Gene Sequence, Soluble Expression and Homologous Comparison of a D-Hydantoinase fromPseudomonas putida YZ-26

作     者：SHI Ya-wei ZHAO Li-xia NIU Li-xi FENG Xia YUAN Jing-ming 

作者机构：Key Laboratory for Chemical Biology and Molecular Engineering of Ministry of Education Institute of Biotechnology Shanxi University Taiyuan 030006 P. R. China 

出 版 物：《Chemical Research in Chinese Universities》 (高等学校化学研究（英文版）)

年 卷 期：2005年第21卷第5期

页      面：552-557页

核心收录：

学科分类：081704[工学-应用化学] 07[理学] 070304[理学-物理化学(含∶化学物理)] 08[工学] 0817[工学-化学工程与技术] 070303[理学-有机化学] 0703[理学-化学] 

基　　金：Supported by the Natural Science Foundation of Shanxi Province(No.20031042) 

主　　题：D-Hydantoinase Gene sequence Soluble expression Homologous comparison Purification Mass spectrum 

摘      要：A 1440bp open-reading frame encoding D-hydantoinase from Pseudomonas putida YZ-26 was cloned and sequenced（ GenBank AY387829）. The DNA fragment was inserted into Nde and BamHI sites of vector pET-3a, yielding a recombinant plasmid, pET-HDT. After being transferred into the host strain, the artificial strain, pET-HDT/ E. coli BL21 can express the D-hydantoinase as the soluble form in the Lura-Bertani medium without addition of any inducers. The activity of the enzyme toward substrate DL-hydantoin can reach 3000-4000 IU per cells from one-liter bacterial culture incubated at 30 ℃ for 10-12 h. By the comparison of amino acid sequence homology, hydrophobic residues analysis and secondary structure prediction, it was found that D-hydantoinase reported herein is quite similar to that from Pseudomonas putdia CCRC12857, and alike to that from Pseudomonas putdia DSM84 or other bacteria. A rapid and efficient purification procedure of the enzyme was performed by a three-step procedure： ammonium sulfate fractionation, phenyl Sepharose hydrophobic interaction chromatography and Sephacryl S-200 gel filtration. The molecular mass of the monomeric enzyme is 52042 Da as determined by MALDI-TOF mass spectrometry.