毕赤酵母发酵 ppt课件

巴斯德毕赤酵母(Pichia pastoris)表达系统综述27页PPT

诱导的醇氧化酶(AOX1)启动子，可严格控制外源基因的表达
营养要求简单，生长快速，适合高密度大规模培养，很少产生有毒物质，毒性比细菌小，用甲醇不易染菌，可以减少污染。
高的调控功能，可用于外源基因的表达调控。甲醇营养型酵母表达系统以巴斯德毕赤酵母(Pichia pastoris)表达系统最为常用。
（3）裂殖酵母(Schizogenesis pombe) 表达系统
只能以分裂和产孢子的方式繁殖的一类酵母，因此定名为裂殖酵母。与前面几种酵母相比，它具有更多的与高等真核生物相似的特性：线粒体结构、启动子结构、转录机制和对蛋白2端酰基化功能均更接近于哺乳类细胞，因而正逐渐成为研究真核细胞分子生物学的模式生物，它作为外源基因表达系统也开始受到人们的关注。目前，已经有多种蛋白利用此系统进行了表达，如人蛋白凝血因子)G、细胞色素 V6:8、人白细胞介素 YL’D等。此系统表达的外源蛋白更接近于它们的天然形式。
的真核生物，其全序列的测定已于 2019年完成。酿酒酵母难于高密度培养，分泌效率低，几乎不分泌分
子量大于30 kD 的外源蛋白质，也不能使所表达的外源蛋白质正确糖基化，而且表达蛋白质的C 端往往被截短。因此，一般不用酿酒酵母做重组蛋白质表达的宿主菌。酿酒酵母本身含有质粒，其表达载体可以有自主复制型和整合型两种。值得注意的是，酿酒酵母表达的外源蛋白质往往被高度糖基化，糖链上可以带有40 个以上的甘露糖残基，糖蛋白的核心寡聚糖链仅含有末端1, 3 甘露糖，产物的抗原性明显增强。所以，酿酒酵母常常用来制备亚单位疫苗(如默克乙肝疫苗、口蹄疫疫苗等)。
1993年，Philip Petroleum公司将毕赤酵母表达系统的专利卖给 Research Corporation Technologies公司，并委托Invitrogea公司进行有关产品销售。

毕赤酵母发酵

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1.2过氧物酶体(Peroxisome)：遍布于真核生物的细胞器中, 用来去除有
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1. 3GFP-SKL（过氧化物酶体定位蛋白）,该载体含有融合了过氧化物酶体定位信号1(PTS1)的绿色荧光蛋白报告分子GFP-SKL编码基因成功地用于描述不同时间进程的AOX1启动子和过氧物酶体生物合成因素的特征
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2.3：补料分批培养条件
研究了三种不同发酵条件下的PpAOX1启动子（ YGLY29325 ）和过氧化物酶体生物合成（ YGLY31884 ）的调控。诱导阶段分批补料条件 ML:甲醇的起始供应速率为2.6gl/lh然后在0.0063/h的基础上以幂指的形式增加 SML:甲醇培养20h用50%的葡萄糖15g/h的速度培养8h作为一个周期共培养3个周期 OL:甲醇进料保持反应器中甲醇1％，每当DO迅速增加，这表明甲醇消耗。 DO级联被打开，关闭和搅拌速度被减小到实现氧气的限制
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2.4 GFP表达的定量和可视化：约107固定的酵母细胞经PBS洗后用mounting solution重悬制作切片用 Axioscope 2 Plus 显微镜来观察OpenLAB软件来实现相机控制和图像收集Volocity软件用来量化GFP在细胞中的强度
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2.5抗体效价的测定在培养上清液中于280nm处抗体浓度用二极管阵列检测器和蛋白A亲和柱与HPLC系统
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3.3毕赤酵母AOX1启动子（YGLY29325）根据三种不同的补料分批条件规制 - 甲醇限制（ML），切换与葡萄糖和甲醇（SML）和氧气限制（OL）补料分批条件。（a） GFP荧光表达图像（b）不同诱导时间的荧光蛋白相对密度。对于ML和OL，时间表示分批阶段（T1），甘油补料分批阶段诱导前（T2）和诱导期（T3，10±2小时; T4中， 36±2小时; T5，62±2小时; T6，84±2小时）。对与SML条件下，细胞生长是在T2，T4 和T66/28/2014 葡萄糖阶段，诱导是在T3和T5甲醇阶段

