied to a single column for 8 consecutive adsorption-desorption cycles. Uptake increased from an initial 31.3 μmol/g to 47.8 μmol/g at cycle 7. The potential for selective metal recovery was demonstrated using two biosorption columns in series. Copper was accumulated and recovered most efficiently. Zinc, cobalt and cadmium were displaced to the second column. Copper bound preferentially to zinc at a ratio of 6:1. Copper bound preferentially to cobalt at a ratio of 4:1. Cadmium was only displaced at a ratio of 2:1. The successful transfer of the bioremediation technology from the laboratory to an industrial application has yet to be realized. Bioremediation of a Plaatjiesvlei Black Mountain mine effluent, which contained copper, zinc, lead and iron, was investigated in this project. The removal of the metals was most effective at pH 4. A combined strategy of pH adjustment and bioremediation using immobilized S. cerevisiae decreased the copper concentration by 92.5%, lead was decreased by 90% and zinc was decreased by 60%. Iron was mostly precipitated from solution at pH ≥ 4. An ageing pond at the mine with conditions such as; pH, water volume and metal concentration, which were more conducive to biological treatment was subsequently identified. The investigation indicated a possible application of the biomass as a supplement to chemical remediation. The metal removal capability of a waste brewer's yeast was subsequently investigated. A yeast conditioning step increased metal uptake up to 100% and enhanced reproducibility. Metal removal from solution was rapid and pH dependent. The metals were efficiently removed from solution at pH ≥ 4. Uptake was substantially inhibited at pH ≤ 3. The waste brewer's yeast accumulated metals with maximum binding capacities in the order of copper (25.4 μmol/g) > lead (19.4 μmol/g) > iron (15.6 μmol/g) > zinc (12.5 μmol/g). No correlation between cell physiology and metal uptake was observed. Uptake of the four metals was confirmed by energy dispersive X-ray microanalysis. The interference of lead, zinc and iron on copper uptake by the waste brewer's yeast, and the interference of copper on the uptake of lead, zinc and iron was investigated. Maximum copper uptake was not decreased in the presence of lead. The Bmax remained constant at approximately 25 μmol/g. The dissociation constants increased with increasing lead concentrations. Lead bioaccumulation was significantly decreased in the presence of copper. The type of inhibition was dependent on the initial copper concentrations. Zinc had a slight synergistic effect on copper uptake. The copper Bmax increased from 30.8 μmol/g in a single-ion system to 34.5 μmol/g in the presence of 200 μmol/l of zinc. Zinc uptake was severely inhibited in the presence of copper. The maximum uptake and dissociation constant values were decreased in the presence of copper, which suggested an uncompetitive inhibition. The affinity of copper was substantially higher than zinc. The presence of higher levels of copper than zinc in the yeast cells was confirmed by energy dispersive microanalysis. Copper uptake was decreased in the presence of iron, with the copper Bmax being decreased from 25.4 μmol/g in a single-ion system to 20.1 μmol/g in the presence of 200 μmol/l iron. Iron Bmax values remained constant at 16.0 μmol/g. Combined biosorption and EDXA results suggested the iron bound at a higher affinity than copper to the cell wall. Total copper removal was higher as larger quantities of copper were deposited in the cell cytoplasm. Metal removal from the Plaatjiesvlei effluent by free cell suspensions of the waste brewer's yeast was satisfactory. Copper levels were decreased by 96%, iron by 42%, lead 25% and zinc 2%. Waste brewer's yeast is a cheap source of biomass in South Africa, and could potentially provide the basis for the development of an innovative purification system for metal-contaminated waters.
33
Da, Serra Maria Fatima. "Fungal and substrate-associated factors affecting lignocellulolytic mushroom cultivation on wood sources available in South African [i.e. Africa]." Thesis, Rhodes University, 1997. http://hdl.handle.net/10962/d1004080.
Full textAbstract:
Vast- quantities of lignocellulosic materials, representing potential substrates for the cultivation of speciality mushrooms, are produced annually in South Africa. A number of these materials are derived as waste products of the timber and agricultural industries, e.g. Maranti (Shorea spp.) and Port Jackson Willow (Acacia longifolia) respectively. The screening of various wood-degrading fungi, which are cultivated worldwide for their production of speciality mushrooms, indicated that under the environmental conditions considered, certain species were adapted to cultivation on these lignocellulosic wastes (Pleurotus species) whereas others were not (Lentinus edodes and Flammulina velutipes). Furthermore, intra- and interspecies specific differences in the growth and production potential of the various lignocellulolytic fungi investigated on synthetic and natural medium were discovered. Biochemical and genetical investigations of these strains indicated differences between and within species which were often significant. Species varied qualitatively and quantitatively in the lignocellulolytic enzymes produced, which was loosely correlated with productivity on the different media investigated. Genetical studies, using RAPD fingerprinting, indicated that the Pleurotus genus is highly variable which supports the observed differences in growth, yield and enzymatic activity between different strains and species.
34
Giraldo, Marielle Aleixo. "Purificação e caracterização bioquímica da invertase extracelular produzida pelo fungo filamentoso Aspergillus terreus /." Araraquara [s.n.], 2011. http://hdl.handle.net/11449/87963.
Full textAbstract:
Orientador: Luis Henrique Souza Guimarães<br>Banca: Rosimeire Cristina Linhari Rodrigues Pietro<br>Banca: Douglas Chodi Masui<br>Resumo: Os microrganismos, de modo especial os fungos filamentosos, possuem papel fundamental na decomposição de matéria orgânica, sendo interessantes como modelos para realização de diferentes estudos biológicos. Além disso, são de fácil manejo e as condições de cultivo podem ser facilmente adaptadas em laboratório. Outra vantagem é, geralmente, o baixo custo e o fácil acesso aos nutrientes necessários para o crescimento dos mesmos. Entre os microrganismos, os fungos filamentosos têm se destacado na obtenção de enzimas de interesse biotecnológico, como é o caso das invertases, as quais podem ser empregadas nas indústrias de alimentos e bebidas. As invertases (EC 3.2.1.26) são hidrolases que podem ser encontradas em uma grande variedade de organismos, realizando a hidrólise da ligação - D-frutofuranosídica, agindo sobre a sacarose, gerando como produtos D-glicose e D-frutose, em quantidades equimolares. Essa mistura é conhecida como açúcar invertido e é geralmente utilizada pelas indústrias de alimentos. Desta maneira, o objetivo deste trabalho foi estudar a produção de invertase extracelular pelo fungo filamentoso Aspergillus terreus, em fermentação submersa e em fermentação em substrato sólido, bem como purificar e caracterizar a enzima bioquimicamente. Entre os diversos meios de cultura testados em FSbm, a maior produção invertásica foi obtida em meio M5 mantido em agitação orbital (100 rpm) a uma temperatura de 30ºC, por um período de 48 horas de incubação. Já na FSS, o melhor período de incubação foi de 72 horas, também a 30ºC. Entre todas as fontes de carbono testadas, a maior produção invertásica foi obtida utilizandose farinha de centeio para a FSbm e soja moída para FSS. A enzima iv extracelular obtida em FSbm foi purificada 139 vezes com uma recuperação de 11%. A invertase extracelular... (Resumo completo, clicar acesso eletrônico abaixo)<br>Abstract: The microorganisms, especially filamentous fungi, have a fundamental role in the decomposition of organic matter, and they are interesting as models for carrying out different biological studies. Moreover, they are easy to handle, and their growing conditions can be easily adapted in the laboratory. Another advantage is, generally, the low cost and easy access to the nutrients needed for their growth. Among the microorganisms, filamentous fungi have been essential in obtaining enzymes of biotechnological interest, such as invertase, which may be employed in the food and beverage industries. The invertases (EC 3.2.1.26) are hydrolases that can be found in a great variety of organisms, performing the hydrolysis of the -D fructofuranosidic bond acting on sucrose, generating products such as D-glucose and D-fructose, in equimolar amounts. This mixture is known as inverted sugar and it is commonly used by food industries. This way, the aim of this work was to study the production of extracellular invertase by the filamentous fungus Aspergillus terreus in submerged fermentation and solid state fermentation, as well as its purification and biochemical characterization. Among the various media tested in FSbm, the highest yield of invertase was obtained by using M5 medium under orbital agitation (100 rpm) at 30°C for 48 hours of incubation. In the FSS, the best incubation period was 72 hours, also at 30°C. Among all carbon sources tested, the highest invertase production was obtained using rye flour for FSbm and soybean meal for FSS. The extracellular enzyme obtained from FSbm was purified 139 fold with 11% recovery. The extracellular invertase of A. terreus is a heterodimer of native vi molecular mass of 74.67 kDa consisting of two subunits, one of 46.77 kDa and another of 26.92 kDa determined... (Complete abstract click electronic access below)<br>Mestre
35
Sindle, Astrid Elizabeth. "Evaluation of the effect of morphological control of dimorphic Mucor circinelloides on heterologous enzyme production." Thesis, Link to the online version, 2006. http://hdl.handle.net/10019/1207.
