嗜热古细菌Sulfolobus tokodaii脱卤酶在D-乳酸生产中的应用  

Application of L-2-Haloacid Dehalogenase from Thermophilic Archaea Sulfolobus Tokodaii in the Production of D-Lactic Acid

作　　者：解桂秋[1] 潘东[2] 何文龙[2] 高贵[2] 高仁钧[2] 

机构地区：[1]吉林大学药学院,长春130021 [2]吉林大学生命科学学院、分子酶学工程教育部重点实验室,长春130012

出　　处：《高等学校化学学报》2015年第4期698-703,共6页Chemical Journal of Chinese Universities

基　　金：国家自然科学基金(批准号:20772046);吉林省科技研究和发展计划项目(批准号:201105011)资助~~

摘　　要：将来源于嗜热古菌Sulfolobus tokodaii的脱卤酶(L-HADST)基因克隆到载体p ET28b,转化大肠杆菌BL21(DE3)进行表达,在蛋白的N末端带有6个组氨酸融合标签,纯化后经聚丙烯酰胺凝胶电泳显示融合蛋白的分子量约为25000.融合蛋白催化2-氯丙酸(2-CPA)的最适反应温度为70℃,最适p H值为9.5.以外消旋2-CPA为底物生产D-乳酸,利用HPLC检测反应液中2-CPA及乳酸的变化,发现L-HADST只催化L-2-CPA脱氯反应.对酶催化反应条件进行了优化,结果表明,在p H值为9.5,温度为60℃的条件下,当反应体系中缓冲液浓度为3 mol/L,底物浓度为0.5 mol/L,酶浓度为3×104U/L时有较高的底物转化率及乳酸生成量.依据条件优化结果可知,影响反应速度的因素有底物浓度、缓冲液浓度以及酶浓度,其中底物浓度变化对转换率的影响最明显.Optical pure lactic acid is a useful chiral intermediate in the synthesis of chiral drugs and materials engineering. For the purpose of environmental-friendly and high stereoselectivity,enzymes were used to resolve racemic mixtures. In this study,we cloned and expressed L-2-haloacid dehalogenase（ ST2570） from thermophilic Archaea Sulfolobus tokodaii in Escherichia coli BL21（ DE3）. The recombinant enzyme was purified to a single band using heat treatment and Ni2＋-NTA affinity chromatography. The optimum temperature and p H value of ST2570 were 70 ℃ and 9.5,respectively. The specific activity of purified ST2570 for the hydrolysis of 2-chloropropionic acid was 2963. 33 U / mg. For the high enatioselectivity of ST2570,only L-2-chloropropionic acid can be hydrolyzed to produce D-lactic acid during the reaction. With the optimization,the best conditions to produce D-lactic acid were p H of 9.5,temperature of 60 ℃,3 mol / L of buffer,0. 5 mol / L of 2-chloropropionic acid and 3×104U / L of ST2570.

关 键 词：L-2-卤代酸脱卤酶 2-氯丙酸 乳酸 

分 类 号：O621.25[理学—有机化学]