Heterologous expression and characterization of antifungal chitinase and glucanase genes from Trichoderma viride in Escherichia coli
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Date
2019
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Punjab Agricultural University, Ludhiana
Abstract
The endochitinase and β-1,3-glucanase genes isolated from Trichoderma spp. were expressed in Escherichia coli strains Rosetta-gami 2 (DE3) and BL21 (DE3) pLysS using His-tagged pET28a+ and pET6xHN-C vectors. The expression of endochitinase and β-1,3-glucanase recombinant proteins was optimized by varying the incubation conditions, such as incubation temperature, incubation time and inducer concentration. The maximum expression (86.23 ng/ml) of endochitinase was observed with 0.4 mM IPTG at 37 ºC for 4 h in Rosetta-gami 2 (DE3), and maximum expression for β-1,3-glucanase (89.23 ng/ml) was observed in Rosetta-gami 2 (DE3) with 0.6 mM IPTG at 37 ºC for 6 h. The recombinant endochitinase and β-1,3-glucanase proteins were purified from E. coli using Ni-NTA affinity columns. The purified recombinant proteins were verified by SDS-PAGE, Western blotting and ELISA. The verified recombinant proteins were then tested for antifungal activity by confronting with Rhizoctonia solani casual organism of rice sheath blight and Phytophothora parasitica casual organism of citrus foot rot. The inhibition ratio of R. solani growth by endochitinase protein was 30±2.08 mm and by β-1,3-glucanase was 22.6±0.44 mm. This inhibition was higher than clotrimazole fungicide (21.85±0.47 mm). The recombinant endochitinase did not inhibit growth of P. parasitica whereas, β-1,3-glucanase revealed inhibition ratio of 32.6±0.47 mm against the pathogen. The hydrolytic action of the recombinant endochitinase and β-1,3-glucanase proteins on the cell morphology of R. solani was observed through scanning electron microscopy revealing breakage and pores on hyphal cell walls of R. solani whereas, β-1,3-glucanase confronted hyphae were prone to desiccation and have wrinkles and globular structures on hyphal walls eventually leading to hyphal lysis.
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