Full text
36
Muller, Christo A. "Monitoring the spreading of commercial wine yeasts in the vineyard." Thesis, Stellenbosch : Stellenbosch University, 2003. http://hdl.handle.net/10019.1/53505.
Full textAbstract:
Thesis (MSc)--Stellenbosch University, 2003.<br>Full text to be digitised and attached to bibliographic record.<br>ENGLISH ABSTRACT: Traditionally, wine has been produced by the spontaneous fermentation of grape
juice by yeast that originate from the grapes and winery equipment. Research has
shown that the population composition and dynamics of these yeasts and other
microorganisms are very complex. Kloeckera and its anamorph, Hanseniaspora,
dominate the yeast population found on the surfaces of grapes, although prevailing
Saccharomyces cerevisiae strains complete the fermentation process.
The yeast S. cerevisiae is an important factor contributing to the quality of wines
and, therefore, the improvement of wine yeasts receives considerable attention
worldwide. Apart from classical yeast breeding studies, genetic engineering and
recombinant DNA techniques are increasingly being used in strain development
research programmes. These techniques might enable the wine yeasts to produce
heterologous enzymes that degrade polysaccharides, convert malic acid to lactic
acid, increase glycerol production, release roam and flavour compounds, secrete
antimicrobial peptides, etc. The release of recombinant yeast strains (genetically
modified organisms, GMOs) is subject to statutory approval. Therefore, it is important
to answer several questions prior to the use of such genetically improved yeast in the
commercial production of wine. For example, will recombinant yeast strains be able
to multiply and spread in nature, and will this GMO be able to out-compete the
natural microflora because of its newly acquired genetic traits. Since existing
commercial wine yeasts are used in the abovementioned strain development
research, it is essential to determine already at this early stage to what extent these
wine yeast strains survive and spread in nature and to what extent they influence the
fermentations of the following vintages.
This study is divided into two sections. The aim of the first section is to sample a
representative number of yeast strains from various vineyards in different
climatological areas, mainly in the Western Cape, South Africa. These yeast strains
were identified mainly by electrophoretic karyotyping (contour-clamped homogenous
electric field electrophoresis; CHEF).
The second part of the study summarises the results obtained when Fourier
transform infrared (FT-NIR) spectroscopy was used to differentiate commercial wine
yeast strains. Sets of data, containing the spectra of the mostly used commercial
wine yeast strains, were constructed and used as a reference library. The spectra of
the isolated yeast strains were then compared to the reference dataset with specific
FT-NIR computer software using mathematical calculations.
In conclusion, the two methods used in conjunction with one another proved that
the commercial wine yeast strains do not easily disperse from the cellar into the
vineyard. The commercial wine yeast strains are also more likely to be found near
the cellar and the places where the grape skins are dumped. Therefore, should a
recombinant yeast strain be used in winemaking, it would not be dispersed into the
vineyard. It therefore appears that the commercial use of genetically improved yeast does not pose a high risk in terms of dominance of the indigenous microbial
population in the environment<br>AFRIKAANSE OPSOMMING: Wyn is tradisioneel gemaak deur die natuurlike gisting van druiwesap deur giste wat
op die druiwe en keldertoerusting voorkom. Navorsing het getoon dat die
samestelling en dinamika van die gispopulasie en ander mikro-organismes baie
kompleks is. Kloeckera en sy anamorf, Hanseniaspora, domineer die inheemse
gispopulasie op druiwedoppe, terwyl Saccharomyces cerevisiae in baie klein getalle
op die druiwedoppe voorkom, maar later die fermentasie oorheers en uiteindelik
voltooi.
Die gis S. cerevisiae speel 'n baie belangrike rol in die kwaliteit van wyn en
daarom geniet die verbetering van wyngiste wêreldwyd besondere aandag.
Benewens die klassieke gistelingstudies, word genetiese manipuleringstegnieke
toenemnd in navorsingsprojekte gebruik wat daarop gefokus is om wyngisrasse te
verbeter. Hierdie tegnieke mag die giste in staat stelom heteroloë ensieme te
produseer wat polisakkariedes afbreek, appelmelksuur afbreek, gliserolproduksie
verhoog, smaak- en geurkomponente vrystel, antimikrobiese peptiede afskei, ens.
Voordat sulke geneties gemanipuleerde giste het egter in kommersiële wynproduksie
gebruik sal kan word, is daar heelwat wetlike vereistes waaraan voldoen sal moet
word en vrae wat vooraf beantwoord sal moet word. Byvoorbeeld, sal die
rekombinante giste in staat wees om vinniger te vermeerder as gevolg van die nuwe
genetiese eienskappe en sodoende die natuurlike populasies onderdruk? Omdat
kommersiële wyngiste in bogenoemde gisverbeteringprogramme gebruik word, is dit
noodsaaklik om nou reeds die verspreiding van die kommersiële giste te monitor en
te bepaal hoe geredelik hulle in die natuur kan versprei en oorleef, en hoe hulle
wynfermentasies van die daaropvolgende jare beïnvloed.
Die studie is in twee gedeeltes verdeel. Die doel van die eerste gedeelte was om
'n verteenwoordigende aantal gisrasse uit die wingerde van 'n aantal wynplase in
verskillende klimaatstreke te isoleer, spesifiek in die Wes-Kaap, Suid-Afrika. Die
gisrasse was grotendeels deur elektroforetiese kariotipering (kontoer-geklampte
homogene elektriese veld; CHEF) geïdentifiseer.
Die tweede deel van die navorsing was gefokus op die onderskeiding tussen die
mees gebruikte kommersiële wyngiste met 'Fourier-Transform Near Infrared' (FTNIR)
spektroskopie. Eerstens is 'n stel data, bestaande uit die spektrum data oor die
kommersiële wyngiste opgestel om as 'n verwysingsbiblioteek te dien. Tweedens is
die spektrum van data oor die geïsoleerde giste onder presies dieselfde toestande
met die verwysingsbiblioteek vergelyk. Dié tegniek maak dit moontlik om tussen die
kommersiële wyngiste te onderskei.
As die twee metodes saam gebruik word vir identifikasie, kan die afleiding
gemaak word dat kommersiële wyngiste nie maklik vanaf die kelder na die wingerd
versprei nie. Die kommersiële wyngiste is ook meestal naby die kelder en die
dopstortingsterreine gevind. Sou 'n rekombinante gisras dus gebruik word om wyn te maak, sal dit nie maklik versprei nie. Die kommersiële gebruik van geneties
gemanipuleerde wyngiste behoort dus nie In groot omgewingsrisiko in te hou nie.
37
Grove, Heather Lee. "Cloning and characterization of the Pichia Pastoris PMR1 gene." Scholarly Commons, 2005. https://scholarlycommons.pacific.edu/uop_etds/613.
Full textAbstract:
Pichia pastoris, a popular protein expression system, is limited in its ability to secrete heterologous proteins. The PMR1 gene, the disruption of which is known to improve the secretion of prochymosin, human prourokinase, and human tissue plasminogen activator in Saccharomyces cerevisiae, was cloned from P. pastoris. The pmr 1 mutant in S. cerevisiae also displayed a slow growth phenotype when grown on low Ca2+ medium. The putative P. pastoris PMR1 gene, encoding for a 924 amino acid P-type Ca2+ ATPase, was disrupted in P. pastoris and the secretion of horseradish peroxidase (HRP) and β-galactosidase (β-gal) analyzed. Secreted HRP activity was determined using 3,3',5,5' tetramethylbenzidine (TMB) colorimetric assay and western analysis. β-gal expression and secretion was determined by western analysis. Secretion in P. pastorius Δpmr1 for both heterologous proteins showed no appreciable difference compared to wild type, nor did P. pastoris Δpmr1 display the slow growth phenotype seen in S. cerevisiae Δpmr1 (Rudolph H. et al., 1989).
38
Hansson, Guy Robert 1974. "Cell differentiation in response to nutrient availability : the repressor of meiosis, RME1, positively regulates invasive growth in Saccharomyces cerevisiae." Thesis, Stellenbosch : Stellenbosch University, 2003. http://hdl.handle.net/10019.1/53322.
Full textAbstract:
Thesis (MSc)--University of Stellenbosch, 2003.<br>ENGLISH ABSTRACT: Yeasts, like most organisms, have to survive in highly variable and hostile
environments. Survival therefore requires adaptation to the changing external
conditions. On the molecular level, specific adaptation to specific environmental
conditions requires the yeast to be able: (i) to sense all relevant environmental
parameters; (ii) to relay the perceived signals to the interior of the cell via signal
transduction networks; and (iii) to implement a specific molecular response by
modifying enzyme activities and by regulating transcription of the appropriate genes.
