响应面法优化以重组大肠杆菌发酵生产类人胶原蛋白过程

In order to improve the production of human-like collagen Ⅲ (HLC Ⅲ) by fed-batch culture of recom-binant Escherichia coli BL21, the Plackett-Burman and Box-Behnken design were applied to optimize the fermen-tation process parameters.Three variables (induction time, inoculum age and pH), which have significant effects on HLC Ⅲ production, were selected from eight variables by Plackett-Burman design.With the regression coefficient analysis in the Box-Behnken design, a relationship between HLC Ⅲ production and three significant factors was obtained, and the optimum levels of the three variables were as follows: induction time 3.2h, inoculum age 12.6 h and pH 6.7.The 3D response surface plots and 2D contour plots created by the Box-Behnken design showed that the interaction between induction time and pH and that between innoculum age and pH were significant.An aver-age 9.68 g·L~(-1)HLC Ⅲ production was attained in the validation experiment under optimized condition, which was 80% higher than the yield of 5.36 g·L~(-1) before optimization.     

响应面方法分析代谢产物对类人胶原蛋白表达的影响

Recombinant Escherichia coli BL21 is used to produce human-like collagen. The key constituents of media are optimized using response surface methodology (RSM). Before thermal induction, the highest biomass production and the lowest production of some hazardous by-products, especially acetic acid, were obtained in the media containing 0.085 mol∙L1 glucose and 0.019 mol∙L1 nitrogen (carbon-nitrogen ratio, 4.47︰1). After thermal induction, when the concentrations of glucose and nitrogen in the media were 0.065 mol∙L1 and 0.017 mol∙L1, respectively (carbon-nitrogen ratio, 3.82︰1), the productivity of human-like collagen per cell was the highest while that of acetic acid was the lowest. The extended analysis showed that the production of lactic acid and propionic acid increased while that of some intermediate acids of the tricarboxylic acid cycle decreased if the dose of glucose in-creased.     

丙酸盐影响利迪链霉菌AS 4.2501生产利迪链菌素及其碳流分布

To achieve higher antibiotic streptolydigin productivity through metabolic regulation, propionate was fed during the fermentation of Streptomyces lydicus AS 4.2501. The effects of propionate feeding on streptolydigin production and intracellular fluxes were investigated. The highest streptolydigin production yield of 95.10mg·L^-1 was obtained when 2mmol·L^-1 of sodium propionate was added at 60h of cultivation into shake-flask culture. This yield is 23.06% higher when compared to that of a batch culture without propionate supplementation. It was also found that when propionate was added, much more organic acids were excreted. Metabolic flux analysis was performed and it demonstrated that the carbon fluxes of the pentose phosphate pathway and the anaplerotic reaction were significantly increased after propionate feeding. The carbon flux from pyruvate to acetyl-CoA was determined to be 24.7, which was 12.27% higher than that in the batch culture. This study indicated that the glucose-6-phosphate and pyruvate nodes were potential bottlenecks for increasing streptolydigin productivity. Potential targets and strategies that could be manipulated through genetic and process engineering to increase the production of streptolydigin were also suggested.     

Optimization of Propionic Acid Feeding for Production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Fed-Batch Culture of Ralstonia eutropha

The feeding method of propionic acid for production of poly(3-hydroxybutyrate-co-3-hydro xyvalerate) [P(3HB-co-3HV)] by fed-batch culture of Ralstonia eutropha was optimized to achieve high cell density and high 3HV yield. Effects of different feeding strategies of propionic acid on the production of P(3HB-co-3HV) were investigated. A decline of specific synthesis rate of copolymer and the yield of 3HV unit from propionic acid were observed due to the propionic acid accumulation in culture broth when the feeding solution with high P/G(propionic acid to glucose) ratio was employed. It was further confirmed by controlling propionic acid concentration at a low level in the separate feeding of propionic acid. An optimal feeding strategy was demonstrated to reduce the propionic acid accumulation. The cell concentration, P(3HB-co-3HV) productivity and 3HV unit fraction reached to 163.9kg.m-3, 1.8kg.m-3.h-1, and 10.6%(by mass), respectively, resulting in a yield of 0.33g HV per g propionic acid.     

