产青霉素G酰化酶工程菌的构建

Construction of penicillin G acylase-producing engineering strain

【目的】构建表达巨大芽孢杆菌青霉素G酰化酶(PGA)的枯草芽孢杆菌工程菌.【方法】首先采用PCR从巨大芽孢杆菌基因组DNA中扩增出长度为2 409bp的PGA编码基因,经琼脂糖凝胶电泳检测及DNA测序验证后,再将其与枯草芽孢杆菌穿梭表达载体PMA5(7.5kb)连接后导入到枯草芽孢杆菌10397中表达,并通过聚丙烯酰胺琼脂糖凝胶电泳(SDS-PAGE)检验其能否表达.【结果】构建了9.9kb大小的PMA5-PGA重组质粒,经SDS-PAGE验证,青霉素酶基因在枯草芽孢杆菌10 397中作出表达.基因工程菌在37℃、220r/min培养36h发酵条件下,所产PGA的酶活可达到12.426U/mL,比巨大芽孢杆菌发酵产PGA酶提高了5.26倍.【结论】所构建的枯草芽孢杆菌是青霉素G酰化酶的高产工程菌.

【Objective】This study was to construct the penicillin G acylase-producing engineerng strain from Bacillus subtilis.【Method】Firstly,the PGA gene of B.megaterium was amplified by PCR and tested by agarose gel electrophoresis.Then the target gene was linked with expression vector PMA5 and transfected into expression host B.subtilis 10 397.【Result】Finally the PMA5-PGA（about 9.9kb）was constructed and transfected into B.subtilis 10 397.The SDS-PAGE showed that the target gene was expressed successfully.As a result,when the engineering strain was cultured at 37℃,220r/min for 36 h,the activity of penicillin G acylase reached 12.426 U/mL,which was 5.26 times more than B.megaterium was cultured.【Conclusion】The B.subtilis engineering strain can obtain high penicillin G acylase.