毕赤酵母发酵手册

Version B Pichia Fermentation Process GuidelinesOverviewIntroduction Pichia pastoris, like Saccharomyces cerevisiae, is particularly well-suited forfermentative growth. Pichia has the ability to reach very high cell densities duringfermentation which may improve overall protein yields.We recommend that only those with fermentation experience or those who have accessto people with experience attempt fermentation. Since there are a wide variety offermenters available, it is difficult to provide exact procedures for your particular case.The guidelines given below are based on fermentations of both Mut+ and Mut S Pichiastrains in a 15 liter table-top glass fermenter. Please read the operator's manual for yourparticular fermenter before beginning. The table below provides an overview of thematerial covered in these guidelines.Step Topic Page1 Fermentationparameters 12 Equipment needed and preparation of medium 23 Measurement and use of dissolved oxygen (DO) in the culture 34 Growth of the inoculum 45 Generation of biomass on glycerol in batch and fed-batch phases 4-56 Induction of expression of Mut+ and Mut S recombinants in themethanol fed-batch phase6-77 Harvesting and lysis of cells 88 References 9-109 Recipes 11Fermentation Parameters It is important to monitor and control the following parameters throughout thefermentation process. The following table describes the parameters and the reasons for monitoring them.Parameter Reason Temperature (30.0°C) Growth above 32°C is detrimental to protein expression Dissolved oxygen (>20%) Pichia needs oxygen to metabolize glycerol andmethanolpH (5.0-6.0 and 3.0) Important when secreting protein into the medium andfor optimal growthAgitation (500 to 1500 rpm) Maximizes oxygen concentration in the mediumAeration (0.1 to 1.0 vvm*for glass fermenters)Maximizes oxygen concentration in the medium whichdepends on the vesselAntifoam (the minimumneeded to eliminate foam)Excess foam may cause denaturation of your secretedprotein and it also reduces headspaceCarbon source (variablerate)Must be able to add different carbon sources at differentrates during the course of fermentationcontinued on next pageOverview, continuedRecommended Equipment Below is a checklist for equipment recommendations.• A jacketed vessel is needed for cooling the yeast during fermentation, especially during methanol induction. You will need a constant source of cold water (5-10°C). This requirement may mean that you need a refrigeration unit to keep the water cold. • A foam probe is highly recommended as antifoam is required.• A source of O2--either air (stainless steel fermenters at 1-2 vvm) or pure O2(0.1-0.3 vvm for glass fermenters).• Calibrated peristaltic pumps to feed the glycerol and methanol.• Automatic control of pH.Medium Preparation You will need to prepare the appropriate amount of following solutions:• Fermentation Basal Salts (page 11)• PTM1Trace Salts (page 11)• ~75 ml per liter initial fermentation volume of 50% glycerol containing 12 ml PTM1 Trace Salts per liter of glycerol.• ~740 ml per liter initial fermentation volume of 100% methanol containing 12 mlPTM1Trace Salts per liter of methanol.Monitoring the Growth of Pichia pastoris Cell growth is monitored at various time points by using the absorbance at 600 nm (OD600) and the wet cell weight. The metabolic rate of the culture is monitored by observing changes in the concentration of dissolved oxygen in response to carbon availability (see next page).Dissolved Oxygen (DO) MeasurementIntroduction The dissolved oxygen concentration is the relative percent of oxygen in the mediumwhere 100% is O2-saturated medium. Pichia will consume oxygen as it grows, reducing the dissolved oxygen content. However, because oxygen is required for the first step ofmethanol catabolism, it is important to maintain the dissolved oxygen (DO) concentra-tion at a certain level (>20%) to ensure growth of Pichia on methanol. Accuratemeasurement and observation of the dissolved oxygen concentration of a culture willgive you important information about the state and health of the culture. Therefore, it isimportant to accurately calibrate your equipment. Please refer to your operator's manual.Maintaining the Dissolved Oxygen Concentration (DO) 1. Maintaining the dissolved oxygen above 20% may be difficult depending on theoxygen transfer rates (OTR) of the fermenter, especially in small-scale glassvessels. In a glass vessel, oxygen is needed to keep the DO above 20%, usually~0.1-0.3 vvm (liters of O2per liter of fermentation culture per minute). Oxygen consumption varies and depends on the amount of methanol added and the protein being expressed.2. Oxygen can be used at 0.1 to 0.3 vvm to achieve adequate levels. This can beaccomplished by mixing with the air feed and can be done in any glass fermenter.For stainless steel vessels, pressure can be used to increase the OTR. Be sure toread the operator's manual for your particular fermenter.3. If a fermenter cannot supply the necessary levels of oxygen, then the methanol feedshould be scaled back accordingly. Note that decreasing the amount of methanol may reduce the level of protein expression.4. To reach maximum expression levels, the fermentation time can be increased todeliver similar levels of methanol at the lower feed rate. For many recombinantproteins, a direct correlation between amount of methanol consumed and theamount of protein produced has been observed.Use of DO Measurements During growth, the culture consumes oxygen, keeping the DO concentration low. Note that oxygen is consumed whether the culture is grown on glycerol or methanol. The DO concentration can be manipulated to evaluate the metabolic rate of the culture and whether the carbon source is limiting. The metabolic rate indicates how healthy the culture is. Determining whether the carbon source is limiting is important if you wish to fully induce the AOX1 promoter. For example, changes in the DO concentrations (DO spikes) allow you to determine whether all the glycerol is consumed from the culture before adding methanol. Secondly, it ensures that your methanol feed does not exceed the rate of consumption. Excess methanol (> 1-2% v/v) may be toxic.Manipulation of DO If carbon is limiting, shutting off the carbon source should cause the culture to decrease its metabolic rate, and the DO to rise (spike). Terminate the carbon feed and time how long it takes for the DO to rise 10%, after which the carbon feed is turned back on. If the lag time is short (< 1 minute), the carbon source is limiting.Fermenter Preparation and Glycerol Batch PhaseInoculum Seed Flask Preparation Remember not to put too much medium in the baffled flasks. Volume should be 10-30% of the total flask volume.1. Baffled flasks containing a total of 5-10% of the initial fermentation volume ofMGY or BMGY are inoculated with a colony from a MD or MGY plate or from a frozen glycerol stock.2. Flasks are grown at 30°C, 250-300 rpm, 16-24 hours until OD600= 2-6. Toaccurately measure OD600> 1.0, dilute a sample of your culture 10-fold before reading.Glycerol Batch Phase 1. Sterilize the fermenter with the Fermentation Basal Salts medium containing 4%glycerol (see page 11).2. After sterilization and cooling, set temperature to 30°C, agitation and aeration tooperating conditions (usually maximum rpm and 0.1-1.0 vvm air), and adjust the pH of the Fermentation Basal Salts medium to 5.0 with 28% ammonium hydroxide(undiluted ammonium hydroxide). Add aseptically 4.35 ml PTM1trace salts/liter of Fermentation Basal Salts medium.3. Inoculate fermenter with approximately 5-10% initial fermentation volume from theculture generated in the inoculum shake flasks. Note that the DO will be close to 100% before the culture starts to grow. As the culture grows, it will consumeoxygen, causing the DO to decrease. Be sure to keep the DO above 20% by adding oxygen as needed.4. Grow the batch culture until the glycerol is completely consumed (18 to 24 hours).This is indicated by an increase in the DO to 100%. Note that the length of timeneeded to consume all the glycerol will vary with the density of the initial inoculum.5. Sampling is performed at the end of each fermentation stage and at least twice daily.We take 10 ml samples for each time point, then take 1 ml aliquots from this 10 mlsample. Samples are analyzed for cell growth (OD600and wet cell weight), pH, microscopic purity, and protein concentrations or activity. Freeze the cell pellets and supernatants at -80°C for later analysis. Proceed to Glycerol Fed-Batch Phase,page 5.Yield A cellular yield of 90 to 150 g/liter wet cells is expected for this stage. Recombinant protein will not yet be produced due to the absence of methanol.Introduction Once all the glycerol is consumed from the batch growth phase, a glycerol feed isinitiated to increase the cell biomass under limiting conditions. When you are ready toinduce with methanol, you can use DO spikes to make sure the glycerol is limited.Glycerol Fed-Batch Phase 1. Initiate a 50% w/v glycerol feed containing 12 ml PTM1trace salts per liter of glycerol feed. Set the feed rate to 18.15 ml/hr /liter initial fermentation volume.2. Glycerol feeding is carried out for about four hours or longer (see below). A cellularyield of 180 to 220 g/liter wet cells should be achieved at the end of this stage while no appreciable recombinant protein is produced.Note The level of expressed protein depends on the cell mass generated during the glycerolfed-batch phase. The length of this feed can be varied to optimize protein yield. A rangeof 50 to 300 g/liter wet cells is recommended for study. A maximum level of 4%glycerol is recommended in the batch phase due to toxicity problems with higher levelsof glycerol.Important If dissolved oxygen falls below 20%, the glycerol or methanol feed should bestopped and nothing should be done to increase oxygen rates until the dissolvedoxygen spikes. At this point, adjustments can be made to agitation, aeration, pressure oroxygen feeding.Proteases In the literature, it has been reported that if the pH of the fermentation medium islowered to 3.0, neutral proteases are inhibited. If you think neutral proteases aredecreasing your protein yield, change the pH control set point to 3.0 during the glycerolfed-batch phase (above) or at the beginning of the methanol induction (next page) andallow the metabolic activity of the culture to slowly lower the pH to 3.0 over 4 to 5 hours(Brierley, et al., 1994; Siegel, et al., 1990).Alternatively, if your protein is sensitive to low pH, it has been reported that inclusion ofcasamino acids also decreases protease activity (Clare, et al., 1991).Introduction All of the glycerol needs to be consumed before starting the methanol feed to fullyinduce the AOX1 promoter on methanol. However, it has been reported that a "mixedfeed" of glycerol and methanol has been successful to express recombinant proteins(Brierley, et al., 1990; Sreekrishna, et al., 1989). It is important to introduce methanolslowly to adapt the culture to growth on methanol. If methanol is added too fast, it willkill the cells. Once the culture is adapted to methanol, it is very important to use DOspikes to analyze the state of the culture and to take time points over the course ofmethanol induction to optimize protein expression. Growth on methanol also generates alot of heat, so temperature control at this stage is very important.Mut+ Methanol Fed-Batch Phase 1. Terminate glycerol feed and initiate induction by starting a 100% methanol feedcontaining 12 ml PTM1trace salts per liter of methanol. Set the feed rate to 3.6 ml/hr per liter initial fermentation volume.2. During the first 2-3 hours, methanol will accumulate in the fermenter and thedissolved oxygen values will be erratic while the culture adapts to methanol.Eventually the DO reading will stabilize and remain constant.3. If the DO cannot be maintained above 20%, stop the methanol feed, wait for theDO to spike and continue on with the current methanol feed rate. Increaseagitation, aeration, pressure or oxygen feeding to maintain the DO above 20%. 4. When the culture is fully adapted to methanol utilization (2-4 hours), and is limitedon methanol, it will have a steady DO reading and a fast DO spike time (generally under 1 minute). Maintain the lower methanol feed rate under limited conditions for at least 1 hour after adaptation before doubling the feed. The feed rate is then doubled to ~7.3 ml/hr/liter initial fermentation volume.5 After 2 hours at the 7.3 ml/hr/liter feed rate, increase the methanol feed rate to~10.9 ml/hr per liter initial fermentation volume. This feed rate is maintainedthroughout the remainder of the fermentation.6. The entire methanol fed-batch phase lasts approximately 70 hours with a total ofapproximately 740 ml methanol fed per liter of initial volume. However, this may vary for different proteins.Note: The supernatant may appear greenish. This is normal.Yield The cell density can increase during the methanol fed-batch phase to a final level of 350 to 450 g/liter wet cells. Remember that because most of the fermentation is carried out ina fed-batch mode, the final fermentation volume will be approximately double the initialfermentation volume.Fermentation of Mut S Pichia Strains Since Mut S cultures metabolize methanol poorly, their oxygen consumption is very low. Therefore, you cannot use DO spikes to evaluate the culture. In standard fermentations of a Mut S strain, the methanol feed rate is adjusted to maintain an excess of methanol in the medium which does not exceed 0.3% (may be determined by gas chromatography). While analysis by gas chromatography will insure that nontoxic levels of methanol are maintained, we have used the empirical guidelines below to express protein in Mut S strains. A gas chromatograph is useful for analyzing and optimizing growth of Mut S recombinants.continued on next pageMethanol Fed-Batch Phase, continuedMut S Methanol Fed- Batch Phase The first two phases of the glycerol batch and fed-batch fermentations of the Mut S strains are conducted as described for the Mut+ strain fermentations. The methanol induction phases of the Mut+ and Mut S differ in terms of the manner and amount in which the methanol feed is added to the cultures.1. The methanol feed containing 12 ml PTM1trace salts per liter of methanol is initiated at 1 ml/hr/liter initial fermentation volume for the first two hours. It is then increased in 10% increments every 30 minutes to a rate of 3 ml/hr which ismaintained for the duration of the fermentation.2.. The vessel is then harvested after ~100 hours on methanol. This time may be variedto optimize protein expression.Harvesting and Lysis of CellsIntroduction The methods and equipment listed below are by no means complete. The amount of cells or the volume of supernatant will determine what sort of equipment you need.Harvesting Cells and Supernatant For small fermentations (1-10 liters), the culture can be collected into centrifuge bottles (500-1000 ml) and centrifuged to separate the cells from the supernatant.For large fermentations, large membrane filtration units (Millipore) or a Sharples centrifuge can be used to separate cells from the supernatant. The optimal method will depend on whether you need the cells or the supernatant as the source of your protein and what you have available.Supernatants can be loaded directly onto certain purification columns or concentrated using ultrafiltration.Cell Lysis We recommend cell disruption using glass beads as described in Current Protocols inMolecular Biology, page 13.13.4. (Ausubel, et al., 1990) or Guide to ProteinPurification (Deutscher, 1990). This method may be tedious for large amounts of cells.For larger amounts, we have found that a microfluidizer works very well. Frenchpressing the cells does not seem to work as well as the glass beads or the microfluidizer.ReferencesIntroduction Most of the references below refer to papers where fermentation of Pichia wasperformed. Note that some of these are patent papers. You can obtain copies of patentsusing any of the following methods.• Patent Depository Libraries. U. S. patents and international patents granted underthe Patent Cooperation Treaty (PCT) are available on microfilm. These can be copiedand mailed or faxed depending on length. There is a fee for this service. The referencelibrarian at your local library can tell you the location of the nearest Patent DepositoryLibrary.• Interlibrary Loan. If you are not near a Patent Depository Library, you may request acopy of the patent through interlibrary loan. There will be a fee for this service.• U. S. Patent Office. Requests may be made directly to the Patent Office, Arlington,VA. Please call 703-557-4636 for more information on cost and delivery.• Private Library Services. There are private companies who will retrieve and sendyou patents for a fee. Two are listed below:Library Connection: 804-758-3311Rapid Patent Services: 800-336-5010Citations Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A.,Struhl, K., eds (1990) Current Protocols in Molecular Biology. GreenePublishing Associates and Wiley-Interscience, New York.Brierley, R. A., Siegel, R. S., Bussineau, C. M. Craig, W. S., Holtz, G. C., Davis, G. R.,Buckholz, R. G., Thill, G. P., Wondrack, L. M., Digan, M. E., Harpold, M. M.,Lair, S. V., Ellis, S. B., and William, M. E. (1989) Mixed Feed RecombinantYeast Fermentation. International Patent (PCT) Application. Publication No.WO 90/03431.Brierley, R. A., Bussineau, C., Kosson, R., Melton, A., and Siegel, R. S. (1990)Fermentation Development of Recombinant Pichia pastoris Expressing theHeterologous Gene: Bovine Lysozyme. Ann. New York Acad. Sci.589: 350-362.Brierley, R. A., Davis, G. R. and Holtz, G. C. (1994) Production of Insulin-Like GrowthFactor-1 in Methylotrophic Yeast Cells. United States Patent5,324,639.Clare, J. J., Romanos, M. A., Rayment, F. B., Rowedder, J. E., Smith, M. A., Payne, M.M., Sreekrishna, K. and Henwood, C. A. (1991) Production of EpidermalGrowth Factor in Yeast: High-level Secretion Using Pichia pastoris StrainsContaining Multiple Gene Copies. Gene105: 205-212.Cregg, J. M., Tschopp, J. F., Stillman, C., Siegel, R., Akong, M., Craig, W. S.,Buckholz, R. G., Madden, K. R., Kellaris, P. A., Davis, G. R., Smiley, B. L.,Cruze, J., Torregrossa, R., Veliçelebi, G. and Thill, G. P. (1987) High-levelExpression and Efficient Assembly of Hepatitis B Surface Antigen in theMethylotrophic Yeast Pichia pastoris. Bio/Technology5: 479-485.Cregg, J. M., Vedvick, T. S. and Raschke, W. C. (1993) Recent Advances in theExpression of Foreign Genes in Pichia pastoris. Bio/Technology11: 905-910.Deutscher, M. P. (1990) Guide to Protein Purification. In: Methods in Enzymology (J.N. Abelson and M. I. Simon, eds.) Academic Press, San Diego, CA.continued on next pageReferences, continuedCitations, continuedDigan, M. E., Lair, S. V., Brierley, R. A., Siegel, R. S., Williams, M. E., Ellis, S. B., Kellaris, P. A., Provow, S. A., Craig, W. S., Veliçelebi, G., Harpold, M. M. andThill, G. P. (1989) Continuous Production of a Novel Lysozyme via Secretionfrom the Yeast Pichia pastoris. Bio/Technology7: 160-164.Hagenson, M. J., Holden, K. A., Parker, K. A., Wood, P. J., Cruze, J. A., Fuke, M., Hopkins, T. R. and Stroman, D. W. (1989) Expression of Streptokinase inPichia pastoris Yeast. Enzyme Microbiol. Technol.11: 650-656.Laroche, Y., Storme, V., Meutter, J. D., Messens, J. and Lauwereys, M. (1994) High-Level Secretion and Very Efficient Isotopic Labeling of Tick AnticoagulantPeptide (TAP) Expressed in the Methylotrophic Yeast, Pichia pastoris.Bio/Technology12: 1119-1124.Romanos, M. A., Clare, J. J., Beesley, K. M., Rayment, F. B., Ballantine, S. P., Makoff,A. J., Dougan, G., Fairweather, N. F. and Charles, I. G. (1991) RecombinantBordetella pertussis Pertactin p69 from the Yeast Pichia pastoris High LevelProduction and Immunological Properties. Vaccine9: 901-906.Siegel, R. S. and Brierley, R. A. (1989) Methylotrophic Yeast Pichia pastoris Produced in High-cell-density Fermentations With High Cell Yields as Vehicle forRecombinant Protein Production. Biotechnol. Bioeng.34: 403-404.Siegel, R. S., Buckholz, R. G., Thill, G. P., and Wondrack, L. M. (1990) Production of Epidermal Growth Factor in Methylotrophic Yeast Cells. International Patent(PCT) Application. Publication No. WO 90/10697.Sreekrishna, K., Nelles, L., Potenz, R., Cruse, J., Mazzaferro, P., Fish, W., Fuke, M., Holden, K., Phelps, D., Wood, P. and Parker, K. (1989) High LevelExpression, Purification, and Characterization of Recombinant Human TumorNecrosis Factor Synthesized in the Methylotrophic Yeast Pichia pastoris.Biochemistry28(9): 4117-4125.©2002 Invitrogen Corporation. All rights reservedRecipesFermentation Basal Salts Medium For 1 liter, mix together the following ingredients:Phosphoric acid, 85% (26.7 ml)Calcium sulfate 0.93 gPotassium sulfate 18.2 gMagnesium sulfate-7H2O 14.9gPotassium hydroxide 4.13 gGlycerol 40.0g Water to 1 literAdd to fermenter with water to the appropriate volume and sterilize.PTM1 Trace Salts Mix together the following ingredients:Cupric sulfate-5H2O 6.0gSodium iodide 0.08 gManganese sulfate-H2O 3.0gSodium molybdate-2H2O 0.2gBoric Acid 0.02 g Cobalt chloride 0.5 g Zinc chloride 20.0 gFerrous sulfate-7H2O 65.0gBiotin 0.2gSulfuric Acid 5.0 mlWater to a final volume of 1 literFilter sterilize and store at room temperature.Note: There may be a cloudy precipitate upon mixing of these ingredients. Filter-sterilize as above and use.11。

