Identification of a novel endochitinase from a marine bacterium Vibrio proteolyticus strain No. 442

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• 作者：Shiro Itoi ; Yuna Kanomata ; Yuki Koyama ; Kazunari Kadokura ; Shinsuke Uchida ; Toshiyuki Nishio ; Tadatake Oku ; Haruo Sugita
• 关键词：Chitinase ; Chitinolytic activity ; Marine bacteria ; Vibrio proteolyticus
• 刊名：Biochimica et Biophysica Acta - Proteins and Proteomics
• 出版年：2007
• 期刊代码：161_15709639
• 类别：bio
• 出版时间：September 2007
• 卷：1774
• 期：9
• 页码：1099-1107
• 文件大小：1466 K

摘要

Chitin binding proteins prepared from Vibrio proteolyticus were purified and the N-terminal amino-acid sequence of a protein from a 110-kDa band on SDS-PAGE was found to be 85–90%identical to the 22nd–41st residues of the N-termini of chitinase A precursor proteins from other vibrios. We cloned the corresponding gene, which encodes a putative protein of 850 amino acids containing a 26-residue signal sequence. The chitinase precursor from V. proteolyticus was 78–80%identical to those from Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio carchariae. However, the proteolytic cleavage site for C-terminal processing between R⁵⁹⁷ and K⁵⁹⁸ in the chitinase precursor of other vibrios was not observed in the amino acid sequence of V. proteolyticus, which instead had the sequence R⁶⁰⁰ and A⁶⁰¹. Subsequently, full-length and truncated chitinases were generated in Escherichia coli. The specific activity of full-length chitinase expressed in E. coli was 17- and 20-folds higher for colloidal and α-chitins (insoluble substrate), respectively, than that of the C-terminal truncated enzyme. However, both recombinants showed similar hydrolysis patterns of hexa-N-acetyl-chitohexaose (soluble substrate), producing di-N-acetyl-chitobiose as major product on TLC analysis. We showed that the C-terminus of the V. proteolyticus chitinase A was important for expression of high specific activity against insoluble chitins.