Academic literature on the topic 'Amylases. Saccharomyces cerevisiae'

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Journal articles on the topic "Amylases. Saccharomyces cerevisiae"

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Viktor, Marko J., Shaunita H. Rose, Willem H. van Zyl, and Marinda Viljoen-Bloom. "Raw starch conversion by Saccharomyces cerevisiae expressing Aspergillus tubingensis amylases." Biotechnology for Biofuels 6, no. 1 (2013): 167. http://dx.doi.org/10.1186/1754-6834-6-167.

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Gronchi, Nicoletta, Lorenzo Favaro, Lorenzo Cagnin, Silvia Brojanigo, Valentino Pizzocchero, Marina Basaglia, and Sergio Casella. "Novel Yeast Strains for the Efficient Saccharification and Fermentation of Starchy By-Products to Bioethanol." Energies 12, no. 4 (February 22, 2019): 714. http://dx.doi.org/10.3390/en12040714.

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The use of solid starchy waste streams to produce value-added products, such as fuel ethanol, is a priority for the global bio-based economy. Despite technological advances, bioethanol production from starch is still not economically competitive. Large cost-savings can be achieved through process integration (consolidated bioprocessing, CBP) and new amylolytic microbes that are able to directly convert starchy biomass into fuel in a single bioreactor. Firstly, CBP technology requires efficient fermenting yeast strains to be engineered for amylase(s) production. This study addressed the selection of superior yeast strains with high fermentative performances to be used as recipient for future CBP engineering of fungal amylases. Twenty-one newly isolated wild-type Saccharomyces cerevisiae strains were screened at 30 °C in a simultaneous saccharification and fermentation (SSF) set up using starchy substrates at high loading (20% w/v) and the commercial amylases cocktail STARGEN™ 002. The industrial yeast Ethanol Red™ was used as benchmark. A cluster of strains produced ethanol levels (up to 118 g/L) significantly higher than those of Ethanol Red™ (about 109 g/L). In particular, S. cerevisiae L20, selected for a scale-up process into a 1-L bioreactor, confirmed the outstanding performance over the industrial benchmark, producing nearly 101 g/L ethanol instead of 94 g/L. As a result, this strain can be a promising CBP host for heterologous expression of fungal amylases towards the design of novel and efficient starch-to-ethanol routes.
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Jacobus, Ana Paula, Jeferson Gross, John H. Evans, Sandra Regina Ceccato-Antonini, and Andreas Karoly Gombert. "Saccharomyces cerevisiae strains used industrially for bioethanol production." Essays in Biochemistry 65, no. 2 (July 2021): 147–61. http://dx.doi.org/10.1042/ebc20200160.

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Abstract Fuel ethanol is produced by the yeast Saccharomyces cerevisiae mainly from corn starch in the United States and from sugarcane sucrose in Brazil, which together manufacture ∼85% of a global yearly production of 109.8 million m3 (in 2019). While in North America genetically engineered (GE) strains account for ∼80% of the ethanol produced, including strains that express amylases and are engineered to produce higher ethanol yields; in South America, mostly (>90%) non-GE strains are used in ethanol production, primarily as starters in non-aseptic fermentation systems with cell recycling. In spite of intensive research exploring lignocellulosic ethanol (or second generation ethanol), this option still accounts for <1% of global ethanol production. In this mini-review, we describe the main aspects of fuel ethanol production, emphasizing bioprocesses operating in North America and Brazil. We list and describe the main properties of several commercial yeast products (i.e., yeast strains) that are available worldwide to bioethanol producers, including GE strains with their respective genetic modifications. We also discuss recent studies that have started to shed light on the genes and traits that are important for the persistence and dominance of yeast strains in the non-aseptic process in Brazil. While Brazilian bioethanol yeast strains originated from a historical process of domestication for sugarcane fermentation, leading to a unique group with significant economic applications, in U.S.A., guided selection, breeding and genetic engineering approaches have driven the generation of new yeast products for the market.
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YÁÑEZ, Esther, A. Teresa CARMONA, Mercedes TIEMBLO, Antonio JIMÉNEZ, and María FERNÁNDEZ-LOBATO. "Expression of the Schwanniomyces occidentalis SWA2 amylase in Saccharomyces cerevisiae: role of N-glycosylation on activity, stability and secretion." Biochemical Journal 329, no. 1 (January 1, 1998): 65–71. http://dx.doi.org/10.1042/bj3290065.

