(S)-羰基还原酶II于酿酒酵母孢子中表达与功能

Expression and functions of (S)-carbonyl reductase II in Saccharomyces cerevisiaes pores

【目的】使近平滑假丝酵母(Candida parapsilosis CCTCC M203011)的(S)-羰基还原酶II表达并包埋于酿酒酵母(Saccharomyces cerevisiae AN120)孢子中,实现了重组酶高效催化生产(S)-苯基乙二醇的转化过程。【方法】采用PCR扩增技术,从近平滑假丝酵母基因组中克隆(S)-羰基还原酶II基因,于酿酒酵母AN120中表达,以醋酸钾为唯一碳源诱导培养产生孢子,包埋(S)-羰基还原酶II。以该孢子为生物催化剂,2-羟基苯乙酮为底物进行生物转化反应,经HPLC分析,计算产物的光学纯度和得率。考察了孢子催化转化反应的最适温度和p H值,温度和p H稳定性以及多批次使用性能。【结果】在最适反应温度40℃和p H6.0条件下,10%(W/V)子囊孢子催化6 g/L 2-羟基苯乙酮,产物(S)-苯基乙二醇的光学纯度和得率均高达99%以上。与重组大肠杆菌相比较,重组孢子合成(S)-苯基乙二醇的得率由89.7%提高到99.0%,反应时间由48h缩短为4 h;连续使用10批次后,其催化产物的光学纯度几乎不变,得率保持在85%以上。【结论】该研究首次实现了氧化还原酶在酵母孢子内的异源表达,为手性化合物的高效制备奠定了坚实的研究基础。

[ Objective ] To realize efficient biotransformation of （S）-l-phenyl-1,2-ethanediol by recombinant （S）- carbonyl reductase II, we expressed （S）-carbonyl reductase Ⅱ from Candida parapsilosis CCTCC M203011 and embedded it in the spores of Saccharomyces cerevisiae AN120. [Methods] （S）-carbonyl reductase Ⅱ gene was cloned from C. parapsilosis genome and expressed in S. cerevisiae AN120 by PCR amplification. When cultured with potassium acetate as the sole carbon source, the yeast spores were produced, and embedded the recombinant （S）-carbonyl reductase II. Using 10% W/V spores as biocatalysts, 6 g/L 2-hydroxyacetophenone as substrate, the biotransformation was carried out and the optical purity and yield of products were analyzed by HPLC. During the biotransformation of 2-hydroxyacetophenone to （S）-l-phenyl-1,2-ethanediol, the optimal temperature and pH, stability and reusability of the recombinant spores were determined. [ Results ] The recombinant yeast spores showed excellent performance to give （S）-l-phenyl-1, 2- ethanediolwith a high optical purity of 99.3% and a high yield of 99.0% at the optimal temperature（40 ℃ ） and pH （6.0）. Compared with the recombinant Escherichia coli, the spores improved the yield of （S）-1-phenyl-1,2-ethanediol from 89.7% to 99.0% , and shortened the biotransformation duration from 48 h to 4 h. After being reused for 10 times, the recombinant spores biotransformed（S）-1-phenyl-1,2-ethanediol with a stable optical purity of about 99% and a yield over 85%. [ Conclusion] The heterologous expression of oxidoreductases was first realized in yeast spores, which laid a solid foundation for efficient preparation of chiralcompounds.