重组大肠杆菌产角质酶-CBM摇瓶发酵优化及分泌表达研究

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,K₂HPO₄ 13.7 g/L,KH₂PO₄ 1.53 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。

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

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.