Relevant bibliographies by topics / Pichia Pastoris Fermentation

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Academic literature on the topic 'Pichia Pastoris Fermentation'

Author: Grafiati
Published: 4 June 2021
Last updated: 7 September 2023

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Journal articles on the topic "Pichia Pastoris Fermentation"

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Wang, Bo, Jun Liu, Ameng Yu, and Haibo Wang. "Development and Optimization of a Novel Soft Sensor Modeling Method for Fermentation Process of Pichia pastoris." Sensors 23, no. 13 (June 29, 2023): 6014. http://dx.doi.org/10.3390/s23136014.

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This paper introduces a novel soft sensor modeling method based on BDA-IPSO-LSSVM designed to address the issue of model failure caused by varying fermentation data distributions resulting from different operating conditions during the fermentation of different batches of Pichia pastoris. First, the problem of significant differences in data distribution among different batches of the fermentation process is addressed by adopting the balanced distribution adaptation (BDA) method from transfer learning. This method reduces the data distribution differences among batches of the fermentation process, while the fuzzy set concept is employed to improve the BDA method by transforming the classification problem into a regression prediction problem for the fermentation process. Second, the soft sensor model for the fermentation process is developed using the least squares support vector machine (LSSVM). The model parameters are optimized by an improved particle swarm optimization (IPSO) algorithm based on individual differences. Finally, the data obtained from the Pichia pastoris fermentation experiment are used for simulation, and the developed soft sensor model is applied to predict the cell concentration and product concentration during the fermentation process of Pichia pastoris. Simulation results demonstrate that the IPSO algorithm has good convergence performance and optimization performance compared with other algorithms. The improved BDA algorithm can make the soft sensor model adapt to different operating conditions, and the proposed soft sensor method outperforms existing methods, exhibiting higher prediction accuracy and the ability to accurately predict the fermentation process of Pichia pastoris under different operating conditions.
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Huang, Wenjing, Yanjie Tong, Wangxiang Huang, Ke Wang, Qiming Chen, Yuanxin Wu, and Shengdong Zhu. "Influence of 1-butyl-3-methylimidazolium Chloride on the Ethanol Fermentation Process of Pichia pastoris GS115." Open Biotechnology Journal 9, no. 1 (August 25, 2015): 109–12. http://dx.doi.org/10.2174/1874070701509010109.

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To evaluate the influence of 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) on the ethanol fermentation process of Pichia pastoris GS115, this paper investigated the yeast growth, ethanol formation and the fermentable sugars consumption during the ethanol fermentation process of Pichia pastoris GS115 at different [Bmim]Cl concentrations in the medium. The results indicated that the [Bmim]Cl had no influence on the ethanol fermentation process at its concentration less than 0.0001 g.L-1. The [Bmim]Cl inhibited the yeast growth and had a negative effect on ethanol formation at its concentration higher than 0.0001 g.L-1. The final biomass and ethanol concentration, and the overall ethanol yield from the fermentable sugars all decreased with its concentration increasing. The yeast growth was very slow and nearly no ethanol formed when its concentration reached 5 g.L-1. Compared to Saccharomyces cerevisiae, the growth of Pichia pastoris GS115 was more sensitive to the [Bmim]Cl, and its ethanol fermentation had lower final ethanol concentration and overall ethanol yield from fermentable sugars at the same [Bmim]Cl concentration. This work provides useful information on selecting suitable strains for ethanol fermentation containing the [Bmim]Cl in the medium.
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Shahid, Iqra, Ghulam Hussain, Mehwish Anis, Muhammad Umar Farooq, Muhammad Usman, Yasser Fouad, and Jaroslaw Krzywanski. "Enzymatic Co-Fermentation of Onion Waste for Bioethanol Production Using Saccharomyces cerevisiae and Pichia pastoris." Energies 16, no. 5 (February 24, 2023): 2181. http://dx.doi.org/10.3390/en16052181.

