Salinivibrio sp.YH4胞外蛋白酶EYHⅠ的分离纯化、酶学性质及基因克隆

doi:10.6040/j.issn.1671-9352.0.2015.609

山东大学学报（理学版） ›› 2016, Vol. 51 ›› Issue (5): 36-42.doi: 10.6040/j.issn.1671-9352.0.2015.609

Salinivibrio sp.YH4胞外蛋白酶EYHⅠ的分离纯化、酶学性质及基因克隆

武翠玲^1,2,刘丹¹,杨兴昊¹,吴日帮¹,黄嘉丰¹,张姜¹,伦梓丰¹,何海伦^1*

1. 中南大学生命科学学院, 医学遗传学国家重点实验室, 湖南长沙 410013;2.长治医学院生物化学教研室, 山西长治 046000

收稿日期:2015-12-17 出版日期:2016-05-20 发布日期:2016-05-16
通讯作者: 何海伦(1976— ),女,博士,教授,博士生导师,研究方向为微生物酶学.E-mail: helenhe@csu.edu.cn E-mail:clw928@163.com
作者简介:武翠玲(1979— ),女,硕士,讲师,研究方向为蛋白质结构与功能. E-mail:clw928@163.com
基金资助:
国家自然科学基金资助项目(31370104);国家星火计划面上项目(2013GA770009);长治医学院科研启动基金项目(QDZ201517)

Purification, characterization and gene cloning of the extracellular protease EYHⅠ from Salinivibrio sp.YH4

WU Cui-ling^1,2, LIU Dan¹, YANG Xing-hao¹, WU Ri-bang¹, HUANG Jia-feng¹, ZHANG Jiang¹, LUN Zi-feng¹, HE Hai-lun^1*

1.School of Life Sciences, State Key Laboratory of Medical Genetics, Central South University, Changsha 410013, Hunan, China;
2. Department of Biochemistry, Changzhi Medical College, Changzhi 046000, Shanxi, China

Received:2015-12-17 Online:2016-05-20 Published:2016-05-16

摘要/Abstract

摘要： 从中度嗜盐菌Salinivibrio sp.YH4发酵的粗酶液中分离纯化出蛋白酶EYHⅠ,对其进行酶学性质分析、串联质谱鉴定及全基因克隆。结果表明, EYHⅠ属于金属蛋白酶,最适温度55 ℃,热稳定性较好。最适pH为9.0,碱性条件下较稳定;在4 mol/L 的NaCl溶液中EYHⅠ仍保持较高活性,EYHⅠ全基因序列共1 836 bp,蛋白序列共611个氨基酸。比对发现EYHⅠ氨基酸序列与Salinivibrio sp. AF-2004所产Zn金属蛋白酶前体(ABI93183)同源性最高(96%)。结构分析表明,EYHⅠ由一个FTP结构域,一个PepSY结构域,一个M4中性蛋白酶和一个PPC结构域组成。本研究为嗜盐菌及其胞外蛋白酶的生产和应用奠定了理论基础。

关键词: Salinivibrio sp.YH4, 分离纯化, 基因克隆, 蛋白酶, 酶学性质

Abstract: The extracellular proteases from moderate halophilic strain, Salinivibrio sp. YH4 was isolated, purified, identified and characterized to provide data for enzyme system of Salinivibrio sp.. Strain was cultured in liquid fermentation medium, the extracellular proteases was purified by using HiTrap Capto DEAE column chromatography and Gel filtration chromatography. The protease was identified by using mass spectrometry(MS)and designated as EYHⅠ. The metalloprotease EYHⅠ had optimal activity at 55 ℃ and pH 9.0, stabilized at 60 ℃ and pH 7.0~11.0. EYHⅠshowed higher proteolytic activity at 4 mol/L NaCl. The nucleotide sequence contains 1836 bp, encoding EYHⅠof 611 amino acid residues, which is highly conserved to zinc metalloprotease precursor in Salinivibrio sp. AF-2004 with 96% sequence similarity. Analysis of the structure showed that EYHⅠcontained an FTP domain,a PepSY domain, M4 neutral protease and a PPC domain. These results may provide important data for the study of Salinivibrio sp. and the application of protease EYHⅠ.

Key words: purification, enzymic properties, gene cloning, Salinivibrio sp., proteases

中图分类号:

Q936

武翠玲,刘丹,杨兴昊,吴日帮,黄嘉丰,张姜,伦梓丰,何海伦. Salinivibrio sp.YH4胞外蛋白酶EYHⅠ的分离纯化、酶学性质及基因克隆[J]. 山东大学学报（理学版）, 2016, 51(5): 36-42.

WU Cui-ling, LIU Dan, YANG Xing-hao, WU Ri-bang, HUANG Jia-feng, ZHANG Jiang, LUN Zi-feng, HE Hai-lun. Purification, characterization and gene cloning of the extracellular protease EYHⅠ from Salinivibrio sp.YH4[J]. JOURNAL OF SHANDONG UNIVERSITY(NATURAL SCIENCE), 2016, 51(5): 36-42.

参考文献

[1] LI Qing, LI Yi, MAREK P, et al. Commercial proteases: present and future [J]. FEBS Letters, 2013, 587(8):1155-1163.
[2] RAO M B, TANKSALE A M, GHATGE M S, et al. Molecular and biotechnological aspect of microbial protease[J]. Microbiol Mol Bio Rev, 1998, 62(3):597-635.
[3] GUPTA R, BEG Q K, LORENZ P. Bacterial alkaline proteases: molecular approaches and industrial applications [J]. Appl Microbiol Biotechnol, 2002, 59(1):15-32.
[4] SHIKHA, SHARAN A, DARMWAL N S. Improved production of alkaline protease from a mutant of alkalophilic Bacillus pantotheneticus using molasses as a substrate [J]. Bioresour Technol, 2007, 98(4):881-885.
[5] RAVICHANDRA P, SUBHAKAR C, ANNAPURNA J. Alkaline protease production by submerged fermentation in stirred tank reactor using Bacillus licheniformis NCIM-2042: effect of aeration and agitation regimes[J]. Biochem Eng J, 2007, 34(2):185-192.
[6] VENTOSA A, NIETO J J, OREN A. Biology of moderately halophilic aerobic bacteria [J]. Microbiol Mol Biol Rev, 1998, 62(2): 504-544.
[7] CRISTINA S P, ENCARNACION M, ANTHONY P P, et al. The haloprotease cpⅠproduced by the moderately halophilic bacterium pseudoalteromonas ruthenica is secreted by the typeⅡ secretion pathway[J]. Applied and Environmental Microbiology, 2009, 75(12):4197-4201.
[8] 东秀珠,蔡妙英. 常见细菌系统鉴定手册[M]. 北京:科学出版社,2001:370-390. DONG Xiuzhu, CAI Miaoying. Manual of Systemmatic Bacteriology[M]. Beijing: Science Press, 2001:370-390.
[9] LIU Dan, YANG Xinghao, HUANG Jiafeng, et al. In situ demonstration and characteristic analysis of the protease components from marine bacteria using substrate immersing zymography[J]. Appl Biochem Biotechnol, 2015, 175(1):489-501.
[10] LIU Yaoguang, WHITTIER R F. Thermal asymmetric interlaced PCR: Automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking[J]. Genomics, 1995, 25(3):674-681.

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Salinivibrio sp.YH4胞外蛋白酶EYHⅠ的分离纯化、酶学性质及基因克隆

Purification, characterization and gene cloning of the extracellular protease EYHⅠ from Salinivibrio sp.YH4

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