响应面法优化重组巴斯德毕赤酵母合成ECH42的培养基组分及其重组酶降解几丁质的研究

采用响应面法在摇瓶水平对重组巴斯德毕赤酵母合成内切几丁质酶的培养基组分进行优化,并探讨重组内切几丁质酶降解几丁质的最佳反应条件。首先对培养基中显著影响内切几丁质酶活力的关键组分通过Plackett-Burman试验设计进行筛选;然后通过Box-Behnken试验设计和响应面法确定关键组分的最佳浓度。结果筛选出3个具有显著效应的关键组分为酵母膏、油酸和吐温-80,最佳浓度分别为：2.45%、0.17%和0.62%。优化后的最佳培养基组成为：2.45%酵母膏、2.00%蛋白胨、0.50%酵母氮碱（YNB）、0.50%甲醇、0.17%油酸、0.62%吐温-80和0.40% PTM₁。在该培养基中,重组巴斯德毕赤酵母在摇瓶水平（25mL/250mL）发酵生产内切几丁质酶的活力高达92.26U/mL。重组内切几丁质酶催化几丁质降解的最佳反应条件为：粉末几丁质浓度为4%,pH和温度分别为7.0和30℃,反应时间为10h。研究结果为后期在发酵罐中大规模生产内切几丁质酶和几丁寡糖提供了基础。     

重组巴斯德毕赤酵母发酵生产几丁质酶的条件优化研究

研究了利用重组巴斯德毕赤酵母诱导表达重组几丁质酶的条件。在摇瓶水平上研究了诱导时间、pH、甲醇流加量、油酸等因素对重组几丁质酶表达的影响。结果发现诱导108h蛋白表达量最高;偏酸性环境不利于蛋白表达,维持在pH5.5～6.0最佳;甲醇最佳诱导浓度为1%;添加0.05%的油酸有助于提高蛋白表达量。在此基础上通过正交试验设计优化了培养基配方,在优化条件下,蛋白表达量达171.99mg/L,酶活达49.58U/mL。     

重组人抗凝血酶发酵条件的优化

目的:通过实验室发酵条件优化工作,实现在巴斯德毕赤酵母中高效表达重组人抗凝血酶。方法:在摇瓶培养条件下,应用正交实验方法考查人抗凝血酶重组毕赤酵母菌pPIC9K-AT-02的培养温度、培养基pH值、接种比例、甲醇补加间隔时间及甲醇补加浓度等5种因素对重组人抗凝血酶活性的影响,确定最优发酵条件。结果与结论:筛选出的最终发酵条件为培养温度30℃、培养基pH6.0、接种比例20%、甲醇补加间隔时间24 h、甲醇补加浓度2%,重组人抗凝血酶在巴斯德毕赤酵母中表达活性为4098 U/L,比原始活性提高了150%。     

里氏木霉内切-β-葡聚糖酶Ⅱ基因在毕赤酵母中的表达及酶学性质研究

采用外显子拼接的方法,以里氏木霉Trichoderma reesei基因组 DNA 为模板,克隆出内切-1,4-β-D-葡聚糖酶II基因egl2的全编码序列,将其插入巴斯德毕赤酵母Pichia pastoris表达载体pPIC9K中,并位于α-因子信号肽序列的下游,获得重组质粒pQY2025。重组质粒线性化后用电穿孔法导入毕赤酵母Pichia pastoris菌株GS115中,经大量筛选,获得高效分泌表达内切葡聚糖酶II的毕赤酵母工程菌株Gp2025。用甲醇诱导培养基进行摇瓶发酵,培养基中内切葡聚糖酶II的活力可达1573.0U/mL,同时对重组内切葡聚糖酶II的性质进行了初步研究。     

毕赤酵母表达巴曲酶发酵条件的优化研究

目的对表达重组巴曲酶的巴斯德毕赤酵母的发酵条件进行优化,确定最佳的发酵控制条件以获得重组巴曲酶的最高表达量。方法通过多因素正交实验确定巴曲酶发酵培养的最适培养条件。结果表达温度在25℃,pH值为7．0,加入甲醇的量为10g／L时为最优发酵条件,诱导表达时间为84-96h。结论重组巴曲酶可以开发为止血药,代替临床应用的从蛇毒中提取的巴曲酶。     