发酵过程优化与控制PPT课件

菌种生产性能越高，其生产条件越难满足。
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发酵过程技术原理
分批发酵补料-分批发酵半连续发酵连续发酵
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分批发酵
几个重要参数：
为比生长速率,h-1； -qs 为比基质消耗速率,(g/g)/h； qp 为比产物形成速率,(g/g)/h 。
uX dX dt
q xX d S dt
补充养分，同时解除/消弱代谢产物的抑制。
不足：
丢失了未利用的养分和处于生长旺盛期的菌体；送去提炼的发酵液体积更大；丢失代谢产生的前体物；利于非产生菌突变株的生长。
实施：海洋微藻合成藻红素和EPA。
需要摸索最佳的培养基更新速率。
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连续发酵
发酵过程中一面补入新鲜的料液，一面以相同的流速放料，维持发酵液原来的体积。（恒化培养）
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发酵过程优化与控制
发酵
狭义——厌氧条件下葡萄糖通过酵解途径生成乳酸或乙醇等的分解代谢过程。
广义——微生物把一些原料养分在合适的发酵条件下经特定的代谢途径转变成所需产物的过程。
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发酵是一个很复杂的生化过程，其好坏涉及诸多因素：菌种性能、培养基组成、原料质量、灭菌条件、种子质量、发酵条件和过程控制等
pH变化会影响酶活，菌对基质的利用效率和细
胞结构，从而影响菌的生长和产物的合成。
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选择最适发酵pH的原则是获得最大比生产速率和
适当的菌量。
分阶段pH控制策略
如何控制发酵液pH？
基础培养基的配方；通过加酸碱或中间补料例如，青霉素发酵，通过调节加糖速率来控制pH；链霉素的生产，补充NH3来控制pH，同时为产物合成提供氮源。
培养液pH可反映菌的生理状况：pH上升超过最适值，意味着菌处于饥饿状态，可加糖调节；糖的过量又使pH下降；用氨水中和有机酸需防止微生物中毒，可通过监测培养液种溶氧浓度的变化来控制。

毕赤酵母发酵手册

毕赤酵母发酵手册毕赤酵母发酵手册总览简介：毕赤酵母和酿酒酵母很相似，都非常适合发酵生长。

毕赤酵母在有可能提高总体的蛋白质产量的发酵中能够达到非常高的细胞浓度，我们建议只有那些有过发酵经验或者能得到有经验的人的指导的人参与发酵。

因为发酵的类型很多，所以我们很难为您的个人案例提高详细的过程。

下面+s两种基因型的毕赤酵母菌株在15L的台式玻璃所给出的指导是基于MutMut和发酵罐中发酵而成。

请在您的发酵开始前先阅读操作员手册。

下面所给出的表就是整个发酵参数：在整个发酵过程中监测和调控下列参数非常重要。

下面的表格描述了这些参数和※设备推荐：下面是所推荐设备的清单：·发酵罐的夹套需要在发酵过程中给酵母菌降温，尤其是在甲醇流加过程中。

你需要一个固定的来源来提供冷却水（5-10℃）。

这可能意味着你需要一个冷冻装置来保持水的冷却。

·一个泡沫探针就像消泡剂一样不可或缺。

·一个氧气的来源——空气（不锈钢的发酵罐需要1-2vvm）或者纯氧（玻璃发酵罐需要0.1-0.3vvm）。

·添加甘油和甲醇的补料泵。

·pH的自动控制。

培养基的准备：你需要准确配置下列溶液：·发酵所需的基本盐类（第11页）·PTM补充盐类（第11页）1·75ml的50％的甘油每升初始发酵液，12ml的PTM补充盐每升甘油。

1·740ml的100％的甲醇每升初始发酵液，12ml的PTM补充盐每升甲醇。

1毕赤酵母生长的测定：在不同的时间点通过测OD600的吸光值和湿细胞的重量来检测毕赤酵母的生长。

培养的代谢速率通过通过观察溶氧浓度对应于有效碳源来测定。

溶氧的测定：简介：溶解氧的浓度时指氧气在培养基中的相关比例，溶氧100％是指培养基中氧达到饱和。

毕赤酵母的生长需要消耗氧气，减少溶解氧的满度。

毕赤酵母在生长时会消耗氧气，减少溶氧的程度。

然而，因为代谢甲醇的最初阶段需要氧气，所以将溶氧浓度维持在一个适当的水平（＞20％）来确保毕赤酵母在甲醇上的生长就至关重要。

发酵工程在食品中的应用 PPT课件

广泛分布于土壤、空气、水等潮湿和偏酸性的环境中
绒毛状，网状、絮状菌丝体
1.产黄青霉
2.紫红曲 3.点青霉 4.黑曲霉 5.米曲霉
部分霉菌菌落形态
泡菜发酵中的有益菌
乳酸菌酵母和醋酸菌
酸奶、奶酪的乳酸发酵剂乳酸菌发酵剂
乳杆菌、乳球菌、链球菌、明串珠菌、双歧杆菌
酵母发酵剂
乳糖发酵型酵母（开菲儿酸奶）
5-7d）→装坛→低温发酵→压榨→煮酒（灭菌）→装坛泥
封→堆置陈化（后熟）→出厂
淋饭酒摊饭酒喂饭酒
黄酒的分类
以酒药、麦曲或米曲、红曲及淋饭
酒母为糖化发酵剂，进行自然的、
传统工艺黄酒多菌种的混合发酵生产而成，发酵取代自然发酵，以大型发酵生产代替小型手工操作，生产过程简化，原料利用率高，去除了笨重的体力劳动
长些，皮薄的时间短些）； ⑤压榨：液体进行后发酵（20-25℃，一周），澄清，取
上部液体，其中低挡的装瓶，较高档次的换桶（最好是橡木桶，橡木能产生抑菌物质，同时具有一种天然植物香）陈化三年（5℃）；渣进行蒸馏，调香，制成白兰地（酒精多）。
葡萄酒的再加工
起泡葡萄酒：是一种含CO2的葡萄酒，以葡萄酒为基础，通过加糖发酵产生或人工压入CO2。
3.出芽短梗霉
4.多孢丝抱酵母
5.荚复膜孢酵
2.产肮假丝酵母 13.大型罗伦隐球酵母
红酵
母1.酿酒酵母
12.玫红法佛酵母
6.解脂复膜孢
14.美极梅奇酵母
11.深红酵母
啤
酒 10.鲁氏酵母
15.浅红酵7母.季也蒙有孢汉逊酵
酵
母
8.碎囊汉逊酵母
9.卡氏酵母
（3）霉菌/丝状真菌
传统发酵食品生产淀粉酶、蛋白酶、纤维素酶等不利方面：食品发霉变质，引起病害