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The role of N-linked glycosylation on the biological activity of Schwanniomyces occidentalis SWA2 α-amylase, as expressed in Saccharomyces cerevisiae, was analysed by site-directed mutagenesis of the two potential N-glycosylation sites, Asn-134 and Asn-229. These residues were replaced by Ala or Gly individually or in various combinations and the effects on the activity, secretion and thermal stability of the enzyme were studied. Any Asn-229 substitution caused a drastic decrease in activity levels of the extracellular enzyme. In contrast, substitutions of Asn-134 had little or no effect. The use of antibodies showed that α-amylase was secreted in all the mutants tested, although those containing substitutions at Asn-229 seemed to have a lower rate of synthesis and/or higher degradation than the wild-type strain. α-Amylases with substitution at Asn-229 had a 2 kDa lower molecular mass than the wild-type protein, as did the wild-type protein itself after treatment with endoglycosidase F. These findings indicate that Asn-229 is the single glycosylated residue in SWA2. Thermostability analysis of both purified wild-type (T50 = 50 °C, where T50 is the temperature resulting in 50% loss of activity) and mutant enzymes indicated that removal of carbohydrate from the 229 position results in a decrease of approx. 3 °C in the T50 of the enzyme. The Gly-229 mutation does not change the apparent affinity of the enzyme for starch (Km) but decreases to 1/22 its apparent catalytic efficiency (kcat/Km). These results therefore indicate that glycosylation at the 229 position has an important role in the extracellular activity levels, kinetics and stability of the Sw. occidentalis SWA2 α-amylase in both its wild-type and mutant forms.
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Yamada, Ryosuke, Syun-ichi Yamakawa, Tsutomu Tanaka, Chiaki Ogino, Hideki Fukuda, and Akihiko Kondo. "Direct and efficient ethanol production from high-yielding rice using a Saccharomyces cerevisiae strain that express amylases." Enzyme and Microbial Technology 48, no. 4-5 (April 2011): 393–96. http://dx.doi.org/10.1016/j.enzmictec.2011.01.002.

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Arifiyanti, Nanda Ayu, Dewi Nafisatul Aqliyah, and Mu'tasim Billah. "Bioetanol Dari Biji Nangka Dengan Proses Likuifikasi dan Fermentasi Menggunakan Saccharomyces Cerevisiae." ChemPro 1, no. 01 (March 31, 2020): 51–55. http://dx.doi.org/10.33005/chempro.v1i01.47.

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Potensi biji nangka (Arthocarphus heterophilus) belum dieksploitasi secara optimal. Biji nangka dapat diolah menjadi bahan baku pembuatan bioetanol. Penelitian ini menggunakan metode hidrolisis enzimatis dengan enzim α-amylase dan glukoamilase untuk memecah pati menjadi glucose dan metode fermentasi dapat mengubah glucose menjadi bioetanol menggunakan bakteri saccharomyces cerevisiae. Penelitian ini bertujuan untuk menentukan komposisi variasi penambahan enzim α-amylase dan glukoamilase yang relative baik, waktu fermentasi yang relative baik pada pembentukan bioethanol. Pembuatan bioethanol dilakukan dengan biji nangka 50gram, selanjutnya dikeringkan dengan oven suhu 150 0C selama 1jam, kemudian digiling, kemudian masuk ke proses hidrolisis dengan variabel komposisi penambahan enzim α-amylase dan glukoamilase (20; 30; 40; 50; 60ml) selanjutnya diuji menggunakan refraktometer brix. Selanjutnya, proses fermentasi dilakukan dengan menambahkan yeast Saccharomyces cereviseae dengan variasi lama fermentasi 24; 36; 48; 60; 72 jam. Hasil analisa menunjukkan kadar glukosa yang relatif baik diperoleh pada volume enzim alfa-amilase dan gluko-amilase sebanyak 60ml dengan kadar sebesar 14%. Pada proses fermentasi diperoleh kadar alkohol sebesar 40% dengan waktu fermentasi 60jam.
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Moon, Hee Chul, Sol Han, João Borges, Tamagno Pesqueira, Hyunwoo Choi, Sang Yeong Han, Hyeoncheol Cho, Ji Hun Park, João F. Mano, and Insung S. Choi. "Enzymatically degradable, starch-based layer-by-layer films: application to cytocompatible single-cell nanoencapsulation." Soft Matter 16, no. 26 (2020): 6063–71. http://dx.doi.org/10.1039/d0sm00876a.

