高分子量腈水合酶工程菌生产烟酰胺的工艺建立  

Establishment of the process of nicotinamide produced by Escherichia coli expressing high molecular mass nitrile hydratase

作　　者：郭军玲[1] 王哲 刘中美[1] 周哲敏[1] GUO Jun-ling;WANG Zhe;LIU Zhong-mei;ZHOU Zhe-min(Key Laboratory of Industrial Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《食品与发酵工业》2018年第2期8-14,共7页Food and Fermentation Industries

基　　金：国家重点研发计划政府间国际科技创新合作重点专项(2016YFE0127400);国家自然科学基金青年科学基金项目(31400078)

摘　　要：为提高表达玫瑰色红球菌(Rhodococcus rhodochrous J1)来源的高分子量腈水合酶大肠杆菌Escherichia coli BL21(DE3)/pET-24a(+)-nhh Brbs Arbs G(BAG)的菌体酶活,建立了合适的烟腈全细胞催化工艺以及重组工程菌的高密度发酵工艺。采用正交试验L_9(3~4)对摇瓶培养基中各组分进行优化,比较烟腈底物流加和底物分批补料工艺,对重组菌进行分批补料高密度发酵培养。培养基优化后其菌体生物量提高到4.42 gDCW/L,细胞比酶活提高到30.91 U/mgDCW;与烟腈底物流加相比,烟腈分批补料工艺更适合烟酰胺的生产,通过此工艺的反应,摇瓶发酵后的BAG能够生产390 g/L烟酰胺;对BAG进行5 L发酵罐分批补料扩大培养,菌体生物量最高达到73.97 gDCW/L(OD600=200),细胞比酶活最高为42.70 U/mgDCW,单位发酵液酶活最高为2 813 U/mL,用较高密度的发酵罐培养后重组菌进行烟腈催化反应,产物质量浓度能达到537 g/L,是已报道的大肠杆菌重组表达腈水合酶产烟酰胺所得终质量浓度的最高水平。To increase the biomass and the activity oi Escherichia coli BL21(DE3)/pET-24a(+)-nhhBrbsArb-sG(BAG)expressing high molecular mass nitrile hydratase from Rhodococcus rhodochrous J1 and establish proper process of whole-cell catalysis of 3-cyanopytidine and high cell-density cultivation,we used orthogonal experiment(L9(34))to optimize elements in culture media.After comparison of substrate-flow and substrate fed-batch process,technology of fed-batch was applied to achieve high cell-density cultivation.After the optimization of culture media,the biomass and the specific activity of BAG in the flask reached 4.42 g DCW/L and 30.91 U/mgDCW,respective-ly.As a more appropriate process,substrate fed-batch process was applied to produce nicotinamide using BAG,and the yield of nicotinamide reached 390 g/L.After the high cell-density cultivation of BAG in 5 L bioreactor,the bio-mass of cell attained 73.97gDCW/L(OD600=200),the specific activity and the total activity reached 42.70 U/mg DCW and 2813 U/mL,respectively.When high density of BAG after fermentation in the tank was used to catalyze 3-cyanopytidine,537 g/L of nicotinamide was obtained,which was the highest yield of nicotinamide among all the re-ported production from recombinant E.coil expressing nitrile hydratase.

关 键 词：腈水合酶 大肠杆菌 全细胞催化 底物分批补料 高密度发酵 

分 类 号：TQ920.6[轻工技术与工程—发酵工程]