酿酒微生物讲稿PPT课件

主要产生乙醇、甘油、杂醇油等，同时产生少量酯类、酸类和醛酮类物质。
细菌的发酵机制与代谢产物
发酵机制
细菌发酵多采用与酵母菌类似的厌氧呼吸方式，将糖类物质转化为乳酸、乙酸等。
代谢产物
主要产生乳酸、乙酸、琥珀酸等，同时可能产生一些具有特殊风味的物质。
霉菌的发酵机制与代谢产物
发酵机制
霉菌发酵主要通过好氧呼吸将糖类物质氧化为水和二氧化碳。
02
根据酿酒用途和发酵类型，酿酒微生物可分为酒精发酵微生物、乳酸发酵微生物、白酒发酵微生物等。
酿酒微生物在酿酒过程中的作用
01
02
03
糖化作用
霉菌能够分解淀粉、蛋白质等大分子物质，生成可发酵性糖，为后续发酵提供必要的营养物质。
酒精发酵
酵母菌利用可发酵性糖进行酒精发酵，生成酒精和二氧化碳，是酿酒的主要环节。
酿酒酵母、毕赤酵母、假丝酵母等。
特性
具有发酵能力，能够将糖类物质转化为酒精和二氧化碳。
作用
在葡萄酒和啤酒等发酵酒的酿造过程中，酵母菌是必不可少的微生物。
细菌的种类与特性
乳酸菌
主要存在于酸奶和泡菜等发酵食品中，能够产生乳酸，抑制其他微生物的生长。
醋酸菌
梭状芽胞杆菌
存在于土壤和动物肠道中，能够产生芽孢，抵抗不良环境。
件，以获得所需微生物；对分离纯化的结果进行验证和鉴定，确保获得
的微生物为酿酒相关。
酿酒微生物的鉴定方法与技术
形态学鉴定
生化鉴定
通过观察微生物的形态、大小、颜色、运动性等特征，初步鉴定微生物种类。
利用微生物对不同底物的代谢反应进行鉴定，如糖发酵试验、氧化发酵试验等。
分子生物学鉴定

毕赤酵母的摇瓶发酵方法

毕赤酵母的‎摇瓶发酵方‎法：一、摇瓶发酵方‎法:毕赤酵母摇‎瓶发酵方法‎分为两个阶‎段，1、酵母菌株生‎长阶段；2、脂肪酶诱导‎表达阶段。

1、酵母生长阶‎段。

准备试剂：1000m‎l BMGY培‎养基，1000m‎l BMMY培‎养基，10X的甲‎醇，摇瓶1L（灭菌），温控摇床，50ml离‎心管（灭菌）。

紫外分光光‎度计，石英比色皿‎。

以下所有操‎作均在超净‎台内或者无‎菌条件下完‎成。

（1）往灭好菌的‎IL摇瓶中‎加入100‎mlBMG‎Y培养基，然后加入约‎1ml 脂肪‎酶菌株（培养基：菌液=100：1），用透气膜封‎口（透气，但是细菌不‎能透过）。

置于温控摇‎床上，温度调至3‎00C，转速为25‎0-300rp‎m/min，使酵母生长‎，OD600‎=2.0-6.0，时间约为1‎5-24小时。

（2）将发酵液转‎入50ml‎离心管，1500g‎-3000g‎离心5mi‎n。

去掉上清，用BMMY‎培养基将菌‎体浓度稀释‎至OD60‎0=1.0，约有500‎ml左右。

将稀释后的‎发酵液分别‎加入到1L‎的药瓶中，每个摇瓶1‎50ml 发‎酵液（绝不能超过‎200ml‎）。

（3）将摇瓶置于‎温控摇床上‎，温度调至3‎00C，转速为25‎0-300rp‎m/min，使酵母表达‎脂肪酶，每24小时‎加入一次5‎%的甲醇，使甲醇的终‎浓度为0.5%。

连续诱导表‎达48小时‎。

（4）将发酵液进‎行1200‎0rpm/min离心‎5min，取上清（若上清仍混‎浊，可反复离心‎）；进行酶活分‎析和蛋白含‎量分析。

BMGY培‎养基的配制‎（1000m‎l）：20g蛋白‎胨（pepto‎ne）,10g酵母‎提取物（Yeast‎ Extra‎ct）,加水至70‎0ml；1210C‎高温灭菌2‎0min。

然后分别在‎无菌条件下‎加入10X‎ YNB 100ml‎，10X 磷酸钾缓冲‎液（PH6.0）100ml‎，10X甘油‎ 100ml‎。

毕赤酵母发酵罐发酵资料讲解

毕赤酵母发酵罐发酵微生物发酵罐发酵（毕赤酵母）灭菌前：室温下校准PH电极，先校6.86零点再4.0斜率（若的发酵pH很长时间是酸性的（如酵母发酵）用6.86校正零点，4.0校正斜率；若你的发酵pH很长时间是碱性的（如某些细菌发酵）用6.86校正零点，9.18校正斜率）；室温下校准溶氧电极，1.0点在不接溶氧电极时候标定，100%点接上溶氧电极，放置在空气中较定；或2.0点在灭菌过程中，温度达到121度左右压力0.12mpa左右时候标定，100%在灭菌结束，降温至发酵温度并稳定，转速在发酵初始转速，通气量在发酵初始通气量时候标定灭菌：1.灭菌，先将各排气阀打开，将蒸汽引入夹套或蛇管进行预热，待罐温升至80~90℃，将排气阀逐渐关小。

接着将蒸汽从进气口、排料口、取样口直接通入罐中（如有冲视罐也同时进汽），使罐温上升到118~120℃，罐压维持在0.09~0.1Mpa（表压），并保持30min左右。

2.保温结束后，依次关闭各排汽、进汽阀门，待罐内压力低于空气压力后，向罐内通入无菌空气，在夹套或蛇管中通冷却水降温，使培养基的温度降到所需的温度，进行下一步的发酵和培养。

（注意压力：灭菌时，总蒸汽管道压力要求不低于0.3~0.35Mpa，使用压力不低于0.2Mpa。

）灭菌后:A.消耗甘油阶段1.灭菌后冷却30℃时：2.冷却至30℃时，开启搅拌(转速最大)和通气(0.1-1.0vvm),接通28%氨水(未稀释)调PH5.0;每升加4.35ml的无菌PTM1基础盐；3.从摇瓶中接种种子液，DO值为100%，开始培养后会消耗，导致DO值下降，通氧气以确保DO值超过20％，速率先为0.1vvm。

4. 发酵过夜甘油被完全消耗（18-24h），标志为DO值增加到100％。

【每天至少两次取样，测OD600，湿重，显微观察. 将菌体和上清（离心后）在-80℃下保藏，用于后面的分析。

】5. 这个阶段所期望达到的细胞产量为90-150g/L湿细胞。

酵母及其应用ppt课件

外膜：含磷脂代谢的酶内膜和嵴：呼吸链组分，ATP合成酶、琥珀酸脱氢酶等
。膜间腔：腺苷酸激酶、磷酸腺苷酸激酶嵴间腔：TCA循环的酶等
.
内质网
内质网：是存在于细胞质中的、由膜构成的、呈游离或广泛互相连续的囊泡状的结构。
种类: 粗糙型内质网（rough ER）光滑型内质网（smooth ER）
.
线粒体
1.一种半自主的细胞器，呈球形或棒状, 0.31×0.5—3um，分散在细胞质中。双层单位膜包围的细胞器；其中含脂类、蛋白质、少量RNA和环状DNA。
2.其DNA可自主复制，不受核DNA控制。决定线粒体的某些遗传性状。
3.生物氧化中心、能量转换的基地
.
线粒体的功能
▪ 构造
外膜、内膜、嵴、膜间腔、嵴间腔
调节渗透压
.
海藻糖
海藻糖由两分子的吡喃葡萄糖单体以α-1,1糖苷键连接而成主要应用： 1：食品方面：改善食品风味、抗氧化、保鲜、延长保藏期等。 2：生物工程方面：酶保护剂、医药药品的保存等。 3：农业领域：抗旱、抗寒植物、 4：其他方面：化妆品（保湿）等
.
其他结构
质粒 2 um质粒是一个环状、周长2 um的6kb双链DNA分子。可用于研究基因调控、染色体复制的理想系统，也可作为酵母菌转化的有效载体，并组建基因工程菌。
生理功能：起物质传递的作用，另外还有合成脂类和
脂蛋白，与出芽起始有关。
.
核糖体
位于游离的细胞质中或附着在内质网上。化学组成：与细菌类似结构：核糖体的沉降系数为80s，它由60s和40s的两个亚基组成
。功能是按照mRNA的指令将氨基酸合成蛋白质多肽链。
.
高尔基体 (高尔基复合体)

