利用恒溶氧．补料分批技术高密度培养大肠杆菌生产重组人骨形成蛋白-2A

对表达人骨形成蛋白－2A(BMP－2A)的重组大肠杆菌YK537／pDH－B2m在500ml摇瓶中进行了培养条件的摸索实验,继后用5L自控发酵罐进行分批培养和分批补料培养,以获取rhBMP－2A两种培养方式结果比较表明,在培养过程中保持30％～40％左右的溶解氧和限制性流加葡萄糖可以使BMP-2A的含量达到2．78g／L,最终菌体密度为OD₆₀₀53(相当于干菌21．2g／L),重组蛋白的表达量占菌体总蛋白的25％。该培养技术的关键是：(1)在培养过程中保持适当的溶解氧;(2)限制性流加葡萄糖;(3)42℃起始诱导的时间控制在对数生长中期,持续表达时间为4h;(4)细菌持续生长的比生长速率控制在0．3h -1左右。     

重组人酸性成纤维细胞生长因子改构体发酵的补料-分批策略研究

人酸性成纤维细胞生长因子(haFGF)是一类对来源于中胚层和神经外胚层的多种类型的细胞具有广泛生物学活性的细胞生长因子。研究了乙酸浓度对重组人酸性成纤维细胞生长因子(rhaFGF)改构体表达体系Escherichia coliBL21(DE3)/pET3C-haFGF的生长和表达的影响,探索了几种高密度培养重组大肠杆菌的流加分批发酵技术。通过比较几种不同的补料策略间歇流加、间歇-静态DO平衡、静态溶氧平衡-葡萄糖饥饿法、静态pH,有效的避免了发酵过程中,尤其是诱导表达阶段乙酸的积累。菌体密度OD600nm=22左右,可溶性重组人酸性成纤维细胞生长因子纯化后水平为450mg/L。     

利用指数流加补料高密度发酵产β-呋喃果糖苷酶重组大肠杆菌

【目的】对重组大肠杆菌BL21(DE3)/pET22b-β-ffase进行高密度发酵产β-呋喃果糖苷酶工艺研究。【方法】比较溶氧反馈补料和指数流加补料对重组菌发酵产酶的影响,对不同比生长速率和诱导时机进行优化。【结果】确定了双阶段指数流加过程中重组菌生长的比生长速率,分别控制诱导前期比生长速率为0.20 h~(-1),诱导后期比生长速率为0.13 h~(-1),诱导时机为指数中期。获得细胞干重约为51 g/L,最高酶活达到1.79×10~5 U/L,单位菌体产酶量为3 510 U/g,单位产酶速率达到3.58×10~4 U/(L·h),生物量、单位菌体产酶量和产酶速率分别是指数流加未优化前的1.8、1.7和3.0倍。【结论】双阶段指数流加补料工艺能有效提高β-呋喃果糖苷酶的产酶量,为β-呋喃果糖苷酶的进一步工业化奠定基础。     

表达重组葡激酶-水蛭素融合蛋白的大肠杆菌工程菌的高密度培养

采用溶氧反馈的分批培养流加补料的方法高密度培养重组大肠杆菌BL21(DE3)生产重组葡激酶-水蛭素融合蛋白。通过摇瓶培养对菌种和培养条件的初步筛选,采用溶氧反馈的流加补料策略,进行了5L发酵罐的合成培养基和复合培养基的发酵工艺的研究。通过对培养条件的不断优化,重组葡激酶-水蛭素融合蛋白在大肠杆菌BL21(DE3)里得到了高效表达,菌体密度最终达到115g/L(WCW)以上,可溶性重组融合蛋白占菌体总蛋白的30%以上,含量约为1.1~1.2g/L。5L发酵罐的发酵工艺参数在40L发酵罐中进行了放大培养,结果表明该工艺能有效的放大,可适用于工业生产。     

牡蛎金属硫蛋白重组毕赤酵母菌株高效诱导表达工艺研究

金属硫蛋白(MT)作为一类广泛存在并高度可诱导的多功能内源性蛋白,具有重金属解毒、抗辐射、清除自由基等功能,近年来已成为研究和开发的热点。为解决MT原料来源的瓶颈问题,本文以一株海洋源MT重组巴斯德毕赤酵母菌株(GS~(-1)15-MT)为研究对象,对其进行培育条件(温度、pH、溶氧、接种量)、诱导表达条件(溶氧、甲醇浓度、菌株生产状况、诱导时间、诱导金属及浓度)综合优化,以获得MT高效表达最佳工艺。结果显示,GS~(-1)15-MT的最佳生长条件为培育温度30℃,培养基初始pH为9,摇床转速240r·min~(-1),接种量4%;最佳MT诱导发酵条件为摇床转速为220r·min~(-1),甲醇浓度为5%,菌液OD_(600)为9.7,诱导24h,Cu~(2+)诱导浓度50μmol·L~(-1)。在以上优化条件下,MT表达量达0.328mg·mL~(-1)。本结果可为海洋源MT的规模化工业生产提供一定理论依据。     

