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Abstract Bacterium QDC01 producing chitinase was isolated from a Rhopilema esculenta in Qingdao sea water. It was identified as Aeromonas hydrophila by morphological and cultural characteristics as well as 16S rDNA sequence analysis. The chitinase gene, named as ahchi (GenBank accession number: JX863407), was cloned from QDC01. Analysis of the gene sequence and its encoded protein sequence showed that the open reading frame (ORF) of ahchi was 2 100 bp in size and the chitinase gene encoded a protein composed of 699 amino acids. The molecular weight of Ahchi was 74.875 kD and the predicted theoretical isoelectric point was 5.81. Sequence alignment and homology analysis indicated that the gene ahchi had the highest similarity (98%) to the chitinase gene (GenBank accession number: CP000462) of A. hydrophila subsp. hydrophila ATCC7966T and the corresponding amino acid sequence homology was also 98%. The phylogenetic analysis of the conserved domains of Ahchi and related chitinases revealed that Ahchi belongs to the typeⅠchitinase and glycosyl hydrolase family 19. The prokaryotic expression vector pET-30a(+)-ahchi was constructed by the method of restriction enzymes (BamHⅠand HindⅢ) and induced with IPTG, and the ahchi gene was successfully expressed in Escherichia coli BL21. Grown in the basic fermentation medium, the chitinase activity of QDC01 was 0.21 U/mL, while in the optimized fermentation medium, the chininase activity was 0.58 U/mL which was much higher than that (0.39 U/mL) of A. hydrophila strain SWCH-6 and also higher than that(0.41 U/mL) of Aeromonas sp. strain CJ-5 reported as chininase producers in the previous literature, suggesting that A. hydrophila strain QDC01 is another good resource for producing chitinase.
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Received: 25 December 2012
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