苯乙酮酸脱羧酶基因的克隆与表达及静息细胞生物转化乙基香兰素的研究

Cloning and Expression of Benzoylformate Decarboxylase Gene and Study on Biotransformation of Ethyl Vanillin by Resting Cell

摘要:

对恶臭假单胞杆菌(Pseudomonas putida ATCC12633)中的苯乙酮酸脱羧酶基因mdlC进行克隆,导入质粒载体pET28a中,将构建得到的重组质粒pET28a-mdlC转化于宿主细胞E.coliBL21(DE3),重组大肠杆菌E.coli BL21(DE3)(pET28a-mdlC)经IPTG诱导,SDS-PAGE分析得到相对分子质量约为57 000的蛋白质条带。将E.coli BL21(DE3)(pET28a-mdlC)和E.coli BL21(DE3)(pET30a-mdlB)两株重组菌以混合静息细胞的形式作为生物催化剂,利用各自胞内的重组酶对3-乙氧基-4-羟基苯乙醇酸(乙基扁桃酸)脱氢氧化、脱羧合成乙基香兰素。未经优化,催化24 h后反应液中乙基香兰素的质量浓度可达1.94 g/L,且没有副产物产生。同时研究表明,该混合静息细胞重复使用3次能保持90%以上的催化活力,还有效缩短了反应时间。

Abstract:

Benzoylformate decarboxylase gene(mdlC) from Pseudomonas putida ATCC12633 was inverted into Escherichia coli(E.coli) strain BL21(DE3) and was efficiently expressed after induction with IPTG.The recombinant strain together with E.coli/pET30a-mdlB converted successfully(S)-4-hydroxy-3-ethoxymandelic acid(EMA) to ethyl vanillin in the forms of mixed resting cells.Without optimization,all the(S)-EMA was consumed to form ethyl vanillin(1.94 g/L) and no by product was obtained with the initial substrate concentration 5 g/L by after 24 h.The cells could maintain their enzyme activity in repeated utilization at least three times and shortened bioconversion time efficiently.