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目的:在脱氮副球菌PD1222中表达山梨糖脱氢酶(SDH)。方法:从质粒pMD-18T上复制氨苄西林抗性基因Ampr,从酮古龙酸菌中复制SDH基因sdh,先后酶切连接到pIND4质粒上,构建pIND4-Ampr-sdh穿梭质粒;再把pIND4-Ampr-sdh电转入大肠杆菌S17-1λpir作为供体菌,脱氮副球菌PD1222为受体菌进行双亲本接合转移;挑取壮观霉素和氨苄西林双抗平板上的接合子进行培养,菌液PCR复筛接合子,测序鉴定,通过DCIP法和非变性聚丙烯酰胺凝胶电泳法检测阳性克隆的SDH活性。结果:构建的质粒pIND4-Ampr-sdh成功转入脱氮副球菌PD1222中,SDH获得表达并检测到其蛋白活性。结论:实现了SDH在脱氮副球菌中的表达,为在脱氮副球菌中研究SDH的下游电子传递链奠定了基础。 相似文献
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2—酮—L—古龙酸还原酶分离纯化及其理化,酶学性质的研究 总被引:5,自引:1,他引:5
从发酵L-山梨糖的Gluconobacter oxydans和Bacillus megaterium2980混和菌株的无细胞抽提液中分离到了2-酮-L-古龙酸还原酶(KGR),测得其分子量为90kDa。动力学性质研究表明它为一个典型的Michaelis-Menten氏酶,对2-酮-L-古龙酸作用的Km值为3.42×10^-3mol,最适作用pH为6.5,最适作用温度为30℃。2-酮-L-古龙酸还原 相似文献
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利用大肠杆菌-生黑醋杆菌穿梭载体pUG18构建了山梨糖脱氢酶基因sdh和氨甲酰化酶基因hyuC的融合表达质粒pUG18-SDH-HyuC.在JM109/pUG18-SDH-HyuC中检测到山梨糖脱氢酶(SDH)和氨甲酰化酶(HyuC)的酶活性,大量的SDH和HyuC不以融合蛋白形式存在.将质粒pUG18-SDH-HyuC电转化至生黑醋杆菌H24,检测到HyuC活性,但尚未检测到SDH酶活性.从生黑醋杆菌转化子中提取质粒,重新转化至JM109,又可同时检测到SDH和HyuC活性,传代实验和对质粒的鉴定结果也说明该质粒在H24中稳定存在. 相似文献
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Fidaleo M Charaniya S Solheid C Diel U Laudon M Ge H Scriven LE Flickinger MC 《Biotechnology and bioengineering》2006,95(3):446-458
We developed a novel <50-microm thick nano-porous bi-layer latex coating for preserving Gluconobacter oxydans, a strict aerobe, as a whole cell biocatalyst. G. oxydans was entrapped in an acrylate/vinyl acetate co-polymer matrix (T (g) approximately 10 degrees C) and cast into 12.7-mm diameter patch coatings (cellcoat) containing approximately 10(9) CFU covered by a nano-porous topcoat. The oxidation of D-sorbitol to L-sorbose was used to investigate the coating catalytic properties. Intrinsic kinetics was studied in microbioreactors using a pH 6.0 D-sorbitol, phosphate, pyruvate (SPP) non-growth medium at 30 degrees C, and the Michaelis-Menten constants determined. By using a diffusion cell, cellcoat and topcoat diffusivities, optimized by arresting polymer particle coalescence by glycerol and/or sucrose addition, were determined. Cryo-FESEM images revealed a two-layer structure with G. oxydans surrounded by <40-nm pores. Viable cell density, cell leakage, and oxidation kinetics in SPP medium for >150 h were investigated. Even though the coatings were optimized for permeability, approximately 50% of G. oxydans viability was lost during cellcoat drying and further reduction was observed as the topcoat was added. High reaction rates per unit volume of coating (80-100 g/L x h) were observed which agreed with predictions of a diffusion-reaction model using parameters estimated by independent experiments. Cellcoat effectiveness factors of 0.22-0.49 were observed which are 20-fold greater than any previously reported for this G. oxydans oxidation. These nano-structured coatings and the possibility of improving their ability to preserve G. oxydans viability may be useful for engineering highly reactive adhesive coatings for multi-phase micro-channel and membrane bioreactors to dramatically increase the intensity of whole-cell oxidations. 相似文献