毕赤酵母表达Q8008发酵条件的优化研究

目的对表达巴斯德毕赤酵母的发酵条件进行优化,确定最佳的发酵控制条件,以获得重组textilinin-1(Q8008)最高表达量。方法通过多因素正交实验确定Q8008发酵培养的最适发酵条件。结果表达温度在22℃,pH值为7.0,加入甲醇量为5g/L时为最优发酵条件,诱导表达时间为84~108h。结论优化巴斯德毕赤酵母的发酵条件,可提高Q8008表达量,为开发抗纤溶酶止血药应用于临床具有重要意义。     

里氏木霉Cel5A基因优化及其在毕赤酵母中的高效表达

内切纤维素酶Cel5A缺乏是限制纤维素酶制剂高效酶解天然纤维素的关键因素。本文尝试构建高效表达里氏木霉Cel5A的毕赤酵母重组菌株以弥补目前Cel5A的天然分泌不足,通过基因密码子偏好性优化里氏木霉Cel5A基因和构建表达载体p PIC9K-eg2,并将其电转入毕赤酵母GS115以构建重组子,利用浓度梯度平板和摇瓶发酵筛选获得一株高产毕赤酵母Pichia pastoris菌株GS115-EGⅡ。重组酶的酶学性质分析显示,该酶分子量50 k Da、最适p H(p H 4.5)略有降低及最适反应温度为60℃,专一性地作用于非结晶纤维素,与天然里氏木霉Cel5A并无明显区别。通过摇瓶发酵的初步优化,该菌摇瓶培养条件:培养温度28℃、起始p H 5.0、接种量2%、每24 h添加甲醇1.5%(V/V)、每24 h添加山梨醇4 g/L及吐温80添加4 g/L,发酵192 h重组酶酶活达到24.0 U/m L。进一步上罐(5 L)发酵180 h,该重组酶Cel5A酶活高达270.9 U/m L,蛋白含量达到4.16 g/L。重组毕赤酵母P.pastoris GS115-EGⅡ是一株适合于外源表达Cel5A的工程菌,该重组酶可替代天然分泌Cel5A适用于当前酶基生物炼制模式下木质纤维素基质高效水解中。     

宇佐美曲霉木聚糖酶基因xynⅡ在不同毕赤酵母中的分泌表达

将宇佐美曲霉E001的内切-1,4-木聚糖酶基因克隆到毕赤酵母表达载体pPIC9K中,得到重组质粒pPXY-NII,将其经SalⅠ线性化后分别转化2株毕赤酵母GS115和KM71,xynⅡ基因通过同源重组被整合到毕赤酵母染色体上,并处于酵母α因子的下游,经筛选获得阳性重组菌PXGL98(Mut+)和PXKL29(Muts)。该木聚糖酶基因在2株毕赤酵母中均实现了分泌表达。同时对工程菌的发酵条件进行了优化,在甲醇诱导下,PXGL98与PXKL29培养物上清液中的酶活力分别可达1156.92 U/mL和1646.03 U/mL。     

里氏木霉内切-β-甘露聚糖酶基因在毕赤酵母中的表达

采用PCR方法从里氏木霉(Trichoderma reesei)基因组中获得含有两个内含子的内切-β-甘露聚糖酶全长基因,末端重叠延伸PCR去除内含子后,将其插入到巴斯德毕赤酵母(Picher pastoris)表达载体pPIC9K中,位于α-因子信号肽序列的下游,并与之同框,获得重组质粒pM242。重组质粒线性化后用电击法转化毕赤酵母菌株GS115。经大量筛选,获得高效分泌表达内切甘露聚糖酶的毕赤酵母工程菌株Gpmf25。摇瓶发酵结果表明,培养基中甘露聚糖酶的活力可达12.5IU/mL。重组酶最适pH和最适反应温度分别为5.0和80℃,在pH5.0～6.0时酶活稳定,在pH5.4时70℃保温30min酶活维持50％以上。     

枯草芽孢杆菌β-甘露聚糖酶在毕赤酵母中的分泌表达

目的:研制高效分泌表达枯草芽孢杆菌β-甘露聚糖酶的毕赤酵母基因工程菌株。方法与结果:将优化设计的枯草芽孢杆菌MA139β-甘露聚糖酶基因用EcoRⅠ/XbaⅠ双酶切,克隆到诱导型表达载体pPICzαA中α因子信号肽编码序列的下游,转化大肠杆菌筛选重组质粒,转化毕赤酵母X-33感受态细胞,经Zeocin筛选,获得重组表达菌株X-33/mann。将重组菌株在10L全自动发酵罐中进行高密度发酵培养,甲醇诱导72h发酵活力达到2100U/mL。重组甘露聚糖酶的最适催化温度为40℃,最适催化pH值为6.0。结论:枯草芽孢杆菌β-甘露聚糖酶在毕赤酵母中获得了高效分泌表达,具有开发作为饲料添加剂的潜能。     

