固定化天冬氨酸酶制备(R)-3-氨基丁酸  

Preparation of(R)-3-aminobutyric acid by immobilized aspartase

作　　者：张飞龙 杨卫华 陈明亮 严燕兵 唐云平 肖延铭 ZHANG Feilong;YANG Weihua;CHEN Mingliang;YAN Yanbing;TANG Yunping;XIAO Yanming(Zhejiang Engineering Research Center of Industrial Biocatalysis and Transformation,Changxing Pharmaceutical Co.,Ltd.,Changxing,313100,China;School of Food and Pharmacy,Zhejiang Ocean University,Zhoushan 316022,China)

机构地区：[1]长兴制药股份有限公司工业生物催化与转化浙江省工程研究中心,浙江长兴313100 [2]浙江海洋大学食品与医药学院,浙江舟山316022

出　　处：《生物加工过程》2020年第5期556-560,共5页Chinese Journal of Bioprocess Engineering

基　　金：国家高技术研究发展计划(863计划)(2015AA021004)。

摘　　要：从芽孢杆菌Bacillus sp.YM55-1基因组中克隆得到天冬氨酸酶基因,以pET-28a(+)为载体构建天冬氨酸酶基因的表达载体pET-28a(+)-Asp,将天冬氨酸酶基因进行定点突变,在E.coli BL21(DE3)系统中实现了天冬氨酸酶的异源表达。利用重组的天冬氨酸酶,以氨水、(NH 4)2SO 4为辅料,将底物巴豆酸转化为(R)-3-氨基丁酸。将天冬氨酸酶的工程菌制备成固定化细胞,通过反应条件的优化研究,提高底物的转化率。结果表明:天冬氨酸酶最适pH为9.0,最适反应温度为40℃。在此反应条件下,加入30 g/L固定化细胞,转化22 h,(R)-3-氨基丁酸质量浓度达到220 g/L,对映体过量值e.e.s≥99.95%,底物转化率达到98%,固定化细胞重复使用次数不低于24次。Aspartate ammonia-lyase gene of Bacillus sp.YM55-1 was cloned into the expression vector pET-28a(+).By site-directed mutagenesis of aspartate ammonia-lyase gene,the heterologous expression was obtained in E.coli BL21(DE3)cells.Conversion of crotonic acid to(R)-3-aminobutyric acid was performed by the recombinant aspartate ammonia-lyase in the presence of ammonia and ammonium sulfate.The aspartate ammonia-lyase engineering bacteria were prepared into immobilized cells,and then enzymatic reaction conditions were optimized to achieve higher production of(R)-3-aminobutyric acid.The optimum pH and reaction temperature was 9.0 and 40℃,respectively.Under these conditions,immobilized cells of 30 g/L were added and incubated for 22 h.Finally,the production of(R)-3-aminobutyric acid was 220 g/L,enantiomeric excess was more than 99.95%,conversion up to 98%and immobilized cells could be reused no less than 24 times.

关 键 词：天冬氨酸酶 巴豆酸 (R)-3-氨基丁酸 固定化 

分 类 号：Q814.2[生物学—生物工程]