洋葱伯克霍尔德氏菌Lu10-1脂肪酶的异源表达

Study on Heterologous Expression of Burkholderia cepacia Lu10-1 Lipase

通过PCR扩增出洋葱伯克霍尔德氏菌Lu10-1脂肪酶基因,将基因片段分别克隆到大肠杆菌、毕赤酵母和枯草杆菌的表达载体,转入表达菌株中。结果表明,重组脂肪酶在大肠杆菌中主要以包涵体形式表达,在毕赤酵母中未有表达,而在枯草杆菌中实现了胞外分泌表达,测得发酵上清液酶活为13.8 U/m L。对重组枯草杆菌发酵条件进行了摇瓶初步优化和3 L的反应器分批培养。当以TB为出发培养基,初始p H 6.5,温度为37℃时,在3 L的发酵罐上最终酶活达到34.5 U/m L,是野生菌表达量的4.2倍。

The lipase gene was isolated from Burkholderia cepacia Lu10-1 by PCR amplification and separately inserted into different vectors, which was then transformed to Escherichia coli ,Pichia pastoris and Bacillus subtilis ,respectively. The recombinant iipase was expressed as inclusion bodies in E. coil,while no expression of lipase was detected in P. pastoris. In B. subtilis ,the lipase was extracellular expressed with an enzyme activity of 13.8 U/mL in the supernatant after fermentation. In addition,the optimization of culture condition for the recombinant B. subtilis was investigated in flask and 3 L stirred tank fermentor,respectively. After cultivation at 37 ℃ with the initial pH of 6.5 and TB used as the starting medium,the lipase activity in 3 L fermentor reached to 34.5 U/mL,a 4.2-fold to that of the original strain.