巨大芽孢杆菌青霉素酰化酶在枯草杆菌中的表达及其分离纯化

青霉素酰化酶（ＰＧＡ）在医药工业起着重要的作用，它能够水解青霉素Ｇ产生６－氨基青霉烷酸（６－ＡＰＡ）和苯乙酸，６－ＡＰＡ是半合成青霉素的关键中间体．该酶广泛存在于各种微生物中如真菌和细菌中．国际上对Ｅ．ｃｏｌｉ、Ａｒｔｈｒｏｂａｃｔｅｒｖｉｓｃｏｓｕ...

Expression and Purification of the PGA gene of Bacillus megaterium in Bacillus subtilis

Penicillin G acylase(PGA) gene of Bacillus megaterium was inserted into the plasmid of pMA5 E.coli B.subtilis shuttle vector,then the recombinant plasmid pMAGA was transformed into B.subtilis BCL1050.This is a secrete system,in which PGA can be expressed and secreted into culture medium directly,providing a convenient means of obtaining relatively pure enzyme in large quantity.After culturing for 24 hours in rich medium,the enzyme activity reached about 1.4 U/ml.PGA expressed in B.subtilis BCL1050 was purified approximately 600 fold by phenylalanine hydrophobic chromatography,ammonia sulfate precipitation,size exclusion chromatography and DEAE Sephadex A 50 chromatography.The purified enzyme,with an approximate molecular weight of 82 kD,appeared to be homogeneous judged by SDS PAGE analysis.The mass of α and β subunits determined by SDS PAGE was 54.95 kD and 21.88 kD respectively.