农业生物技术学报2013，Vol.21Issue(6)：734-744,11.
海洋细菌QDC01的鉴定及其几丁质酶基因的克隆与分析
Identification of Chitinase-producing Marine Bacterium QDC01 and Cloning and Analysis of the Chitinase Gene
摘要
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 699amino 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 type Ⅰ chitinase and glycosyl hydrolase family 19.The prokaryotic expression vector pET-30a(+)-ahchi was constructed by the method of restriction enzymes (BamH Ⅰ and Hind Ⅲ) 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 C J-5 reported as chininase producers in the previous literature,suggesting that A.hydrophila strain QDC01 is another good resource for producing chitinase.关键词
嗜水气单胞菌/分类鉴定/几丁质酶基因/克隆与分析Key words
Aeromonas hydrophila/ Taxonomic indentification/ Chitinase gene/ Clone and sequence analysis引用本文复制引用
王振东,罗春艳,杨晨,张殿朋,吴慧玲,刘伟成..海洋细菌QDC01的鉴定及其几丁质酶基因的克隆与分析[J].农业生物技术学报,2013,21(6):734-744,11.基金项目
北京市农林科学院科技创新基金(No.KJCX201101001)、北京市科技计划课题(No.Z121100001212002)和北京市自然科学基金项目(No.6101001) （No.KJCX201101001）
