嗜热踝节菌脂肪酶(TTL)的重组表达及催化合成普瑞巴林手性中间体  

Overexpression of Talaromyces thermophilus lipase(TTL) and its application in biosynthesis of pregabalin intermediate

作　　者：吴欣玮 郑仁朝[1] 郑裕国[1] 

机构地区：[1]浙江工业大学生物工程学院,浙江杭州310014

出　　处：《生物加工过程》2016年第5期37-44,共8页Chinese Journal of Bioprocess Engineering

基　　金：浙江省重大科技专项社会发展项目(2012C03005-2)

摘　　要：以催化合成普瑞巴林中间体（3 S）-2-羧乙基-3-氰基-5-甲基己酸为目标,通过基因挖掘得到来源为嗜热踝节菌的脂肪酶氨基酸序列,构建了基因工程菌Escherichia coli BL21/p ET28b-TTL,在16℃成功诱导表达了嗜热踝节菌脂肪酶TTL。分离纯化得到TTL电泳纯酶,其最适反应温度为50℃,在50℃的半衰期为14.5 d,表明TTL具有良好的热稳定性;TTL最适p H为8.0,在p H 7.0-8.5时稳定性好。TTL在50℃、p H 8.0条件下,催化100 mmol/L外消旋2-羧乙基-3-氰基-5-甲基己酸乙酯水解转化率达42%以上,产物的对映体过量值（e.e.值）〉94%。这表明TTL在化学-酶法合成普瑞巴林中具有较大应用潜力。（ 3S）-2-carboxyethyl-3-cyano-5-methylhexanoic acid is the key intermediate of pregabalin. In this study,a novel lipase（ TTL） was exploited from genome of Talaromyces thermophiles by gene mining for efficient synthesis of（ 3S）-2-carboxyethyl-3-cyano-5-methylhexanoic acid. TTL was overexpressed in Escherichia coli BL21 at low temperature. TTL was purified to electrophoretic homogeneity and characterized. The obtained TTL exhibited the maximum activity at 50 ℃ and showed excellent thermostability with a half-life of 14. 5 d.TTL was stable in the p H range from 7. 0 to 8. 5 and the optimal p H was 8. 0. Kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester by TTL was carried out at p H 8. 0,50 ℃.A conversion of 42% with e. e. 94% was obtained after 20 h at a substrate loading of 100 mmol / L. The obtained results demonstrate that TLL is a promising biocatalyst in chemoenzymatic synthesis of pregabalin.

关 键 词：嗜热踝节菌 脂肪酶 低温表达 酶学性质 普瑞巴林 

分 类 号：Q936[生物学—微生物学]