中文摘要：

海洋微生物是几丁质酶的重要来源。本研究从青岛海域海蜇（Rhopilema esculenta）体中分离到一株产几丁质酶的细菌QDC01，通过形态、培养特征以及16SrDNA序列同源性分析，将该菌株鉴定为嗜水气单胞菌（Aeromonas hydrophila）。根据已报道的嗜水气单胞菌几丁质酶基因相关序列设计引物，克隆了QDC01几丁质酶基因，命名为ahchi（GenBank：JX863407）。应用生物信息学软件对获得的基因序列及编码的蛋白序列进行分析，结果显示，ahchi开放阅读框（ORF）长2100bp，无内含子，其编码的蛋白由699个氨基酸组成，分子量为74．875kD，等电点为5．81。序列比对和同源性分析结果表明，该基因与嗜水气单胞（Aaeromonas hydrophila subsp．hydrophila）ATCC 7966T几丁质酶基因（GenBank登录号：CP000462）的相似性最高，为98％；相应的氨基酸序列同源性为98％。系统进化分析以及Ahchi的保守结构域分析表明，Ahchi属于Ⅰ型几丁质酶，糖苷水解酶19家族。采用限制性内切酶（BamHI和Hind Ⅲ）双酶切法构建了原核表达载体pET-30a（＋）．ahchi，并经IPTG诱导在大肠杆菌（Escherichia coli）中成功表达。在基础产酶发酵条件下，菌株QDC01所产粗酶液酶活力达0．21U／mL，采用文献报道的优化产酶发酵条件，菌株QDC01所产粗酶液酶活力可达0．58U／mL，是前人报道的产几丁质酶嗜水气单胞菌SWCH-6所产酶活力的1．49倍，同时也高于己报道的气单胞（Aeromonas sp．） CJ-5的酶活力（0．41U／mL），为微生物源几丁质酶开发应用提供了又一优良种质资源。

英文摘要：

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 openreading 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 was5.81. 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, hydrophilaATCC7966T 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 chitinaseand glycosyl hydrolase family 19. The prokaryotic expression vector pET-3Oa（＋）-ahchi was constructed by the method of restriction enzymes （BamH Ⅰ and Hind Ⅲ） and induced with IPTG, and the ahchi gene wassuccessfully 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 was0.58 U/mL which was much higher than that （0.39 U/mL） ofA. hydrophila strain SWCH-6 and also higher than that（0.41 U/mL） ofAeromonas 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.