毕赤酵母发酵 ppt课件

二：实验材料和方法
2.1：质粒的构建
表达GFP- SKl pGLY11245或pGLY13173 转化YGLY27355或YGLY31884 YGLY14836生产单克隆IgG1抗体
表达Ub-R-GFP pGLY10148转化 YGLY19309或YGLY29325 生产单克隆IgG1抗体 2.2：转化：毕赤酵母感受态的制备和电转化的方法（参见nvitrogen公司的Pichia
• 最大量的生产重组蛋白高度依赖甲醇消耗速率
文章来源：Journal of biotechnology
• 1背景介绍 • 2实验材料和方法 • 3实验结果 • 4结论与分析
背景介绍
1.1醇氧化酶AOX1基因启动子作为甲醇诱导的强启动子,在Pichia pastoris表达系统生产重组蛋白中得到广泛应用。P.pastoris中的转录因子通过特异的顺式作用元件与启动子相互作用而影响基因的转录,因此通过调节AOX1基因启动子的活性可以控制下游基因的表达
2.6:细胞裂解
从毕赤酵母细胞释放细胞外的DNA使用 Quant-iT™ PicoGreen® dsDNA Assay Kit 双链DNA检测试剂盒检测
3实验结果与讨论 3.1:
通过荧光显微术 UB - R- GFP和GFP- SKL 被正确地定位于细胞质和过氧化物酶体，符合预期
3.2:YGLY29325的三种不同的补料
分批发酵条件的取样数据(a) 甲醇
限制 (ML); (b) 葡萄糖培养8h甲醇培养(20 h) (SML); (c) 氧气限制并
额外增加1%甲醇（OL）DO (%) ：
黑色直线，甘油或葡萄糖：蓝色,甲醇:红色。细胞湿重：黑色三角
3.3毕赤酵母AOX1启动子（YGLY29325）根据三种不同的补料分批条件规制 - 甲醇限制（ML），切换与葡萄糖和甲醇（SML）和氧气限制（OL）补料分批条件。（a） GFP荧光表达图像（b）不同诱导时间的荧光蛋白相对密度。对于ML和OL，时间表示分批阶段（T1），甘油补料分批阶段诱导前（T2）和诱导期（T3，10±2小时; T4中， 36±2小时; T5，62±2小时; T6，84±2小时）。对与SML条件下，细胞生长是在T2，T4 和T6 葡萄糖阶段，诱导是在T3和T5甲醇阶段