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This paper evaluates the feasibility of bioethanol production from onion waste by Saccharomyces cerevisiae and Pichia pastoris and their novel co-culture through fermentation. The process parameters were optimized for each strain and their combination to observe the synergistic effect of co-fermentation. A dinitro salicylic acid (DNS) test was conducted to study the reducing sugar content of samples at different time intervals. Fourier transform infrared (FTIR) spectroscopic analysis was used to compare results for functional groups of samples before and after fermentation, and gas chromatography with flame ionization detection (GC-FID) analysis was performed to measure the bioethanol concentration obtained at different combinations of pH (5, 5.5, 6), temperature (20 °C, 30 °C, 40 °C), and time (24–110 h). The maximum bioethanol concentration was achieved through a monoculture of Saccharomyces cerevisiae, i.e., 30.56 g/L. The ethanol productivity was determined based on the ethanol concentration and fermentation time ratio. The energy content was determined using the obtained ethanol value and the specific energy content of ethanol, i.e., 30 kJ/g. The productivity and energy of bioethanol obtained at this maximum concentration were 0.355 g/L h and 916.8 kJ/L, respectively, after 86 h of fermentation at 30 °C and pH 5. Pichia pastoris produced a maximum of 21.06 g/L bioethanol concentration with bioethanol productivity and energy of 0.264 g/L h and 631.8 kJ/L, respectively, after 72 h of fermentation at 30 °C and pH 5. The coculture fermentation resulted in 22.72 g/L of bioethanol concentration with bioethanol productivity and energy of 0.264 g/L h and 681.6 kJ/L, respectively, after 86 h of fermentation at 30 °C and pH 5. The results of reducing sugars also supported the same conclusion that monoculture fermentation using Saccharomyces cerevisiae was the most effective for bioethanol production compared to Pichia pastoris and co-culture fermentation.
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Borshchevskaya, L. N., T. L. Gordeeva, and S. P. Sineoky. "Increase in the Production of Endo-1,4-β-Xylanase from Paenibacillus brasilensis in Pichia pastoris." Biotekhnologiya 35, no. 6 (2019): 30–38. http://dx.doi.org/10.21519/0234-2758-2019-35-6-30-38.

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A Pichia pastoris yeast strain producing endo-l,4-β-xylanase from Paenibacillus brasilensis with an activity of 54,400 U/mL after 140 h of fermentation in a laboratory fermenter has been obtained. A number of approaches were used to increase the level of the xylanase production in this strain: optimization of the target gene codon composition, multiple integration of the expression cassette into the recipient strain chromosome using the Cre-lox recombination system, and also improving the heterologous protein folding via the overexpression of the HAC1i gene from Pichia pastoris. xylanase, xylan, Cre-lox system, HAC1p transcriptional activator, multicopy strain, Paenibacillus brasilensis, Pichia pastoris The work was performed with the financial support of the Ministry of Education and Science of Russia (Unique Project Identifier RFMEFI60717X0180) using the Multipurpose Scientific Installation of «All-Russian Collection of Industrial Microorganisms» National Bio-Resource Center, NRC «Kurchatov Institute» - GosNIIgenetika.
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Rosenbergová, Zuzana, Kristína Kántorová, Martin Šimkovič, Albert Breier, and Martin Rebroš. "Optimisation of Recombinant Myrosinase Production in Pichia pastoris." International Journal of Molecular Sciences 22, no. 7 (April 1, 2021): 3677. http://dx.doi.org/10.3390/ijms22073677.

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Myrosinase is a plant defence enzyme catalysing the hydrolysis of glucosinolates, a group of plant secondary metabolites, to a range of volatile compounds. One of the products, isothiocyanates, proved to have neuroprotective and chemo-preventive properties, making myrosinase a pharmaceutically interesting enzyme. In this work, extracellular expression of TGG1 myrosinase from Arabidopsis thaliana in the Pichia pastoris KM71H (MutS) strain was upscaled to a 3 L laboratory fermenter for the first time. Fermentation conditions (temperature and pH) were optimised, which resulted in a threefold increase in myrosinase productivity compared to unoptimised fermentation conditions. Dry cell weight increased 1.5-fold, reaching 100.5 g/L without additional glycerol feeding. Overall, a specific productivity of 4.1 U/Lmedium/h was achieved, which was 102.5-fold higher compared to flask cultivations.
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Zhang, Biao, Baizhi Li, Dai Chen, Jie Zong, Fei Sun, Huixin Qu, and Chongyang Liang. "Transcriptional Regulation of Aerobic Metabolism in Pichia pastoris Fermentation." PLOS ONE 11, no. 8 (August 18, 2016): e0161502. http://dx.doi.org/10.1371/journal.pone.0161502.

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Tolner, Berend, Lisa Smith, Richard H. J. Begent, and Kerry A. Chester. "Production of recombinant protein in Pichia pastoris by fermentation." Nature Protocols 1, no. 2 (August 2006): 1006–21. http://dx.doi.org/10.1038/nprot.2006.126.