Effect of oleic acid and Tween-80 on lysine synthesis by the culture Corynebacterium glutamicum]

The effect of oxidized and unoxidized oleic acid and Tween-80 on the growth and lysine synthesis by the producers C. glutamicum strains 95, 8, 28 was investigated. Surface active substances like oxidized and unoxidized oleic acid and Tween-80 during cultivation of the lysine producers on the glucose medium (the synthetic medium) and the medium with molasses and corn extract either inhibited the culture growth, thus reducing lysine yield, or accelerated the culture growth, thus increasing lysine yield. Oxidized and unoxidized oleic acid produced the greatest effect when added to the nutrient medium on the 48th cultivation hour. The increment of synthesized lysine was 120-150% of the control. Tween-80 proved to be very effective when added at early stages of fermentation (20 hours).     

Optimization of medium for lipase production by Acinetobacter haemolyticus from healthy human skin

A lipase producing Acinetobacter haemolyticus TA106 was isolated from healthy human skin of tribal population. The maximum activity of 55 U/ml was observed after medium optimization using the "one variable at a time" and the statistical approaches. The optimal composition of the medium was determined as (% w/v or v/v): tryptone--1, yeast extract--0.5, sodium chloride-1, olive oil-1, Tween-80 1, manganese sulphate--5 mM, sucrose--1, pH-7. It was found that maximum production occurred in late log phase, i.e., after 72 h and at 200 rpm. From factorial design and statistical analysis, it was found that pH, temperature, salt, inoculum density and aeration significantly affected the lipase production. It was also noted that inoculum density of 3% (v/v), sucrose (1% w/v) and manganese sulphate (5 mM) displayed maximum lipase activity of 55 U/ml by conventional as well as statistical method. Optimization studies also indicated the increase in specific activity from 0.2 U/mg to 6.7 U/mg.     

Significant improvement of Geobacillus thermoleovorans CCR11 thermoalkalophilic lipase production using response surface methodology

The medium optimization for the production of the Geobacillus thermoleovorans CCR11 thermoalkalophilic lipase was carried out in shake flask cultures using safflower high oleic oil. In the first step of optimization, a two level fractional factorial design allowed the identification of the concentration of nutrient broth and temperature as the main variables significantly affecting lipase production (P<0.05). In a second step, a D-optimal design was applied to determine the variables optimal values, defined as those yielding maximal lipase production in shaken flasks, thus demonstrating that the optimal concentration of nutrient broth was 3.8 g/l and the optimal culture temperature was 39.5°C. The model was experimentally validated, yielding a lipase production of 2283.70 ± 118.36 U/mL which represents a 6.7-fold increase in comparison to the non-optimized medium.     

极地适冷菌Pseudoalteromonas sp. QI-1产适冷蛋白酶发酵条件的优化

对极地适冷菌Pseudoalteromonas sp. QI-1产适冷蛋白酶的发酵条件进行优化。结果表明:菌株QI-1的最适生长和产酶温度均为5℃;最佳接种量为1%;发酵培养基的最适初始pH和最佳装样量分别为5和10%;盐度为2%时对菌株的生长和产酶最为有利;麸皮和醋酸钠分别为最佳N源和C源;添加0.75%酪蛋白时菌株QI-1胞外蛋白酶的活性最高;10 mmol/L Mg2+和0.5%Tween-80有利于产酶。正交试验结果表明:菌株Pseudoalteromonassp. QI-1产蛋白酶较佳培养基配方(g/L)为麸皮5,酵母粉2.5,酪蛋白3,MgCl2.6H2O 3,KCl 1.5;发酵液比酶活为166.20 U/mL,较优化前提高了约56%。     

假单胞菌属No.2120生产D-甘露糖异构酶发酵培养基的优化

通过单因子实验、Plackett-Burman实验设计、响应面分析法对假单胞菌属No.2120产D-甘露糖异构酶的培养基进行优化,确定发酵优化条件:果糖15.26 g/L,牛肉膏20 g/L,酵母膏2 g/L,K2HPO42 g/L,MgSO4.7H2O0.5 g/L,NaCl 0.5 g/L,Tween-80 1.54 g/L。采用优化配方异构酶比酶活可以达到68.28 U/mL。     

Statistical Optimization of Medium and Fermentation Conditions of Recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis> for the Production of Xylanase