The availability of nutrients is one of the major trophic factors for all unicellular
organisms, including yeast. Saccharomyces cerevisiae senses the nutritional
composition of the media and implements a specific developmental choice in response
to the level of essential nutrients. In conditions in which ample nutrients are available,
S. cerevisiae will divide mitotically and populate the growth environment. If the
nutrients are exhausted, diploid S. cerevisiae cells can undergo meiosis, which
produces four ascospores encased in an ascus. These ascospores are robust and
provide the yeast with a means to survive adverse environmental conditions. The
ascospores can lie dormant for extended periods of time until the onset of favourable
growth conditions, upon which the spores will germinate, mate and give rise to a new
yeast population. However, S. cerevisiae has a third developmental option, referred to
as pseudohyphal and invasive growth. In growth conditions in which nutrients are
limited, but not exhausted, the yeast can undergo a morphological switch, altering its
budding pattern and forming chains of elongated cells that can penetrate the growth
substrate to forage for nutrients.
The focus of this study was on elements of the signal transduction networks
regulating invasive growth in S. cerevisiae. Some components of the signal
transduction pathways are well characterised, while several transcription factors that
are regulated via these pathways remain poorly studied. In this study, the RMEt gene
was identified for its ability to enhance starch degradation and invasive growth when
present on a multiple copy plasmid. Rme1 p had previously been identified as a
repressor of meiosis and, for this reason, the literature review focuses on the
regulation of the meiotic process. In particular, the review focuses on the factors
governing entry into meiosis in response to nutrient starvation and ploidy. Also, the
transcriptional regulation of the master initiator of meiosis, IMEt, and the action of
Ime1 p are included in the review.
The experimental part of the study entailed a genetic analysis of the role of Rme1 p
in invasive growth and starch metabolism. Epistasis analysis was conducted of
Rme1 p and elements of the MAP Kinase module, as well as of the transcription
factors, Mss11p, Msn1p/Mss10p, Tec1p, Phd1p and F108p. Rme1p is known to bind
to the promoter of CLN2, a G1-cyclin, and enhances its expression. Therefore, the cell cyclins CLN1 and CLN2 were included in the study. The study revealed that Rme1 p
functions independently or downstream of the MAP Kinase cascade and does not
require Cln1 p or Cln2p to induce invasive growth. FL011/MUC1 encodes a cell wall
protein that is required for invasive growth. Like the above-mentioned factors, Rme1 p
requires FL011 to induce invasive growth. We identified an Rme1 p binding site in the
promoter of FL011. Overexpression of Rme1p was able to induce FL01t expression,
despite deletions of mss11, msn1, ttos, tee1 and phd1. In the inverse experiment,
these factors were able to induce FL011 expression in an rme1 deleted strain. This
would indicate that Rme1 p does not function in a hierarchical signalling system with
these factors, but could function in a more general role to modify transcription.<br>AFRIKAANSE OPSOMMING: Die natuur is hoogs veranderlik en alle organismes, insluitende gis, moet by die
omgewing kan aanpas om te kan oorleef. Baie eksterne faktore beïnvloed die
ontwikkeling van die gissel. Vir die gis om by spesifieke omgewingstoestande aan te
pas, moet die gis op 'n molekulêre vlak: (i) al die omgewingsparameters waarneem; (ii)
die waargenome omgewingsparameters as seine na die selkern deur middel van
seintransduksieweë gelei; en (iii) transkripsie van gene aktiveer of onderdruk en
ensiemaktiwiteit reguleer om sodoende die gepaste molekulêre respons te
implementeer.
Die beskikbaarheid van voedingstowwe in die omgewing is een van die
belangrikste omgewingseine wat eensellige organismes moet kan waarneem.
Saccharomyces cerevisiae kan spesifieke ontwikkelingsopsies, na gelang van die
voedingstowwe wat beskikbaar is, uitoefen. In groeiomstandighede waar daar 'n
oorvloed van voedingstowwe is, verdeel S. cerevisiae d.m.v. mitose en vesprei dit
deur die omgewing. Sodra die voedingstowwe uitgeput is, word mitose onderdruk.
Diploïede S. cerevisiae inisieer meiose, wat aanleiding tot die vorming van vier spore
gee. Hierdie spore bevat slegs die helfte van die ouer se chromosome en kan
gevolglik met 'n ander spoor paar om weer 'n diploïede gissel te vorm. Die spore is
bestand teen strawwe omgewingstoestande en kan vir lang tye oorleef. Wanneer die
spoor aan gunstige groeitoestande blootgestel word, ontkiem dit om aan 'n nuwe
giskolonie oorsprong te gee. S. cerevisiae het egter 'n derde ontwikkelingsopsie,
naamlik pseudohife-differensiëring. Wanneer die beskikbaarheid van voedingstowwe
in die omgewing afneem, maar nog nie uitgeput is nie, ondergaan die gis 'n
morfologiese verandering. Hierdie verandering word gekenmerk deur selverlenging,
nl. botselle wat slegs aan die een punt van die gissel vorm en dogterselle wat aan die
moerderselle geheg bly. Dit lei tot die vorming van kettings van selle wat van die
giskolonie af weggroei. Voorts kan die selkettings ook die groeisubstraat binnedring.
Dit staan as penetrasie-groei bekend en laat die gis toe om na nuwe voedingsbronne
te soek.
Hierdie studie het op die elemente van seintransduksieweë, wat by
penetrasiegroei betrokke is, gefokus. Sekere komponente van die seintransduksieweë
is reeds goed gekarakteriseer, terwyl ander komponente nog grootliks onbekend is. In
hierdie studie, word 'n rol vir RME1 in die verbetering van styselafbraak en
penetrasiegroei geïdentifiseer. Aangesien Rme1 p voorheen as 'n onderdrukker van
meiose geïdentifiseer is, is 'n litetaruurstudie oor die inisiasie van meiose saamgestel.
Die faktore wat meiose induseer, naamlik 'n gebrek aan voedingstowwe en die sel se
ploïedie, word bespreek. Die regulering van die meester inisieerder van meiosie,
IME1, asook die proteïene waarmee Ime1p reageer, is ook in die studie ingesluit. Die eksperimentele deel van die studie behels die genetiese analise van Rme1 p
tydens penetrasiegroei en styselhidroliese. 'n Epistase-analise tussen Rme1 p en
elemente van die MAP-Kinasemodule, asook van die transkripsie faktore Mss11 p,
Msn1p/Mss10p, Tec1p, Phd1p en F108p, is onderneem. Rme1p is bekend om aan die
promotor van CLN2 te bind en transkripsie te induseer. Daarom is die selsikliene
CLN1 en CLN2 in die studie ingesluit. Die studie dui daarop dat Rme1 ponafhanklik
van die MAP-Kinasemodule funksioneer en nie Cln1 p en Cln2p benodig om
penetrasiegroei te induseer nie. FL011/MUC1 kodeer vir 'n selwandproteïen wat
noodsaaklik vir pentrasiegroei is. Soos in die geval van die bogenoemde faktore,
benodig Rme1 p FL011 om penetrasiegroei te kan induseer. Ten spyte van mss11-,
msn1-, ttos-, tec1- en phd1- delesies, kan ooruitdrukking van Rme1p die transkripsie
van FL011 induseer. In die omgekeerde eksperiment kon die bogenoemde faktore
FL011-transkripsie ten spyte van 'n rme1 delesie induseer. Die resultate dui daarop
dat Rme1 p nie in 'n hiërargiese pad funksioneer nie, maar dat dit waarskynlik 'n meer
algemene rol deur transkripsiemodifisering vervul.
39
Marques, Marina Paganini. "Estudo da hidrólise do bagaço de cana-de-açúcar por fungos filamentosos /." Araraquara : [s.n.], 2010. http://hdl.handle.net/11449/88050.