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Chen, Yinliang, Jeffrey Krol, Julia Cino, David Freedman, Christopher White, and Elizabeth Komives. "Continuous Production of Thrombomodulin from a Pichia pastoris Fermentation." Journal of Chemical Technology & Biotechnology 67, no. 2 (October 1996): 143–48. http://dx.doi.org/10.1002/(sici)1097-4660(199610)67:2<143::aid-jctb561>3.0.co;2-s.

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He, Li Yan, Gui Bin Wang, Fu Liang Cao, Lin Guo Zhao, and Yong Xin Ji. "Cloning of Laccase Gene from Coriolus Versicolor and Optimization of Culture Conditions for Lcc1 Expression in Pichia Pastoris." Advanced Materials Research 236-238 (May 2011): 1039–44. http://dx.doi.org/10.4028/www.scientific.net/amr.236-238.1039.

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A laccase cDNA lcc1 (GenBank accession number HM137002), without native signal peptide, was cloned by RT-PCR from total RNA of Coriolus versicolor. Recombination expression vector pPICZαA-lcc1 was constructed and transformed into Pichia pastoris KM71H after lineared. Recombination laccase was expressed at a higher level. Single factors of fermentation conditions of Pichia pastoris KM71H for laccase production were optimized. The results showed optimal culture conditions were as follows: medium initial pH 7.5, Cu2+ concentration 0.5mmol/L, methanol additive amount 1.0% and shaker rotate speed 210r/min. Furthermore, induction at low temperature was more suitable for lcc1 secretion. And addition of appropriate amount peptone and tyrosine in culture medium could enhanced lcc1 yields and reduce its degradation.
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Wang, Bo, Xingyu Wang, Mengyi He, and Xianglin Zhu. "Study on Multi-Model Soft Sensor Modeling Method and Its Model Optimization for the Fermentation Process of Pichia pastoris." Sensors 21, no. 22 (November 17, 2021): 7635. http://dx.doi.org/10.3390/s21227635.

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The problems that the key biomass variables in Pichia pastoris fermentation process are difficult measure in real time; this paper mainly proposes a multi-model soft sensor modeling method based on the piecewise affine (PWA) modeling method, which is optimized by particle swarm optimization (PSO) with an improved compression factor (ICF). Firstly, the false nearest neighbor method was used to determine the order of the PWA model. Secondly, the ICF-PSO algorithm was proposed to cooperatively optimize the number of PWA models and the parameters of each local model. Finally, a least squares support vector machine was adopted to determine the scope of action of each local model. Simulation results show that the proposed ICF-PSO-PWA multi-model soft sensor modeling method accurately approximated the nonlinear features of Pichia pastoris fermentation, and the model prediction accuracy is improved by 4.4884% compared with the weighted least squares vector regression model optimized by PSO.
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Dissertations / Theses on the topic "Pichia Pastoris Fermentation"

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Cochran, Keith Jacob. "Combined fermentation and recovery using expanded bed chromatography." Worcester, Mass. : Worcester Polytechnic Institute, 2006. http://www.wpi.edu/Pubs/ETD/Available/etd-081806-184321/.

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Zhang, Wei. "Microbubble fermentation of recombinant Pichia pastoris for human serum albumin production." Thesis, Virginia Tech, 2003. http://hdl.handle.net/10919/33395.