Ralstonia eutropha批式流加培养生产羟基丁酸-丙酸混合聚酯中丙酸进料优化研究

The feeding method of propionic acid for production of poly (3-hydroxybutyrate-co-3-hydro xyvalerate)[P(3HB-co-3HV)]by fed-batch culture of Ralstonia eutropha was optimized to achieve high cell density and high 3HV yield,Effects of different feeding strategies of propionic acid on the production of P(3HB-co-3HV) were investigated,A decline of specific synthesis rate of copolymer and the yield of 3HV unit from propionic acid were observed due to the propionic acid accumulation in culture broth when the feeding solution with high P/G(ropionic acid to glucose) ration was employed,It was further confirmed by controlling propionic acid concentration at a low leve in the separate feeding of propionic acid.An optimed feeding strategy was demonstrated to reduce the propionic acid accumulation ,The cell concentration,P(3HB-co-3HV) productivity and 3HV unit fraction reached to 163.9 kg.m^-3,1.8kg.m^-3.h^-1,and 10.6%(by mass),respectively,resulting in a yield of 0.33g HV per g propionic acid.     

氮源对利迪链菌素生产及相关次级代谢物分布的影响

The effects of nitrogen sources on streptolydigin production and distribution of secondary metabolites were investigated for flask cultured S.lydicus AS 4.2501.When peptone,asparamide,and glutamic acid were ex- amined as the nitrogen source,respectively,liquid chromatography-mass spectrometry（LC-MS）and photodiode array（PDA）analyses revealed the formation of two analogues of streptolydigin in the fermentation broth.When soybean meal was used as the source of nitrogen,three analogues of streptolydigin were detected.The use of am- monium sulfate as a source of nitrogen resulted in a lower pH value of the fermentation system,thus inhibiting streptolydigin biosynthesis and changing the metabolic profiling.Among the nitrogen sources that were made use of,glutamic acid was most favorable to the formation of streptolydigin.Simultaneously,this study also showed that the changing nitrogen sources resulted in altering the production and relative ratios of streptolydigin and its analogues.     

多策略代谢工程大肠杆菌高效生产辅酶Q10

Escherichia coli BW25113 was metabolically engineered for CoQ10 production by replacing ispB with ddsA from Gluconobacter suboxydans.Effects of precursor balance and reduced nicotinamide-adenine dinucleotide phosphate (NADPH) availability on CoQ10 production in E.coli were investigated.The knockout of pykFA along with pck overexpression could maintain a balance between glyceraldehyde 3-phosphate and pyruvate,increasing CoQ10 production.Replacement of native NAD-dependent gapA with NADP-dependent gapC from Clostridium acetobutylicum,together with the overexpression of gapC,could increase NADPH availability and then enhanced CoQ10 production.Three effects,overexpressions of various genes in CoQ biosynthesis and central metabolism,different vectors and culture conditions on CoQ10 production in E.coli,were all investigated.The investigation of different vectors indicated that low copy number vector may be more beneficial for CoQ10 production in E.coli.The recombinant E.coli (△ispB::ddsA,△pykFA and △gapA::gapC),harboring the two plasmids encoding pck,dxs,idi and ubiCA genes under the control of PT5 on pQE30,ispA,ddsA from Gluconobacter suboxydans and gapC from Clostridium acetobutylicum under the control of PBAD on pBAD33,could produce CoQ10 up to 3.24 mg·g-1 dry cell mass simply by changing medium from M9YG to SOB with phosphate salt and initial culture pH from 7.0 to 5.5.The yield is unprecedented and 1.33 times of the highest production so far in E.coli.     

黑曲霉Aspergillus niger P-6021浆态发酵常山胡柚皮渣产果胶霉

The peel of Citrus changshan-huyou, coupled with wheat bran, could be utilized by Aspergillus niger P-6021 in slurry-state fermentation to produce pectinase with suitable enzyme composition for application in apple juice processing. The production of pectinase is improved by additional nitrogen source substances and mineral supplements. The ratio of carbon source substances to nitrogen source substances in the medium also has significant effect on the pectinase production by A. niger P-6021 in slurry-state fermentation. In the optimized medium composition, the maximal enzyme activity could reach 42 U.L^- 1 （polymethylgalacturonase）, 6.7 U.L^- 1 （polymethygalacturatesterase）, and 4.3 U.L^-1 （polymethylgalacturonate lyase）, respectively, after 3 days at 180 r.min^- 1 and 30℃. The crude pectinase shows significant effect to improve the yield and clarification of apple juice. Keywords Aspergillus niger, slurry-state fermentation, pectinase, Citrus changshan-huyou, apple juice     

甲醇营养型重组毕赤酵母高效表达外源蛋白过程的控制与优化(英文)

The methylotrophic yeast Pichia pastoris is a highly successful system for production of a variety of heterologous proteins due to its unique features/abilities for effective protein expression, and tremendous efforts have been made to increase heterologous protein productivity by P. pastoris in recent years. When new engineered yeast strains are constructed and are ready to use for industrial protein production, process control and optimization techniques should be applied to improve the fermentation performance in the following aspects: (1) increase recombinant cell concentrations in fermentor to high density during growth phase; (2) effectively induce heterologous proteins by enhancing/stabilizing titers or concentrations of the proteins during induction phase; (3) decrease operation costs by relieving the working loads of heat-exchange and oxygen supply. This article reviews and discusses the key and commonly used techniques in heterologous protein production by P. pastoris, with the focus on optimizations of fermentation media and basic operation conditions, development of optimal glycerol feeding strategies for achieving high density cultivation of P. pastoris and effective heterologous protein induction methods by regulating specific growth rate, methanol concentration, temperatures, mixture ratio of multi-carbon substrates, etc. Metabolic analysis for recombinant protein production by P. pastoris is also introduced to interpret the mechanism of sub-optimal heterologous protein production and to explore further optimal expression methods.     