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Starch-based layer-by-layer (LbL) nanofilms are formed and enzymatically degraded on individual Saccharomyces cerevisiae in a highly cytocompatible fashion. Their enzymatic degradation by α-amylase is also exploited for the controlled release of DNA.
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Terashima, M., S. Katoh, B. R. Thomas, and R. L. Rodriguez. "Characterization of rice ?-amylase isozymes expressed by Saccharomyces cerevisiae." Applied Microbiology and Biotechnology 43, no. 6 (November 1995): 1050–55. http://dx.doi.org/10.1007/bf00166924.

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Sakai, A., Y. Shimizu, and F. Hishinuma. "Isolation and characterization of mutants which show an oversecretion phenotype in Saccharomyces cerevisiae." Genetics 119, no. 3 (July 1, 1988): 499–506. http://dx.doi.org/10.1093/genetics/119.3.499.

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Abstract We have isolated mutants responsible for an oversecretion phenotype in Saccharomyces cerevisiae, using a promoter of SUC2 and the gene coding for alpha-amylase from mouse as a marker of secretion. These mutations defined two complementation groups, designated as ose1 (over secretion) and rgr1 (resistant to glucose repression). The ose1 mutant produced an oversecretion of amylase by 12- to 15-fold under derepressing conditions; however, the amylase mRNA was present at nearly the same amount as it was in the parent cells. No expression of the amylase gene was detected under repressing conditions. The rgr1 mutant oversecreted amylase by 11- to 13-fold under repressing conditions by 15- to 18-fold under derepressing conditions. The rgr1 mutant showed pleiotropic effects on the following cellular functions: (1) resistance to glucose repression, (2) temperature-sensitive lethality, (3) sporulation deficieny in homozygous diploid cells, and (4) abnormal cell morphology. The rgr1 mutation was not allelic with ssn6 and cyc9, and failed to suppress snf1.
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Valkonen, Mari, Merja Penttilä, and Markku Saloheimo. "Effects of Inactivation and Constitutive Expression of the Unfolded- Protein Response Pathway on Protein Production in the Yeast Saccharomyces cerevisiae." Applied and Environmental Microbiology 69, no. 4 (April 2003): 2065–72. http://dx.doi.org/10.1128/aem.69.4.2065-2072.2003.

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ABSTRACT One strategy to obtain better yields of secreted proteins has been overexpression of single endoplasmic reticulum-resident foldases or chaperones. We report here that manipulation of the unfolded-protein response (UPR) pathway regulator, HAC1, affects production of both native and foreign proteins in the yeast Saccharomyces cerevisiae. The effects of HAC1 deletion and overexpression on the production of a native protein, invertase, and two foreign proteins, Bacillus amyloliquefaciens α-amylase and Trichoderma reesei endoglucanase EGI, were studied. Disruption of HAC1 caused decreases in the secretion of both α-amylase (70 to 75% reduction) and EGI (40 to 50% reduction) compared to the secretion by the parental strain. Constitutive overexpression of HAC1 caused a 70% increase in α-amylase secretion but had no effect on EGI secretion. The invertase levels were twofold higher in the strain overexpressing HAC1. Also, the effect of the active form of T. reesei hac1 was tested in S. cerevisiae. hac1 expression caused a 2.4-fold increase in the secretion of α-amylase in S. cerevisiae and also slight increases in invertase and total protein production. Overexpression of both S. cerevisiae HAC1 and T. reesei hac1 caused an increase in the expression of the known UPR target gene KAR2 at early time points during cultivation.
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Dissertations / Theses on the topic "Amylases. Saccharomyces cerevisiae"

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Ramachandran, Nivetha. "Development of improved [alpha]-amylases /." Link to the online version, 2005. http://hdl.handle.net/10019.1/1102.

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Ramachandran, Nivetha. "Development of improved α-amylases." Thesis, Stellenbosch : University of Stellenbosch, 2005. http://hdl.handle.net/10019.1/1102.

