枯草芽胞杆菌PnbA酯酶选择性拆分制备l-薄荷醇

陶惟一,徐娴,李霜

(南京工业大学 生物与制药工程学院, 江苏南京211800)

摘要：利用重组大肠杆菌表达来源于枯草芽胞杆菌CICC 20034中的PnbA酯酶，不对称催化水解dl-薄荷醇丙酸酯制备l-薄荷醇。考察助溶剂种类、助溶剂添加浓度、温度、催化剂用量、底物浓度以及pH等对反应的影响。结果表明：添加25%(体积分数)助溶剂乙醇可显著提升该酯酶对l-薄荷醇的立体选择性，对映选择率(E)由2.4提高到99.43，为不加乙醇条件下的40倍。酶催化最佳条件：25%乙醇作为助溶剂，反应温度37 ℃，缓冲液为0.1 mol/L Tris-HCl(pH 8.0)并保持pH 8.0反应条件，底物量50 mmol/L，反应体系中酶的添加量750 U/mL，在此条件下，酶促反应30 min后，l-薄荷醇转化率可达34%，产物光学纯度对映体过量值(e.e.p)达95%。

关键词：PnbA酯酶；对映选择率；l-薄荷醇；助溶剂；选择性拆分

doi:10.3969/j.issn.1672-3678.2015.06.008

收稿日期：2015-03-11

基金项目：国家重点基础研究发展计划(973计划)(2011CB710800)；国家自然科学基金青年基金(21406111)

作者简介：陶惟一(1990—)，女，江苏南京人，硕士研究生，研究方向：生物化工；李霜(联系人)，教授，lishuang@njtech.edu.cn

中图分类号：TQ033

文献标志码：A

文章编号：1672-3678(2015)06-0043-06

Abstract：The PnbA esterase from Bacillus subtilis CICC 20034 was expressed in recombinant Escherichia coli and used to produce l-menthol through enantioselective hydrolysis of dl-menthyl propionate. The effects of co-solvents,temperature,pH,substrate titers and enzyme dosage on the enantioselective hydrolysis reaction were studied. Results showed that ethanol was the most suitable co-solvent; adding 25%(V/V) ethanol could significantly improve the optical purity of l-menthol,enantiomeric selectivity(E) enhanced from 2.4 to 99.43,about 40 times higher than that of the control.The optimized conditions for hydrolysis of dl-menthyl propionate to prepare l-menthol by PnbA were 25%(V/V) ethanol,0.1 mol/L Tris-HCl buffer (pH8.0), 50 mmol/L dl-menthyl piopionate,750 U/mL crude enzyme solution,37 ℃ and pH 8.0. Under these conditions,l-menthol could be obtained with the conversion rate obove 34% and the optical purity above 95% in 30 min reaction.

Keywords：PnbA esterase; enantiomeric selectivity; l-menthol; co-solvent; enantioselective hydrolysis

Enantioselective hydrolysis of dl-menthyl propionate in enzymatic preparation of l-menthol by PnbA esterase from Bacillus subtilis

TAO Weiyi,XU Xian,LI Shuang

(College of Biotechnology and Pharmaceutical Engineering,Nanjing Tech University,Nanjing 211800,China)

l-薄荷醇是天然薄荷醇的立体构象，具有特有的薄荷香气和清凉效果，已经广泛应用于食品、医药和日用品等行业。目前，薄荷醇的生产主要是天然提取，但受环境、天气等因素影响，天然薄荷醇质量和产量都难以满足日益增长的消费需求。因此，近年来采用脂肪酶或酯酶不对称拆分制备l-薄荷醇成为研究热点。用于拆分制备l-薄荷醇的酯酶或脂肪酶的微生物主要有枯草芽胞杆菌(Bacillussubtilis)、产碱假单胞菌(P￣s￣e￣u￣d￣o￣m￣o￣n￣a￣salcaligenes)、洋葱布克氏菌(Burkholderiac￣e￣p￣a￣c￣i￣a)等。……