An Experimental Investigation of Hydrogen Production from Biomass

In gaseous products of biomass steam gasification, there exist a lot of CO, CH4 and other hydrocarbons that can be converted to hydrogen through steam reforming reactions. There exists potential hydrogen production from the raw gas of biomass steam gasification. In the present work, the characteristics of hydrogen production from biomass steam gasification were investigated in a small-scale fluidized bed. In these experiments, the gasifying agent (air) was supplied into the reactor from the bottom of the reactor and the steam was added into the reactor above biomass feeding location. The effects of reaction temperature, steam to biomass ratio, equivalence ratio (ER) and biomass particle size on hydrogen yield and hydrogen yield potential were investigated. The experimental results showed that higher reactor temperature, proper ER, proper steam to biomass ratio and smaller biomass particle size will contribute to more hydrogen and potential hydrogen yield.     

溶氧探测补料技术发酵生产类人胶原蛋白的研究

在重组大肠杆菌发酵生产类人胶原蛋白(HLC)过程中,快速确定不同反应器中的补料速率,抑制乙酸产生,高水平表达HLC.采用溶氧探测补料技术,在不同规模反应器中,分批-补料培养重组大肠杆菌生产HLC,根据脉冲补料方式时溶氧信号的响应特征来辩识是否产生乙酸并确定补料速率.在实验室规模获得的最终细胞密度和HLC质量浓度分别为6...     

营养物及补料方式对重组酵母产α-淀粉酶的影响

研究了两段培养中基质浓度、配比及天然氮源添加方式对重组酵母发酵生产(-淀粉酶的影响。结果表明,重组酵母发酵适宜的初糖浓度为7g·L-1,葡萄糖和基础氮配比为7:4,生长期补料碳氮比为5:1,且产酶期供给天然氮源以少量多次脉冲补加酵母膏为宜。于2L发酵罐中培养,证明脉冲补加酵母膏、流动添加基本营养物的补料分批培养方式是有效的,指数流加基本营养物较之恒流量流加更能提高菌体浓度和(-淀粉酶表达水平。培养60小时细胞量达24.5g·L-1,酶活力达97.3U·mL-1。     

重组大肠杆菌VG1(pTU14)产PHB的补料分批培养

利用已构建的同时含有透明颤菌血红蛋白基因 (vgb)、λ噬菌体裂解基因 (S-RRz)和PHB合成基因(phbCAB)的产PHB基因工程大肠杆菌VG1(pTU14 ) ,在 5L发酵罐里进行补料分批培养 .研究发现 :碳氮比(C/N) ,DO(溶氧 )及碳源和氮源的浓度是影响菌体生长和PHB积累的重要因素 ;补料时间可以通过DO和 pH值两个参数的变化在线综合识别 ;流加策略应使发酵前期菌体大量生长 ,发酵后期PHB大量积累 .在优化条件下 ,对VG1(pTU14 )进行 6 0h的补料分批培养 ,菌体细胞浓度、PHB浓度和PHB占细胞干质的分数分别高达2 15 .9g·L^-1、 193.7g·L^-1和 89.7% .     

产朊假丝酵母流加发酵法生产谷胱甘肽

研究了产朊假丝酵母在不同葡萄糖浓度下的谷胱甘肽(GSH)分批发酵过程,在此基础上进一步考察了重复补料、恒速流加和指数流加等不同培养方式对GSH发酵生产的影响. 结果发现,这几种培养方式都可以实现酵母细胞和GSH的高产. 综合比较,无论是从细胞还是从GSH的产量、得率和生产强度的角度来看,指数流加都是较为理想的选择. 经过48 h的指数流加培养,细胞干重达到40.9 g/L, GSH产量和胞内GSH含量分别达到857.2 mg/L和2.25%.     

