乙内酰脲水解酶基因在大肠杆菌中的克隆表达

Expression of Hydantoin Hydrolase Gene in Escherichia coli

节杆菌BT80 1的乙内酰脲酶系能够水解 5 苄基乙内酰脲生成L 苯丙氨酸 ,其中乙内酰脲水解酶负责乙内酰脲的水解开环。乙内酰脲水解酶的表达对于乙内酰脲酶的催化机制研究及氨基酸的生物不对称合成都具有重要意义。通过PCR技术扩增得到乙内酰脲水解酶基因 (hyuH) ,置于表达载体pT2 2 1的T7启动子下游 ,将构建的重组质粒引入大肠杆菌BL2 1(DE3)。SDS PAGE分析在相对分子量 5 0kD处有一较强的表达带 ,经薄层扫描分析目的蛋白占全菌蛋白的 4 0 % ,主要以可溶性形式存在 。

Hydantoin hydrolase with responsibility for the ring opening of hydantoin is one of the components of hydantoin utility enzymes of Arthrobacter BT801 which can convert 5-benzylhydantoin into L-phenylalanine. The expression of hydantoin hydrolase gene (hyuH) is very important in elucidation of mechanisms of bio-catalysis and its application in asymmetry synthesis of amino acids. To improve the production and activity of the enzyme, the hydantoin hydrolase gene was amplified by PCR and cloned into E. coli by using vector pT221. The hydantoin hydrolase gene was highly expressed in E. coli BL21(DE3) under the control of T7 promoter. A protein band about 50kD was detected by SDS-PAGE in the recombinant cell lysate. The objective protein in BL21(DE3)/pT221-hyuH accounted for 40% of total cellular protein, mostly in soluble form. The products in the recombinant strain showed biological activity.