新型中性植酸酶在大肠杆菌中的高效表达、纯化及酶学性质

食品科学 ›› 2012, Vol. 33 ›› Issue (21): 153-156.

新型中性植酸酶在大肠杆菌中的高效表达、纯化及酶学性质

路国伟1，许伟2,*，邵荣2，云志1

1.南京工业大学化学化工学院 2.盐城工学院化学与生物工程学院

收稿日期:2011-09-17 修回日期:2012-09-21 出版日期:2012-11-15 发布日期:2012-11-09
通讯作者: 许伟 E-mail:xuweiyc@163.com
基金资助:
国家自然科学基金项目;江苏省高校自然科学研究项目

Expression of a New Neutral Phytase in Escherichia coli and Its Purification and Enzymatic Characterization

Received:2011-09-17 Revised:2012-09-21 Online:2012-11-15 Published:2012-11-09
Contact: XU Wei E-mail:xuweiyc@163.com

摘要/Abstract

摘要： 运用基因工程技术，将克隆到的淀粉液化芽孢杆菌Bacillus amyloliquefaciens DSM 1061新型中性植酸酶基因(GenBank登录号为HM747163)构建到表达载体pET22b(＋)上，并在E. coli BL21(DE3)中进行高效表达。电泳结果表明该酶分子质量约为42kD，目的蛋白占大肠杆菌可溶性蛋白30%左右。利用Ni-NTA琼脂糖凝胶进行亲和纯化，酶学性质研究表明，其最适温度为60℃，最适pH值为7.0，最大酶比活力为15U/mg。60℃时以植酸钠为底物的Km值为0.30mmol/L。

关键词: 淀粉液化芽孢杆菌, 高效表达, 中性植酸酶, 酶学性质

Abstract: A new neutral phytase gene (phyc) from Bacillus amyloliquefaciens DSM 1061 (GenBank: HM747163) was cloned into expression vector pET22b(+). The phytase was expressed in Escherichia coli BL21(DE3). The molecule weight of the phytase protein was about 42 kD determined by SDS-PAGE. The expressed phytase was about 30% of the total soluble protein of E. coli. The recombinant protein was purified by 6×His-Tagged Protein Purification Kit. Optimal pH value and temperature of the phytase were 7.0 and 60 ℃, respectively. Under the optimum conditions, the activity was 15 U/mg. The Km value of the phytase for hydrolysis of sodium phytate under 60 ℃ was 0.30 mmol/L.

Key words: Bacillus amyloliquefaciens, overexpression, neutral phytase, enzymatic properties

中图分类号:

Q814

路国伟1，许?伟2,*，邵?荣2，云?志1. 新型中性植酸酶在大肠杆菌中的高效表达、纯化及酶学性质[J]. 食品科学, 2012, 33(21): 153-156.

参考文献

[1]. Singh B, Kunze G, Satyanarayana T. Developments in biochemical aspects and biotechnological applications of microbial phytases. Biotechnol Mol Biol Rev, 2011, 6(3): 69-87.
[2]. Fu S J, Sun J Y, Qian L C, et al. Bacillus phytases: present scenario and future perspectives. Appl Biochem Biotechnol, 2008, 151(1): 1-8.
[3]. Ling C, Wang W M,Yang C G, et al. Application of microbial phytase in fish feed. Enzyme and Microbial Technology, 2007, 40(4): 497-507.
[4]. Roongsawang N, Promdonkoy P, Wongwanichpokhin M, et al. Coexpression of fungal phytase and xylanase utilizing the cis-acting hydrolase element in Pichia pastoris. FEMS Yeast Res , 2010, 10(5): 909-916.
[5]. Viader-Salvadó J M, Gallegos-López J A, Carreón-Trevino J G, et al. Design and overproduction of thermostable beta-propeller phytases in Pichia pastoris with a broad pH activity range. Appl Environ Microbiol , 2010, 76(2): 6423-6430.
[6]. Tran T T, Hashim S O, Gaber Y, et al. Thermostable alkaline phytase from bacillus sp. MD2: effect of divalent metals on activity and stability. J Inorg Biochem, 2011, 105(7): 1000-1007.
[7]. Gulati H K, Chadha B S, Saini H S. Production and characterization of thermostable alkaline phytase from bacillus laevolacticus isolated from rhizosphere soil. J Ind Microbiol Biotechnol , 2007, 34(4): 91–98.
[8]. Kim Y O, Lee J K, Kim H K, et al. Cloning of the thermostable phytase gene (phy) from Bacillus sp. DS11 and its overexpression in Escherichia coli. FEMS Microbial Lett, 1998, 162(1): 185-191.
[9]. Kerovuo J, Tynkkynen S. Expression of Bacillus subtilis phytase in Lactocacillus plantarum 755. Letters in Applied Microbiology, 2000, 30(4): 325-329.
[10]. 李朝霞, 王爱民, 李小敏. 中性植酸酶高产菌株的筛选及产酶条件研究. 微生物学通报, 2007, 34(4): 633-641.
Li Z X, Wang A M, Li X M. Studies on screening and conditions of strains highly producing neutral phytase. Microbiology, 2007, 34(4): 633-641.
[11]. Miksch G, Kleist S, Friehs K, et al. Overexpression of the phytase from Escherichia coli and its extracellular production in bioreactors. Appl Microbiol Biotechnol. 2002, 59(1): 685-694.
[12]. Guo M J, Hang H F, Zhu T C, et al. Effect of glycosylation on biochemical characterization of recombinant phytase expressed in Pichia pastoris. Enzyme and Microbial Technology , 2008, 42(4): 340-345.
[13]. Liang J F, Han B Z, Robert Nout M J, et al. Effect of soaking and phytase treatment on phytic acid, calcium, iron and zinc in rice fractions. Food Chemistry, 2009, 115(3): 789-794.
[14]. 姚斌，袁铁铮，王元火，等.来源于Bacillus subtilis的中性植酸酶基因的克隆及在大肠杆菌的表达. 生物工程学报，2001，17 (1)：11-15.
Yao B, Yuan T Z, Wang Y H, et al.Cloning of neutral phytase gene nphy from bacillus subtilis and its expressions in Escherichia coli. Chinese Journal of Biotechnology, 2001, 17 (1): 11-15.
[15].陈艳，孙健义，赵学新，等.Bacillus amyloliquef aciens 中性植酸酶基因的原核表达及蛋白纯化和性质. 食品与生物技术学报，2005，24(2): 60-65.
Chen Y, Sun J Y, Zhao X X, et al. Expression of neutral phytase gene from bacillus amyloliquef aciens in Escherichia coli. Journal of Food Science and Biotechnology, 2005, 24(2): 60-65.
[16]. Rao D E, K.V.Rao, V.D.Reddy. Cloning and expression of Bacillus phytase gene (phy) in Escherichia coli and recovery of active enzyme from the inclusion bodies. Journal of Applied Microbiology. 2008, 105(3): 1128-1137.
[17]. 许伟，仇明，余晓红，等.芽孢杆菌植酸酶基因的克隆及生物信息学分析. 食品科学，2011，32(17)：202-206.
Xu W, Qiu M, Yu X H, et al. Gene cloning and bioinformatics analysis of phytase from bacillus amyloliquefaciens. Food Science, 2011, 32(17): 202-206.
[18]. Sambrook J, Fristch E F, Maniatis T. Molecular Cloning: A Laboratory Manual.2 rd ed. New York: Cold Spring Harbor Laboratory Press, 1989.
[19]. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976, 72(1): 248-254.