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Thesis (DSc (Microbiology))--University of Stellenbosch, 2005.
The technological advancement of modern human civilisation has, until recently, depended on extensive exploitation of fossil fuels, such as oil, coal and gas, as sources of energy. Over the last few decades, greater efforts have been made to economise on the use of these nonrenewable energy resources, and to reduce the environmental pollution caused by their consumption. In a quest for new sources of energy that will be compatible with a more sustainable world economy, increased emphasis has been place on researching and developing alternative sources of energy that are renewable and safer for the environment. Fuel ethanol, which has a higher octane rating than gasoline, makes up approximately two-thirds of the world’s total annual ethanol production. Uncertainty surrounding the longterm sustainability of fuel ethanol as an energy source has prompted consideration for the use of bioethanol (ethanol from biomass) as an energy source. Factors compromising the continued availability of fuel ethanol as an energy source include the inevitable exhaustion of the world’s fossil oil resources, a possible interruption in oil supply caused by political interference, the superior net performance of biofuel ethanol in comparison to gasoline, and a significant reduction in pollution levels. It is to be expected that the demand for inexpensive, renewable substrates and cost-effective ethanol production processes will become increasingly urgent. Plant biomass (including so-called ‘energy crops’, agricultural surplus products, and waste material) is the only foreseeable sustainable source of fuel ethanol because it is relatively low in cost and in plentiful supply. The principal impediment to more widespread utilisation of this important resource is the general absence of low cost technology for overcoming the difficulties of degrading the recalcitrant polysaccharides in plant biomass to fermentable sugars from ethanol can be produced. A promising strategy for dealing with this obstacle involves the genetic modification of Saccharomyces cerevisiae yeast strains for use in an integrated process, known as direct microbial conversion (DMC) or consolidated bioprocessing (CBP). This integrated process differs from the earlier strategies of SHF (separate hydrolysis and fermentation) and SSF (simultaneous saccharification and fermentation, in which enzymes from external sources are used) in that the production of polysaccharide-degrading enzymes, the hydrolysis of biomass and the fermentation of the resulting sugars to ethanol all take place in a single process by means of a polysaccharidefermenting yeast strain. The CBP strategy offers a substantial reduction in cost if S. cerevisiae strains can be developed that possess the required combination of substrate utilisation and product formation properties. S. cerevisiae strains with the ability to efficiently utilise polysaccharides such as starch for the production of high ethanol yields have not been described to date. However, significant progress towards the development of such amylolytic strains has been made over the past decade. With the aim of developing an efficient starch-degrading, high ethanol-yielding yeast strain, our laboratory has expressed a wide variety of heterologous amylase-encoding genes in S. cerevisiae. This study forms part of a large research programme aimed at improving these amylolytic ‘prototype’ strains of S. cerevisiae. More specifically, this study investigated the LKA1- and LKA2-encoded α-amylases (Lka1p and Lka2p) from the yeast Lipomyces kononenkoae. These α-amylases belong to the family of glycosyl hydrolases (EC 3.2.1.1) and are considered to be two of the most efficient raw-starch-degrading enzymes. Lka1p functions primarily on the α-1,4 linkages of starch, but is also active on the α-1,6 linkages. In addition, it is capable of degrading pullulan. Lka2p acts on the α-1,4 linkages. The purpose of this study was two-fold. The first goal was to characterise the molecular structure of Lka1p and Lka2p in order to better understand the structure-function relationships and role of specific amino acids in protein function with the aim of improving their substrate specificity in raw starch hydrolysis. The second aim was to determine the effect of yeast cell flocculence on the efficiency of starch fermentation, the possible development of high-flocculating, LKA1-expressing S. cerevisiae strains as ‘whole-cell biocatalysts’, and the production of high yields of ethanol from raw starch. In order to understand the structure-function relationships in Lka1p and Lka2p, standard computational and bioinformatics techniques were used to analyse the primary structure. On the basis of the primary structure and the prediction of the secondary structure, an N-terminal region (1-132 amino acids) was identified in Lka1p, the truncation of which led to the loss of raw starch adsorption and also rendered the protein less thermostable. Lka1p and Lka2p share a similar catalytic TIM barrel, consisting of four highly conserved regions previously observed in other α-amylase members. Furthermore, the unique Q414 of Lka1p located in the catalytic domain in place of the invariant H296 (TAKA amylase), which offers transition state stabilisation in α-amylases, was found to be involved in the substrate specificity of Lka1p. Mutational analysis of Q414 performed in the current study provides a basis for understanding the various properties of Lka1p in relation to the structural differences observed in this molecule. Knowing which molecular features of Lka1p contribute to its biochemical properties provides us with the potential to expand the substrate specificity properties of this α-amylase towards more effective processing of its starch and related substrates. In attempting to develop ‘whole-cell biocatalysts’, the yeast’s capacity for flocculation was used to improve raw starch hydrolysis by S. cerevisiae expressing LKA1. It was evident that the flocculent cells exhibited physicochemical properties that led to a better interaction with the starch matrix. This, in turn, led to a decrease in the time interval for interaction between the enzyme and the substrate, thus facilitating faster substrate degradation in flocculent cells. The use of flocculation serves as a promising strategy to best exploit the expression of LKA1 in S. cerevisiae for raw starch hydrolysis. This thesis describes the approaches taken to investigate the molecular features involved in the function of the L. kononenkoae α-amylases, and to improve their properties for the efficient hydrolysis of raw starch. This study contributes to the development of amylolytic S. cerevisiae strains for their potential use in single-step, cost-effective production of fuel ethanol from inexpensive starch-rich materials.
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Hudečková, Helena. "Studie možnosti využití odpadního pečiva k bioprodukci vybraných metabolitů." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2014. http://www.nusl.cz/ntk/nusl-217057.

