海洋链霉菌<em>Streptomyces olivaceus</em> FXJ7.023来源多功能几丁质酶的克隆、表达及鉴定

doi:10.13523/j.cb.20140808

中国生物工程杂志

2014, Vol. 34

Issue (8): 47-53 DOI: 10.13523/j.cb.20140808

研究报告

海洋链霉菌Streptomyces olivaceus FXJ7.023来源多功能几丁质酶的克隆、表达及鉴定

岳昌武¹, 李园园¹, 吕玉红¹, 王苗¹, 邵美云¹, 刘明皓², 黄英²

1. 遵义医学院医学与生物学研究中心贵州省微生物资源及药物开发特色重点实验室遵义 563003;
2. 中国科学院微生物研究所微生物资源前期开发国家重点实验室北京 100101

Isolation, Expression and Identification of Multifunctional Chitosanase from Marine Streptomyces olivaceus FXJ7.023

YUE Chang-wu¹, LI Yuan-yuan¹, LV Yu-hong¹, WANG Miao¹, SHAO Mei-yun¹, LIU Ming-hao², HUANG Ying²

1. Guizhou Key Laboratory of Microbial Resources & Drug Development, Zunyi Medical College, Zunyi 563003, China;
2. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China

全文: PDF(737 KB) HTML

摘要：

碳水化合物水解酶家族在自然界碳素循环及农业废弃物中几丁质、纤维素等碳水化合物的生物质转化利用中发挥了重要作用。通过PCR技术从海洋链霉菌Streptomyces olivaceus strain FXJ 7.023 的fosmid基因组文库中成功克隆得到1个全长885bp的编码295个氨基酸残基的包含1个19个氨基酸残基的N-末端信号肽的壳聚糖酶完全编码区。系统进化分析表明该基因编码蛋白与已报道的Streptomyces sp. SirexAA-E来源壳聚糖酶csnA同源性为71%，与Streptomyces coelicolor A3（2）来源的csn46A同源性为70%。将该编码区重组入原核表达质粒载体pET32a并转化大肠杆菌表达菌株BL21（DE3）plysS，添加IPTG 在18℃条件下振荡诱导该蛋白表达，Ni²⁺-NTA亲和纯化获得分子量为50.3 kDa融合表达蛋白TrxA-SoCsn。该融合重组蛋白在最适反应条件下对底物胶体壳聚糖和羧甲基纤维素的最大酶活分别为3.673U/mg和1.302U/mg，最适反应温度分别为37℃和50℃，最适反应pH分别为pH5.0和pH6.0。TrxA-SoCsn相关的研究结果表明该酶在农业废弃物生物质转化等方面具有一定的应用潜力。

关键词： 几丁质酶; 海洋链霉菌; 异源表达; 生物催化

Abstract:

A predicted chitosanase was cloned from a fosmid genomic library of Streptomyces olivaceus strain FXJ 7.023 by PCR. The coding fragment of 885 bp encodes of 295 amino acids was fused to the expression vector pET32a (+) and transformed into Escherischia coli strains BL21 (DE3) plysS. A novel fusion protein with molecular weight of 50.3 kDa was obtained by induced the engineered strain with 1mmol/L IPTG under 18℃ and purified with the affinity chromatography of Ni²⁺-NTA. The recombinant chitosanase showed multifunctional catalytic activity of hydrolyze colloidal chitosan and carboxymethylcellulose with the maximal catalytic activity of 3.673U/mg and 1.302U/mg respectively. Due to it has multifunctional catalytic activity, such protein may have a potential application for the recycling of the carbohydrates and sugars in the waste.

Key words: Chitosanase Marine streptomycete Heterologous expression Biocatalysis

收稿日期: 2014-06-12 出版日期: 2014-08-25

ZTFLH:

Q786

基金资助:

国家基础研究规划（2011CB808800）、国家自然科学基金（31160004）、国家海洋矿物资源研究与开发协会（DY125-15-R-02）、贵州省科学技术基金[（2010）2156，（2012）2348]资助项目

通讯作者: 黄英，E-mail：huangy@im.ac.cn；岳昌武，E-mail：changwuyue@hotmail.com E-mail: huangy@im.ac.cn；changwuyue@hotmail.com

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岳昌武, 李园园, 吕玉红, 王苗, 邵美云, 刘明皓, 黄英. 海洋链霉菌Streptomyces olivaceus FXJ7.023来源多功能几丁质酶的克隆、表达及鉴定[J]. 中国生物工程杂志, 2014, 34(8): 47-53.

YUE Chang-wu, LI Yuan-yuan, LV Yu-hong, WANG Miao, SHAO Mei-yun, LIU Ming-hao, HUANG Ying. Isolation, Expression and Identification of Multifunctional Chitosanase from Marine Streptomyces olivaceus FXJ7.023. China Biotechnology, 2014, 34(8): 47-53.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140808 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I8/47

[1] Zhu X F, Tan H Q, Zhu C, et al. Cloning and overexpression of a new chitosanase gene from Penicillium sp. D-1. AMB Express, 2013, 2(1):13.

