节杆菌BT801N-氨甲酰氨基酸水解酶基因的克隆与表达  

Cloning and Expression of L-N-Carbamoylase Gene from Arthrobacter BT801 in Escherichia coli

作　　者：郝淑凤[1] 张惟材[1] 李迎丽[1] 袁红杰[1] 黄留玉[1] 

机构地区：[1]军事医学科学院生物工程研究所

出　　处：《生物工程学报》2003年第2期174-177,共4页Chinese Journal of Biotechnology

摘　　要：通过PCR从质粒pUC18 16 9中扩增得到N 氨甲酰氨基酸水解酶基因 (hyuC) ,置于原核表达载体pQE6 0的T5启动子下游构成表达质粒pQE6 0 hyuC ,并在大肠杆菌M15中实现了该基因的高表达。SDS PAGE检测表达产物 ,在相对分子量 44kD处有一表达带 ,经薄层扫描分析目的蛋白占全菌蛋白的 40 % ,主要以可溶性形式存在。酶活性分析结果表明 ,工程菌M15 pQE6 0 hyuC的N 氨甲酰氨基酸水解酶的比活分别比原始菌株ArthrobacterBT80 1和亚克隆DH5α pUC18 16 9提高了 5 2倍和 72倍。在节杆菌BT80 1和大肠杆菌DH5α pUC18 16 9的反应体系中加入等量菌体的工程菌M15 pQE6 0 hyuC ,可使乙内酰脲酶总比活分别提高 8 1倍和 3 0倍。Hydantoin-utility-enzyme is widely used in enzymic production of various amino acids.One of its component,carbamoylase,is responsible for the conversion of N-carbamylamino acids to corresponding amino acids,which is crucial for the stereoselectivity and rate limiting.To improve the production of the enzyme,an L-N-carbamoylase gene from Arthrobacter BT801,a hydantoinase producting strain being able to convert 5-benzylhydantoin to phenylalanine,was cloned into E.coli. The gene was highly expressed in E.coli M15 under control of T5 promoter.A protein band about 44kD was detected by SDS-PAGE in the recombinant cell lysate.The objective product,which is principally in soluble form,represented 40% of total cell protein.The N-carbamoylase specific activity of the recombinant M15/pQE60-hyuC is 53 times higher than that of Arthrobacter BT801.The total biotransformation activity increased 8.1 times when.M15/pQE60-hyuC was added into the Arthrobacter BT801 reaction system.The successful expression of the enzyme is significant for the application of the hydantoinase producing strain or the enzyme thereof.

关 键 词：N-氨甲酰氨基酸水解酶 基因表达 苯丙氨酸 克隆 节杆菌 

分 类 号：Q78[生物学—分子生物学]