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

Nadiawati Alias, Nor Muhammad Mahadi, Abdul Munir Abd. Murad, Farah Diba Abu Bakar, Nik Azmi Nik Mahmood, Rosli Md Illias

Research output: Contribution to journalArticle

6 Citations (Scopus)

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 Mg 2+ 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.

Original languageEnglish
Pages (from-to)561-572
Number of pages12
JournalWorld Journal of Microbiology and Biotechnology
Volume25
Issue number4
DOIs
Publication statusPublished - Apr 2009

Fingerprint

Chitinases
Trichoderma
Escherichia coli
Enzymes
Chitin
Genes
Isopropyl Thiogalactoside
Introns
Open Reading Frames
Polyacrylamide Gel Electrophoresis
Hydrolysis
Complementary DNA
Molecular Weight
High Pressure Liquid Chromatography
Ions
Amino Acids
Temperature

Keywords

  • Characterization
  • Expression
  • Inclusion bodies
  • Recombinant chitinase
  • Trichoderma virens

ASJC Scopus subject areas

  • Biotechnology
  • Physiology
  • Applied Microbiology and Biotechnology

Cite this

Expression and characterization of Trichoderma virens UKM-1 endochitinase in Escherichia coli. / Alias, Nadiawati; Mahadi, Nor Muhammad; Abd. Murad, Abdul Munir; Abu Bakar, Farah Diba; Mahmood, Nik Azmi Nik; Illias, Rosli Md.

In: World Journal of Microbiology and Biotechnology, Vol. 25, No. 4, 04.2009, p. 561-572.

Research output: Contribution to journalArticle

Alias, Nadiawati ; Mahadi, Nor Muhammad ; Abd. Murad, Abdul Munir ; Abu Bakar, Farah Diba ; Mahmood, Nik Azmi Nik ; Illias, Rosli Md. / Expression and characterization of Trichoderma virens UKM-1 endochitinase in Escherichia coli. In: World Journal of Microbiology and Biotechnology. 2009 ; Vol. 25, No. 4. pp. 561-572.
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