毕赤酵母发酵手册

Version B Pichia Fermentation Process GuidelinesOverviewIntroduction Pichia pastoris, like Saccharomyces cerevisiae, is particularly well-suited forfermentative growth. Pichia has the ability to reach very high cell densities duringfermentation which may improve overall protein yields.We recommend that only those with fermentation experience or those who have accessto people with experience attempt fermentation. Since there are a wide variety offermenters available, it is difficult to provide exact procedures for your particular case.The guidelines given below are based on fermentations of both Mut+ and Mut S Pichiastrains in a 15 liter table-top glass fermenter. Please read the operator's manual for yourparticular fermenter before beginning. The table below provides an overview of thematerial covered in these guidelines.Step Topic Page1 Fermentationparameters 12 Equipment needed and preparation of medium 23 Measurement and use of dissolved oxygen (DO) in the culture 34 Growth of the inoculum 45 Generation of biomass on glycerol in batch and fed-batch phases 4-56 Induction of expression of Mut+ and Mut S recombinants in themethanol fed-batch phase6-77 Harvesting and lysis of cells 88 References 9-109 Recipes 11Fermentation Parameters It is important to monitor and control the following parameters throughout thefermentation process. The following table describes the parameters and the reasons for monitoring them.Parameter Reason Temperature (30.0°C) Growth above 32°C is detrimental to protein expression Dissolved oxygen (>20%) Pichia needs oxygen to metabolize glycerol andmethanolpH (5.0-6.0 and 3.0) Important when secreting protein into the medium andfor optimal growthAgitation (500 to 1500 rpm) Maximizes oxygen concentration in the mediumAeration (0.1 to 1.0 vvm*for glass fermenters)Maximizes oxygen concentration in the medium whichdepends on the vesselAntifoam (the minimumneeded to eliminate foam)Excess foam may cause denaturation of your secretedprotein and it also reduces headspaceCarbon source (variablerate)Must be able to add different carbon sources at differentrates during the course of fermentationcontinued on next pageOverview, continuedRecommended Equipment Below is a checklist for equipment recommendations.• A jacketed vessel is needed for cooling the yeast during fermentation, especially during methanol induction. You will need a constant source of cold water (5-10°C). This requirement may mean that you need a refrigeration unit to keep the water cold. • A foam probe is highly recommended as antifoam is required.• A source of O2--either air (stainless steel fermenters at 1-2 vvm) or pure O2(0.1-0.3 vvm for glass fermenters).• Calibrated peristaltic pumps to feed the glycerol and methanol.• Automatic control of pH.Medium Preparation You will need to prepare the appropriate amount of following solutions:• Fermentation Basal Salts (page 11)• PTM1Trace Salts (page 11)• ~75 ml per liter initial fermentation volume of 50% glycerol containing 12 ml PTM1 Trace Salts per liter of glycerol.• ~740 ml per liter initial fermentation volume of 100% methanol containing 12 mlPTM1Trace Salts per liter of methanol.Monitoring the Growth of Pichia pastoris Cell growth is monitored at various time points by using the absorbance at 600 nm (OD600) and the wet cell weight. The metabolic rate of the culture is monitored by observing changes in the concentration of dissolved oxygen in response to carbon availability (see next page).Dissolved Oxygen (DO) MeasurementIntroduction The dissolved oxygen concentration is the relative percent of oxygen in the mediumwhere 100% is O2-saturated medium. Pichia will consume oxygen as it grows, reducing the dissolved oxygen content. However, because oxygen is required for the first step ofmethanol catabolism, it is important to maintain the dissolved oxygen (DO) concentra-tion at a certain level (>20%) to ensure growth of Pichia on methanol. Accuratemeasurement and observation of the dissolved oxygen concentration of a culture willgive you important information about the state and health of the culture. Therefore, it isimportant to accurately calibrate your equipment. Please refer to your operator's manual.Maintaining the Dissolved Oxygen Concentration (DO) 1. Maintaining the dissolved oxygen above 20% may be difficult depending on theoxygen transfer rates (OTR) of the fermenter, especially in small-scale glassvessels. In a glass vessel, oxygen is needed to keep the DO above 20%, usually~0.1-0.3 vvm (liters of O2per liter of fermentation culture per minute). Oxygen consumption varies and depends on the amount of methanol added and the protein being expressed.2. Oxygen can be used at 0.1 to 0.3 vvm to achieve adequate levels. This can beaccomplished by mixing with the air feed and can be done in any glass fermenter.For stainless steel vessels, pressure can be used to increase the OTR. Be sure toread the operator's manual for your particular fermenter.3. If a fermenter cannot supply the necessary levels of oxygen, then the methanol feedshould be scaled back accordingly. Note that decreasing the amount of methanol may reduce the level of protein expression.4. To reach maximum expression levels, the fermentation time can be increased todeliver similar levels of methanol at the lower feed rate. For many recombinantproteins, a direct correlation between amount of methanol consumed and theamount of protein produced has been observed.Use of DO Measurements During growth, the culture consumes oxygen, keeping the DO concentration low. Note that oxygen is consumed whether the culture is grown on glycerol or methanol. The DO concentration can be manipulated to evaluate the metabolic rate of the culture and whether the carbon source is limiting. The metabolic rate indicates how healthy the culture is. Determining whether the carbon source is limiting is important if you wish to fully induce the AOX1 promoter. For example, changes in the DO concentrations (DO spikes) allow you to determine whether all the glycerol is consumed from the culture before adding methanol. Secondly, it ensures that your methanol feed does not exceed the rate of consumption. Excess methanol (> 1-2% v/v) may be toxic.Manipulation of DO If carbon is limiting, shutting off the carbon source should cause the culture to decrease its metabolic rate, and the DO to rise (spike). Terminate the carbon feed and time how long it takes for the DO to rise 10%, after which the carbon feed is turned back on. If the lag time is short (< 1 minute), the carbon source is limiting.Fermenter Preparation and Glycerol Batch PhaseInoculum Seed Flask Preparation Remember not to put too much medium in the baffled flasks. Volume should be 10-30% of the total flask volume.1. Baffled flasks containing a total of 5-10% of the initial fermentation volume ofMGY or BMGY are inoculated with a colony from a MD or MGY plate or from a frozen glycerol stock.2. Flasks are grown at 30°C, 250-300 rpm, 16-24 hours until OD600= 2-6. Toaccurately measure OD600> 1.0, dilute a sample of your culture 10-fold before reading.Glycerol Batch Phase 1. Sterilize the fermenter with the Fermentation Basal Salts medium containing 4%glycerol (see page 11).2. After sterilization and cooling, set temperature to 30°C, agitation and aeration tooperating conditions (usually maximum rpm and 0.1-1.0 vvm air), and adjust the pH of the Fermentation Basal Salts medium to 5.0 with 28% ammonium hydroxide(undiluted ammonium hydroxide). Add aseptically 4.35 ml PTM1trace salts/liter of Fermentation Basal Salts medium.3. Inoculate fermenter with approximately 5-10% initial fermentation volume from theculture generated in the inoculum shake flasks. Note that the DO will be close to 100% before the culture starts to grow. As the culture grows, it will consumeoxygen, causing the DO to decrease. Be sure to keep the DO above 20% by adding oxygen as needed.4. Grow the batch culture until the glycerol is completely consumed (18 to 24 hours).This is indicated by an increase in the DO to 100%. Note that the length of timeneeded to consume all the glycerol will vary with the density of the initial inoculum.5. Sampling is performed at the end of each fermentation stage and at least twice daily.We take 10 ml samples for each time point, then take 1 ml aliquots from this 10 mlsample. Samples are analyzed for cell growth (OD600and wet cell weight), pH, microscopic purity, and protein concentrations or activity. Freeze the cell pellets and supernatants at -80°C for later analysis. Proceed to Glycerol Fed-Batch Phase,page 5.Yield A cellular yield of 90 to 150 g/liter wet cells is expected for this stage. Recombinant protein will not yet be produced due to the absence of methanol.Introduction Once all the glycerol is consumed from the batch growth phase, a glycerol feed isinitiated to increase the cell biomass under limiting conditions. When you are ready toinduce with methanol, you can use DO spikes to make sure the glycerol is limited.Glycerol Fed-Batch Phase 1. Initiate a 50% w/v glycerol feed containing 12 ml PTM1trace salts per liter of glycerol feed. Set the feed rate to 18.15 ml/hr /liter initial fermentation volume.2. Glycerol feeding is carried out for about four hours or longer (see below). A cellularyield of 180 to 220 g/liter wet cells should be achieved at the end of this stage while no appreciable recombinant protein is produced.Note The level of expressed protein depends on the cell mass generated during the glycerolfed-batch phase. The length of this feed can be varied to optimize protein yield. A rangeof 50 to 300 g/liter wet cells is recommended for study. A maximum level of 4%glycerol is recommended in the batch phase due to toxicity problems with higher levelsof glycerol.Important If dissolved oxygen falls below 20%, the glycerol or methanol feed should bestopped and nothing should be done to increase oxygen rates until the dissolvedoxygen spikes. At this point, adjustments can be made to agitation, aeration, pressure oroxygen feeding.Proteases In the literature, it has been reported that if the pH of the fermentation medium islowered to 3.0, neutral proteases are inhibited. If you think neutral proteases aredecreasing your protein yield, change the pH control set point to 3.0 during the glycerolfed-batch phase (above) or at the beginning of the methanol induction (next page) andallow the metabolic activity of the culture to slowly lower the pH to 3.0 over 4 to 5 hours(Brierley, et al., 1994; Siegel, et al., 1990).Alternatively, if your protein is sensitive to low pH, it has been reported that inclusion ofcasamino acids also decreases protease activity (Clare, et al., 1991).Introduction All of the glycerol needs to be consumed before starting the methanol feed to fullyinduce the AOX1 promoter on methanol. However, it has been reported that a "mixedfeed" of glycerol and methanol has been successful to express recombinant proteins(Brierley, et al., 1990; Sreekrishna, et al., 1989). It is important to introduce methanolslowly to adapt the culture to growth on methanol. If methanol is added too fast, it willkill the cells. Once the culture is adapted to methanol, it is very important to use DOspikes to analyze the state of the culture and to take time points over the course ofmethanol induction to optimize protein expression. Growth on methanol also generates alot of heat, so temperature control at this stage is very important.Mut+ Methanol Fed-Batch Phase 1. Terminate glycerol feed and initiate induction by starting a 100% methanol feedcontaining 12 ml PTM1trace salts per liter of methanol. Set the feed rate to 3.6 ml/hr per liter initial fermentation volume.2. During the first 2-3 hours, methanol will accumulate in the fermenter and thedissolved oxygen values will be erratic while the culture adapts to methanol.Eventually the DO reading will stabilize and remain constant.3. If the DO cannot be maintained above 20%, stop the methanol feed, wait for theDO to spike and continue on with the current methanol feed rate. Increaseagitation, aeration, pressure or oxygen feeding to maintain the DO above 20%. 