大肠杆菌发酵生产重组人颗粒溶素

目的:在FUS-50L生物反应器中利用补料分批培养技术培养表达含重组质粒pET28a( )-GNLY的大肠杆菌BL21(DE3)pLysS株,生产重组颗粒溶素(GNLY)。方法:选取含pET28a( )-GNLY的BL21(DE3)pLysS菌株单个克隆分级培养,将制备的二级种子液接种于发酵罐中。在发酵过程中,控制溶氧为30%～50%,温度为37℃;在基础培养基内生长4h后,补加以甘油为碳源的补料,继续生长到11h;加入葡萄糖至终浓度为1%,30℃诱导表达6h;收集菌体,纯化制备目的蛋白。利用Western印迹检测重组蛋白的抗原性,用CFU方法检测其生物学活性。结果:发酵液中最终菌体密度达80g/L;纯化所得重组蛋白约占菌体总蛋白的5%,含量为60mg/L;经鉴定所获重组颗粒溶素有较好的免疫学活性和生物学活性。结论:用含重组质粒pET28a( )-GNLY的大肠杆菌BL21(DE3)pLysS表达系统,可得到具有生物活性的重组颗粒溶素,为大批量生产提供了条件。     

重组铜绿假单胞菌外毒素A工程菌的高密度发酵与表达

高密度培养表达大肠杆菌生产重组铜绿假单胞菌外毒素A(rEPA)。用上海高机公司的30L自控发酵罐,采用分批补料培养技术,维持葡萄糖的浓度始终处于较低的水平,并分批补加氮源,同时溶氧控制在30%～40%,pH自动调控至7.0,培养至对数中期进行诱导。重组菌最终发酵液光密度(A600)未诱导时达到44,诱导时达到36,在上清液中表达的rEPA蛋白的含量占总蛋白的28.1%,在菌体中表达蛋白含量占总蛋白的4.7%。本实验为rE-PA的大规模生产奠定了基础。     

表达血管生长抑制素的重组毕赤酵母在诱导阶段混合碳源的流加

为进行高密度发酵并实现外源基因的高表达,在表型为Mut^S的重组毕赤酵母（Pichia pastoris）表达人血管生长抑制素的诱导阶段,采用了甘油甲醇混合补料的培养方式。以溶氧水平作为甘油代谢指针来控制甘油限制性流加既可维持一定菌体生长,又不会发生发酵液中残余甘油及有害代谢产物（乙醇）阻遏蛋白表达。当表达阶段的菌体平均比生长速率控制于0.012h^-1,菌体浓度达150 g/L,血管生长抑制素浓度最高达到108 mg/L,血管生长抑制素的平均比生产速率为0.02 mg/(g·h),菌体关于甘油的表观得率为0.69 g/g,菌体关于甲醇的表观得率为0.93g/g,较没有采用甘油限制性流加时都有所提高。     

碳源对重组大肠杆菌发酵生产GLP-1融合蛋白的影响

以一株表达人胰高血糖素样肽-1融合蛋白的重组大肠杆菌为研究对象,首先通过摇瓶实验对碳源种类进行了初步选择,发现葡萄糖和甘油对菌体生长以及GLP-1融合蛋白表达较为适宜。进一步在5 L反应器上对初始葡萄糖及甘油浓度进行了考察,发现高浓度碳源有利于菌体生长却抑制GLP-1融合蛋白表达,但能提高GLP-1融合蛋白的体积得率。在0.25%初始葡萄糖或甘油存在的条件下,在培养过程中流加葡萄糖或甘油维持其在发酵液中的浓度,比较了两者对菌体生长以及GLP-1融合蛋白表达的影响,结果发现,以甘油为碳源时,菌体生长以及GLP-1融合蛋白的表达量均高于以葡萄糖为碳源的结果,最终发酵液的菌浓(OD_(600))可达到25.4,较葡萄糖为碳源时19.1提高了33.0%,GLP-1融合蛋白表达水平和体积得率分别可达到22.4%和1.051 g/L,较葡萄糖为碳源的15.8%和0.504 g/L分别提高41.8%和108.5%。该结果对GLP-1融合蛋白表达菌株发酵条件的进一步优化提供了依据。     

