Abstract:

To develop enzymatic proteins for the efficient degradation of gossypol, a strain of B. amyloliquefaciens XP-13 was screened from soil, the gene BmLac was amplified from its genome, then the recombinant plasmid Ppic9k-BmLac was constructed and introduced into Pichia pastoris GS115 for heterologous expression. The characteristics of the laccase BmLac and the degradation effect of gossypol were studied. The results showed that the enzyme activity was 101.56 U/mL at pH 4 and 55℃ under the optimal reaction conditions with ABTS as the substrate; the relative enzyme activity was retained over 70% after incubation at 90℃ for 1 h; the relative enzyme activity was retained over 96% after incubation at pH 3-7 for 1 h. The enzyme activity increased 1.93 times by adding 1 mmol/L Cu²⁺, but high concentrations（10 mmol/L）of Fe²⁺, Fe³⁺ and Al³⁺ completely inhibited the enzyme activity. After reaction at 55℃ for 2 h, the degradation rate of gossyrol by this enzyme reached 94.57%（without mediator）and 98.73%（with ABTS）. In conclusion, the laccase BmLac has good thermal stability and wide pH adaptability and can effectively degrade gossypol, and this enzyme provides the basic support for the effective degradation of free gossypol in the cottonseed meal.

Key words: free gossypol, Bacillus amyloliquefaciens, laccase, enzymatic characterization, cottonseed meal