Full textAbstract:
Orientador: Sandra Regina Pombeiro Sponchiado<br>Banca: Rubens Monti<br>Banca: Hamilton Cabral<br>Resumo: O bagaço de cana de açúcar (BCA) é o principal subproduto das indústrias de açúcar e álcool no Brasil e em função da sua abundância e baixo custo poderia ser usado para aumentar a produção de etanol, fornecendo vantagens econômicas e ambientais. A hidrólise enzimática é um caminho promissor para obter açúcares de BCA, mas a baixa acessibilidade enzimática da celulose nativa é a chave do problema neste processo. Por este motivo, o pré tratamento da biomassa é exigido para remover a lignina e a hemicelulose. O objetivo principal deste trabalho foi avaliar o potencial biotecnológico de fungos filamentosos capazes de produzir grandes quantidades de enzimas celulolíticas utilizando o BCA como substrato com pré tratamento térmico e termo ácido. As espécies fúngicas selecionadas para este estudo foram: Aspergillus nidulans (mutante melanizado), Trichophyton terrestre e Aspergillus niger. Para o cultivo em estado sólido, o bagaço pré tratado foi inoculado com micélios ou esporos dos fungos e incubados a temperatura ambiente durante 15, 30 e 60 dias. A eficácia da hidrólise microbiana foi avaliada em relação ao açúcar total (AT), ao açúcar redutor (AR), as concentrações de fenol (Fe) e a atividade das enzimas celulolíticas (celulase, avicelase e CMCase). Os resultados mostraram que a quantidade de AT, AR e Fe foi mais elevada quando os fungos foram crescidos sobre o BCA com pré tratamento termo ácido comparado ao BCA com pré tratamento térmico. Dentre os fungos estudados, o filtrado da cultura do A. niger crescido sobre o BCA com pré tratamento termo ácido por 60 dias pode ser usado como o meio fermentativo para a produção de etanol devido a uma elevada concentração açúcar redutor (1,16% m/v ou 11,6 g.L 1 ) e de baixas concentrações de fenol (0,003% m/v ou 0,03 g.L 1 ). O nível o mais elevado de celulase... (Resumo completo, clicar acesso eletrônico abaixo)<br>Abstract: The sugar bagasse cane (SCB) is the major by product of the sugar and alcohol industries in Brazil and in function of its abundance and low cost could be used to increase ethanol production, providing economic and environmental advantages. Enzymatic hydrolysis is a promising way for obtaining sugars from SCB, but the low enzymatic accessibility of the native cellulose is a key problem in this processes. For this reason, the pretreatment of biomass is required to remove lignin and hemicellulose. The main objective of this work was to evaluate the biotechnological potential of filamentous fungi to produce large amounts of cellulolitic enzymes using thermal and/or acid pretreated SCB as substrate. The selected fungal species for this study were: Aspergillus nidulans (melanized mutant), A. niger and Trychophyton terrestre. For cultivation in solid state, the pretreated bagasse was inoculated with mycelia or spores of the fungi and incubate at room temperature during 15, 30 and 60 days. The effectiveness of the microbial hydrolysis was evaluated in terms of total sugar (TS), reducing sugar (RS) and phenol (Phe) concentrations and also activity of cellulolitic enzymes (cellulase, avicelase and CMCase). The results showed that the amount of TS, RS and Phe was higher when the fungi grown on acid and thermal pretreated SCB compared to thermal pretreated SCB. Among fungi studied, the filtrate of A. niger culture grown on acid and thermal pretreated SCB for 60 days could be used as fermentative medium for production of ethanol due to presence of high concentration of reducing sugar (1,16% m/v or 11,6 g.L 1 ) and low amounts of phenol (0,003% m/v or 0,03 g.L 1 ). The highest level of cellulase, corresponding to 0,74 IU.mL 1 , was obtained in the A. nidulans culture after 15 days of growth. For CMCase and avicelase, the maximum activity was exhibited respectively... (Complete abstract click electronic access below)<br>Mestre
40
Mukasa-Mugerwa, Thomas Tendo. "The role of arbuscular mycorrhizal fungi in the biotransformation of coal and application in dump rehabilitation." Thesis, Rhodes University, 2007. http://hdl.handle.net/10962/d1004059.
Full textAbstract:
Fundamental processes underpinning the biotransformation of coal by fungal biocatalysts have been intensively investigated, however, limited large-scale industrial applications using such systems have been reported. The un-anticipated sporadic growth of Cynodon dactylon on the surface of un-rehabilitated discard coal dumps has been noted and this was found to be coupled with the breakdown of coal into a humic soil-like material in the top 1.5 metres of the dumps. Extensive fungal growth was observed to be associated with the Cynodon dactylon root system and examination of plant roots indicated the presence of mycorrhizal fungi. Analysis of the Cynodon dactylon plant roots around which coal biotransformation was occurring confirmed the presence of arbuscular mycorrhizal colonisation with the species Glomus clarum, Paraglomus occultum, Gigaspora gigantea and Glomus mosseae identified to be associated with the plants. Further molecular characterisation of non-mycorrhizal rhizospheric fungi showed the presence of fungal species with coal-degrading capabilities that most likely played a role in the coal biotransformation observed. The discard coal dump environment was simulated in pot and column studies and coal biotransformation was reproduced, with this process enhanced by the addition of mycorrhizal and non-mycorrhizal rhizospheric fungal inocula to the environment. Mycorrhizal and non-mycorrhizal species in the inoculum were re-isolated from the simulated environment fulfilling a number of Koch’s postulates and indicating a causal role in the biotransformation of coal. An inversion of conventional mycorrhizal colonisation was demonstrated in this system with reduction in extraradicular presence and an increase in intracellular colonisation compared to soil controls. A descriptive model was formulated suggesting a two-part fungal system involving organic carbon and nutrient exchange between the plant, mycorrhizal fungi and non-mycorrhizal coal-degrading rhizospheric fungi ultimately resulting in the biotransformation of coal. The biotransformation observed was comparable to reports of “rock-eating fungi”. Results suggest that the biological degradation of coal in situ with the production of a soil-like substrate could provide a feasible method of discard coal dump rehabilitation as well as provide a humic-rich substrate that can be utilised in further industrial applications.
41
De, Koker T. H. (Theodorus Hermanus) 1965. "Genetic and enzymatic characterisation of wood degrading strains of Phanerochaete species." Thesis, Stellenbosch : Stellenbosch University, 2000. http://hdl.handle.net/10019.1/51775.
Full textAbstract:
Thesis (PhD)--University of Stellenbosch, 2000.<br>ENGLISH ABSTRACT: White rot fungi are of interest in the paper and pulp industry because of their
removal of lignin from wood. In this study over 600 Basidiomycete fungi were
isolated from indigenous forests as well as from commercial Eucalyptus spp.
and Pinus spp. plantations in South Africa. One hundred isolates were
identified to genus level. Biochemical tests were done to screen the fungal
cultures for characteristics that are favourable for biopulping, e.g. low
cellulase activity with concomitant high activity of ligninolytic enzymes.
Various Phanerochaete isolates with potentially high ligninolytic activity were
identified.
Although Phanerochaete chrysosporium Burds. has previously been isolated
from the indigenous forest at Knysna in South Africa, this study showed that
P. chrysosporium was a natural coloniser of wood chip piles in South Africa,
indicating potential for application in industry. A possible new species of
Phanerochaete, viz. Phanerochaete pseudomagnoliae nom. provo (strain
PP25) from decayed wood collected in Stellenbosch, South Africa, was
described and illustrated. It differs from previously described Phanerochaete
species in having smaller basidiospares, and in the formation of few
chlamydospores on malt extract agar but more on xylose containing media.
The potential of using internal transcribed spacer DNA sequences (ITS) to
infer phylogenetic relationships among species of the genus Phanerochaete
was investigated. Consensus phylogenetic trees could be presented, but the
presence of ambiguous aligning sequences within the ITS made inferring of
phylogenetic relationships within the whole genus difficult.
Fifty-five South African strains of P. chrysosporium were screened for lignin
peroxidase (liP), manganese peroxidase (MnP) and glyoxal oxidase (GLOX)
enzymes. Manganese peroxidase activity was quantified on agar media. The
liP and GLOX activities of 13 selected strains, including control strains and
P. pseudomagnoliae (PP25), were also quantified on agar media. Differences
in MnP and GLOX activities existed among the strains. Preliminary
biochemical characterisation of strain PP25 indicated that the most important
difference was the apparent unique regulation of ligninolytic enzymes. Under
low nitrogen, liP activity of the selected strains showed no significant variation, whereas strain PP25 had significantly increased liP levels under
high nitrogen conditions. Restriction fragment length polymorph isms of the
lignin and manganese peroxidase gene DNA fragments showed variability
among strains, whereas there was probably only allelic variation for the glox
gene DNA fragments.
Previous research has indicated xylose oxidation activity within
P. chrysosporium. To investigate whether GLOX can oxidise xylose, a purified
recombinant GLOX (rGLOX) from P. chrysosporium BKM-F-1767 Burds. was
used in this study. This rGLOX oxidised D-xylose and D-glucose (D-xylose >
D-glucose) to produce H202. Xylose was oxidised to xylono-1 ,4-lactone with a
1:1 stoichiometric relationship between H202 produced and xylose used.
Xylono-1,4-lactone was converted non-enzymatically to xylonate. This
suggested that the furanose form of xylose, rather than the pyranose form, is
a substrate of GLOX. The production of H202 and the removal of inhibitory
compounds by GLOX could enhance ligninolytic activity. .
To conclude, unique strains of P. chrysosporium have been isolated from
South Africa with potential biotechnological use in paper manufacturing. The
relationship of P. pseudomagnoliae nom. provo to other Phanerochaete
species was evaluated and light was shed on the possible role of GLOX in
lignin degradation.<br>AFRIKAANSE OPSOMMING: Witvrot swamme is van belang vir die papier en pulp industrie omdat hulle
lignin vanaf hout kan verwyder. Meer as 600 Sasidiomiseet fungi, afkomstig
vanaf inheemse woude asook kommersiële Eucalyptus spp. en Pinus spp.
plantasies, IS geïsoleer. Een honderd isolate is tot op genusvlak
geïdentifiseer. Die isolate is biochemies vir eienskappe wat voordelig vir
"bioverpulping" kon wees, bv. die gelyktydige produksie van lae sellulosemaar
hoë ligninolitiese ensiemaktiwiteit, getoets. Verskeie isolate met
potensieel hoë vlakke van ligninolitiese aktiwiteit is verkry.
