青霉素酰化酶基因重组大肠杆菌在两水相体系中的转化反应

PENICILLIN BIOCONVERSION IN AOUEOUS TOW-PHASE SYSTEMS BY RECOMBINANT ESCHERICHIA COLI WITH INTRACELLULAR PENICILLIN ACYLASE

以青霉素酰化酶基因重组大肠杆菌Ａ５６（ｐＰＡ２２），经戊二醛交联（防止细胞自溶）后，在ＰＥＧ２０００－ＤｅｘｔｒａｎＴ７０两水相体系中进行转化反应，生产６－氨基青霉烷酸（６－ＡＰＡ）。实现含酶菌体在两水相体系中的单侧分配。考察了菌体加量对青霉素转化率的影响。在两水相体系中进行５批半连续转化反应，底物浓度为７％，转化率在９０％以上，反应时间为４０分钟，比生产速率为３·６～４·６μｍｏｌ／ｍｌ·ｍｉｎ，下相酶活为５２ｕ／ｍｌ，并从上相中直接结晶６－ＡＰＡ，６－ＡＰＡ的纯度为９６·２％。实现了高底物浓度，高转化率，缩短反应时间和产物不需浓缩直接结晶之目的。在两水相体系中生产６－ＡＰＡ成为又一新的青霉素酰化酶应用形式。

In this paper,Penicillin bioconversion in PEG20000-De- xtran T70 aqueous two-phase system has been studied.The bacterial part-itioning was totally biased to the bottom phase under suitable conditions.The optimum reaction conditions were found to be:7%(w/v)substrate(Penicillin G),enzyme activity in the bottom phase 52 u/ml, pH7.8,te-mperature 37℃,reaction time 40 min. Repeated batch conversion was ca-rried out four times by replacing the top phase at optimum condition. The average conversion ratio of Penicillin exceeded 90%。Specific produc-tivity based on the bottom phase was 3.6～4.6μmol/ml·min,The product,6-APA could directly be crystallized from top phase with a purity of 96.2%