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The aim of this diploma thesis was to study the possibility of using waste bread to bioproduction of selected metabolites. As bakery waste was used waste bread that came from coffee-house “Zastávka”. Bread was pre-treated by grinding into small particles and then it was made to form 15% w/v suspension, which was subjected to enzymatic hydrolysis. For the hydrolysis has been used the -amylase for liquefaction of the substrate and that was followed by a glucoamylase which sacharificated the substrate. There have been several methods of hydrolysis from which was chosen the optimal method for pre-treatment of the substrate prior to fermentation. The effectivity and a process of hydrolysis were determined spectrophotometrically by Somogyi-Nelson method. Final yields of glucose from hydrolysis were determined by HPLC method. Enzymatic hydrolysis was followed by fermentation, which was carried out in two ways, namely by adjusting the pH of the hydrolyzate to pH 5, and without pH adjustment. During the fermentation was carried out sampling in which was determined the content of glucose, glycerol and ethanol by HPLC method. The yeasts Saccharomyces cerevisiae were used for the fermentation which was performed at 30 °C. High yield of glucose was achieved by hydrolysis in two steps (70,28 gl-1), but for the fermentation was used mixed hydrolysis (second method of mixed hydrolysis) with yield 67,94 gl-1. High ethanol yield was achieved during fermentation without treatment pH, namely 31,5 gl-1.
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Gonçalves, Louise Garbelotti [UNESP]. "Produção de amilases de Rhizopus microsporus var. oligosporus e hidrólise enzimática do bagaço de mandioca visando a produção de etanol por Saccharomyces cerevisiae." Universidade Estadual Paulista (UNESP), 2016. http://hdl.handle.net/11449/138917.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O desenvolvimento de bioprocessos para produção de alimentos e energia, que utilizam resíduos agroindustriais como matéria-prima, é alvo de intensas pesquisas no mundo devido às limitações de novas fronteiras agrícolas e de fontes de energia renováveis. A utilização de enzimas nesses processos produtivos surge como uma forte tendência, dentre elas, as amilases. O objetivo desse estudo foi a otimização da produção de amilases empregando o fungo Rhizopus microsporus var. oligosporus, cultivado em Fermentação em Estado Sólido (FES), tendo como substrato os resíduos agroindustriais: farelo de trigo, bagaço de mandioca e bagaço de cana de açúcar. O hidrolisado obtido da hidrólise do bagaço de mandioca foi avaliado quanto à sua fermentabilidade por meio da fermentação alcoólica em batelada alimentada por Saccharomyces cerevisiae. A melhor fonte de carbono como substrato para formulação do meio de cultivo foi aquele contendo 100% de farelo de trigo. Em relação às fontes de nutrientes para produção de amilases, a maior atividade amilolítica (418 U/g) foi para a solução de nutrientes contendo (%): 1,34 de sulfato de amônio, 0,54 de monofosfato de amônio (MAP) e 1,86 de ureia. Porém, foi observado que, ao acrescentar um mínimo de 0,01 % (m/m) de extrato de levedura na solução de nutrientes, obteve-se aumento na atividade amilolítica de 418 U/g para 551 U/g. A hidrólise enzimática do bagaço de mandioca foi feita com a associação de celulases comerciais (Novozyme) (10 FPU/g no tempo zero horas) e com o extrato amilolítico (12 U/g no tempo 10 horas de reação) produzido em FES para a obtenção de um hidrolisado concentrado. Com estas condições foi possível converter 63% de bagaço em açúcares redutores com predomínio de glicose. Por meio da fermentação alcoólica em batelada alimentada por Saccharomyces cerevisiae, obteve-se eficiências de 74,6 e 76,8%, e produtividades etanólicas com 3,14 g L-1h-1 e 1,61 g L-1h-1 em 12 e 24 h, respectivamente. A produção de etanol a partir de um hidrolisado de bagaço de mandioca nas condições de hidrólise proposta foi possível, o que corresponde a uma alternativa à tecnologia da cana-de-açúcar para produção deste biocombustível.
The development of bioprocesses for food and energy production, which makes use of agricultural waste as raw material, is focus of intense research around the world due to limitations on new agricultural frontiers and on renewable energy sources. The use of enzymes in these processes emerges as a strong trend, among them, amylases. The aim of this study was the amylase production optimization employing fungus Rhizopus microsporus var. oligosporus, grown by Solid Station Fermentation (SSF), using as substrate these agricultural waste: wheat bran, cassava bagasse and sugarcane bagasse. The hydrolizate obtained from cassava bagasse hydrolysis was evaluated with regards to its fermentability through fed-batch alcoholic fermentation by Saccharomyces cerevisiae. The best carbon source as substrate for the culture medium formulation was the one containing 100% wheat bran. Regarding salts sources for amylases production, the higher amylolytic activity (418 U/g) was for the nutrient solution containing (%): ammonium sulfate 1.34; ammonium monophosphate 0.54 and urea 1.86. However, the addiction of 0.01% (w/w) yeast extract in salts solution, amylolytic activity increased from 418 U/g to 551 U/g. The enzymatic hydrolysis of cassava bagasse was performed with the combination of commercial cellulases (Novozyme) (10 FPU/g at time zero hours) and the amylolytic extract (12 U/g at time 10 hours) produced by SSF to obtain a hydrolizate concentrate. With these conditions it was possible to convert 63% of bagasse in reducing sugars predominantly glucose. Through the fed-batch alcoholic fermentation tests by Saccharomyces cerevisiae, efficiencies were determined as 74.6 and 76.8%, and the ethanolic productivities with 3.14 gL-1h-1 and 1.61 gL-1h-1, at 12 and 24 h, respectively. Ethanol production from cassava bagasse hydrolyzate in hydrolysis conditions proposal was possible, which corresponds to an alternative technology of sugarcane for production of biofuel.
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Juge, Nathalie. "Expression de deux isoenzymes d'alpha-amylase d'orge chez la levure saccharomyces cerevisiae : apport de la recombinaison homéologue à l'étude de la sécrétion et des relations structure-fonction." Aix-Marseille 3, 1993. http://www.theses.fr/1993AIX30060.