Alhoewel Phanerochaete chrysosporium Surds. vantevore in die Knysna
inheemse woud in Suid-Afrika geïsoleer is, het hierdie studie gewys dat
P. chrysosporium natuurlik op hope houtblokkies voorgekom, met moontlike
toepasing in die industrie. Isolaat PP25, geisoleer vannaf verrottende hout te
Stellenbosch, Suid Afrika, is as 'n potensieel nuwe spesie van die genus
Phanerochaete beskryf en as Phanerochaete pseudomagnoliae nom. provo
benoem. Hierdie isolaat verskil van ander Phanerochaete-spesies daarin dat
dit kleiner basidiospore vorm en nie klamydospore op moutekstrakagar
produseer nie, maar wel op media wat xilose bevat. Die potensiaal van intern
getranskribeerde spasieerder ONS opeenvolging vir die aflei van filogenetiese
verhoudings tussen spesies van die genus Phanerochaete is ondersoek.
Konsensus filogenetiese bome kon bepaal word, maar die teenwoordigheid
van varieerbare areas het die afleiding van filogenetiese verwantskappe vir
die hele genus bemoeilik.
Vyf-en-vyftig Suid-Afrikaanse isolate van P. chrysosporium is vir die
teenwoordigheid van lignienperoksidase- (liP), mangaanperoksidase- (MnP)
en glioksaaloksidase (GLOX)-aktiwiteit getoets. Vlakke van MnP-aktiwiteit is
op agarplate gekwantifiseer. Vlakke van LiP- en GLOX-ensieme op agarplate
is vir 13 geselekteerde isolate, insluitend kontroles en ras PP25,
gekwantifiseer. Aktiwiteit van MnP en GLOX het statisties betekenisvol tussen
isolate verskil. Lignienperoksidase-aktiwiteit onder lae stikstof toestande het
nie statisties betekenisvol van mekaar verskil nie. Onder hoë stikstof
toestande het isolaat PP25 wel verhoogde liP-aktiwiteit getoon. Restriksie
fragment polimorfismes van die lignien- en mangaanperoxidase-gene het variasie getoon, terwyl waarskynlik slegs alleliese variasie vir die glox geen
waargeneem IS.
Rekombinante GLOX (GLOX vanaf P. chrysosporium BKM-F-1767) het xilose
en glukose (D-xilose > D-glukose) geoksideer met meegaande produksie van
H202. Xilose is na xilono-1,4-laktoon geoksideer met 'n 1:1 stoigiometrie
tussen H202-produksie en xilose verbruik. Xilono-1,4-laktoon is nieensiematies
na xilonaat omgeskakel. Bogenoemde resultaat dui aan dat die
furanose vorm van xilose die werklike substraat vir GLOX is. Deur die
meegaande produksie van H202 en die verwydering van inhiberende produkte
word lignoliese aangehelp.
Ten slote, unieke P. chrysosporium rasse met potensiële gebruik in
papiervervaardiging is in Suid-Afrika geisoleer. Die genetiese diversiteit van 'n
nuwe spesie, P. pseudomagnoliae, is bepaal en nuwe lig is op die potensiële
rol van GLOX in lignienafbraak gewerp.
42
Campos, Katherine Helen de Sa. "Identification and characterization of components that overcome secretion limitations of the yeast Pichia pastoris : a thesis." Scholarly Commons, 2001. https://scholarlycommons.pacific.edu/uop_etds/844.
Full text
43
Lennartsson, Patrik. "Zygomycetes and cellulose residuals : hydrolysis, cultivation and applications." Doctoral thesis, Högskolan i Borås, Institutionen Ingenjörshögskolan, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:hb:diva-3608.
Full textAbstract:
Zygomycetes is a class of fungi living worldwide as saprobes, as part of mycorrhizae, and as parasites. Humans have used some zygomycetes for centuries in the production of traditional foods, e.g. Indonesian tempe. In the present thesis, the experimental focus was on two zygomycetes strains, Mucor indicus CCUG 22424 and Rhizopus sp. IT. One of the distinguishing features of M. indicus is its dimorphism. The different cell forms were influenced by the culturing conditions. After inoculation, when the initial spore concentration was high (6-8×106 spores/ml), yeast-like growth dominated under anaerobic conditions. With a smaller inoculum, yielding 1-2×105 spores/ml, and access to oxygen, filamentous forms dominated. Only negligible differences in ethanol yield (390-420 mg/g hexoses), productivity (3-5 g/l/h), and inhibitor tolerance were observed. Differential expressions of probably four genes were observed between the yeast-like and filamentous growth forms. Lignocelluloses are a suitable substrate for cultivating zygomycetes, as they occur in abundance, particularly since zygomycetes, unlike Saccharomyces cerevisiae, can utilise pentoses. Lignocelluloses require pretreatment to achieve efficient hydrolysis of the cellulose. N-methylmorpholine-N-oxide (NMMO) was tested for pretreatment of spruce and birch. Reducing wood chip size and/or prolonged pretreatment, promoted hydrolysis yield. Best yields were achieved from <2 mm chips and 5 h pretreatment. The hydrolysate was used for fermentation with M. indicus, resulting in 195 and 175 mg ethanol/g wood, and 103 and 86 mg fungal biomass/g wood, from spruce and birch respectively. Orange peel is another potential substrate. However, the hydrolysate contained 0.6 % (v/v) D-limonene, ten times higher than the concentration inhibiting S. cerevisiae. M. indicus was more resistant and successfully fermented the hydrolysate, producing 400 mg ethanol/g hexoses and 75 mg fungal biomass/g sugars. Both M. indicus and Rhizopus sp. grew in 1.0 % and 2.0 % D-limonene, although the latter was unable to grow in the hydrolysate. A third substrate was also used, spent sulphite liquor (SSL), which is a by-product from sulphite paper pulp mills. The SSL was diluted to 50 % and used for airlift cultivations of Rhizopus sp. In 1.0 vvm aeration, up to 340 mg biomass/g sugars was produced. Prolonged cultivations generally decreased the protein (from 500 to 300 mg/g) and lipid (from 70 to 20 mg/g) contents. In contrast, the cell wall fraction, measured as alkali-insoluble material (AIM), increased (160-280 mg/g), as did the glucosamine (GlcN) content (220-320 mg GlcN/g AIM). The produced fungal biomass could serve as animal feed, e.g. for fish.<br><p>Akademisk avhandling som för avläggande av teknologie doktorsexamen vid Chalmers tekniska högskola försvaras vid offentlig disputation den 9 februari 2012, klockan 10.00 i KS101, Kemigården 4, Göteborg.</p>
44
Garcia, Alexandre Kopte. "Avaliação da atividade lipolítica de fungos filamentosos da costa brasileira." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-24032011-170215/.
Full textAbstract:
A produção de compostos biologicamente ativos por micro-organismos marinhos tem atraído crescente interesse de biotecnólogos e microbiologistas nas últimas décadas. No presente trabalho 162 isolados de fungos filamentosos recuperados de amostras de água do mar e de dez diferentes macro-organismos marinhos foram avaliados quanto a produção de lipases pelo método de High Throughput Screening (HTS). Destes, 45 isolados tiveram a produção de lipases confirmada individualmente. A atividade lipolítica foi quantificada, e os resultados sugerem que fungos filamentosos oriundos de ambientes marinhos tem maior potencial para produção de lipases estáveis em pH alcalino. Entre estes, Fusarium sp. CBMAI 1227, Aspergillus parasiticus CBMAI 1228 e Trichoderma sp. CBMAI 1229 apresentaram atividade expressiva e de interesse biotecnológico em pH 8,0 (23,1, 12,7 e 12,2 U respectivamente). Os resultados deste trabalho demonstram o potencial dos fungos provenientes de ambiente marinho para aplicações biotecnológicas, e estimulam novos estudos envolvendo lipases.<br>The production of biologically active compounds by marine microorganisms has been drawing increasing interest of biotechnologists and microbiologists in the last decades. In the present work 162 filamentous fungi isolates recovered from water samples and ten different marine macroorganisms were evaluated for the production of lipases by the method of High Throughput Screening (HTS). Out of those, 45 had the lipase production individually confirmed. The lipolytic activity was quantified, and the results suggest that marine-derived filamentous fungi have a greater potential for the production of lipases stable under alkaline pH. Among these, Fusarium sp. CBMAI 1227, Aspergillus parasiticus CBMAI 1228 and Trichoderma sp. CBMAI 1229 showed expressive and biologically interesting activity in pH 8,0 (23,1, 12,7 and 12,2 U/min respectively). The results of the present work show the potential of marine-derived fungi in biotechnological applications, and stimulate new studies involving lipases.