恒pH葡萄糖流加方法培养重组大肠杆菌生产人肿瘤坏死因子-α及其动力学研究

研究了一种新型的流加方法──恒pH流加葡萄糖法,用于培养重组大肠杆菌生产人肿瘤坏死因子-α。流加后培养液中菌体OD(600)达到9．0,是在LB培养基培养的15倍,而α-肿瘤坏死因子的比活保持（1．05±0．11）×105u/mg,并建立了菌体生长的动力学方程。     

pH及流加葡萄糖对酵母分批发酵生产谷胱甘肽的影响

在5 L的发酵罐中研究了pH及流加葡萄糖对酵母分批发酵生产谷胱甘肽(GSH)的影响。实验考察了不同浓度的流加葡萄糖和不同的恒pH值的分批发酵对于酵母生产GSH产量的变化。实验结果表明,当pH值控制为5.0,流加葡萄糖流速为5g.L-1.h-1,连续流加30 h,可使GSH产量最高,与之前未流加葡萄糖和控制pH相比,其产量提高了6倍。     

Kinetics of batch single cell protein production from rice polishings with Candida utilis in continuously aerated tank reactors

Single cell protein was produced from the defatted rice polishings by fermentation with Candida utilis in an aerated 14-L fermentor to optimize bioprocess variables. Maximum values of specific growth rate coefficient (mu, h(-1)), cell mass yield (Y(X/S), g/g) and cell mass productivity (g/Lh) were 0.31, 0.65, and 1.24, respectively under optimized conditions of aeration rate (1 v.v(-1) m(-1)), dissolved oxygen (50%), corn steep liquor (5%), temperature (35 degrees C), and substrate concentration (90 g rice polishings/L) in yeast salt medium (pH 6.0). The kinetic parameters for 50-L fermentor under same conditions were 0.33 h(-1), 0.66 g/g, 1.33 g/Lh, 2.25 g/Lh, 1.23 g/Lh, 0.45 g/g substrate and 0.20 g/g cell h for mu, Y(X/S), Q(X), Q(S), Q(CP), Y(TP/S), and q(CP), respectively and were significantly higher than their respective values reported on C. utilis in batch culture studies. This biomass protein contained 23.6%, 32.75%, 11.50%, 12.95%, 10.5%, and 0.275% true protein, crude protein, crude fiber, ash, cellulose and RNA content respectively. This implied that the fermentation process could be up scaled to manufacture animal feed. Gross metabolizable energy content of dried SCP was 29,711 kcal/kg and indicated that the SCP could serve both as energy as well as a protein source. Yeast can replace expensive feed ingredients currently being incorporated in poultry feed and can reduce cost of poultry ration by 0.33 US dollars-0.51 US dollars/100 kg bag and improve the economics of feed production in our country.     

高密度培养重组大肠杆菌发酵生产胆固醇氧化酶的策略

以重组大肠杆菌发酵生产胆固醇氧化酶,依据溶解氧的变化控制底物的流加速率,实现了重组大肠杆菌的高密度培养,最高密度达85（OD600）。在此基础上确定了最佳的诱导时机为发酵中期,菌体产酶水平达5830．6tJ／L,产酶速率为971．77U·L^-1·h^-1,生产强度为388．71U·L^-1·h^-1,实现了胆固醇氧化酶的高效生产。     

以葡萄糖为生长相基质的重组毕赤酵母表达植酸酶发酵

甲醇营养型毕赤酵母表达外源蛋白是在醇氧化酶（alcohol oxidase,AOX）启动子（PAOXI）严格调控下进行的,然而这种启动子在转录水平受到葡萄糖的阻遏。本文研究了毕赤酵母在葡萄糖替代甘油为生长相碳源时表达重组植酸酶蛋白的发酵特征。结果表明：初始葡萄糖浓度为20dL的细胞得率高,为0．39g[DCW]／g。通过基于实时参数（溶氧和呼吸商）调控的葡萄糖补料策略,生长相40h后细胞密度达到100g[DCW]／L,甲醇诱导100h后植酸酶产量达到2200FTUphytase／mL,甲醇得率系数为0．25FTU phytase／gmethnol。因此,在毕赤酵母高表达重组蛋白培养中葡萄糖能够用作生长相基质,并能实现重组蛋白的高效表达。     

Twenty-four-well plate miniature bioreactor high-throughput system: assessment for microbial cultivations

