共查询到18条相似文献,搜索用时 156 毫秒
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研究了用于谷胱甘肽合成的温度诱导型重组大肠杆菌的补料分批发酵,通过葡萄糖的脉冲补入,结合溶氧反馈控制技术,实时控制发酵过程中乙酸的产生. 在此基础上,进一步考察了在谷胱甘肽合成酶系诱导表达阶段,流加酵母粉和蛋白胨时重组大肠杆菌的补料分批发酵过程. 结果发现,脉冲补料-溶氧反馈控制技术在流加酵母粉和蛋白胨的补料分批发酵中仍能得到很好的应用,乙酸浓度可被有效地控制在2.5 g/L以下. 通过比较诱导表达阶段流加不同组成的补料液成分,发现添加适量酵母粉和蛋白胨可促进谷胱甘肽合成酶系的表达,在42℃诱导4 h收获的重组大肠杆菌酶法合成谷胱甘肽达到2.46 g/L. 相似文献
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人胰高血糖素样肽-1(Glucagon Like Peptide-1,GLP-1)是一种有潜在应用价值的治疗糖尿病的药物。今以一株表达GLP-1融合蛋白的重组大肠杆菌为研究对象,在分批培养条件下,通过流加甘油并在诱导前0.5h添加氮源使菌体密度、融合蛋白表达水平、融合蛋白体积得率和融合蛋白细胞得率分别提高了138.0%、29.5%、216.9%和29.6%。在此基础上,进一步考察了甘油补加策略、氮源补料方式以及补氮液浓度等过程因素对菌体生长以及GLP-1融合蛋白表达的影响,发现氮源流加过程是提高融合蛋白表达水平的关键因素。进而通过对补料过程的优化,建立了高密度高表达的分批补料培养工艺,最终使菌体密度、融合蛋白表达水平、融合蛋白体积得率和融合蛋白细胞得率分别达到51.93g·L-1、34.6%、2.937g·L-1和0.057g·g-1cell,较分批培养分别提高了314.5%、100.0%、784.6%和111.1%。研究结果对GLP-1融合蛋白的产业化开发有一定的指导意义。 相似文献
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应用溶氧反馈控制高密度培养重组大肠杆菌过程中乙酸的产生 总被引:4,自引:0,他引:4
在培养重组大肠杆菌的过程中,乙酸的产生会抑制细菌的生长,也不利于重组蛋白的表达.研究了补料培养重组大肠杆菌过程中,应用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.当大肠杆菌以0.1 h-1的比生长速率生长时,补料过程中发酵液的乙酸含量很低,溶氧pO2的响应信号随脉冲补料而振荡.而在诱导表达重组人表皮生长因子时,大肠杆菌的呼吸能力下降,过量补入的葡萄糖使重组菌产生大量的乙酸,同时pO2的响应信号失去振荡的特征.根据pO2响应信号的特征反馈控制葡萄糖的流加速率,有效地避免了重组蛋白表达过程中乙酸的产生,实现了重组大肠杆菌的高密度培养,菌体干重达到48 g·L-1,重组人表皮生长因子的表达水平提高了45%. 相似文献
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混合补料诱导高拷贝重组毕赤酵母高效表达X-HBsAg 总被引:2,自引:2,他引:0
目的对高拷贝重组毕赤酵母表达X-HBsAg的诱导策略进行优化。方法在摇瓶中分别诱导1、4、8拷贝数的重组毕赤酵母,ELISA检测X-HBsAg的表达量;在15L发酵罐中对8拷贝的重组毕赤酵母进行诱导表达,分别考察甘油浓度对菌体比生长速率的影响、过渡阶段混合补料对菌体生长及细胞相对活性的影响、诱导阶段比生长速率对目的蛋白表达的影响以及诱导阶段混合补料对目的蛋白表达的影响。结果 8拷贝的重组毕赤酵母在摇瓶中诱导表达的X-HBsAg量明显高于1和4拷贝重组子。优化的发酵诱导策略为:初始补加甘油阶段控制甘油浓度在15g/L;过渡阶段采用甲醇与甘油混合补料,控制甘油浓度在限制性浓度以下;诱导阶段采用甲醇与甘油混合诱导X-HBsAg表达,诱导初始阶段控制比生长速率在0.015/h以上,中后期在0.002/h左右;发酵92h,X-HBsAg的表达量比单纯甲醇诱导提高约67%。结论混合补料诱导较单纯甲醇诱导策略更有利于高拷贝重组毕赤酵母表达X-HBsAg。 相似文献
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目的建立重组戊型肝炎病毒抗原工程菌的发酵和目的蛋白纯化工艺。方法在三角烧瓶中,探讨不同的诱导剂浓度和培养基对菌体密度和目的蛋白表达量的影响;在10L发酵罐中,探讨诱导时间、补料方式、溶氧量对菌体密度和目的蛋白表达量的影响。根据目的蛋白的理化特性建立纯化工艺。结果重组HEV抗原工程菌在10L发酵罐分批补料培养中,采用0·1mmol/L的IPTG诱导4h,目的蛋白表达量约为25%,目的蛋白产率约为2·88g/L,且以包涵体形式存在。经过对包涵体粗纯、复性、纯化,SDS-PAGE分析,纯度可达95%以上。结论建立了周期短、产率高且稳定可靠的发酵及纯化工艺,为重组HEV抗原的大规模生产奠定了基础。 相似文献
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营养物及补料方式对重组酵母产α-淀粉酶的影响 总被引:1,自引:0,他引:1
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。 相似文献
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目的 建立重组NK4融合基因工程菌的发酵工艺。方法 采用锥形瓶、发酵罐发酵 ,对工程菌生长和表达条件如pH、诱导时间及诱导剂浓度等方面进行优化。确定其最佳诱导表达条件 ,在 15L自控发酵罐中进行分批补料培养。结果 在 15L发酵罐进行工程菌发酵 ,菌体收获量湿重可达 2 4 .6g L± 0 .98g L ,目的蛋白的表达量保持在菌体总蛋白的 5 0 %左右 ,发酵时间为 11h。结论 建立了周期短、产率高且稳定可靠的发酵工艺。 相似文献
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目的克隆碱性纤维素酶基因,构建酵母整合型表达质粒,在巴氏毕赤酵母中表达,并对重组菌的发酵工艺进行优化。方法应用PCR技术从嗜碱性芽孢杆菌ATCC21833中扩增碱性纤维素酶基因,克隆至酵母整合型表达载体pGAPZαA中,构建重组表达质粒pGAPZαA-ATCC21833,并转化至巴氏毕赤酵母GS115。通过单因素实验及正交实验,确定重组酵母的最佳发酵培养基。在20L发酵罐中进行高密度发酵,观察碳源对批式发酵的影响,并检测在4种流加方式(连续恒速流加、间歇匀速流加、间歇递减流加、维持底物浓度流加)下的菌体干重及发酵液中的酶活性。结果重组表达质粒pGAPZαA-ATCC21833经酶切及DNA测序证明构建正确,其基因序列与嗜碱性芽孢杆菌KSM-635的碱性纤维素酶基因序列一致。最佳发酵培养基组成为6%葡萄糖、2%硫酸铵、12g/L磷酸二氢钾。碳源浓度对于重组酵母菌体生长及产酶至关重要。SDS-PAGE表明表达产物的相对分子质量约为103000。维持底物浓度的流加方式可获得最高的菌体干重(29.8g/L)及酶活力(24U/ml)。结论已成功构建了表达碱性纤维素酶的巴氏毕赤酵母工程菌,并确定了维持底物浓度的流加方式为最佳发酵方式。 相似文献
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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... 相似文献
