【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.更多还原

【Abstract】 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.更多还原