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Le travail de recherche presente dans ce memoire porte sur l'etude de deux isoenzymes de l'-amylase d'orge amy1 et amy2 par le biais de leur expression dans la levure saccharomyces cerevisiae. Les deux cdnas amy1 et amy2 sont inseres dans le vecteur d'expression episomal pma91 sous le controle du promoteur constitutif pgk et diriges par leur propre sequence signal. Dans ces conditions, l'isoenzyme recombinant reamy1 est secrete a raison de 2-3 g/ml alors que reamy2 n'est pratiquement pas detecte dans le milieu exterieur. L'analyse moleculaire de l'expression de ces deux isoenzymes a permis de circonscrire l'etape limitante de reamy2 au processus de secretion. La recherche des determinants structuraux responsables de cette difference de secretion a ete entreprise avec pour corollaire l'amelioration de la secretion de reamy2 ou d'une proteine presentant les proprietes d'amy2. L'axe central de cette etude repose sur la construction de molecules hybrides reamy1/reamy2 par recombinaison homologue in vivo chez la levure. Deux types de constructions ont ete realises afin de substituer des regions d'amy2 par les regions correspondantes d'amy1, la premiere consistant a echanger des longueurs croissantes en n-terminal de la molecule et la deuxieme visant a echanger la region c-terminale dans sa totalite. L'analyse moleculaire des differents hybrides obtenus revele une augmentation de la secretion pour les hybrides contenant au moins les 53 premiers residus d'amy1 en n-terminal de la molecule. Inversement, le seul changement de la sequence signal ou de la region c-terminale n'influence pas la secretion. La possibilite de faire secreter ces proteines hybrides a permis d'envisager leur caracterisation biochimique. Dans ce but, une nouvelle construction a ete realisee en vue d'etendre la region n-terminale d'amy1 a la fin du domaine b de la proteine reamy1/reamy2. Trois hybrides ont ete retenus sur la base de leur structure chimerique et analyses. Les resultats biochimiques soulignent l'importance du domaine b dans la liaison a l'inhibiteur basi, l'activite de l'enzyme et la stabilite a ph acide
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Santo, Paulo Filipe Ribeiro do Espírito. "Pesquisa de estirpes de Saccharomyces cerevisiae para expressão de proteína recombinante." Master's thesis, 2013. http://hdl.handle.net/10316/24681.