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12 small-scale bubble columns, each and every column with a total volume of 500 ml, individually controlled distribution of sterile and humidified air and a pH-probe, were operated in an incubation chamber with temperature control. Intermittent feeding of substrate or inducer, as well as of base for parallel pH-control was achieved by connecting a precision syringe pump to a valve distributor with one 2/2-way miniature valve for each bubble column. This new parallel bio-reactor technique was applied for optimization of the feed profile of the chemical inducer isopropyl-β-D-thiogalactoside (IPTG) to improve the expression of a foreign protein (guanosin-5′-diphosphate-α-D-mannose-pyrophosphorylase, GDP-manPP) under the control of the lac promoter in Escherichia coli. A mean cell density of 8 g l–1 of recombinant E. coli was achieved in parallel fed-batch fermentations within 9.5 h due to a sufficient oxygen transfer (kLa of up to 0.2 s–1) and parallel pH-control. The GDP-manPP activity was improved by more than 100 % compared to the standard IPTG pulse within 3 sets of parallel fed-batch fermentations. A Genetic Algorithm was used for optimization of the IPTG feed profile. Scale-up of the optimized fed-batch process into the stirred tank reactor (scale-up factor of 20) was possible, if all reaction conditions were copied exactly with respect to the reactor volume. A final GDP-manPP activity of 715 U l–1 was measured 3 h after IPTG induction was finished. 相似文献
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Vytautas Galvanauskas Norbert Volk Rimvydas Simutis Andreas Lü bbert 《Chemical Engineering Communications》2004,191(5):732-748
A model-aided process design and supervision technique is proposed. Its efficiency is demonstrated by a nontrivial example, the development of a production process for the light chain of the antibody MAK33. This protein was expressed by the genetically modified E. coli B pUBS520 p12023 bacteria under the control of the tac promoter where lactose was used for induction. It is shown in this example that data from a few experiments are sufficient to develop a satisfactory production process. The development of process models, which are prerequisite for a systematic optimization of protein production processes, as well as their use for the determination of quasi-optimal feed-forward control profiles, is discussed. Model-supported closed-loop control is shown to lead to another improvement. Further performance enhancements can be obtained by changing the operational mode from the usually employed fed-batch procedure to a repeated fed-batch mode. 相似文献
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VYTAUTAS GALVANAUSKAS NORBERT VOLK RIMVYDAS SIMUTIS ANDREAS LÜBBERT 《Chemical Engineering Communications》2013,200(5):732-748
A model-aided process design and supervision technique is proposed. Its efficiency is demonstrated by a nontrivial example, the development of a production process for the light chain of the antibody MAK33. This protein was expressed by the genetically modified E. coli B pUBS520 p12023 bacteria under the control of the tac promoter where lactose was used for induction. It is shown in this example that data from a few experiments are sufficient to develop a satisfactory production process. The development of process models, which are prerequisite for a systematic optimization of protein production processes, as well as their use for the determination of quasi-optimal feed-forward control profiles, is discussed. Model-supported closed-loop control is shown to lead to another improvement. Further performance enhancements can be obtained by changing the operational mode from the usually employed fed-batch procedure to a repeated fed-batch mode. 相似文献