Recombinant xylanase (rPcXynC) from Pichia pastoris was produced on large-scale by optimizing production-medium composition using statistical experimental methods. Production medium was optimized through the use of statistical methods such as one factor at a time (OFAT), Plackett-Burman design, fractional factorial design (FFD), steepest ascent method (SAM), and response surface methodology (RSM). The optimum medium composition was established to be (g/L); wheat bran 11.62, yeast extract 30, Tween 60.5, DL-β-Phenylalanine 0.5, Thiamine 0.5, FeSO₄ 0.01, KH₂PO₄ 0.66, and KHSO₄ 0.09. The optimum medium composition yielded 3,051 mU/mL of xylanase activity which was three times higher than that obtained from the initial medium composition. Finally, fermentation conditions were examined using the optimized production medium in a laboratory bioreactor. The optimal fermentation conditions were found to be 25ºC, pH 6, 170 rpm and 1 vvm with intermittent feeding of methanol (67.5 mL) and the xylanase activity was 3,683 mU/mL. In repeated-batch fermentation using optimized production medium and fermentation condition, the xylanase activity was 3,680 mU/mL at the first cycle of 96 h harvesting time using 90% of the culture solution. The activity was similarly maintained until the last cycle of 264 h.     

洋葱伯克霍尔德菌（Burkholderia cepacia）CF-66发酵生产新型抗菌物质CF66I培养基的优化

利用基于统计学的实验设计RSM（Response surface methodology）优化了Burkholderia cepacia CF-66产新型抗菌活性物质CF66I的发酵培养基组成。首先,用部分重复因子实验对培养基组分NH4Cl,MgSO4·7H2O,柠檬酸钠及酵母粉浓度对菌株产CF66I的影响进行评价,找出主要影响因子为柠檬酸钠和酵母粉。两者均为正影响,其他组分对CF66I活性的影响不显著。其次用最陡爬坡路径逼近最大响应区域。最后用中心组合设计及响应面分析确定主要影响因子的最佳浓度。菌株在优化培养基中培养较初始培养基CF66I活性提高了约两倍。     

Optimization of medium composition for the production of exopolysaccharides from Phellinus baumii Pilát in submerged culture and the immuno-stimulating activity of exopolysaccharides

Optimization of medium composition for the production of exopolysaccharides (EPS) from Phellinus baumii Pilát in submerged culture and the immuno-stimulating activity of EPS were carried out. Firstly, the medium components having significant effect on EPS production were screened out to be glucose, yeast extract and diammonium oxalate monohydrate by using a 2⁽⁷⁻³⁾ fractional factorial design. Secondly, the concentrations of the three factors were optimized using central composite design in response surface methodology. As results, a quadratic model was found to fit for EPS production, and the optimal medium composition was determined as following (g/l): 34.12 glucose, 4 peptone, 5.01 yeast extract, 0.88 diammonium oxalate monohydrate, 0.75 MgSO₄ and 1 KH₂PO₄ and 0.0075 thiamine (VB₁). A yield of 2.363 ± 0.04 g/l for EPS was observed in verification experiment. Finally, EPS from P. baumii Pilát was found to have direct immuno-stimulating activity in vitro on splenocyte proliferative response and acid phosphatase activity in peritoneal macrophages in a dose-dependent manner.     

Production and characterization of a low‐molecular‐weight bacteriocin from Bacillus licheniformis MKU3

Aims: Enhancing production and characterization of a low‐molecular‐weight bacteriocin from Bacillus licheniformis MKU3. Methods and Results: The culture supernatant of B. licheniformis MKU3 exhibited bacteriocin‐like activity against Gram‐positive and ‐negative bacteria and different fungi and yeast. SDS–PAGE analysis of the extracellular proteins of B. licheniformis MKU3 revealed a bacteriocin‐like protein with a molecular mass of 1·5 kDa. This bacteriocin activity was found to be stable under a pH range of 3·0–10·0 and at temperatures up to 100°C for 60 min, but inactivated by proteinase K, trypsin or pronase E. An experimental fractional factorial design for optimization of production medium resulted in a maximum activity of bacteriocin (11 000 AU ml^?1) by B. licheniformis MKU3. Conclusions: A low‐molecular‐weight bacteriocin‐like protein from B. licheniformis MKU3 exhibited a wide spectrum of antimicrobial activity against several Gram‐positive bacteria, several fungi and yeast. A 3·6‐fold increase in the production of bacteriocin was achieved using the culture medium optimized through a fractional factorial design. Significance and Impact of the Study: A bacteriocin with wide spectrum of activity against Gram‐positive bacterial pathogens, filamentous fungi and yeast suggested its potential clinical use. Statistical method facilitated optimization of cultural medium for the improved production of bacteriocin.