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目的:从氧化葡糖杆菌H763中克隆sndh-sdh基因簇,在大肠杆菌和氧化葡糖杆菌621H中分别表达山梨酮脱氢酶-山梨糖脱氢酶(SNDH-SDH),并检测其活性。方法与结果:以氧化葡糖杆菌H763基因组DNA为模板,PCR扩增包括启动子、结构基因及终止序列在内的sndh-sdh基因簇,回收3533 bp的扩增产物,连入pMD18T载体,转化至大肠杆菌DH5α中表达;以山梨糖或木糖为底物,DCIP法检测菌体裂解液,DCIP检测液颜色由蓝绿色变为黄色,表明大肠杆菌表达产物具有脱氢酶活性。构建pBBR1MCS2-sndh-sdh载体,通过接合转移导入氧化葡糖杆菌621H,重组葡糖杆菌在以山梨醇或山梨糖为底物的培养基中培养,采用薄层层析检测法检测其培养上清中的代谢产物,层析板上显示了2-酮基-L-古龙酸斑点。结论:重组大肠杆菌DH5α和氧化葡糖杆菌621H中均表达了有脱氢酶活性的SNDH-SDH。 相似文献
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目前,国内维生素C主要采用二步发酵法生产,其中第一步为生黑葡萄酸杆菌(Gluconobacter melano-genus)将D-山梨醇转化为L-山梨糖。考察了该菌株在提高培养基中山梨醇浓度时的发酵特性和发酵条件。实验室摇瓶实验结果显示,通风量、发酵前期及后期pH值控制、接种种液类型都影响高浓度山梨醇摇瓶发酵的转化率。以35%山梨醇浓度发酵液做种子液明显优于生产上采用的三级种子液(12%~17%山梨醇浓度),培养基前期pH值5.0~6.0,后期pH值4.2~3.9,装液量180 mL,发酵周期在30 h之内,山梨醇转化率在98%以上。培养基山梨醇浓度由23%提高到35%,发酵周期延长8 h。上述实验结果对指导生产工艺优化具有重要意义。 相似文献
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Microbial oxidation of D-sorbitol tol-sorbose byAcetobacter suboxydans is of commercial importance since it is the only biochemical process in vitamin C synthesis. The main bottleneck in the batch
oxidation of sorbitol to sorbose is that the process is severely inhibited by sorbitol. Suitable fed-batch fermentation designs
can eliminate the inherent substrate inhibition and improve sorbose productivity. Fed-batch sorbose fermentations were conducted
by using two nutrient feeding strategies. For fed-batch fermentation with pulse feeding highly concentrated sorbitol (600
g/L) along with other nutrients were fed intermittently in four pulses of 0.5 liter in response to the increased DO signal.
The fed-batch fermentation was over in 24 h with a sorbose productivity of 13.40 g/L/h and a final sorbose concentration of
320.48 g/L. On the other hand, in fed-batch fermentation with multiple feeds, two pulse feeds of 0.5 liter nutrient medium
containing 600 g/L sorbitol was followed by the addition of 1.5 liter nutrient medium containing 600 g/L sorbitol at a constant
feed rate of 0.36 L/h till the full working capacity of the reactor. The fermentation was completed in 24 h with an enhanced
sorbose productivity of 15.09 g/L/h and a sorbose concentration of 332.60 g/L. The sorbose concentration and productivity
obtained by multiple feeding of nutrients was found to be higher than that obtained by pulse feeding and was therefore a better
strategy for fed-batch sorbose fermentation. 相似文献
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目的:构建SDH-SV2C-L4融合蛋白表达载体,在大肠杆菌中表达具有山梨糖脱氢酶(SDH)活性的融合蛋白。方法:将C亚型突触囊泡蛋白2大突环L4(SV2C-L4)基因与SDH基因以GGGS柔性接头连接,在大肠杆菌DH5α中表达;用NBT染色和DCIP脱色的方法检测融合蛋白的SDH活性。结果:DNA测序及SDS-PAGE结果显示构建了融合蛋白表达载体,并表达了SDH-SV2C-L4融合蛋白,相对分子质量约80×103;DCIP脱色及NBT染色均检测到融合蛋白的SDH活性。结论:与SV2C-L4融合的SDH仍具有活性,为下一步SV2C-L4活性检测方法的建立及SDH与SV2C-L4的其他相关研究奠定了基础。 相似文献