| Chitin is a linear polymer of (β-1,4) linked N-acetyl glucosamine. It is a major structural component of invertebrates, such as insects, crustaceans, and mollusks, it is also found in fungal cell walls. Chitinase (EC3.2.1.14) catalyze the hydrolysis of the β-1,4-glycosidic bonds of chitin to produce oligomeric derivatives of N-acetyl-D-glucosamine. Chitinase is produced by a wide variety of organisms including microorganism, plants and animals, and contribute to many physiological reaction. Chitinase have received increased attention because it has been long deemed as a promising candidate to protect plants from phytopathogenic fungal and insect pest infections.PCR method was adopted for cloning of chitinase encoding genes chi from Bacillus thuringiensis9602, Bacillus thuringiensis YBT-1221and Bacillus thuringiensis HD-73strains respectively, after sequence analysis, the Bacillus thuringiensis9602strain was choosen for the subsequent research ultimately.The chitinase gene fragment cloned from Bacillus thuringiensis9602was about2031bp, deduced amino acid was about676, and Mr=74.5kDa.The amino acid sequence of chi gene shows90%highly identity to that found in GeneBank. Conserved domain architectures analysis demonstrated that chi was categorized as a member of family18chitinases, it showed a modular structure comprised of four domains:a signal peptide, an N-terminal catalytic domain, a fibronectin type-Ⅲ-like domain (Fn3D) and a C-terminal chitin-binding domain (ChBD).The chi9602gene (without signal peptide) was linked with expression vector pTrcHisB to construct recombinant plasmid pMB332, and expressed in Escherichia coli Top10cells with IPTG induction. The result showed that the protein was successfully expressed in E.coli cells. In addition, the better-induced conditions for expression of the fusion protein were at37℃for6h with induction of lmmol/L IPTG at final concentration. Enzyme analysis indicated the optimum conditions of purified chitinase were37℃and pH7.0with a enzyme activity of30.01U/ml, enzyme kinetic studies gave apparent Km value of85.99mg/ml with a Vmax value of 18.28mg·ml-1·min-1for colloidal chitin at pH7.0and37℃The mature chitinase (Chi9602, without signal peptide) and chitinase catalytic domain (Chi9602c) were successfully displayed onto the surface of B. thuringiensis target cell BMB171using the two repeat N-terminal region of Mbg (Mbgn) as the binding domain, and a sporulation-independent promoter of cry3Aa was be used to instead of the resident Mbg promoter to increase the expression of the (Mbgn)2-Chi fusion protein. Western blot analysis, flow cytometry examinations, and immunofluorescence microscopic assays comfirmed the surface localization of the Chi9602and Chi9602c. The measurements of whole cell chitinase activity indicated the surface displayed recombinant strains showed substantial catalytic activity toward colloidal chitin, the highest chitinase activity were2.95U/ml and3.72U/ml at32h respectively.Furthermore, antifungal activities of the purified chitinase and the recombinant strains were investigated using the Rhizoctonia solania as the test fungi. The results showed that purified chitinase exhibited obviously inhibitory activity toward the hyphal growth of the test fungi. The recombinant strain which displayed the Chi9602showed significant inhibition of hyphal growth. By contraries, the recombinant strain which displayed the Chi9602c showed a very low inhibitory effect on fungi growth in despite of it has a higher chitinase activity.This study suggested that Chi9602showed high catalytic activity and antifungal activity. The fibronectin typeⅢ-like domain and the chitin-binding domain of the Chi9602play an important role in antifungal activity, but not essential for the hydrolytic activity toward colloidal chitin. In addition, the display of Chi9602on the cell surface of B. thuringiensis might provide an attractive method for against fungal phytopathogens in agriculture, and are thought to have a lower impact on the environment than conventional chemical method. |
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