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The high cell density fermentation of recombinant Pichia pastoris for human serum albumin (HSA) production is a high oxygen demand process. The oxygen demand is usually met by increased agitation rate and use of oxygen-enriched air. Microbubble fermentation however can supply adequate oxygen to the microorganisms at relatively low agitation rates because of improved mass transfer of the microbubbles used for the sparging. Conventionally sparged fermentations were conducted for the production of HSA using P. pastoris at agitation rates of 350, 500, and 750 rpm, and were compared to MBD sparged fermentation at 150, 350, and 500 rpm agitation rates. The MBD improved the volumetric oxygen transfer coefficient (kLa) and subsequently increased the cell mass and protein production compared to conventional fermentation. Cell production in MBD fermentation at 350 rpm was 4.6 times higher than that in conventional fermentation at 350 rpm, but similar to that in the conventional 750 rpm. Maximum cell mass productivity in the conventional 350 rpm was only 0.37 g / (Lâ ¢h), while the maximum value in MBD 350 rpm was 2.0 g / (Lâ ¢h), which was similar to 2.2 g / (Lâ ¢h) in the conventional 750 rpm. Biomass yield on glycerol Ys (g cell/ g glycerol) was 0.334 g / g in the conventional 350 rpm, 0.431 g / g in MBD 350 rpm and 0.438 g / g in the conventional 750 rpm. Protein production in MBD 350 rpm was 7.3 times higher than that in the conventional 350 rpm, but similar to the conventional 750 rpm. Maximum protein productivity in the conventional 350 rpm was 0.37 mg / (Lâ ¢h), 2.8 mg / (Lâ ¢h) in MBD 350 rpm, and 3.3 mg / (Lâ ¢h) in the conventional 750 rpm. Protein yield on methanol Yp (mg protein / g methanol) was 1.57 mg /g in the conventional 350 rpm, 5.02 in MBD 350 rpm, and 5.21 in the conventional 750 rpm. The volumetric oxygen transfer coefficient kLa was 1011.9 h-1 in MBD 350 rpm, which was 6.1 times higher than that in the conventional 350 rpm (164.9 h-1) but was similar to the conventional 750 rpm (1098 h-1). Therefore, MBD fermentation results at low agitation of 350 rpm were similar to those in the conventional fermentation at high agitation of 750 rpm. There was considerable improvement in oxygen transfer to the microorganism using MBD sparging relative to the conventional sparging. Conventional fermentations were conducted both in a Biostat Q fermenter (small) at 500 rpm, 750 rpm, and 1000 rpm, and in a Bioflo III fermenter (large) at 350 rpm, 500 rpm, and 750 rpm. At the same agitation rate of 500 rpm, cell production in the large reactor was 3.8 times higher than that in the small one, and no detectable protein was produced in the small reactor at 500 rpm. At the same agitation rate of 750 rpm, both cell production and protein production in the large reactor were 4.6 times higher than the small reactor. Thus, the Bioflo III fermenter showed higher oxygen transfer efficiency than the Biostat Q fermenter, because of the more efficient aeration design of the Bioflo III fermenter.
Master of Science
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Vorlová, Sandra. "Die humane Acetylcholinesterase: Design und Synthese eines optimierten Gens und die Expression in Pichia pastoris." [S.l. : s.n.], 2002. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB9923806.

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Zhong, Shuping. "Study of Operational Strategies and Carbon Source Selection for the Production of Phytase using Pichia pastoris." Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/32204.

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The methylotrophic yeast Pichia pastoris has become an efficient expression system for heterologous protein production. Different methods have been studied to enhance cell growth as well as the production of products of interest. Two of the major strategies for improving the product or biomass yields are optimizing bioprocess controls and cultivation conditions. In this work, the characteristics of this yeast system and of its different promoters are discussed, and the effect of operational strategies on cell growth and recombinant protein expression is also studied. The effect of different feeding strategies were studied and optimized for pGAP (glyceraldehyde-3-phosphate dehydrogenase)-regulated phytase production in P. pastoris. Alternative carbon sources were screened and the feasibility of using citric acid as a carbon source for recombinant protein production was also investigated. The effects of parameters such as the carbon source concentration and culture pH were studied using shake-flasks, and the effect of different feeding profiles on bioreactor performance was also investigated. Three feeding strategies, Stepwise feeding, Exponential feeding and DO-stat feeding were tested and DO-stat was found to be more efficient and led to a high phytase activity. A modified DO-stat method was investigated to overcome the oxygen limited condition in the standard DO-stat method. For the carbon source, citric acid showed promise in improving phytase expression. Further experiments in bioreactors performed with the presence of certain amount of citric acid showed that less glycerol could be used to achieve the same level of phytase activity.
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Khatri, N. K. (Narendar Kumar). "Optimisation of recombinant protein production in Pichia pastoris:single-chain antibody fragment model protein." Doctoral thesis, Oulun yliopisto, 2011. http://urn.fi/urn:isbn:9789514295850.