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Dissertação de mestrado em Bioquímica, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências da Vida Universidade de Coimbra
Recombinant DNA expression constitutes a major approach in gene function studies that naturally complement genetic and genomic research. Expression systems provide an invaluable tool for investigating the roles of novel gens, either in their original cellular environment or in specialized host organisms, to express high quantities of recombinant proteins for biochemical studies and structural determination, or even for industry applications or medical applications. Higher yield of proteins are achieved in prokaryotic systems and E. coli is the traditional host for producing recombinant protein. Its drawback of poor post-translational modifications when compared to a eukaryote organism, gave S.cerevisiae a chance to stand out as expression system. It’s well known genetics, influence on mankind’s economics and culture in beer wine and bread making during centuries, its GRAS status, fast and easy growth to high densities with low cost medium, along with its eukaryote machinery made it one of the most appreciated expression systems used today. Genetic engineering is one of the best tools to improve already existent strains, however yeast libraries harbour huge biodiversity with uncharacterized strains from witch much profit can be taken. New enzymes, new organic molecules and new strains with specific enhanced capacities can be lying in this libraries. In this project, S. cerevisiae library of wine and vineyeard strains was screened to discover and validate a S. cerevisiae expression strain. The screen was prepared first by assembling an expression vector with the pAMT20 shuttle vector as backbone, mus musculus salivary α-Amylase as reporter enzyme, KanMX4 as selection marker, and K. Lactis α-mating factor prepro peptide as secretion leader. Transformation was adapted and carried on in a high throughput manner to screen the library and activity seen as halos formed around colonies in agar plates supplements with starch. Two commercial lab strains were used as reference for protein production levels, and to do so, a large-scale expression in liquid medium was performed and protein purified from supernatant in various steps, and activity measured to set a threshold for later comparison with the yeast candidates selected from the library. From 400 strains, 3 candidates stood out the average and are serious candidates to proceed with validations as a strain to produce recombinant protein.
A expressão de ADN recombinante é uma abordagem abundantemente utilizada para estudar a função de genes complementando as áreas de investigação genética e genómica. Os sistemas de expressão representam ferramentas valiosas para investigar a função de novos genes, expressando-os em grandes quantidades, quer nos hospedeiros aos quais pertencem ou em sistemas heterólogos, para estudos de caracterização bioquímica, determinação de estrutura 3D, e ate para serem usados na indústria ou na área da medicina. Os sistemas procariotas como a tradicional E. coli produzem maiores quantidades de proteína recombinante mas apresentam algumas limitações nas modificações póstraducionais de proteínas. Face às limitações do sistema em E.coli, a S. cerevisiae destacou-se como sistema de expressão heteróloga simples e robusto que permitia modificações pós-traducionais em proteínas. Acresce ainda o facto de ser microrganismo que reúne imensa informação acerca do seu genoma, é usado como modelo de estudo de mecanismos eucariotas em diversas áreas da ciência, teve influência preponderante na cultura e economia humana durante seculos como a produção de pão cerveja ou vinho, o seu estatuto GRAS e o facto de atingir grandes densidades celulares de forma rápida e pouco dispendiosa tornou-o rapidamente num sistema robusto e de uso rotineiro no laboratório. A engenharia genética é uma ferramenta interessante para melhorar as características de algumas estirpes já existentes, no entanto existem outras estipes de leveduras que nunca foram vistas ou caracterizadas, que estão escondidas por de trás da enorme biodiversidade existente em bibliotecas criadas a partir do isolamento de estirpes no seu meio natural. Estas bibliotecas têm um potencial enorme para se descobrir por exemplo novas estirpes com capacidades peculiares e melhores, novas enzimas, novas moléculas orgânicas entre outras. Este projecto, tirou partido do facto de dispor de uma libraria de leveduras isoladas de mostos de vinho ao longo de vários anos, à qual se fez uma seleção de estirpes com capacidade de produzir proteína heteróloga a níveis superiores à média. Para esta selecção criteriosa construiu-se um vetor, baseado no pAMT20, contendo uma alfa amílase salivar de mus musculus que serviu proteína repórter de actividade das colonias recombinantes, a cassete KanMX4 que é uma marca de selecção que confere resistência ao antibiótico G418, e uma sequência do prepro peptído responsável pela secreção de uma feromona com origem no K. lactis. O protocolo de transformação foi ajustado para funcionar em larga escala, e os testes de actividade foram avaliados como o tamanho de halos formados por colonias transformadas em meio solido com amido. Duas colonias de laboratório foram usadas como controlos no que diz respeito à quantidade de proteína produzida. Para isso fez-se uma expressão em meio liquido em larga escala, e purificou-se a proteína recombinante do sobrenadante e mediu-se a actividade. Esta actividade serviria como patamar de expressão proteica para comprara mais tarde com os níveis de produção de proteína recombinante de uma potencial estirpe isolada da biblioteca. De 400 estirpes evidenciaram se 4 estirpes que são fortes fortes candidatos para continuar para a fase de validação de estirpe para expressão de proteína heterologa
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Book chapters on the topic "Amylases. Saccharomyces cerevisiae"

