Cloning,Expression and Enzymatic Activities of chiA73 Gene from Bacillus thuringiensis

doi:10.13560/j.cnki.biotech.bull.1985.2015.08.021

Abstract

Abstract: In order to identify and detect chitinase activities from Bacillus thuringiensis strains, chitinase gene chiA73（GenBank accession number：KJ508093）from genomic DNA of Bt DLD171 producing high-yield chitinase was cloned by PCR. Gene of chitinase chiA73 was expressed and the weight of expression product was approximate 74 kD. The results measured by fluorogenic substrates showed that the expression product of chiA73 hydrolyzed 4-MU-（GlcNAc）₃only, but not 4-MU-GlcNAc. In conclusion, the work indicated that ChiA73 was one of endochitinase, and had the highest activity at pH8 and 40℃.

Key words: Bacillus thuringiensis, chitinase, chiA73, fluorogenic substrates, enzymatic activity

Zhang Yuan, Zhou Guowang, Li Haitao, Liu Rongmei, Zhao Yikui, Gao Jiguo. Cloning,Expression and Enzymatic Activities of chiA73 Gene from Bacillus thuringiensis[J]. Biotechnology Bulletin, 2015, 31(8): 147-152.

References

[1] 陈少波, 吴根福. 几丁质酶研究进展[J]. 科技通报, 2004（3）：258-262.
[2] Barboza-Corona JE, Nieto-Mazzocco E, Velazquez-Robledo R, et al. Cloning, sequencing, and expression of the chitinase gene chiA74 from Bacillus thuringiensis[J]. Applied and Environmental Microbiology, 2003, 69：1023-1029.
[3] 李力, 黄胜元, 关雄. 产几丁质酶的苏云金杆菌菌株筛选及酶合成条件研究[J]. 中国病毒学, 2000, S1：97-100.
[4] 孙慎侠, 付昌斌, 伦永志, 等. 芽胞杆菌菌株产几丁质酶发酵条件的研究[J]. 中国微生态学杂志, 2003（3）：20-22.
[5] 祁红兵, 刘铭, 李红敬. 产几丁质酶苏云金芽胞杆菌的筛选及其对甜菜夜蛾高毒菌株的增效活性[J]. 江苏农业科学, 2003（6）：61-63.
[6] 张利平. 几丁质酶及其在农业生产中的应用[J]. 浙江柑橘, 2013（2）：11-13.
[7] 张新军, 范丽卿, 岳海梅, 等. 几丁质酶产生菌发酵条件初步研究[J]. 中国农学通报, 2010, 24：42-46.
[8] 周樱, 王风平, 肖湘. 豚鼠气单胞菌CB101中几丁质酶基因的克隆及其异源表达的全酶蛋白和酶片段的功能研究[J]. 自然科学进展, 2002（8）：70-74.
[9] 关海宁, 徐桂花, 刁小琴. 微生物产几丁质酶的研究概况及其应用[J]. 中国食物与营养, 2006（8）：33-34.
[10] Chernin L, Ismailov Z, Haran S, et al. Chitinolytic Enterobacter agglomerans antagonistic to fungal plant pathogens[J]. Applied and Environmental Microbiology, 1995, 61：1720-1726.
[11] Susuki K, Sugawara N, Susuki M, et al. Chitinases A, B, and C1 of Serratia marcescens 2170 produced by recombinant Escherichia coli：Enzymatic properties and synergism on chitin degradation[J]. Bioscience, Biotechnology, and Biochemistry, 2002, 66：1075-1083.
[12] 魏巍, 贺淹才, 刘爱花. 几丁质酶酶活测定方法的研究进展[J]. 全国工业催化信息站, 2006, 14：11-14.
[13] 檀建新, 陈忠义, 张杰, 等. 产几丁质酶菌的分离鉴定及其抑菌作用的初步研究[J]. 植物保护, 2007, 27（2）：1-3.
[14] 卢伟, 赵秋敏, 陈艳玲, 等. 几丁质酶在苏云金芽胞杆菌中的分布及抑小麦赤霉菌菌株的筛选[J]. 南开大学学报：自然科学版, 2007（3）：97-101.
[15] Baroy F, Lecadet MM, Deleluse A. Cloning and sequencing of three new putative toxin genes from Clostridium bifermentans[J]. Gene, 1998, 211（2）：293-299.
[16] 乔贝贝, 裘梁, 董德刚, 等. 1株产几丁质酶菌株的鉴定[J]. 贵州农业科学, 2013, 41（7）：132-134.
[17] 宋福平, 张杰, 黄大昉, 等. 苏云金芽胞杆菌cry基因PCR-RFLP鉴定体系的建立[J]. 中国农业科学, 1998, 31（3）：13-18.
[18] 刘东明. 苏云金芽胞杆菌新型cry1基因克隆, 表达及活性分析[D]. 哈尔滨：东北农业大学, 2011.
[19] Barboza-Corona JE, Reyes-Rios DM, Salcedo-Hernández R, et al. Molecular and biochemical characterization of an endochitinase（ChiA-HD73）from Bacillus thuringiensis subsp. kurstaki HD-73[J]. Molecular Biotechnology, 2008, 39（1）：29-37.
[20] Ruiz-Sanchez A, Cruz-Camarillo R, Salcedo-Hernández R, et al. Molecular cloning andpurification of an endochitinase from Serratia marcescens（Nima）[J]. Molecular Biotechnology, 2005, 31：103-112.
[21] Chernin L, Ismailov Z, Haran S, et al. Chitinolytic enterobacter agglomerans antagonistic to fungal plant pathogens[J]. Appl Environ Microbiol, 1995, 61（5）：1720-1726.
[22] 蔡亚君, 袁志明, 胡晓敏, 等. 苏云金芽胞杆菌几丁质酶基因的克隆及诱导表达[J]. 湖北农业科学, 2011, 50（3）：599-603.
[23] 韩苗苗, 肖亮, 蔡峻, 等. 一株抑真菌、对甜菜夜蛾高效的苏云金芽胞杆菌菌株[J]. 微生物学通报, 2008, 35（11）：1750-1754.