β-葡萄糖醛酸苷酶的重组表达及对黄芩苷的生物转化  

Bioconversion of baicalin to baicalein with recombinant β-glucuronidase in Escherichia coli

作　　者：周琪[1] 窦同意 丁乐乐[1] 曲明佳 翁仔淼 吴大畅[1] 侯洁[1] 

机构地区：[1]大连医科大学生物技术系,辽宁大连116044 [2]中国科学院大连化学物理研究所生物技术部药用资源开发组,辽宁大连116023

出　　处：《大连医科大学学报》2017年第2期110-115,共6页Journal of Dalian Medical University

基　　金：国家自然科学基金项目(81273590;81302793);精细化工国家重点实验室开放课题(KF1408)

摘　　要：目的构建表达β-葡萄糖醛酸苷酶(β-glucuronidase,GUS)的重组菌株,得到高纯度β-葡萄糖醛酸酶,进而对黄芩苷进行生物转化获得黄芩素。方法以E.coli K12基因组DNA为模板,采用PCR方法扩增GUS基因,经酶切后连接到表达载体p ET-28a中,获得重组表达载体p ET28a-GUS,将重组载体转化到感受态细胞BL21(DE3)中,对所得基因工程菌进行培养条件和表达条件的优化;后经分离纯化后得到高纯度β-葡萄糖醛酸酶。以黄芩苷为底物进行酶法转化生产黄芩素,并对反应条件进行优化。结果经测序,扩增的重组质粒目的基因序列与数据库中GUS基因序列一致。经IPTG(isopropy-β-D-thiogalactoside)诱导表达后,SDS-PAGE分析获得分子质量70 k Da的单一蛋白条带,工程菌最适培养条件为:37℃、pH7.2、IPTG浓度0.6μmol/L、诱导时间12 h。经过离心破碎、亲和层析、冻干后得到高纯度β-葡萄糖醛酸酶冻干粉。在该酶催化转化下,黄芩苷可转化为黄芩素,在40℃,pH 6.5,酶浓度60μg/m L条件下,反应2.5 h,黄芩苷转化率可达72.5%。结论成功构建了β-葡萄糖醛酸苷酶高表达菌株,并用于黄芩苷的定向水解制备黄芩素,为生物转化法生产黄芩素提供了新思路。Objective To develop a novel and environmental -friendly method for bioconversion of baicalin to baicalein with recombinant β - glueuronidase. Methods The β - glucuronidase encoding gene （GUS） was cloned from Escherichia coli wild strain K12, and transformed into Escherichia coli BL21 （DE3） with pET -28a （ ＋ ） as the vector, β - Glucuroni- dase was overexpressed and then purified by Ni affinity chromatography in one step. The biotransformation of baicalin to ba- icalein was performed by the recombinant β- glucuronidase, and the reaction conditions were optimization with orthogonal design. Results The protein was identified by SDS - PAGE with 70 kDa, and the maximum expression level could be a- chieved after induction for 12 h with O. 6 p, mol/L IPTG, and 320 mg of purified enzyme, which could be obtained from one liter of bacterial culture. With the catalysis of the recombinant β - glueuronidase, the baiealin was transformed to baiealein with 72.5% conversion ratio under optimized conditions. Condusion A novel and environmental - friendly scheme for bio- conversion of baiealin to baicalein with recombinant β - glucuronidase was established. The method would hold great promise for further applications in both scientific research and biomedical industry.

关 键 词：黄芩素 酶解 黄芩苷 Β-葡萄糖醛酸苷酶 

分 类 号：R284[医药卫生—中药学]