Construction and prokaryotic expression of transaminase expression vector of Chromobacterium violaceum
10.13200/j.cnki.cjb.004289
- VernacularTitle:紫色色杆菌转氨酶基因表达载体的构建及其原核表达
- Author:
CHEN Cun
- Publication Type:Journal Article
- Keywords:
Chromobacterium violaceum;
Transaminase;
Escherichia coli(E.coli);
Gene cloning;
Expression;
Transamination reaction
- From:
Chinese Journal of Biologicals
2024;37(9):1070-1074
- CountryChina
- Language:Chinese
-
Abstract:
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 51 000,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
