重组大肠杆菌产角质酶-CBM摇瓶发酵优化及分泌表达研究  

Fermentation Optimization on Flask-scale and Secretional Expression of Recombinant Cutinase-CBM in E.coli

作　　者：郭森[1,2] 吴丹[1,2] 陈晟[1,2] 吴敬[1,2] 陈坚[1,2] 

机构地区：[1]江南大学食品科学与技术国家重点实验室,无锡214122 [2]江南大学生物工程学院工业生物技术教育部重点实验室,无锡214122

出　　处：《中国生物工程杂志》2011年第9期55-61,共7页China Biotechnology

基　　金：国家"863"计划(2009AA02Z204);国家自然科学基金(30970057);国家杰出青年基金(20625619);江苏省六大人才高峰支持计划(08-B-吴敬)资助项目

摘　　要：在TB培养基的基础上,通过单因素分析和正交设计对重组大肠杆菌产角质酶-CBM发酵进行优化,得到最适培养基的组分为:甘油5 g/L,蛋白胨16 g/L,MgSO4.7H2O 2.5 mmol/L,K2HPO413.7 g/L,KH2PO41.53 g/L,菌体生长至对数前中期时添加终浓度为1 g/L乳糖和0.75 g/L甘氨酸,30℃发酵48 h,角质酶-CBM产量可达63 U/ml,较TB培养(20 U/ml)提高了近3倍。考察了热激作用、渗透调节物质及温度两控制对角质酶-CBM分泌表达的影响,在添加Lactose和Glycine后,发现在添加终浓度为75 mmol/L的L-脯氨酸,37℃热激1 h或47℃热激0.5 h,变温至25℃发酵,角质酶-CBM产量可达90 U/ml,较TB恒温培养提高了近四倍。On the basis of TB fermentation medium,an optimized fermentation strategy of cutinase-CBM by E.coli was developed through single factor analyzing and orthogonal design,which can be illustrated as follows： glycerol 5g/L,peptone 16 g/L,MgSO4·7H2O 2.5 mmol/L,K2HPO4 13.7 g/L,KH2PO4 1.53 g/L,1 g/L lactose and 0.75 g/L glycine（final concentration） were added in the prometaphase of logarithmic phase of growth curve,and the fermentation period lasted 48h under 30℃.An extracellular enzyme activity of 63 U/ml was reached,about 3 times that of the control（20 U/ml）.Further efforts were made to investigate the influence of other factors,such as heat-shock,osmotic agent,and temperature,on the secretional expression of cutinase-CBM.It is found that with the addition of 75 mmol/L L-proline,heat shocked for 1h at 37℃ or 0.5h at 47℃,then shifted to 25℃,the final extracellular production of cutinase-CBM can reach 90U/ml,4 times that of the control cultivated at constant temperature.

关 键 词：角质酶-CBM 正交设计 热激作用 渗透调节物质 变温发酵 

分 类 号：Q786[生物学—分子生物学]