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Abstract Potential lethal diarrhoea caused by enterotoxigenic Escherichia coli strains is one of the most common diseases in young pigs. It can be cured by single-chain antibody fragments (scFv), which can be produced in recombinant microorganisms. Pichia pastoris, a methylotrophic yeast, is generally considered an interesting production system candidate, as it can secrete properly folded proteins. These proteins accumulate in high concentrations during fermentation, reducing the cost for product recovery. Strong inducible AOX1 promoter, widely used in P. pastoris for fast, inexpensive production, is typically induced by methanol. The high oxygen demand of methanol metabolism makes oxygen supply a major parameter in cultivations requiring special process design strategies. In standard fed-batch cultivation, dissolved oxygen concentration inside a bioreactor is kept at a certain level by pumping air and pure oxygen into the reactor. There are safety concerns over the handling of oxygen, especially at a large scale. Therefore, there is a need to develop a production process under oxygen-limited conditions. This dissertation studies the development of a cost-efficient production process of scFv in P. pastoris. Both methanol and oxygen parameters influence the production process and the objective was to find a robust production process. Fed-batch cultivations were performed in a 10 L scale bioreactor. The effects of lower oxygen level, methanol concentration, glycerol feeding duration and specific substrate-uptake rates on product formation were studied. A P. pastoris GS115 his4 strain under an AOX1 promoter system expressing scFv was used in this study. The fed-batch fermentations were carried out in a bioreactor with basal salt media. In this doctoral dissertation, a process was developed for a single-chain antibody fragment (scFv) production in P. pastoris. The product levels of 3.5 g L-1 scFv in culture supernatant were achieved and a production process was designed without additional need of pure oxygen, thus relieving safety requirements and lowering the amount of methanol. The process developed during this research may potentially be utilised by both academia and industry having interests in expressing proteins in P. pastoris. The methanol-uptake control strategy is beneficial for those products that suffer from degradation or modification during limited feeding of methanol
Tiivistelmä Enterotoksigeenisten E.coli kantojen aiheuttama ripuli on porsaiden tavallisimpia tauteja, joka voi johtaa jopa kuolemaan. Tautia voidaan hoitaa yhdistelmä-DNA-tekniikalla tuotetuilla vasta-ainefragmenteilla (scFv). Metylotrofista Pichia pastoris hiivaa pidetään kiinnostavana vasta-ainefragmenttien tuottoisäntänä, koska se pystyy erittämään oikealla tavalla laskostuneita proteiineja. Näitä proteiineja kertyy fermentointiprosessissa solujen ulkopuolelle korkeina pitoisuuksina, mikä vähentää tuotteiden talteenottokustannuksia. Vahva metanolilla indusoituva AOX1-promoottori on laajassa käytössä P. pastoris tuottosysteemissä tuoton nopeuden ja alhaisten kustannusten ansiosta. Metanolin aineenvaihdunta vaatii paljon happea, joten riittävän tehokas hapen liuottaminen on tärkeimpiä fermentointiparametreja ja vaatii erityisiä prosessin toteutusstrategioita. Perinteisessä fed-batch-fermentoinnissa liuenneen hapen pitoisuus bioreaktorissa pidetään halutulla tasolla lisäämällä ilmaa ja puhdasta happea reaktoriin. Koska hapen käsittelyyn liittyy turvallisuusriskejä erityisesti teollisuusmittakaavassa, happirajoitteisissa olosuhteissa toimiva tuotantoprosessi olisi hyödyllinen. Tässä väitöstutkimuksessa kehitettiin kustannustehokasta prosessia scFv-:n tuottoon P. pastoris hiivalla. Metanoliin ja happeen liittyvät parametrit ovat olennaisia prosessiin vaikuttavia tekijöitä. Tavoite oli kehittää yksinkertainen ja käytännöllinen prosessi. Työssä tutkittiin alhaisen happitason, metanolin pitoisuuden, glyserolisyötön keston ja substraattien spesifisten kulutusnopeuksien vaikutuksia tuotteen muodostumiseen 10 litran bioreaktorissa. Isäntäkantana oli P. pastoris GS115 his4, jossa scFv-ekspressiota säädeltiin AOX1 promoottorilla. Fed-batch fermentointien kasvatusalustana käytettiin Basal Salt Medium alustaa (BSM). Väitöstyössä kehitettiin tavoitteiden mukainen vasta-ainefragmenttien tuottoprosessi P.pastoris hiivalle. Menetelmällä saavutettiin tuotepitoisuus 3,5 g L-1 kasvatusliemen supernatantissa ilman puhtaan hapen lisäystarvetta, ja siten metanolin kulutus väheni ja prosessiturvallisuus parani verrattuna perinteisiin prosesseihin. Kehitetty prosessi soveltuu käytettäväksi sekä akateemisessa tutkimuksessa että teollisuudessa tuotettaessa erilaisia proteiineja P. pastoris hiivalla. Metanolin kulutuksen säätöstrategia on erityisen hyödyllinen tuotteille, joilla ongelmana on proteolyysi tai muokkautuminen metanolirajoitteisessa fermentoinnissa
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Pérard, Anne-Laure. "Étude de la GFP, Green Fluorescent Protein, dans la levure méthylotrophe Pichia pastoris comme un outil de diagnostic du procédé de fermentation." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape3/PQDD_0021/MQ57423.pdf.

