中文摘要：

为获取全新来源并具有潜在高性能的海因酶，本研究通过对海洋沉积物进行以D-对羟基苯海因作为唯一氮源的选择培养基对潜在菌株进行初步筛选，然后通过双层琼脂法和微孔快速筛选法对初筛菌株进行复筛，并结合分子生物学筛选方法进行终筛，最终得到桔橙小单胞菌（Micromonosporaaurantiaca，GenBank登录号：FJ547135．1）、金色链霉菌（Streptomycesaureofaciens，GenBank登录号：AB326923．1）、桑氏链霉菌（Streptomyces sampsonii，GenBank登录号：GU238264．1）和链霉菌7．145（Streptomycessp．7—145，GenBank登录号：JQ782979．2）4株产D-海因酶的阳性链霉菌。用兼并引物扩增4株阳性菌中的D-海因酶表达序列，转化大肠杆菌（Escherichia coli），并构建表达相应D-海因酶的工程菌株E．coliS1、E．coliS14、E．coli$29和E．coliSl45，提纯4种D-海因酶，并测定酶的酶活和动力学参数。结果显示，链霉菌7．145菌株中表达的海因酶活性最高，比活力为9．7U／mg，催化速度常数Kcat=3．2×10-66／s，Kin=9．5mmol／L。最后利用Swiss—model软件对其进行在线同源模建和CaverAnalyst软件对链霉菌7—145菌株来源的海因酶的催化通道进行结构模拟分析。模拟分析结果显示，本研究中D-海因酶的主要催化通道Tunnel1长度为9．1A，瓶颈氨基酸残基为59位的组氨酸、181位的组氨酸和313位的谷氨酸，瓶颈半径为2．18A；潜在的催化通道Tunnel2长度为13．6A，瓶颈氨基酸为62位的苏氨酸、93位的天冬酰胺和107位的色氨酸，瓶颈半径为1．52A。如果对Tunnel2通道中的62位的苏氨酸、93位的天冬酰胺和107位的色氨酸进行定点突变，有望开发出一种性能更优的D-海因酶。本研究提供了一种全新的海因酶阳性菌筛选体系，通过计算机模拟手段能够更直观了解海因酶的催化机制，为进一步获得性能更优的海因酶奠定基础。

英文摘要：

In order to develop D-hydantoinase with high performance from brand new sources, this research regarded D-phydroxyphenyl hydantion separated primarily from marine Streptomyces library as the only N element to form the medium. Then double agar plate method and rnicroporous rapid screening method were used to do the second screening. At last, the final screening was done with the method of molecular biology. As a result, 4 positive Streptomyces of D-hydantoinase including Micromonospora aurantiaca （GenBank No. FJ547135.1）, Streptomyces aureofaciens （GenBank No. AB326923.1）, Streptomyces sampsonii （GenBank No. GU238264.1） and Streptomyces sp. 7- 145 （GenBank No. JQ782979.2） were obtained. Then the expression engineered strains of E.coli S1, E.coli S14 E.coli $29 and E.coli S145, by transforming Escherichia coli, secreting D-hydantoinase were built through degenerating primer and extending D-hydantoinases from the 4 positive bacterium. The 4 D-hydantoinases were purified, meanwhile the enzyme activity and kinetic parameters were also tested. The result showed that the enzyme from Streptomyces sp. 7-145 was the most active and its enzymatic compare energy was 9.7 U/mg, with Kcat~-3.2x 10-6/s and Km =9.5 mmol/L. Finally, the homology modeling online by Swiss-model and the simulation analysis of structure to the catalytic channel of D-hydantoinase through Caver Analyst were performed. According to simulation analysis, the length of the main catalytic channel, Tunnel_l, of D-hydantoinase was 9.1 /~. The amino acids residue of bottleneck were histidines of 59th and 181st sites, and glutamic acid of 313rd site, and neck radius was 2.18 ~. However, the length of the potential catalytic channel, Tunnel_2, was 13.6/~ long. The amino acids residue of bottleneck were the threonine of 62nd site, asparagine of 93rd site and tryptophan of 107th site, and neck radius was 1.52 A. Conducting site-directed mutagenesis to the threonine of 62nd site, asparagine of 93rd site and tryptophan of 107th site, might be more