High-throughput (HT) miniature bioreactor (MBR) systems are becoming increasingly important to rapidly perform clonal selection, strain improvement screening, and culture media and process optimization. This study documents the initial assessment of a 24-well plate MBR system, Micro (micro)-24, for Saccharomyces cerevisiae, Escherichia coli, and Pichia pastoris cultivations. MBR batch cultivations for S. cerevisiae demonstrated comparable growth to a 20-L stirred tank bioreactor fermentation by off-line metabolite and biomass analyses. High inter-well reproducibility was observed for process parameters such as on-line temperature, pH and dissolved oxygen. E. coli and P. pastoris strains were also tested in this MBR system under conditions of rapidly increasing oxygen uptake rates (OUR) and at high cell densities, thus requiring the utilization of gas blending for dissolved oxygen and pH control. The E. coli batch fermentations challenged the dissolved oxygen and pH control loop as demonstrated by process excursions below the control set-point during the exponential growth phase on dextrose. For P. pastoris fermentations, the micro-24 was capable of controlling dissolved oxygen, pH, and temperature under batch and fed-batch conditions with subsequent substrate shot feeds and supported biomass levels of 278 g/L wet cell weight (wcw). The average oxygen mass transfer coefficient per non-sparged well were measured at 32.6 +/- 2.4, 46.5 +/- 4.6, 51.6 +/- 3.7, and 56.1 +/- 1.6 h(-1) at the operating conditions of 500, 600, 700, and 800 rpm shaking speed, respectively. The mixing times measured for the agitation settings 500 and 800 rpm were below 5 and 1 s, respectively.     

四倍体刺槐无性系K2和K3的组织培养

1997年,北京林业大学从韩国引进了具有速生和饲料用途的刺槐(Robinia pseudoacacia L.)四倍体优良无性系,目前已在全国各省区试推广.与普通刺槐相比,四倍体刺槐具有叶大、速生等优点,且较普通刺槐有更强的适应性,耐干旱、贫瘠、烟尘及盐碱能力强,成林快,是水土保持、防风固沙及退耕还林的良好树种,可作为西北地区造林的先锋树种.     

Kinetics and modelling of kojic acid production by Aspergillus flavus Link in batch fermentation and resuspended mycelial system

An unstructured model based on logistic and Luedeking-Piret equations was proposed to describe growth, substrate consumption and kojic acid production by Aspergillus flavus Link strain 44-1 in batch fermentation and also in a resuspended cell system. The model showed that kojic acid production was non-growth associated. The maximum kojic acid and cell concentrations obtained in batch fermentations using the fermenter with optimized dissolved oxygen control (32.5 g/l and 11.8 g/l, respectively) and using a shake-flask (36.5 and 12.3 g/l, respectively) were not significantly different. However, the maximum specific growth rate and a non-growth-associated rate constant for kojic acid formation (n) for batch fermentation using the fermenter (0.085/h and 0.0125 g kojic acid/g cell.h, respectively) were approximately three and two times higher than the values obtained for fermentation using a shake-flask, respectively. Efficient conversion of glucose to kojic acid was achieved in a resuspended pellet or mycelial system, in a solution containing only glucose with citrate buffer at pH 3.5 and at a temperature of 30 °C. The resuspended cell material in the glucose solution was still active in synthesizing kojic acid after prolonged incubation (up to about 600 h). The rate constant of kojic acid production (n) in a resuspended cell system using 100 g glucose/l was almost constant at an average value of 0.011 g kojic acid/g cell.h up to a cell concentration of 19.2 g/l, above which it decreased. A drastic reduction of n was observed at a cell concentration of 26.1 g/l. However, the yield based on glucose consumed (0.45 g/g) was similar for all cell concentrations investigated.     

Fed-batch fermentation studies with Bacillus thuringiensis H-14 synthesising endotoxin.

Cell yield and toxicity of B. thuringiensis H-14 was improved markedly by adopting a simple fed-batch fermentation technique based on controlling glucose concentration. Maintenance of steady glucose concentration (0.3-0.5%) in the culture medium was achieved by the continuous addition of concentrated glucose solution. Addition of glucose at 3 g/hr/l of culture starting from 3rd hr till 16th hr of fermentation was found to yield cell densities of 80 g/l (wet weight) which represented a nearly 3-fold increase over the batch mode. A fivefold increase in toxicity was obtained by fed-batch fermentation. Cultivation of B. thuringiensis H-14 to high cell densities had no negative effect on sporulation and toxin synthesis. The rate of pH drop and dissolved oxygen level were within manageable limits.     

温度对发酵生产透明质酸的影响

为进一步提高兽疫链球菌透明质酸产量,本实验采用分阶段控制温度工艺,当温度由36℃培养到28 h转入38℃培养至发酵结束,其透明质酸产量有很大提高。经发酵罐验证温度优化结果,其粗品产量由684 g提高到735 g。