Bacillus.sp.bs-1的内切葡聚糖酶在大肠杆菌中表达条件的优化及其活性分析  

OPTIMIZATION OF EXPRESSION CONDITIONS OF ENDOGLUCANASE OF Bacillus. sp. bs-1 in E. Coli AND ANALYSIS OF ITS ACTIVITY

作　　者：宝力德[1] 秦伟[1] 杨嘉萌[1] 邵影[1] 

机构地区：[1]内蒙古农业大学生命科学学院,呼和浩特010018

出　　处：《内蒙古农业大学学报（自然科学版）》2013年第5期77-82,共6页Journal of Inner Mongolia Agricultural University(Natural Science Edition)

基　　金：内蒙古农业大学项目(BJ07-69)

摘　　要：用两个不同的表达载体pET-30a-C和pET-28a-C进行了Bacillus.sp.bs-1的内切葡聚糖酶在大肠杆菌中的诱导表达,确定了最佳可溶性诱导表达条件。pET-30a-C表达的内切葡聚糖酶在25℃,用1mM的IPTG诱导3h就可以达到最佳可溶性表达量。pET-28a-C表达的内切葡聚糖酶在25℃,用0.8mM的IPTG诱导5h能够达到最佳可溶性表达量。对表达的内切葡聚糖酶活性进行了测定,并确定了最适反应条件。用Ni-NTA树脂纯化了表达的内切葡聚糖酶蛋白,得到单一的目的蛋白,并测定了纯化后的内切葡聚糖酶的比活力。pET-30a-C表达的内切葡聚糖酶的粗酶液活性为1489U/ml,比活力为723U/mg,pET-28a-C表达的内切葡聚糖酶的粗酶液活性为683U/ml,比活力为808U/mg。此结果为枯草芽胞杆菌内切葡聚糖酶在工业应用方面提供了理论依据。Using two different expression vectors pET - 30a - C and pET - 28a - C the endoglucanase of Bacillus. sp. bs - 1 have been expressed in E. Coli, and their optimal condition for the soluble over expression has been investigated. The expression of the endoglu- canase using expression vector pET -30a - C has achieved its best result at the condition d25~C, induction with lmM Of IPTG and ex- pression for 3 hours. The expression using the expression vector pET- 28a -C shows the best soluble over expression at the condition of 25~C, induction with 0.8ram of IPTG and expression for 5 hours. The activities of the expressed endoglucanases have been deter- mined, and the optimal reaction conditions of the enzyme have been investigated. The expressed endoglueanases were purified by Ni - NTA column, and the specific activities of purified endoglueanase have been determined. The enzyme expressed by pET - 30a - C shows the best volume activity of 1489U/ml with a specific activity of 723U/mg. The endoglucanase expressed by pET -28a - C shows a volume activity of 683U/ml. and shows a specific activity of 808U/rag. This result provides a theoretical basis and suggestion for the industrial applications of the endoglueanase.

关 键 词：内切葡聚糖酶 表达条件 纯化 

分 类 号：Q816[生物学—生物工程]