青霉素G酰化酶调节基因定点诱变的研究  

Site directed Mutagenesis of Regulatory Gene of Penicillin G Acylase

作　　者：徐京宁[1] 杨运桂[2] 龚毅[2] 杨胜利[2] 俞俊棠[1] 

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237 [2]中国科学院上海生物工程研究中心,上海200233

出　　处：《华东理工大学学报（自然科学版）》2002年第3期241-244,共4页Journal of East China University of Science and Technology

摘　　要：构建了大肠杆菌中青霉素 G酰化酶 ( PAC)的调节基因 ( pac R)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2 4 ku、65 ku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 ,无葡萄糖及分解代谢物阻遏效应 ,因而 pac R对 PAC表达水平存在着影响 ,pac R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时 ,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.

关 键 词：青霉素G酰化酶 定点突变 基因调控 葡萄糖阻遏 

分 类 号：Q786[生物学—分子生物学]