在内置式植物纤维床反应器中用糖蜜固定化发酵制丙酸

开发了一种综合利用制糖工业副产物生产丙酸的新方法,将制糖工业副产物甘蔗渣和糖蜜分别作为固定化载体和碳源,应用于丙酸生产.以甘蔗渣为固定化载体构建了一种内置式植物纤维床反应器(Inner plant fibrous-bed bioreactor,IPFB).以Propionibacterium freudenreichii CCTCC M207015为发酵菌株,对在IPFB中分别用未处理糖蜜及糖蜜水解液为碳原发酵制丙酸进行考察.以未处理糖蜜为碳源发酵,254 h丙酸浓度为52.39 g·L~(-1),丙酸生产速率为0.21 g·(L·h)~(-1);糖蜜水解液为碳源发酵,254 h后丙酸浓度达75.18 g·L~(-1),丙酸生产速率达0.30 g·(L·h)~(-1),丙酸占总有机酸比例达83.10%(w/w).稳定性实验表明以糖蜜水解液为碳源,利用IPFB发酵生产丙酸10批仍然保持较高的丙酸生产效率.扫描电镜显示大量丙酸杆菌的细胞吸附于甘蔗渣表面与间隙中,有效实现了高密度发酵.     

Production of polyhydroxyalkanoates by batch and fed-batch cultivations of <Emphasis Type="Italic">Bacillus megaterium</Emphasis> from acid-treated red algae

Polyhydroxyalkanoates (PHAs) are linear polyesters synthesized by microbial fermentation of various substrates. PHAs are accumulated in microbial cells in order to store carbon and energy for future use. We used acid-pre-treated red alga (Gelidium amansii) as a cheap, abundant carbon source to produce PHA via batch and fed-batch cultivation of Bacillus megaterium KCTC 2194. After acid treatment of 10% (w/v) G. amansii, 25.5 g/L galactose, 3.6 g/L glucose, 6 g/L 5-HMF, and 1.05 g/L levulinic acid were formed. In batch culture at pH 7, the dry cell weight (DCW) and PHA content increased to 5.5 g/L and 51.4%, respectively. The cell concentration was enhanced by fed-batch cultivation using two feeding strategies: pH-stat and intermittent feeding. When the pH-stat feeding strategy was employed to add concentrated hydrolysate to the fermentor, DCW increased to 8.2 g/L, with 53.2% PHA content. When concentrated hydrolysate was fed using the intermittent feeding strategy, higher DCW (10.1 g/L) was obtained, along with a slight increase of PHA content to 54.5%. This study demonstrates that red algae could be used after simple acid treatment, to produce PHA without steps for enzymatic hydrolysis and inhibitor removal.     

基于高密度培养的反复分批发酵法生产丁二酸

引言丁二酸俗称琥珀酸,是三羧酸循环的中间代谢产物和厌氧代谢的终端还原产物,广泛存在于动物、植物及微生物体内。作为重要的C4平台化合物,丁二酸可用于多种大宗化学品以及生物可降解材料的制备。利用微生物发酵生产丁二酸,由于     

珊瑚状氮掺杂多孔碳的制备及其超电容性能

在三聚氰胺为氮源、碳酸钾为活化剂的条件下,由菜籽饼制得了珊瑚状氮掺杂分级多孔碳(CNPCs)。采用场发射扫描电子显微镜、透射电子显微镜、X射线光电子能谱、氮吸脱附等表征手段,研究了三聚氰胺的用量对CNPCs微观形貌、组成及孔隙结构的影响。结果表明,当三聚氰胺的用量为2 g时,所得CNPC₂的比表面积达2050 m²·g^-1。以6 mol·L^-1 KOH为电解液,在0.05 A·g^-1的电流密度下,CNPCs的比容可达274 F·g^-1;当电流密度为50 A·g^-1时,CNPCs的比容为169 F·g^-1,显示了优异的倍率性能。经过10000次充放电测试后,比容保持率达96%,展现了良好的循环稳定性。此工作为从生物质大规模生产高性能储能用多孔碳材料提供了一种简单、绿色的方法。     

培养条件及培养基组分对粘质沙雷氏菌生长及产D-乳酸的影响

通过摇瓶实验,确定粘质沙雷氏菌发酵生产I）-乳酸的培养条件为：温度34℃,pH值6．5,种子液接种量5％,载液量为150mL／500mL,采用前期通气有氧培养至菌体对数生长后期、而后厌氧发酵产酸的两阶段培养工艺。考察培养基各组分对菌体生长及乳酸产量的影响,确定葡萄糖及酵母粉作为碳、氮源,并补充添加适量的无机氮。培养基各组分如下（g·L^-1）：葡萄糖15,酵母粉15,KH2P041．5,K2HP04·3H2O2．0,MgSO4·7H2O1．0,FeSO4·7H2O0．03,MnSO4·H2O0．005,以此培养条件及培养基配方进行摇瓶发酵,D-乳酸产量由（13．5±1．29）g·L^-1提高至（29．0±1．42）g·L^-1,提高一倍以上,光学纯度大于97％。