4. When the culture is fully adapted to methanol utilization (2-4 hours), and is limitedon methanol, it will have a steady DO reading and a fast DO spike time (generally under 1 minute). Maintain the lower methanol feed rate under limited conditions for at least 1 hour after adaptation before doubling the feed. The feed rate is then doubled to ~7.3 ml/hr/liter initial fermentation volume.5 After 2 hours at the 7.3 ml/hr/liter feed rate, increase the methanol feed rate to~10.9 ml/hr per liter initial fermentation volume. This feed rate is maintainedthroughout the remainder of the fermentation.6. The entire methanol fed-batch phase lasts approximately 70 hours with a total ofapproximately 740 ml methanol fed per liter of initial volume. However, this may vary for different proteins.Note: The supernatant may appear greenish. This is normal.Yield The cell density can increase during the methanol fed-batch phase to a final level of 350 to 450 g/liter wet cells. Remember that because most of the fermentation is carried out ina fed-batch mode, the final fermentation volume will be approximately double the initialfermentation volume.Fermentation of Mut S Pichia Strains Since Mut S cultures metabolize methanol poorly, their oxygen consumption is very low. Therefore, you cannot use DO spikes to evaluate the culture. In standard fermentations of a Mut S strain, the methanol feed rate is adjusted to maintain an excess of methanol in the medium which does not exceed 0.3% (may be determined by gas chromatography). While analysis by gas chromatography will insure that nontoxic levels of methanol are maintained, we have used the empirical guidelines below to express protein in Mut S strains. A gas chromatograph is useful for analyzing and optimizing growth of Mut S recombinants.continued on next pageMethanol Fed-Batch Phase, continuedMut S Methanol Fed- Batch Phase The first two phases of the glycerol batch and fed-batch fermentations of the Mut S strains are conducted as described for the Mut+ strain fermentations. The methanol induction phases of the Mut+ and Mut S differ in terms of the manner and amount in which the methanol feed is added to the cultures.1. The methanol feed containing 12 ml PTM1trace salts per liter of methanol is initiated at 1 ml/hr/liter initial fermentation volume for the first two hours. It is then increased in 10% increments every 30 minutes to a rate of 3 ml/hr which ismaintained for the duration of the fermentation.2.. The vessel is then harvested after ~100 hours on methanol. This time may be variedto optimize protein expression.Harvesting and Lysis of CellsIntroduction The methods and equipment listed below are by no means complete. The amount of cells or the volume of supernatant will determine what sort of equipment you need.Harvesting Cells and Supernatant For small fermentations (1-10 liters), the culture can be collected into centrifuge bottles (500-1000 ml) and centrifuged to separate the cells from the supernatant.For large fermentations, large membrane filtration units (Millipore) or a Sharples centrifuge can be used to separate cells from the supernatant. The optimal method will depend on whether you need the cells or the supernatant as the source of your protein and what you have available.Supernatants can be loaded directly onto certain purification columns or concentrated using ultrafiltration.Cell Lysis We recommend cell disruption using glass beads as described in Current Protocols inMolecular Biology, page 13.13.4. (Ausubel, et al., 1990) or Guide to ProteinPurification (Deutscher, 1990). This method may be tedious for large amounts of cells.For larger amounts, we have found that a microfluidizer works very well. Frenchpressing the cells does not seem to work as well as the glass beads or the microfluidizer.ReferencesIntroduction Most of the references below refer to papers where fermentation of Pichia wasperformed. Note that some of these are patent papers. You can obtain copies of patentsusing any of the following methods.• Patent Depository Libraries. U. S. patents and international patents granted underthe Patent Cooperation Treaty (PCT) are available on microfilm. These can be copiedand mailed or faxed depending on length. There is a fee for this service. The referencelibrarian at your local library can tell you the location of the nearest Patent DepositoryLibrary.• Interlibrary Loan. If you are not near a Patent Depository Library, you may request acopy of the patent through interlibrary loan. There will be a fee for this service.• U. S. Patent Office. Requests may be made directly to the Patent Office, Arlington,VA. Please call 703-557-4636 for more information on cost and delivery.• Private Library Services. There are private companies who will retrieve and sendyou patents for a fee. Two are listed below:Library Connection: 804-758-3311Rapid Patent Services: 800-336-5010Citations Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A.,Struhl, K., eds (1990) Current Protocols in Molecular Biology. GreenePublishing Associates and Wiley-Interscience, New York.Brierley, R. A., Siegel, R. S., Bussineau, C. M. Craig, W. S., Holtz, G. C., Davis, G. R.,Buckholz, R. G., Thill, G. P., Wondrack, L. M., Digan, M. E., Harpold, M. M.,Lair, S. V., Ellis, S. B., and William, M. E. (1989) Mixed Feed RecombinantYeast Fermentation. International Patent (PCT) Application. Publication No.WO 90/03431.Brierley, R. A., Bussineau, C., Kosson, R., Melton, A., and Siegel, R. S. (1990)Fermentation Development of Recombinant Pichia pastoris Expressing theHeterologous Gene: Bovine Lysozyme. Ann. New York Acad. Sci.589: 350-362.Brierley, R. A., Davis, G. R. and Holtz, G. C. (1994) Production of Insulin-Like GrowthFactor-1 in Methylotrophic Yeast Cells. United States Patent5,324,639.Clare, J. J., Romanos, M. A., Rayment, F. B., Rowedder, J. E., Smith, M. A., Payne, M.M., Sreekrishna, K. and Henwood, C. A. (1991) Production of EpidermalGrowth Factor in Yeast: High-level Secretion Using Pichia pastoris StrainsContaining Multiple Gene Copies. Gene105: 205-212.Cregg, J. M., Tschopp, J. F., Stillman, C., Siegel, R., Akong, M., Craig, W. S.,Buckholz, R. G., Madden, K. R., Kellaris, P. A., Davis, G. R., Smiley, B. L.,Cruze, J., Torregrossa, R., Veliçelebi, G. and Thill, G. P. (1987) High-levelExpression and Efficient Assembly of Hepatitis B Surface Antigen in theMethylotrophic Yeast Pichia pastoris. Bio/Technology5: 479-485.Cregg, J. M., Vedvick, T. S. and Raschke, W. C. (1993) Recent Advances in theExpression of Foreign Genes in Pichia pastoris. Bio/Technology11: 905-910.Deutscher, M. P. (1990) Guide to Protein Purification. In: Methods in Enzymology (J.N. Abelson and M. I. Simon, eds.) Academic Press, San Diego, CA.continued on next pageReferences, continuedCitations, continuedDigan, M. E., Lair, S. V., Brierley, R. A., Siegel, R. S., Williams, M. E., Ellis, S. B., Kellaris, P. A., Provow, S. A., Craig, W. S., Veliçelebi, G., Harpold, M. M. andThill, G. P. (1989) Continuous Production of a Novel Lysozyme via Secretionfrom the Yeast Pichia pastoris. Bio/Technology7: 160-164.Hagenson, M. J., Holden, K. A., Parker, K. A., Wood, P. J., Cruze, J. A., Fuke, M., Hopkins, T. R. and Stroman, D. W. (1989) Expression of Streptokinase inPichia pastoris Yeast. Enzyme Microbiol. Technol.11: 650-656.Laroche, Y., Storme, V., Meutter, J. D., Messens, J. and Lauwereys, M. (1994) High-Level Secretion and Very Efficient Isotopic Labeling of Tick AnticoagulantPeptide (TAP) Expressed in the Methylotrophic Yeast, Pichia pastoris.Bio/Technology12: 1119-1124.Romanos, M. A., Clare, J. J., Beesley, K. M., Rayment, F. B., Ballantine, S. P., Makoff,A. J., Dougan, G., Fairweather, N. F. and Charles, I. G. (1991) RecombinantBordetella pertussis Pertactin p69 from the Yeast Pichia pastoris High LevelProduction and Immunological Properties. Vaccine9: 901-906.Siegel, R. S. and Brierley, R. A. (1989) Methylotrophic Yeast Pichia pastoris Produced in High-cell-density Fermentations With High Cell Yields as Vehicle forRecombinant Protein Production. Biotechnol. Bioeng.34: 403-404.Siegel, R. S., Buckholz, R. G., Thill, G. P., and Wondrack, L. M. (1990) Production of Epidermal Growth Factor in Methylotrophic Yeast Cells. International Patent(PCT) Application. Publication No. WO 90/10697.Sreekrishna, K., Nelles, L., Potenz, R., Cruse, J., Mazzaferro, P., Fish, W., Fuke, M., Holden, K., Phelps, D., Wood, P. and Parker, K. (1989) High LevelExpression, Purification, and Characterization of Recombinant Human TumorNecrosis Factor Synthesized in the Methylotrophic Yeast Pichia pastoris.Biochemistry28(9): 4117-4125.©2002 Invitrogen Corporation. All rights reservedRecipesFermentation Basal Salts Medium For 1 liter, mix together the following ingredients:Phosphoric acid, 85% (26.7 ml)Calcium sulfate 0.93 gPotassium sulfate 18.2 gMagnesium sulfate-7H2O 14.9gPotassium hydroxide 4.13 gGlycerol 40.0g Water to 1 literAdd to fermenter with water to the appropriate volume and sterilize.PTM1 Trace Salts Mix together the following ingredients:Cupric sulfate-5H2O 6.0gSodium iodide 0.08 gManganese sulfate-H2O 3.0gSodium molybdate-2H2O 0.2gBoric Acid 0.02 g Cobalt chloride 0.5 g Zinc chloride 20.0 gFerrous sulfate-7H2O 65.0gBiotin 0.2gSulfuric Acid 5.0 mlWater to a final volume of 1 literFilter sterilize and store at room temperature.Note: There may be a cloudy precipitate upon mixing of these ingredients. Filter-sterilize as above and use.11。