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"TABLE 3 Major Commercial Fermentation Conditions for Cereal Foods Fermentation conditions Bread Beer Whiskey Soy sauce Miso Main starters Baker's yeast Brewer's yeast Distillery yeast Molds Molds (Saccharomyces (Saccharomyces (Saccharomyces (Aspergillus spp.) (Aspergillus spp.) cerevisiae) cerevisiae) cerevisiae) Saccharomyces rouxii Lactic acid bacteria Lactobacillus delbrueckii Cereals Milled wheat Barley (malted) Corn Soybeans (defatted) Rice Milled rye Sorghum Rye (malted or not) Wheat Barley Minor: Minor: Barley (malted) Minor: Soybeans Barley (malted) Corn Wheat Barley flour Wheat (malted) Rice Wheat Other ingredients Water Water Water Water Salt Salt Hops Salt Hot pepper Sugar Adjuncts Fat (corn syrup, sugar Emulsifiers or starch) Dough strengtheners Preservatives Enzymes Fermentation 1-6h2-10 days 2-3 days (Koji: 3 days at 30°C) (Koji: 2 days at 30°C) conditions 20-42°C 3-24°C 32-35°C 3-12 months 2 days to 1 year Aging: Aging: 15-30°C 30-50°C 3 days-1 month 2-3 years or more 0-13°C 21-30°C baker's yeast is probably the most common of these microorganisms that may be a problem are bacteria (usual-starters; it is commercially produced in liquid, paste (com-ly spore-forming or lactic acid bacteria, especially in some pressed), or dry form. Recently, commercial lactic acid yeast fermentations), wild yeasts, and molds. bacteria starters have been introduced for cereal fermenta-Several spore-forming bacteria (e.g., Bacillus spp.) may tions, but this application is less frequent than their regular produce amylases and degrade hydrated starchy materials. use in dairy or meat fermentations. A close control of the In bread, heat-tolerant spores of Bacillus subtilis (formerly performance of commercial starters is important, since it Bacillus mesentericus) survive the baking process; after a has a major effect on the final products. few days in bread, they produce a spoilage called ropiness, characterized by yellow spots on crumb, putrid pineapple aroma, and stringiness when breaking a piece of bread. The spores of these species, when contaminating flour, may Considering the diversity of the microbial flora that may cause a major problem in bakeries since they are highly re-be present in cereals to be fermented, undesirable microor-sistant in the environment and difficult to eliminate. How-ganisms are likely to be part of this flora and may produce ever, these bacterial infections have become rare in recent problems in the main fermentation process with subse-years, presumably due to improved sanitation. In beer, un-quent adverse effects on the final product. Nowadays these desirable microbial contamination is exhibited by viscosity, problems are lessened by good sanitary practices. Sources appearance, as well as aroma and flavor problems. of these organisms may be the cereals themselves, soil, as Microbial pathogens are usually not a problem for fer-well as any particular ingredient, surface contamination, mented cereals because of the inhibition brought about by and unsanitary handling. acids and ethanol generated by fermenting organisms. A Table 4 summarizes microbial problems likely to occur large proportion of fermented cereals are also eaten shortly during major cereal fermentations. In general, undesirable after complete cooking. However, the biggest problem." In Handbook of Cereal Science and Technology, Revised and Expanded, 765–70. CRC Press, 2000. http://dx.doi.org/10.1201/9781420027228-81.

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