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

authors:

avatar Farnaz Nikzad Jamnani 1 , * , avatar Seyed Omid Ranaei Siadat 2 , avatar Sohrab Moradi 2 , avatar Mohammad Taghi Borjian Boroujeni 2 , avatar Shirin Yousefian 2

Dept. of Biotechnology, Faculty of Agriculture and Natural resources, Science and Research Branch of Tehran, Islamic Azad University, Iran
Dept. 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: November 29, 2013
article type: Research Article
received: May 28, 2013
accepted: September 17, 2013

how to cite: Nikzad Jamnani F, Ranaei Siadat S O, Moradi S, Borjian Boroujeni M T, Yousefian S. Endoglucanase enzyme protein engineering by site-directed mutagenesis to improve the enzymatic properties and its expression in yeast. J Kermanshah Univ Med Sci. 2013;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.

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