酵母菌优秀PPT课件-优秀PPT文档

佛山市高明鸿鹰食品有
限公司
产品名称瓜子仁
荣华花生
开心果
商标
规格
吉昌
散装
散装
旭明
散装
生产日期 200604-07
200603-24
200604-10
主要不合格项目酵母
酵母
霉菌
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4. 酱油、醋
酵母和霉菌嗜好酸性反应，因而能使醋发生霉变。含一定盐分的食品，对一般细菌生长是不利的，但酵母对盐则有抵抗性，而且能够生长，所以酱油也会受到侵犯。
二是微生物指标不合格。微生物指标不合格主要是霉菌、酵母超标。抽查中有个别产品微生物指标不合格。其中有1种产品的酵母实测值为480cfu/g，是标准规定值的近20倍
三是产品标签标注不规范。
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烘炒食品产品质量国家监督抽查部分质量较差的产品及其企业名单
企业名称
广州番禺区越秀食品厂
广州市番禺钟村荣华食品厂
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酵母菌的危害：
➢少数耐高渗的酵母菌和鲁氏酵母、蜂蜜酵母可使蜂蜜和果酱等败坏； ➢有的酵母菌是发酵工业的污染菌，影响发酵的产量和质量； ➢某些酵母菌会引起人和植物的病害，例如白假丝酵母可引起皮肤、粘膜、呼吸道、消化道等多种疾病.
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四、食品中常见的酵母属
（一）酵母属酵母中最重要、应用最广泛的一类： 1.酒类的酿造、面包发酵 2.引起高糖食品（果酱等）变质 3.能抵抗高浓度食盐溶液
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作业题
1. 酵母菌的特征 2. 什么是冷链？
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3
（三）酵母菌菌落特征
啤酒酵母菌落
细菌菌落
4
（三）酵母菌菌落特征
啤酒酵母菌落
红酵母菌落