Expression and characterization of Trichoderma virens UKM-1 endochitinase in Escherichia coli

Alias, Nadiawati and Muhammad Mahadi, Nor and Abdul Murad, Abdul Munir and Abu Bakar, Farah Diba and Nik Mahmood, Nik Azmi and Md Ilias, Rosli (2008) Expression and characterization of Trichoderma virens UKM-1 endochitinase in Escherichia coli. World Journal of Microbiology and Biotechnology, 25 (4). pp. 561-572. ISSN 0959-3993

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Abstract

A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed as soluble and active enzyme at 20 C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time. SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50 C. The enzyme is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60 C. The presence of Mg2 and Ca2 ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis showed the release of (GlcNAc)3, (GlcNAc)2 and GlcNAc, in which (GlcNAc)2 was the main product.

Item Type: Article
Keywords: Trichoderma virens � Recombinant chitinase � Expression � Inclusion bodies � Characterization
Subjects: Q Science > Q Science (General)
Q Science > QR Microbiology
Faculty / Institute: Faculty of Bioresources & Food Industry
Depositing User: Nadiawati Alias
Date Deposited: 30 Nov 2014 02:53
Last Modified: 30 Nov 2014 02:53
URI: http://erep.unisza.edu.my/id/eprint/131

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