45
Marques, Marina Paganini [UNESP]. "Estudo da hidrólise do bagaço de cana-de-açúcar por fungos filamentosos." Universidade Estadual Paulista (UNESP), 2010. http://hdl.handle.net/11449/88050.
Full textAbstract:
Made available in DSpace on 2014-06-11T19:23:06Z (GMT). No. of bitstreams: 0
Previous issue date: 2010-03-30Bitstream added on 2014-06-13T19:08:57Z : No. of bitstreams: 1
marques_mp_me_araiq.pdf: 653823 bytes, checksum: 55d8fe257a4e7fa45682f56f8a5f622d (MD5)<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)<br>O bagaço de cana de açúcar (BCA) é o principal subproduto das indústrias de açúcar e álcool no Brasil e em função da sua abundância e baixo custo poderia ser usado para aumentar a produção de etanol, fornecendo vantagens econômicas e ambientais. A hidrólise enzimática é um caminho promissor para obter açúcares de BCA, mas a baixa acessibilidade enzimática da celulose nativa é a chave do problema neste processo. Por este motivo, o pré tratamento da biomassa é exigido para remover a lignina e a hemicelulose. O objetivo principal deste trabalho foi avaliar o potencial biotecnológico de fungos filamentosos capazes de produzir grandes quantidades de enzimas celulolíticas utilizando o BCA como substrato com pré tratamento térmico e termo ácido. As espécies fúngicas selecionadas para este estudo foram: Aspergillus nidulans (mutante melanizado), Trichophyton terrestre e Aspergillus niger. Para o cultivo em estado sólido, o bagaço pré tratado foi inoculado com micélios ou esporos dos fungos e incubados a temperatura ambiente durante 15, 30 e 60 dias. A eficácia da hidrólise microbiana foi avaliada em relação ao açúcar total (AT), ao açúcar redutor (AR), as concentrações de fenol (Fe) e a atividade das enzimas celulolíticas (celulase, avicelase e CMCase). Os resultados mostraram que a quantidade de AT, AR e Fe foi mais elevada quando os fungos foram crescidos sobre o BCA com pré tratamento termo ácido comparado ao BCA com pré tratamento térmico. Dentre os fungos estudados, o filtrado da cultura do A. niger crescido sobre o BCA com pré tratamento termo ácido por 60 dias pode ser usado como o meio fermentativo para a produção de etanol devido a uma elevada concentração açúcar redutor (1,16% m/v ou 11,6 g.L 1 ) e de baixas concentrações de fenol (0,003% m/v ou 0,03 g.L 1 ). O nível o mais elevado de celulase...<br>The sugar bagasse cane (SCB) is the major by product of the sugar and alcohol industries in Brazil and in function of its abundance and low cost could be used to increase ethanol production, providing economic and environmental advantages. Enzymatic hydrolysis is a promising way for obtaining sugars from SCB, but the low enzymatic accessibility of the native cellulose is a key problem in this processes. For this reason, the pretreatment of biomass is required to remove lignin and hemicellulose. The main objective of this work was to evaluate the biotechnological potential of filamentous fungi to produce large amounts of cellulolitic enzymes using thermal and/or acid pretreated SCB as substrate. The selected fungal species for this study were: Aspergillus nidulans (melanized mutant), A. niger and Trychophyton terrestre. For cultivation in solid state, the pretreated bagasse was inoculated with mycelia or spores of the fungi and incubate at room temperature during 15, 30 and 60 days. The effectiveness of the microbial hydrolysis was evaluated in terms of total sugar (TS), reducing sugar (RS) and phenol (Phe) concentrations and also activity of cellulolitic enzymes (cellulase, avicelase and CMCase). The results showed that the amount of TS, RS and Phe was higher when the fungi grown on acid and thermal pretreated SCB compared to thermal pretreated SCB. Among fungi studied, the filtrate of A. niger culture grown on acid and thermal pretreated SCB for 60 days could be used as fermentative medium for production of ethanol due to presence of high concentration of reducing sugar (1,16% m/v or 11,6 g.L 1 ) and low amounts of phenol (0,003% m/v or 0,03 g.L 1 ). The highest level of cellulase, corresponding to 0,74 IU.mL 1 , was obtained in the A. nidulans culture after 15 days of growth. For CMCase and avicelase, the maximum activity was exhibited respectively... (Complete abstract click electronic access below)
46
Leite, Carla Andréa [UNESP]. "Influência das condições de cultivo e métodos de extração na produção de metabólitos antioxidantes por fungos isolados do litoral paulista." Universidade Estadual Paulista (UNESP), 2010. http://hdl.handle.net/11449/88055.
Full textAbstract:
Made available in DSpace on 2014-06-11T19:23:06Z (GMT). No. of bitstreams: 0
Previous issue date: 2010-02-26Bitstream added on 2014-06-13T19:49:48Z : No. of bitstreams: 1
leite_ca_me_araiq.pdf: 5762801 bytes, checksum: bc09a9c50cb41d4cbe82e881237028f7 (MD5)<br>Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)<br>Vários estudos indicam que os antioxidantes podem prevenir e/ou atenuar o dano oxidativo causado pelos radicais livres, os quais em excesso estão associados com várias doenças neurodegenerativas, como Alzheimer e Parkinson. Nos últimos anos houve um aumento na descoberta de antioxidantes naturais devido à possibilidade de produção em larga escala a um custo menor que a síntese química. Devido ao seu enorme potencial de exploração, os fungos derivados do ambiente marinho são considerados um dos mais importantes recursos para a obtenção de novos agentes terapêuticos, pois um grande número de metabólitos estruturalmente novos e biologicamente ativos tem sido relatado destes organismos. Estudos também mostraram que a quantidade e a diversidade dos metabólitos secundários produzidos pelos fungos podem variar de acordo com o método de extração aplicado e as condições de cultivo. Neste contexto, o objetivo deste estudo foi identificar taxonomicamente algumas espécies de fungos isolados das praias do Cabelo Gordo de Fora (São Sebastião/SP) e Balneário (Peruíbe/SP) e avaliar a influência do solvente, tempo de extração, temperatura de evaporação dos solventes, o meio de cultura (Meio Completo Marinho e Meio Sabouraud) e a fase de crescimento do fungo (exponencial e estacionária) para obtenção de compostos com atividade antioxidante e também analisar cromatograficamente os extratos obtidos. Os fungos estudados foram identificados como: Aspergillus niger, A. versicolor, Aureobasidium pullulans, Cladosporium, Exophiala sp; Madurella grisea, Penicillium sp, Rhizopus oryzae, Trichophyton tonsurans e Trichophyton terrestre. Utilizando análise estatística, os resultados obtidos mostraram que a atividade antioxidante foi significantemente influenciada pelos parâmetros estudados. Em todos os extratos, independente da espécie do fungo, a atividade antioxidante...<br>Various studies indicate that the antioxidants can prevent and/or attenuate the oxidative damage caused by free radicals, which in excess are associated with various neurodegenerative disorders like Alzheimer's and Parkinson's disease. In recent years, there had a growing interest in the discovery of natural antioxidants due to the possibility of largescale production at a lower cost than chemical synthesis. Marine-derived fungi, due to its enormous potential for exploration, have been considered one of the most important resources to obtain of new therapeutic agents because a large number of structurally novel and biologically active metabolites have been reported from these organisms. Studies also have shown that the quantity and diversity of secondary metabolites produced by fungi can vary depending on the applied extraction method and the cultivation conditions. In the context, the objectives of this study were to identify taxonomically some fungi isolated from two locations: Cabelo Gordo de Fora beach (São Sebastião -SP) and Balneário beach, (Peruíbe – SP) and to evaluate the influence of solvent, extraction time, evaporation temperature of the solvents, the culture media (Marine Complete Medium and Sabouraud Medium) and stage of fungal growth (exponential and stationary phases) for obtaining compounds with antioxidant activity and also to analyze chromatographically the extracts obtained. The fungi studied were identified as: Aspergillus niger, A. versicolor, Aureobasidium pullulans, Cladosporium, Exophiala sp; Madurella grisea, Penicillium sp, Rhizopus oryzae, Trichophyton tonsurans and Trichophyton terrestre. Using statistical analysis, the results obtained showed that the antioxidant activity was significantly influenced by parameters studied. In all extracts, independent of the fungal species, the antioxidant activity increased with the longer time of extraction... (Complete abstract click electronic access below)
47
Finimundy, Tiane Cristine. "Efeito biológico de extratos de pleurotus sajor-caju e lentinula edodes em cultivo de células tumorais." reponame:Repositório Institucional da UCS, 2013. https://repositorio.ucs.br/handle/11338/660.
