Abstract： 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 microporous 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 S29 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=3.2×10-6/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_1, 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 ?. Conducting site-directed mutagenesis to the threonine of 62nd site, asparagine of 93rd site and tryptophan of 107th site, might be more likely to develop the better D-hydantoinase. In conclusion, this study provides a new screening system, understanding the catalyst mechanism of hydantoinase more intuitively through computer simulation, which lays a firm foundation for getting D-hydantoinase with better performance and the industrial use of the D-hydantoinase.
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