生物工程学报 25 June 2009, 25(6):863～868
响应面法优化热带假丝酵母104菌株产羰基还原酶发酵培养基
王普, 孙立明, 何军邀
浙江工业大学药学院, 杭州 310032
摘 要: 通过菌种优选得到产高选择性羰基还原酶的热带假丝酵母(Candida tropicalis) 104菌株, 可不对称还原1-(3,5-二三氟甲基)苯基乙酮生成(S)-1-(3,5-二三氟甲基)苯基乙醇, 对映体过量值>99.9%。采用部分因子实验设计考察发酵培养基中各组分对产酶的影响, 结果表明, 酵母粉、葡萄糖和NH4Cl浓度对产酶影响显著。继而采用最陡爬坡路径逼近最大响应区域, 并结合中心组合实验和响应面对3个显著性因素进行分析, 得到优化的发酵培养基组成: 葡萄糖47.14 g/L, 酵母粉13.25 g/L, NH4Cl 2.71 g/L, MgSO4·7H2O 0.4 g/L, KH2PO4 1 g/L和K2HPO4 1 g/L。采用该优化培养基, 供试菌株的羰基还原酶活力达851.13 U/L, 较优化前提高了65.2%。
关键词: 热带假丝酵母, 羰基还原酶, (S)-1-(3,5-二三氟甲基)苯基乙醇, 响应面, 培养基优化
Medium optimization for enhanced production of carbonyl reductase by Candida tropicalis 104 by response surface methodology
Pu Wang, Liming Sun, and Junyao He
College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, China
Abstract: Using response surface method, we optimized the medium for the asymmetric whole cell biotransformation by Candida tropicalis 104. This strain was used for microbial reduction of 1-[3,5-bis(trifluoromethyl)phenyl] ethanone to (S)-1-[3,5-bis(trifluoromethyl)phenyl] alcohol, with enantiomeric excess(e.e.) reached more than 99.9%. Fractional factorial design was used to evaluate the effects of medium components on carbonyl reductase activity of Candida tropicalis 104. Yeast extract, glucose and NH4Cl were the most important factors among six tested variables that influence the enzyme activity for the biotransformation process. Based on the experimental results, the path of steepest ascent was undertaken to approach the optimal region of these factors. Central composite design and response surface analysis were subsequently employed for further optimization. The optimal medium for Candida tropicalis 104 was composed of (in g/L): glucose 47.14, yeast extract 13.25, NH4Cl 2.71, MgSO4·7H2O 0.4, KH2PO4 1, K2HPO4 1. Under the optimum conditions, the maximum enzyme activity of 852.75 U/L in theory and 851.13 U/L in the experiment were obtained, with an increase of 65.2% compared to the original medium components.
Keywords: Candida tropicalis, carbonyl reductase, (S)-1-[3,5-bis(trifluoromethyl)phenyl] alcohol, response surface methodology, medium optimization
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