Identification of a novel endochitinase from a marine bacterium Vibrio proteolyticus strain No. 442.
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 alpha-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.
Itoi
Shiro
S
Department of Marine Science and Resources, Nihon University, Fujisawa, Kanagawa 252-8510, Japan. sitoi@brs.nihon-u.ac.jp
Kanomata
Yuna
Y
Koyama
Yuki
Y
Kadokura
Kazunari
K
Uchida
Shinsuke
S
Nishio
Toshiyuki
T
Oku
Tadatake
T
Sugita
Haruo
H
D000595:Amino Acid Sequence; D002688:Chitinases / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D014733:Vibrio / Q000201:enzymology*
D009844:Oligosaccharides; D011994:Recombinant Proteins; C050523:chitohexaose; D002688:Chitinases