紫色色杆菌转氨酶基因表达载体的构建及其原核表达

Construction and prokaryotic expression of transaminase expression vector of Chromobacterium violaceum

目的利用紫色色杆菌(Chromobacterium violaceum)的转氨酶(CV2025)基因序列构建重组质粒pET-28aCV2025,并在大肠埃希菌(Escherichia coli,E.coli)中进行原核表达。方法将CV2025基因序列与pET-28a(+)载体连接,构建重组质粒pET-28a-CV2025,转化E.coli TOP10中进行阳性克隆子筛选,经双酶切及测序验证后,转化E.coli BL21(DE3)和Rosetta,诱导表达目的蛋白;将重组菌28a-CV2025-BL21活化后接种2TY液体培养基,IPTG诱导表达制备粗酶液后,以异丙胺为氨基供体,1-(4-甲氧苯基)乙酮、邻氟苯乙酮、苯乙酮为氨基受体,采用薄层色谱法监测反应,初步检验转氨酶活性,高效液相色谱法进行进一步分析。结果重组质粒pET-28a-CV2025经双酶切及测序鉴定证明构建正确;重组菌28a-CV2025-BL21、28a-CV2025-Rosetta的表达产物均可见相对分子质量约51000的目的蛋白条带,且28a-CV2025-BL21表达的蛋白大部分以可溶性形式存在;酶液催化后,1-(4-甲氧苯基)乙酮与异丙胺发生转氨基化反应,生成对应的手性胺1-(4-甲氧苯基)乙胺,具有一定的催化活性。结论在E.coli中成功表达了CV2025,初步检验上清中可溶性蛋白具有酶活性,为后期转氨酶的分离、纯化以及增强产物转化率奠定了基础。

Objective To construct the recombinant plasmid pET-28a-CV2025 by using the transaminase(CV2025)gene sequence of Chromobacterium violaceum and express it in Escherichia coli(E.coli).Methods The recombinant plasmid pET-28a-CV2025 was constructed by linking the gene sequence of CV2025 with pET-28a(+)vector,which was transformed into E.coli TOP10 for screening of positive clones.After double enzyme digestion and sequencing verification,the recombinant plasmid was transformed into E.coli BL21(DE3)and Rosetta,and was induced to express the target protein.The recombinant strain 28a-CV2025-BL21 was activated and inoculated into 2TY liquid medium.After IPTG induced expression,with isopropylamine as the amino donor and 1-(4-methoxyphenyl)acetophenone,o-fluoroacetophenone and acetophenone as the amino receptor,the reaction was monitored by thin layer chromatography,and the transaminase activity was preliminarily tested.Finally,the reaction was further identified and analyzed by high-performance liquid chromatography.Results The recombinant plasmid pET-28a-CV2025 was constructed correctly as identified by double enzyme digestion and sequen-cing.The expression products of 28a-CV2025-BL21 and 28a-CV2025-Rosetta showed target protein bands with the relative molecular mass of about 51000,and most of the proteins expressed by 28a-CV2025-BL21 existed in soluble form.The enzyme solution catalyzed the transamination reaction of 1-(4-methoxyphenyl)acetophenone with isopropylamine to generate the corresponding chiral amine 1-(4-methoxyphenyl)ethylamine,which had certain catalytic activity.Conclusion In this study,CV2025 was successfully expressed in E.coli,and the soluble protein in the supernatant was preliminarily tested to have catalytic activity,which lays a foundation for the later separation and purification of transaminase and the enhancement of product conversion rate.