青霉素G酰化酶调节基因定点诱变的研究

构建了大肠杆菌中青霉素G酰化酶（PAC）的调节基因（pacR）翻译起始密码定点突变株。对突变后PAC的表达调控特征进行了研究，结果表明：突变株的pac基因表达后能正常加工成24ku、65ku的α亚基和β亚基；突变后，虽然PAC表达仍需要苯乙酸诱导，但是，可诱导性提高，形成低组成型表达，无葡萄糖有分解代谢物阻遏效应，因而pacR对PAC表达水平存在着影响，pacR基因是葡萄糖以及它的分解代谢物在分子水平上影响PAC表达的另一作用因素。采用突变株进行发酵生产中，PAC产量较亲株提高3倍，而且可以采用葡萄糖作为碳源，有利于改善PAC的生产。

Site-directed Mutagenesis of Regulatory Gene of Penicillin G Acylase

Site directed mutagenesis was performed at the start codon ATG of the regulatory gene of the penicillin G acylase operon ( pacR ) in E.coli HB101 (pPA6). The CAT was substituted by CTC. A plasmid pPA52 was constructed containing the mutant at the start codon ATG of pacR . The pPA52 was transformed into E.coli HB101. The regulation of penicillin G acylase gene ( pac ) expression from the mutant strain was studied. It can be founded that the posttranslational modifications of PAC in E.coli HB101 (pPA52) is similar to that in E.coli HB101 (pPA6). Although pacR translation was deleted from E.coli HB101 (pPA52), the expression of pac is still induced by PAA. The activities of PAC in E.coli HB101 (pPA52) is more than that in E.coli HB101(pPA6), with 0.05%PAA, 0.2%PAA or without PAA. The expression of pac is repressed by glucose in E.coli HB101 (pPA6), not in E.coli HB101 (pPA52). Site directed mutagenesis of pacR at start codon has improved production of PAC.