Expression of 42 kDa 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 42 kDa 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 5 mM Zn2+ or 1% SDS, whereas it remained about haft under the effect of 1 M urea, 1% Triton X-100 or 5 mM Cu2+. Except for ions such as Mn2+ and Ca2+ at 5 mM that have enhanced chitinolytic activity; 5 mM of Na+, Fe2+ or Mg2+ ions or 1 mM EDTA negatively impacted the enzyme. Ta-CHI42 at 60 U/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.

Enhancement of resistance against fungal pathogens in peanut (Arachis hypogaea L.) cultivar L14 by heterologous expression of gene encoding chitinase 42 kDa from Trichoderma asperellum SH16

“…Chi42 (HM191683.1) is a wild-type gene from T. asperellum SH16 (Loc et al 2011). Both syncodChi42-1 (MT083802.1) and syncodChi42-2 (MT083803.1) are synthetic genes derived from the Chi42 gene optimized for codon usage for plant expression (Luong et al 2021). Chitinase genes were driven by the rootspeci c Asy promoter from peanut (Geng et al 2014).…”

“…The non-speci c binding on the blot was blocked by 5% skim milk (Sigma-Aldrich, St. Louis, MO, USA). The primary antibody was a mouse anti-Ta-CHI42 polyclonal antibody diluted 1:2000 in Tris-buffered saline with Tween 20 (TBST), Ta-CHI42 is recombinant chitinase 42 kDa derived from fungus T. asperellum SH16 (Luong et al 2021). The secondary antibody was a 1:5000 dilution of goat anti-mouse IgG antibody conjugated with alkaline phosphatase (AbD Serotec-currently Bio-Rad Antibodies, Bio-Rad Laboratories, Hercules, CA, USA).…”

“…As a consequence, the present study might be the rst to employ Agrobacterium-mediated transformation to transfer the chitinase gene from Trichoderma to peanuts in order to increase their antifungal activity. Different chitinase genes were employed in this study, including one wild-type gene, Chi42, from T. asperellum SH16, which encodes chitinase 42 kDa, and two synthetic genes (syncodChi42-1 and syncodChi42-2) generated from the Chi42 gene by optimizing codon usage for plant expression (Luong et al 2021). The goal of this study is to evaluate the level of expression of the wild-type gene and the two synthetic genes regulated by the tissue-speci c promoter (pAsy) in peanut roots, as well as their antifungal e cacy.…”

OPTIMIZING THE PRODUCTION OF A FUNCTIONAL TYPE A RECOMBINANT ENDOCHITINASE FROM Trichoderma asperellum IN Escherichia coli

“…Imidazole was eliminated from eluent fractions, and CHI42 was suspended in PBS (buffer exchange) by molecular weight cutoff centrifugal filters (Amicon, 10 kDa cutoff). Purified soluble CHI42 was analyzed by Western blot using anti-CHI42 polyclonal antibodies as a probe (Luong et al, 2021).…”

“…The pre-punched holes were loaded with 30 L PBS containing 300 g of purified soluble CHI42 or control (cellular extract from pre-induced E. coli) and incubated at 4 °C for 8 hrs for the enzyme to diffuse to the surrounding agar, and then at 28°C for 6 hrs for chitinolysis. Finally, the plate was stained with 0.1% Lugol's solution to detect substrate hydrolysis (Luong et al, 2021). The experiment was repeated thrice and the diameters of hydrolysis rings were compared statistically.…”