Full textAbstract:
Produtos naturais são cada vez mais vendidos como suplementos dietéticos devido a várias das suas propriedades terapêuticas, enfatizando a pesquisa para novos medicamentos. Este trabalho mostra que extratos aquosos de Lentinula edodes, Pleurotus sajor-caju e Agaricus blazei exercem atividade citotóxica, através do ensaio da redução do sal de brometo tetrazólio, nas linhagens celulares humanas de carcinoma de laringe (Hep-2) e carcinoma cervical (HeLa). Os extratos foram obtidos inicialmente em três diferentes temperaturas (4C, 22ºC e 50C). Entretanto, a temperatura ambiente foi escolhida para a realização dos outros experimentos já que apresentou os melhores resultados na redução da viabilidade celular. Ensaios bioquímicos realizados em paralelo indicaram uma maior quantidade de polifenóis nos extratos do L. edodes nas três temperaturas de extração e também uma melhor capacidade de eliminação do radical 2,2-difenil-1-picrilhidrazilo. Os resultados para a atividade citotóxica revelaram que o extrato de P. sajor-caju foi mais eficiente do que os extratos de L. edodes e A. blazei, nas linhagens testadas. Modificações morfológicas observadas nas células foram confirmadas pela coloração de Giemsa, após o tratamento com os extratos, sugerindo a inibição da proliferação e indução da apoptose dose/dependente. O tipo de morte celular foi testada com o método da anexina V acoplada a um fluorocromo mais iodeto de propídio, confirmando que tanto os extratos de L. edodes e P. sajor-caju induzem a apoptose tardia. Ensaios da composição química obtidos por cromatografia gasosa acoplada ao espectrômetro de massas mostraram a presença de ácidos graxos nos extratos, com uma prevalência do ácido palmítico no extrato de L. edodes e P. sajorcaju e ácido esteárico no extrato de A. blazei. A quantificação dos carboidratos e proteínas indicaram uma maior quantidade de carboidratos no L. edodes, e o A. blazei apresentou um maior teor de proteínas. Estes resultados indicam que os extratos aquosos dos cogumelos L. edodes, P. sajor-caju e A. Blazei são potenciais fontes antioxidantes, ricos em proteínas e ácidos graxos e possuem atividade anticancerígena por indução da apoptose. No entanto, novos estudos são necessários para explorar seus usos terapêuticos e para elucidar o seu modo de ação no organismo.<br>Submitted by Marcelo Teixeira (mvteixeira@ucs.br) on 2014-06-11T12:32:57Z
No. of bitstreams: 1
Dissertacao Tiane Cristine Finimundy.pdf: 150195 bytes, checksum: 16fa4de06cc7be6b6a285105c8c35610 (MD5)<br>Made available in DSpace on 2014-06-11T12:32:57Z (GMT). No. of bitstreams: 1
Dissertacao Tiane Cristine Finimundy.pdf: 150195 bytes, checksum: 16fa4de06cc7be6b6a285105c8c35610 (MD5)<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior<br>Natural products are increasingly sold as dietary supplements due to several of its therapeutic properties, emphasizing the search for new drugs. This work shows that aqueous extracts of Lentinula edodes, Pleurotus sajor-caju and Agaricus blazei, exert cytotoxic activity by testing the reduction of tetrazolium bromide salt in human cell lines of laryngeal carcinoma (Hep-2) and cervix carcinoma (HeLa). The extracts were obtained initially at three different temperatures (4C, 22ºC and 50C), however ambient temperature was chosen to carry out other experiments that have shown the best results in the reduction of cell viability. Biochemical assays performed in parallel showed an increased amount of polyphenols in the extracts of L. edodes the three extraction temperatures and also a better ability to eliminate the radical 2,2-diphenyl-1- picrylhydrazyl. The results for the cytotoxic activity revealed that the extract of P. sajor-caju was more efficient than that of L. edodes and A. blazei, the strains tested. Observed morphological changes in the cells was confirmed by Giemsa staining after treatment with the extracts, suggesting that the inhibition of proliferation and induction of apoptosis dose/dependent. The type of cell death was assayed by the method of annexin V bound to a fluorochrome and propidium iodide further confirming that both of L. edodes and P. sajor-caju induce apoptosis late. Testing the chemical composition obtained by gas chromatography mass spectrometer showed the presence of fatty acids in the extracts, with a prevalence of palmitic acid in the extract of L. edodes and P. sajor-caju and stearic acid extract of A. blazei. The quantification of carbohydrates and proteins indicated a greater amount of the carbohydrates L. edodes and A. blazei showed a higher protein content. These results indicate that the aqueous extracts of the mushroom L. edodes , P. sajor-caju and A. blazei, are potential sources antioxidants, high in protein and fatty acids and have anticancer activity by induction of apopt sis. However, further studies are needed to explore their therapeutic use and to elucidate its systemic effects.
48
Stoll, Anita. "Bioaccumulation of heavy metals by the yeast S. cerevisiae and the bioremediation of industrial waste water." Thesis, Rhodes University, 1997. http://hdl.handle.net/10962/d1004075.
Full textAbstract:
Water is an essential element in all aspects of life and is vital for both domestic and industrial purposes regarding both the quality and quantity thereof. Similar to many other drought stricken countries, South Africa requires water for the socio-economic growth of the country, yet is faced with the problem of maintaining the quality of its drinking water as well as protecting the dwindling supplies. In an attempt to prevent the deterioration of South African water supplies the treatment, purification and recycling of industrial and mining waste water has recently become of prime importance. Many industrial and mining waste waters contain heavy metals in toxic quantities. The conventional processes that have been used till recently to address this problem, are often expensive or contain chemical agents which compound the environmental problem. As an alternative biological methods of metal accumulation appear to offer an economic and efficient alternative to these methods. An advantage to the South African scenario is the commercial production of the yeast, S. cerevisiae as a readily inexpensive by-product from some fermentation industries, Yeast cells, and in particular S. cerevisiae have proven to be capable of accumulating heavy metals, and therefore exhibit potential application in the bioremediation of waste water. The aim of this project was twofold. The initial part of this work attempted to define the mechanisms of metal accumulation by the yeast cells and cellular components. The information obtained from these initial studies provided a data base required for the development of a bioremediation system. Initial contact with the metal ions occurs at the wall interface of the yeast cell. Metal accumulation appears to be a function of all the cell wall components. The isolated cell wall components are better metal chelators then the intact cell walls. An apparent affinity series of mannan > chitin> glucan > intact cell walls exists. However, these components differ in their affinities for metal ions. Storage of metal ions within the cell occurs predominantly in the vacuole. The present study concluded that metal accumulation by the vacuole could be related to size. Metal accumulation occurred in the order of Cu2+ > Co2+ > Cd2+ with a corresponding decrease in atomic radii of Cd2+ > C02+ > Cu2+. Vacuolar ion deposition occurs at an early stage during the internalization of metal ions within the yeast cells. At the onset of vacuolar saturation, depositions of metal ions as granules within the cytosol occurs. In the presence of heavy metal cations viable yeast cells can be shown to exhibit two types of cellular responses. Uptake of Cu2+ and Cd2+ causes the loss of intracellular physiological cations from within the yeast cell. In comparison, uptake of Co2+ into the cell does not have this effect. All three heavy metal cations initiate plasma cell membrane permeability, thus the Cu2+ and Cd2+ induced loss of the intracellular cations, occurs. ~ a result of ion-exchange mechanisms and not due to cation leakage brought about by membrane permeabilization. Uptake of heavy metals by viable yeasts appears to be generally non-selective though the amount of metals accumulated are largely affected by the ratio of ambient metal concentration to biomass quantity. In addition, the energy dependent nature of internalization necessitates the availability of an external energy source for metal uptake by viable yeast cells. For these reasons metal removal from industrial waste water was investigated using non-viable biomass. By immobilizing the yeast cells additional mechanical integrity and stability was conferred apon the biomass. The three types of biomass preparations developed in this study, viz. polyvinyl alcohol (PV A) Na-alginate, PV A Na-orthophosphate and alkali treated polyethylenimine (PEI):glutaraldehyde (GA) biomass pellets, all fulfilled the necessary physical requirements. However, the superior metal accumulating properties of the PEI:GA biomass determined its selection as a biosorbent for bioremediation purposes. Biosorption of heavy metals by PEI:GA biomass is of a competitive nature, with the amount of metal accumulated influenced by the availability of the metal ions. This availability is largely determined by the solution pH. At low pH values the affinity of the biomass for metals decreases, whilst enhanced metal biosorption occurs at higher pHs, ego pH 4.5 - 6.0. PEI:GA biomass pellets can be implemented -as a biosorbent for the bi9remediaiton of high concentration, low-volume metal containing industrial waste. Several options regarding the bioremediation system are available. Depending on the concentration of the metals in the effluent, the bioremediation process can either be used independently or as part of a biphasic remediation system for the treatment of waste water. Initial phase chemical modification may be required, whilst two types of biological systems can be implemented as 'part of the second phase. The PEI:GA biomass can either be contained within continuous-flow fixed bed tanks or continuous-flow stirred bioreactor tanks. Due to the simplicity of the process and the ease with which scale-up is facilitated, the second type of system shows greater application potential for the treatment of this type of industrial waste water than the fixed-bed systems.
