Endoglucanase enzyme protein engineering by site-directed mutagenesis to improve the enzymatic properties and its expression in yeast

Farnaz Nikzad Jamnani; Seyed Omid Ranaei Siadat; Sohrab Moradi; Mohammad Taghi Borjian Boroujeni; Shirin Yousefian

Journal of Kermanshah University of Medical Sciences

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Endoglucanase enzyme protein engineering by site-directed mutagenesis to improve the enzymatic properties and its expression in yeast

Author(s):

Farnaz Nikzad Jamnani^1,*,

Seyed Omid Ranaei Siadat²,

Sohrab Moradi²,

Mohammad Taghi Borjian Boroujeni²,

Shirin Yousefian²

1Dept. of Biotechnology, Faculty of Agriculture and Natural resources, Science and Research Branch of Tehran, Islamic Azad University, Iran

2Dept. of Biotechnology, Nanobiotechnology Engineering Laboratory, Faculty of New Technologies Engineering, Shahid Beheshti University, Tehran, Iran

Journal of Kermanshah University of Medical Sciences:Vol. 17, issue 8; e74388

Published online:Nov 29, 2013

Article type:Research Article

Received:May 28, 2013

Accepted:Sep 17, 2013

How to Cite:Farnaz Nikzad JamnaniSeyed Omid Ranaei SiadatSohrab MoradiMohammad Taghi Borjian BoroujeniShirin YousefianEndoglucanase enzyme protein engineering by site-directed mutagenesis to improve the enzymatic properties and its expression in yeast.J Kermanshah Univ Med Sci.17(8):e74388.

Abstract

Introduction: Fossil fuel is an expensive and finite energy source. Therefore, the use of renewable energy and biofuels production has been taken into consideration. One of the most suitable raw materials for biofuels is cellulosic compounds. Only microorganisms that contain cellulose enzymes can decompose cellulose and fungus of Trichodermareesei is the most important producer of this enzyme.

Methods: In this study the nucleotide sequence of endoglucanase II, which is the starter of attack to cellulose chains, synthesized from amino acid sequence of this enzyme in fungus T.reesei and based on codon usage in the host; yeast Pichiapastoris. To produce optimized enzyme and to decrease the production time and enzyme price, protein engineering will be used. There are some methods to improve the enzymatic properties like site-directed mutagenesis in which amino-acid replacement occur. In this study two mutations were induced in endoglucanase enzyme gene by PCR in which free syctein positions 169 and 393 were switched to valine and histidine respectively. Then this gene was inserted into the pPinka expression vector and cloned in Escherichia coli. The recombinant plasmids were transferred into P.pastoris competent cells with electroporation, recombinant yeasts were cultured in BMMY medium and induced with methanol.

Results: The sequencing of gene proved the induction of the two mutations and the presence of recombinant enzyme was confirmed by dinitrosalicilic acid method and SDS-PAGE.

Conclusion: Examination of biochemical properties revealed that the two mutations simultaneously decreased catalytic power, thermal stability and increased the affinity of enzyme and substrate.

Keywords

endoglucanase

yeast

protein engineering

site-directed mutagenesis

thermal stability

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