醇脱氢酶的纯化、酶学性质及其不对称催化  

Purification and characterization of alcohol dehydrogenase for asymmetric catalysis

作　　者：程书兰[1] 魏淼[1] 许琳[1] 严明[1] 

机构地区：[1]南京工业大学生物与制药工程学院,江苏南京211800

出　　处：《生物加工过程》2016年第1期8-13,共6页Chinese Journal of Bioprocess Engineering

基　　金：国家重点基础研究发展计划(973计划)(2011CBA00804);国家高技术研究发展计划(863计划)(2012AA022101);江苏高校优势学科建设工程

摘　　要：为了研究木兰假丝酵母(Candida magnolia)中的醇脱氢酶(alcohol dehydrogenase)3(ADH3)的突变酶(AIDI)的纯化、酶学性质以及不对称还原合成(3R,5S)-6-氯-3,5-二羟基己酸叔丁酯的能力。首先对AIDI进行(NH_4)_2SO_4沉淀、透析以及DEAE Sepharose离子交换层析。将纯化后的酶用于最适温度、pH、有机溶剂耐受等酶学性质的研究,同时考察底物浓度对催化效率的影响。结果发现:通过纯化获得了电泳纯的AIDI。酶的最适温度为40℃,最适pH为7.0。该醇脱氢酶的热稳定性较好,对环境pH的变化不敏感,在pH 4~8之间较为稳定。部分金属离子对该酶的活性具有抑制作用,特别是Fe2+对其具有显著抑制作用。在0.1 mol/L、pH 7.0的Na_3PO_4缓冲液中,底物质量浓度为50 g/L,30℃转化24 h,转化率能达到94%。A mutant（ AIDI） of alcohol dehydrogenase 3（ ADH3） was derived from Candida magnolia.We characterized AIDI from E. coli and optimized the condition of purification and studied diastereoselective R-ketoreductase activity of the mutant towards（ 3R, 5S）-6-chloro-3, 5-dihydroxyhexanoate. AIDI was purified through ammonium sulphate precipitation,dialysis,and anion exchange chromatography. AIDI were characterized for its optimum temperature,pH,resistance to organic solvents. Effects of concentration of substrate on enzymatic catalysis were explored. The optimal pH and temperature of AIDI were 7. 0 and 40 ℃,respectively. AIDI is a heat-stable alcohol dehydrogenase and insensitive to environmental pH. Fe^（2＋） could significantly inhibit activity of AIDI. Under the optimal conditions of 30 ℃,pH 7. 0 in sodium phosphate buffer,94% of 50 g / L tert-butyl（ S）-6-chloro-5-hydroxy-3-oxo-hexanoate was converted to（ 3R,5S）-6-chloro-3,5-dihydroxyhexanoate in 24 h.

关 键 词：醇脱氢酶 (3R 5S)-6-氯-3 5-二羟基己酸叔丁酯 酶学性质 

分 类 号：Q78[生物学—分子生物学]