49
Cheeke, Tanya Elizabeth Amy. "An Evaluation of the Nontarget Effects of Transgenic Bacillus thuringiensis Maize on Arbuscular Mycorrhizal Fungi in the Soil Ecosystem." PDXScholar, 2013. https://pdxscholar.library.pdx.edu/open_access_etds/1027.
Full textAbstract:
My dissertation research examined the effect of the cultivation of insect-resistant Bacillus thuringiensis (Bt) maize on the soil environment with a goal of understanding how to obtain a balance between technological advancement and maintenance of a healthy soil ecosystem. Although Bt plants may help to reduce pesticide use, conferring benefits to farm workers and the environment, there are still unresolved questions about how the cultivation of Bt plants affects soil organisms. For this dissertation project, I used 14 different genotypes of Bt maize and non-Bt maize (Zea mays) to investigate the effects of transgenic Bt plants on the colonization ability, abundance, and diversity of symbiotic arbuscular mycorrhizal fungi (AMF) in the soil ecosystem over time. My greenhouse studies demonstrated that Bt maize plants exhibited reduced AMF colonization across multiple Bt genotypes and that effects were most pronounced when fertilizer levels were limited and spore density was high. In addition, I found that although differences in AMF colonization between Bt and non-Bt maize were difficult to detect in the field, spore density was reduced in Bt field plots after just one growing season. When I tested the effect of plot history on AMF and plant growth, I found that Bt and non-Bt maize plants had higher leaf chlorophyll content when grown in plots previously cultivated with the same maize line as the previous year, indicative of a positive feedback effect. I also examined potential mechanisms contributing to the reduced AMF colonization observed in Bt maize in greenhouse studies and determined that follow-up experiments should continue to investigate differences in root apoplastic invertase activity and root permeability in Bt and non-Bt maize. Future investigations would also benefit from examining potential differences in root exudate profiles and volatile organic compounds between Bt and non-Bt cultivars. Taken together, my dissertation results suggest that, while difficult to detect in the field, reductions in AMF colonization in Bt maize roots may be ecologically significant as they could lead to a decrease in the abundance of AMF propagules in the soil over time, potentially impacting soil structure and function in areas where Bt crop cultivation is high.
50
Domingo, Jody L. (Jody Lawren). "Stationary phase-specific expression of dominant flocculation genes for controlled flocculation of yeast." Thesis, Stellenbosch : Stellenbosch University, 2003. http://hdl.handle.net/10019.1/49788.
Full textAbstract:
Thesis (MSc)--University of Stellenbosch, 2003.<br>ENGLISH ABSTRACT: Flocculation can be defined as the asexual aggregation of yeast cells in a liquid
environment. This aggregation of cells, also referred to as "floc formation", will in most
cases lead to rapid settling or sedimentation. However, in so-called top-fermenting yeast
strains, the floes can move to the surface of the liquid growth substrate to form a thin layer,
called a "velum", that has been compared to other microbial biofilms.
The factors that trigger flocculation can be divided into two groups, physical/chemical
(e.g. sugar content, the presence of inorganic salts, organic solvents, ethanol
concentration, pH, agitation etc.) and genetic factors (genes that encode for proteins that
are either directly or indirectly involved in flocculation). In top-fermenting yeast strains,
several physical and chemical factors that trigger the process have been described,
including ethanol concentration, the presence of organic solvents, the absence of
molecular oxygen and the presence of inorganic salts (Ca2+ and Mg2+). These factors
appear to affect the cell hydrophobicity and the cell surface charge. As for genetic factors,
no specific genes have thus far been associated with flocculation in top fermenting yeast
strains.
In bottom-fermenting yeast strains, the physical and chemical factors that affect the
process are similar to the ones described for top-fermenting yeast strains, but include,
more specifically, the concentration of hexoses in the media (mannose or glucose), which
may inhibit the process. Indeed, flocculation in bottom-fermenting yeast strains has been
divided into the NewFlo type (inhibited by both mannose and glucose) and the Fl01 type
(inhibited by mannose) on the basis of the inhibitory effect of specific sugars. Various
genes have been associated with the flocculation of bottom-fermenting yeast strains.
Through genetic analysis, the genes have been categorised into dominant genes, semidominant
genes and recessive genes.
In order to better understand the role of some of the proteins responsible for
flocculation in S. cerevisiae, and to create strains whose flocculation properties would
correspond to those wanted in the wine and beer industries, three of the dominant
flocculation genes, FL01, FL05 and FL011, were placed under the control of the
promoters of the stationary phase-induced genes, ADH2 and HSP30. This was achieved
by replacing the native promoters of the flocculation genes with the heterologous
promoters through homologous recombination. The laboratory strain FY23, which is nonflocculent
due to the absence of the transcription factor that is required for flocculation,
F108p,was used as a model system.
Some of the transformed strains showed high flocculation, especially when the genes
were placed under control of the ADH2 promoter. In addition to this, the strains carrying a
modified FL011 gene showed increased adhesion to solid agar media and were able to
invade the growth substrate. These strains also showed an increased velum-forming ability
when grown in media containing only non-fermentable carbon sources.<br>AFRIKAANSE OPSOMMING: Flokkulasie kan gedefinieër word as die ongeslagtelike aggregasie van gisselle in 'n
vloeibare medium. Hierdie aggregasie van selle, kan ook na verwys word as flok formasie,
en in meeste gevalle lei dit tot In vinnige sedimentering. In oppervlak-fermenterende giste,
beweeg die flokke na die oppervlakte van die vloeibare medium om sodoende 'n flor -lagie
te vorm. Hierdie verskynsel was ook al gevind in ander organismes.
Verskeie faktore is verantwoordelik vir die effektiwiteit van flokkuklasie. Hierdie faktore
kan in twee groepe verdeel word, nl. fisiese en chemiese faktore (byv. suikerkonsentrasie,
die teenwoordigheid van anorganiese soute, organiese oplossings, etanol konsentrasie,
pH, ens.) en genetiese faktore (gene wat kodeer vir die proteïene wat of direk of indirek
betrokke is by flokkulasie). In oppervlak-fermenterende giste is daar al heelwat informasie
beskikbaar omtrent fisies en chemiese faktore se effekte op flokkulasie. Van die faktore
waarvan heelwat informasie beskikbaar is sluit in, etanol konsentrasie, die
teenwoordigheid van organiese oplossings, die afwesigheid van molekulêre suurstof en
die teenwoordigheid van anorganiese soute (Ca2+ en Mg2+). Hierdie faktore toon 'n effek of
hidrofobisiteit en elektriese lading op die seloppervlakte. Geen genetiese faktore kon tot
dusver gekoppel word aan flokkulasie in oppervlak-fermenterende giste nie.
Benede-oppervlak fermenterende giste se fisies en chemiese faktore wat effektiwiteit
van flokkulasie beïnvloed is dieselfde as die van oppervlak-fermenterende giste, maar sluit
in meer spesifiek, die konsentrasie van heksoses in die media (nl. mannose en glukose),
wat 'n inhiberende effek het op flokkulasie. Die benede-oppervlak fermenterende giste se
flokkulasie kan in twee segmente verdeel word nl. die NewFlo tipe (word geïnhibeer deur
die teenwoordigheid van mannose en glukose) en die Flo1-tipe (word geïnhibeer deur
slegs die teenwoordigheid van mannose). Verskeie gene was ook al geidentifiseer wat die
effektiwiteit van flokkulasie beïnvloed in benede-oppervlak fermenterende giste. Hierdie
gene kan in drie kategorieë opverdeel word, nl dominante-, semi-dominante- en
ressessiewe flokkulerende gene.
Ten orde 'n beter begrip te kry rondom die proteïene verantwoordelik vir die meeste
effektiwiteit ten opsigte van flokkulasie in S. cerevisiae, asook om giste te manipuleer om
spesifieke flokkulasie eienskappe te toon volgens die belange van die wyn en bierindustrieë,
was drie dominante flokkulerende gene, nl. FL01, FL05, en FL011, onder
regulering van stationêre fase-geïnduseerde promotors, PADH2 en PHSP30, geplaas. Dit was
verkry deur die vervanging van die wilde tipe promotors van die drie gene met die
stationêre fase-geïnduseerde promotors deur middel van homoloë rekombinasie. Die
laboratorium gisras, FY23, wat 'n nie-flokkulerende gisras is vanweë die afwesigheid van
'n transkripsionele faktor, Flo8p, wat verantwoordelik is vir die aktivering van belangrike
gene in flokkulasie, was gebruik as 'n wilde tipe ras.
Sommige van die transformante het In hoë mate van flokkulasie getoon, veral wanneer
onder die regulering van die PADH2. Tesame met laasgenoemde verskynsel, was daar gevind dat FL011-transformante 'n verhoging in hul vermoeë het om te bind aan die agar
en ook om die agar te penetreer. Laasgenoemde gisrasse het ook die vermoë getoon om
'n flor-lagie te vorm bo-op die oppervlakte van die medium, maar slegs wanneer dit in niefermenteerbare
koolstofbronbevattende media opgegroei word.