alexa Cloning and Characterization of Physarum Endo-Beta-1,3-1,4-Glucanase-1 Expression in E. coli and Trichoderma reesei | OMICS International | Abstract
ISSN: 2155-952X

Journal of Biotechnology & Biomaterials
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Research Article

Cloning and Characterization of Physarum Endo-Beta-1,3-1,4-Glucanase-1 Expression in E. coli and Trichoderma reesei

Jian-Hua Zhang1,2#, Xiao-qing Li1#, Yong-Xia Zhang1, Miao Xing1,2, Sheng-Li Tian1* and Shi-De Liu1*

1Shenzhen Key Laboratories of Microbial Genetic Engineering and Marine Sciences, College of Life Sciences Shenzhen University, Shenzhen 518060, China

2College of Life Sciences South China Agricultural University, Guangzhou 510642, China

#The authors made equal contribution to this work

Corresponding Author:
Sheng-Li Tian
Shenzhen Key Laboratories of Microbial
Genetic Engineering and Marine Sciences
College of Life Sciences Shenzhen
University, Shenzhen 518060, China
Tel: +86-755-26557245
Fax: +86-755- 26534274
E-mail: [email protected]

Shi-De Liu
Shenzhen Key Laboratories of Microbial
Genetic Engineering and Marine Sciences
College of Life Sciences Shenzhen University
Shenzhen 518060, China
Tel: +86-755-26557245
Fax: +86-755-26534274
E-mail: [email protected]

Received date: November 28, 2014; Accepted date: March 26, 2015; Published date: April 03, 2015

Citation: Zhang JH, Li XQ, Zhang YX, Xing M, Tian SL et al. (2015) Cloning and Characterization of Physarum Endo-Beta-1,3-1,4-Glucanase-1 Expression in E. coli and Trichoderma reesei. J Biotechnol Biomater 5:173. doi:10.4172/2155-952X.1000173

Copyright: ©2015 Zhang JH, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

The endo-β-1,3-1,4-glucanases have been found widely in bacteria, fungi, algae and plants, and have been applied to the hydrolysis of barley β-1,3-1,4-glucans in the beer brewing industry. Here, we report a novel endo-β-1,3- 1,4-glucanase gene (PEGase1) isolated from Physarum polycephalum and expressed in E. coli, and Trichoderma reesei. The molecular weights of the proteins are 34 and 66 kDa respectively by SDS-PAGE analysis. Enzymatic assays show that recombinant PEGase1 expressed by T. reesei was more efficient when hydrolyzing barley β-1,3- 1,4-glucans specifically, suggesting that modifications have a dramatic effect on the activity of PEGase1. We found that recombinant PEGase1 can be enriched by 40-70% ammonium sulfate precipitation and purified by HiTrapTM CaptoTM Q ion-exchange chromatography. Measurements of the enzymatic activity of purified PEGase1 under a range of conditions show that its optimal pH is around 5.5, and its optimal temperature is 55°C, with Km = 0.882 mg/ ml. PEGase1 activity is more stable in acidic solution and at low temperatures than it is in alkaline solution and at high temperatures. Metal ions and their concentration are able to affect the enzymatic activity, some metal ions, for example, Cu2+, Ni2+, Zn2+, Fe2+, Ba2+, Mg2+ and Ca2+ can enhance the enzymatic activity at 1 mmol/L concentration and inhabit the activity at 5 mmol/L concentration, but metal ion Mo2+and Co2+show inhabit the enzymatic activity at both concentration.

Keywords

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