[2] Thadathil N, Velappan S P. Recent developments in chitosanase research and its biotechnological applications: A review. Food Chem, 2014, 150:392-399.

[3] Zhang J, Sun Y.Molecular cloning, expression and characterization of a chitosanase from Microbacterium sp. Biotechnol Lett, 2007, 29(8):1221-1225.

[4] Saito A, Ooya T, Miyatsuchi D, et al. Molecular characterization and antifungal activity of a family 46 chitosanase from Amycolatopsis sp. CsO-2. FEMS Microbiol Lett, 2009, 293(1):79-84.

[5] Jiang, X, Chen D, Chen L, et al. Purification, characterization and action mode of a chitosanase from Streptomyces roseolus induced by chitin. Carbohydrate Research, 2012, 355:40-44.

[6] Heggset E B, Tuveng T R, Hoell I A, et al. Mode of action of a family 75 chitosanase from Streptomyces avermitilis. Biomacromolecules, 2012, 13(6):1733-1741.

[7] Lacombe-Harvey M E, Fortin M, Ohnuma T, et al. A highly conserved arginine residue of the chitosanase from Streptomyces sp. N174 is involved both in catalysis and substrate binding.BMC Biochem, 2013, 14:23.

[8] Takasuka T E, Bianchetti C M, Tobimatsu Y, et al.Structure-guided analysis of catalytic specificity of the abundantly secreted chitosanase SACTE_5457 from Streptomyces sp. SirexAA-E.Proteins. DOI:10.1002/prot.24491.

[9] Somashekar D, Joseph R. Chitosanases properties and applications: A review. Bioresource Technology, 1996, 55(1):35-45.

[10] Kouzai Y, Mochizuki S, Saito A, et al.Expression of a bacterial chitosanase in rice plants improves disease resistance to the rice blast fungus Magnaporthe oryzae. Plant Cell Reports, 2012, 31(4):629-636.

[11] Zhang H, Sang Q, Zhang W. Statistical optimization of chitosanase production by Aspergillus sp. QD-2 in submerged fermentation. Annals of Microbiology, 2012, 62(1):193-201.

[12] Wang S L, Tseng W N, Liang T W. Biodegradation of shellfish wastes and production of chitosanases by a squid pen-assimilating bacterium, Acinetobacter calcoaceticus TKU024. Biodegradation, 2011, 22(5):939-948.

[13] Shirling E B, Gottlieb D. Methods for characterization of Streptomyces species. Int J Syst Bacteriol, 1966, 16:3313-3340.

[14] Sambrook J, Russel D W. Molecular Cloning. 3^rd ed. New York:Cold Spring Harbor Laboratory Press, 2001.

[15] Altschul S F, Madden T L, Schäffer A A, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res, 1997, 25:3389-3402.

[16] Larkin M A, Blackshields G, Brown N P, et al.ClustalW and ClustalX version 2.0. Bioinformatics, 2007, 23(21):2947-2948.

[17] Tamura K, Stecher G, Peterson D, et al. MEGA 6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 2013, 30: 2725-2729.

[18] Petersen T N, Brunak S, von Heijne G, et al. Signal P4.0: discriminating signal peptides from transmembrane regions. Nature Methods, 2011, 8:785-786.

[19] Marchler-Bauer A, Zheng C, Chitsaz F, et al.CDD: conserved domains and protein three-dimensional structure. Nucleic Acids Res, 2013, 41:348-352.

[20] Wilkins M R, Gasteiger E, Bairoch A, et al.Protein identification and analysis tools in the ExPASy server. Methods Mol Biol, 1999, 112:531-552.

[21] Cantarel B L, Coutinho P M, Rancurel C, et al.The carbohydrate active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res, 2009, 37:233-238.

[22] Marcotte E M, Monzingo A F, Ernst S R, et al. X-ray structure of an anti-fungal chitosanase from Streptomyces N174. Nat Struct Biol, 1996, 3(2):155-162.

[23] Monzingo A F, Marcotte E M, Hart P J, et al.Chitinases, chitosanases, and lysozymes can be divided into procaryotic and eucaryotic families sharing a conserved core. Nat Struct Biol, 1996, 3(2):133-140.

[24] Robertus J D, Monzingo A F, Marcotte E M, et al. Structural analysis shows five glycohydrolase families diverged from a common ancestor. J Exp Zool, 1998, 282(1-2):127-132.

[25] Fukamizo T. Chitinolytic enzymes: catalysis, substrate binding, and their application. Curr Protein Pept Sci, 2000, 1(1):105-124.

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