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Improved catalytic and antifungal activities of Bacillus thuringiensis cells with surface display of Chi9602ΔSP

Tang, M., Sun, X., Zhang, S., Wan, J., Li, L., Ni, H.
Journal of applied microbiology 2017 v.122 no.1 pp. 106-118
Bacillus thuringiensis, Fusarium oxysporum, Physalospora, Western blotting, antifungal properties, bacteria, biocatalysts, biological control agents, catalytic activity, chitin, chitinase, enzyme activity, flow cytometry, fluorescent antibody technique, gametolysin, molecular weight, mycelium, pH, plant pathogenic fungi, radiation resistance, spores, thermal stability, ultraviolet radiation, vegetative cells
AIMS: The cell‐surface display of chitinase Chi9602ΔSP was constructed on Bacillus thuringiensis BMB171 by using two repeat N‐terminal regions of autolysin (Mbgn)₂ as the anchoring motif. This study aimed to prepare a chitinolytic whole‐cell B. thuringiensis biocatalyst with improved catalytic and antifungal activities. METHODS AND RESULTS: The chitinase Chi9602ΔSP was displayed on the cell surface of B. thuringiensis BMB171 by using (Mbgn)₂ as the anchoring motif. Immunofluorescence microscopic assays and flow cytometry confirmed that Chi9602ΔSP was anchored onto the surface of vegetative cells and spores. Western blot analysis further confirmed that the (Mbgn)₂‐Chi9602ΔSP fusion protein was of the predicted molecular size of 94·2 kDa. A whole‐cell‐specific chitinase activity assay demonstrated that catalytic activities towards colloidal chitin of the recombinant strain (MB333) were significantly improved compared with those of wild inactive B. thuringiensis BMB171. The pH tolerance, thermostability and ultraviolet (UV) radiation resistance of Chi9602ΔSP also slightly improved compared with the unanchored Chi9602ΔSP displayed on the surface of MB333 cells and cultured for 24 h. After continuous culturing for 120 h, MB333 chitinase activity showed narrow pH tolerance and slightly decreased thermostability. In contrast, the UV radiation resistance capacity was obviously enhanced. An antifungal assay indicated that the recombinant strain exhibited a high inhibitory effect towards the mycelial growth of two phytopathogenic fungi, Fusarium oxysporum FB012 and Physalospora piricola FB016. CONCLUSIONS: We successfully developed a cell‐surface display system for Chi9602ΔSP on the surface of B. thuringiensis BMB171. The cell‐surface display system can enhance the stability of Chi9602ΔSP and has the potential of becoming a whole‐cell biocatalyst for agricultural applications. SIGNIFICANCE AND IMPACT OF THE STUDY: Results demonstrated that the bacterial cell display system can be used to express biocontrol agents on the cell surface of B. thuringiensis and provides an attractive method for inhibiting phytopathogenic fungi.