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Carneiro, F??bio Correia. "Otimiza????o de t??cnicas de cultivo em biorreator aplicado ?? produ????o do inibidor de tripsina ILTI em Komagataella phaffii (Pichia pastoris)." Universidade Cat??lica de Bras??lia, 2018. https://bdtd.ucb.br:8443/jspui/handle/tede/2378.

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Protease inhibitors have a broad biotechnological application, which goes since the development of several drugs to your utilization as a bioinsecticide, antifungal and as an antibacterial agent. However, those are found in small quantities in their natural sources, which unfeasible it utilization in industrial scale. Therefore, the heterologous production ends up as a method that allows the increase of scale production of those proteins. The Inga laurina Trypsin Inhibitor (ILTI) previously characterized showed an inhibitory effect in proteases extracted from the midgut of insects, besides reducing its larval developments by up to 84%, therefore, boing a candidate to be used as a potential bioinsecticide. Thus, the present work aimed at the heterologous production of ILTI in Komagataella phaffii (Pichia pastoris), followed by the optimization of the culture modes in a bioreactor with the objective of maximizing the production of the recombinant protein. For this, the gene that codifies ILTI were cloned in the expression vector pPIC9K, followed by your insertion on the strain GS115 by electroporation. PCR analysis showed that the recombinant vector was integrated into the genome of the yeast, and all the clones obtained had MutS genotype. The expression was performed for 96 hours by adding 0.5% methanol. An analysis of the proteins on the supernatant of the recombinant strain culture, by SDS-PAGE, confirmed the production of a protein with a size close to 20 KDa. Data from MALDI-TOF confirmed that the obtained protein is, in fact, the recombinant ILTI. Furthermore, inhibitory assays showed that the produced protein had activity against trypsin. Thus, culture in bioreactors was performed to optimize the production of this heterologous protein. In order to increase its expression, fed-batch was performed where on the batch phase the biomass production was favored, and the feeding phase was programmed to continuously supply methanol, based on the methanol specific consumption and its specific growth velocity, using methanol as carbon source. During the fermentations, 351.27 UIT were obtained in the extract of crude fermentation broth, and a specific activity of 2.07 UIT/mg protein. Although widely used as a host for the production of heterologous proteins, studies of the production of protease inhibitors in K. phaffii are still very limited. Until the moment, there is no report in the optimization of the production of serine protease inhibitors in K. phaffii, making this study pioneering and essential for the beginning of scaling up the process of this technology.
Os inibidores de protease possuem uma ampla aplica????o biotecnol??gica que vai desde o desenvolvimento de diversos f??rmacos at?? sua utiliza????o como bioinseticidas, antif??ngicos e como agentes antibacterianos. Por??m, estes s??o encontrados em pequenas quantidades nas suas fontes naturais, o que inviabiliza sua utiliza????o em escala industrial. Sendo assim, a produ????o heter??loga acaba sendo um m??todo que permite o aumento da escala de produ????o dessas prote??nas. O inibidor de tripsina de Inga laurina (ILTI) foi caracterizado previamente e mostrou possuir efeito inibit??rio em proteases extra??das do trato digestivo de insetos, al??m de diminuir em at?? 84% seu desenvolvimento larval, sendo, portanto, um candidato a ser utilizado como um poss??vel bioinseticida. Dessa forma, o presente trabalho teve como objetivo a produ????o heter??loga do inibidor ILTI, em Komagataella phaffii (Pichia pastoris), seguido da otimiza????o dos modos de cultivo em biorreator com o objetivo de maximizar a produ????o da prote??na recombinante. Para isso, o gene que codifica o ILTI foi clonado no vetor de express??o pPIC9K seguindo de sua inser????o na cepa GS115 por eletropora????o. An??lises de PCR mostraram que o vetor recombinante foi integrado ao genoma da levedura e que todos os clones obtidos possu??am gen??tipo MutS. A indu????o da express??o foi realizada durante 96 horas por meio da adi????o de metanol ?? 0,5%. A an??lise de prote??nas presentes no sobrenadante da cultura da cepa recombinante, por meio de SDS-PAGE, confirmou a produ????o de uma prote??na com tamanho pr??ximo a 20 KDa. Dados obtidos em MALDI-TOF confirmaram que a prote??na obtida ?? de fato ILTI recombinante. Al??m disso, ensaios inibit??rios mostraram que a prote??na produzida possui atividade contra tripsina. Dessa forma, cultivo em biorreatores foram realizados com a finalidade de otimizar a produ????o dessa prote??na heter??loga. A fim de aumentar sua express??o foram realizadas bateladas alimentadas, onde durante a fase de batelada foi favorecida a produ????o de biomassa, e a fase de alimenta????o programada para fornecer metanol de forma cont??nua, com base nos dados de velocidade espec??fica de consumo de metanol e velocidade de crescimento espec??fica nesta fonte de carbono. Ao longo das fermenta????es realizadas foi obtido 351,27 UIT no extrato bruto do caldo fermentado e uma atividade espec??fica de 2,07 UIT/mg de prote??na. Apesar de ser amplamente utilizada como hospedeira para a produ????o de prote??nas heter??logas, estudos da produ????o de inibidores em K. phaffii ainda s??o muito limitados. At?? o momento n??o existem relatos de otimiza????o da produ????o de inibidores de serinoproteases em K. phaffii, sendo esse estudo pioneiro e essencial para o in??cio do processo de escalonamento dessa tecnologia.
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Rotticci-Mulder, Johanna C. "Expression and Mutagenesis studies of Candida antactica lipase B." Doctoral thesis, KTH, Biotechnology, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3493.

