Expression of 42kDa chitinase of Trichoderma asperellum (Ta-CHI42) from a synthetic gene in Escherichia coli.

Abstract

Chitinases are enzymes that catalyze the degradation of chitin, a major component of the cell walls of pathogenic fungi and cuticles of insects, gaining increasing attention for the control of fungal pathogens and insect pests. Production of recombinant chitinase in a suitable host can result in a more pure product with less processing time and a significantly larger yield than that produced by native microorganisms. The present study aimed to express the synthetic chi42 gene (syncodChi42), which was optimized from the chi42 gene of Trichoderma asperellum SH16, in Escherichia coli to produce 42kDa chitinase (Ta-CHI42); then determined the activity of this enzyme, characterizations and in vitro antifungal activity as well as its immunogenicity in mice. The results showed that Ta-CHI42 was overexpressed in E. coli. Analysis of the colloidal chitin hydrolytic activity of purified Ta-CHI42 on an agar plate revealed that this enzyme was in a highly active form. This is a neutral chitinase with pH stability in a range of 6-8 and has an optimum temperature of 45°C with thermal stability in a range of 25-35°C. The chitinolytic activity of Ta-CHI42 was almost completely abolished by 5mM Zn2+ or 1% SDS, whereas it remained about haft under the effect of 1M urea, 1% Triton X-100 or 5mM Cu2+. Except for ions such as Mn2+ and Ca2+ at 5mM that have enhanced chitinolytic activity; 5mM of Na+, Fe2+ or Mg2+ ions or 1mM EDTA negatively impacted the enzyme. Ta-CHI42 at 60U/mL concentration strongly inhibited the growth of the pathogenic fungus Aspergillus niger. Analysis of western blot indicated that the polyclonal antibody against Ta-CHI42 was greatly produced in mice. It can be used to analyze the expression of the syncodChi42 gene in transgenic plants, through immunoblotting assays, for resistance to pathogenic fungi.