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

关键词:  黄芩素  酶解  黄芩苷  β-葡萄糖醛酸苷酶

Key words:  baicalein  biosynthesis  baicalin  β-glucuronidase

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pET-28a中，获得重组表达载体pET28a-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 kDa的单一蛋白条带，工程菌最适培养条件为：37 ℃、pH7.2、IPTG浓度0.6 μmol/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60 μg/mL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。

Abstract:

Objective To develop a novel and environmental-friendly method for bioconversion of baicalin to baicalein with recombinant β-glucuronidase. 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-Glucuronidase was overexpressed and then purified by Ni affinity chromatography in one step. The biotransformation of baicalin to baicalein 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 achieved after induction for 12 h with 0.6 μmol/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-glucuronidase, the baicalin was transformed to baicalein with 72.5% conversion ratio under optimized conditions. Conclusion A novel and environmental-friendly scheme for bioconversion of baicalin to baicalein with recombinant β-glucuronidase was established. The method would hold great promise for further applications in both scientific research and biomedical industry.