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Recombinant Candida antarctica lipase B was successfullyproduced in the methylotropic yeast Pichia pastoris. Thespecific activities of Candida antarctica lipase B produced inPichia pastoris and commercial Candida antarctica lipase B fromNovozymes were the same. In shake-flask cultivations theexpression levels were about 25 mg L-1. Production levels couldbe increased to 1.5 g L-1, using a fermentor. A model tosimulate growth and oxygen consumption was described. The highcell density growth could be explained by the low maintenancecoefficient of Pichia pastoris. Enrichment of the aeration withoxygen increased the recombinant protein production. The lipasewas also produced as a fusion to a cellulose binding module.The cellulose binding module did not interfere with thespecific activity of the lipase. With this fusion proteincatalytic reactions can be performed in close proximity to acellulose surface. The binding module can also function as anaffinity tag for purification. Establishment of the Candidaantarctica lipase B production system allowed the engineeringof Candida antarctica lipase B variants. Four differentvariants were produced in order to investigate if electrostaticinteractions contributed to enantioselectivity. Theenantioselectivity of two halogenated secondary alcohols wasdoubled for the Ser47Ala variant. Thisimplied thatelectrostatic interactions are important forenantioselectivity. The Trp104His variant showed a decrease inenantioselectivity for all tested substrates. This was causedby an increase in the size of the stereoselectivity pocket.Symmetrical secondary alcohols of different size were used tomap the stereoselectivity pocket. A substituent as large as apropyl or isopropyl could be accommodated in the pocket of theTrp104His variant. In the wild-type lipase thestereoselectivity pocket was estimated to fit an ethyl group.The enzyme variants were subjected to a thermodynamic study, toelucidate changes in the enthalpic and entropic contributionsto enantioselectivity. The enthalpic and entropic contributionschanged for the different lipase variants and werecompensatory. The compensation was not perfect, allowing forchanges in enantioselectivity.

In general one can conclude that rational design of newenzyme properties, in order to change the substrateselectivity, is feasible if based on a thorough model ofsubstrate enzyme interactions.

Key words:Protein expression, Candida antarctica lipaseB, Pichia pastoris, sitedirected mutagenesis, fermentation,selectivity

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Klinková, Lucie. "Vybrané mikrobiální procesy v bioreaktoru." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2014. http://www.nusl.cz/ntk/nusl-217069.

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This thesis focuses on the study of the influence of selected parameters on the course of microbial cultivation and evaluation of bioprocess. It is divided into two parts. The first part deals with the production of mutant forms of the protein cryptogein yeast Pichia pastoris. The theoretical part summarizes the findings of the yeast P. pastoris and its expression. It also deals with cryptogein that induces defense reactions in plants. In the experimental part was produced mutant cryptogein X24, in which the concentration of each fraction and the ability to transfer sterols. The second part of this thesis is focused on aerobic and anaerobic oxidation of elemental sulfur by Acidithiobacillus ferrooxidans. In the theoretical section, our knowledge on A. ferrooxidans, its metabolism and the importance of ATP in cell metabolism was summarized. In the experimental part, the above bioprocess was monitored using pH, biomass concentration, the rate of oxidation of elemental sulfur the cellular ATP content.
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Klein, Cécile. "Production de quatre isoformes de protéines de transfert de lipides du blé dans "Pichia pastoris". Expression des gènes codant ces protéines dans le blé." Montpellier 2, 1998. http://www.theses.fr/1998MON20065.

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Les proteines de transfert de lipides (ltp) sont des proteines des faible masse moleculaire qui sont capables de realiser in vitro un transfert de lipides entre deux membranes. Dans le ble, les genes de ltp forment une famille mutigenique et il existe au moins cinq genes de ltp exprimes dans le grain de ble. Dans un premier temps, nous avons utilise le systeme d'expression levurien pichia pastoris pour produire quatre isoformes de ltp de ble dur (triticum durum) et de ble tendre (triticum aestivum). Ce systeme permet de secreter trois ltp recombinantes solubles dans le milieu de culture en quantite variable (1,1 g/l, 700 mg/l et 80 mg/l). Quand l'acide amine n-terminal de la proteine deduite de l'adnc est un residu alanine et non un residu isoleucine, la maturation de la ltp recombinante correspondante est optimale. Nos resultats ont montre qu'il y a une grande diversite de production selon le clone considere et les conditions de cultures utilisees. Dans un deuxieme temps, nous avons suivi l'expression des genes de ltp de ble au cours de la maturation et de la germination du grain de ble dur, et l'effet de stress abiotiques sur l'expression de ces memes genes dans des plantules de ble dur. Au cours de la maturation, ce sont les transcrits des genes ltp4. 90 et ltp8. 3 qui sont les plus abondants. Apres application des stress abiotiques, une variation de la quantite de transcrits des genes ltp6. 48 et ltpd2 est observee dans les pousses. Chaque gene de ltp de ble etudie semble avoir une expression spatiale et temporelle specifique.
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Book chapters on the topic "Pichia Pastoris Fermentation"

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Liu, Wan-Cang, and Ping Zhu. "Demonstration-Scale High-Cell-Density Fermentation of Pichia pastoris." In Methods in Molecular Biology, 109–16. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-7312-5_9.

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Tolner, Berend, Gaurav Bhavsar, Bride Foster, Kim Vigor, and Kerry Chester. "Production of Recombinant Proteins from Pichia pastoris: Interfacing Fermentation and Immobilized Metal Ion Affinity Chromatography." In Laboratory Protocols in Fungal Biology, 407–20. New York, NY: Springer New York, 2012. http://dx.doi.org/10.1007/978-1-4614-2356-0_37.

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Conference papers on the topic "Pichia Pastoris Fermentation"

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Geethalakshmi, S., and N. Pappa. "Artificial Neural Network Based Soft Sensor for Fermentation of Recombinant Pichia Pastoris." In 2010 International Conference on Advances in Computer Engineering (ACE). IEEE, 2010. http://dx.doi.org/10.1109/ace.2010.56.

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Wang, Xiuhong, Tianjun Guo, Wei Hao, and Qingqiang Guo. "Predicting Model based on LS-SVM for Inulinase Concentration during Pichia Pastoris’ Fermentation Process." In 2019 Chinese Control Conference (CCC). IEEE, 2019. http://dx.doi.org/10.23919/chicc.2019.8866656.

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Guo, Qingqiang, Yingjia Zhang, Yuxi Deng, and Xiuhong Wang. "A Soft-sencor and Parameter Optimization for Predicting Inulinase Concentration at Recombinant Pichia Pastoris Fermentation Process." In The Second International Conference on Materials Chemistry and Environmental Protection. SCITEPRESS - Science and Technology Publications, 2018. http://dx.doi.org/10.5220/0008184700140018.

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Reports on the topic "Pichia Pastoris Fermentation"

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Meagher, Michael M. Fermentation, Recovery, and Purification of the HC Fragments of the Botulinum Neurotoxin from Pichia Pastoris. Fort Belvoir, VA: Defense Technical Information Center, June 2000. http://dx.doi.org/10.21236/ada384855.

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