Enhancement Of Secretion Of Manganese-catalase In Recombinant Bacillus Subtilis

Catalase is able to treat residual hydrogen peroxide in industrial wastewater,so it is an important enzyme in industrial production,but the heat resistance and acid and alkali resistance of traditional catalase are poor,which greatly limits the Its potential for application in industrial production.In the previous study,we screened and expressed a novel manganese catalase,GeoMnCAT?GMC?,which has good heat resistance and acid and alkali resistance,and the maximum enzyme activity is achieved at 75°C and pH=9.0.However,its expression is low and most of the enzyme is in the form of inclusion bodies which are inactive.Therefore,the search for suitable host strains and gene expression components to improve the GMC expression of enzymes is an urgent problem to be solved.In this study,the GMC gene was constructed in the shuttle plasmid pBS-S and expressed by the host strain Bacillus subtilis RIK1285.Meanwhile,48 individual B.subtilis homologous signal peptides?45 Sec pathway signal peptides,3 Tat pathway signal peptides?which are suitable for GMC expression were screened from the signal peptide library.Subsequently,three different types of promoters(constitutive promoter P₄₃,inducible promoter P_glv,and phase-specific promoter P_aprE)were inserted into the recombinant shuttle plasmid respectively and acquire the most suitable promoter for the enhancement of expression of GMC.According to the results,the signal peptide from the Sec secretion pathway can promote the expression of heterogeneous proteins more efficiently,and the signal peptide yoqH can increase the expression of GMC to 46.08 U/mL.Meanwhile,the physicochemical properties of each signal peptides were calculated by SingalP software.By analysis of the correlation with the secretion level of the protein,D-score which presents the of efficiency of combination of the N-terminal amino acid sequence and signal peptide is inversely related to the expression level of protein.After optimization of the promoter,the constitutive promoter P₄₃ was able to maximally increase the expression of GMC to 143.77 U/mL.At the same time,the inducible promoter P_glv and the period-specific promoter P_aprE also increased the expression of manganese catalase GMC to 115.33 U/mL and 107.6 U/mL,respectively.The GMC expressed in Bacillus subtilis optimized by the signal peptide and promoter was up to 4.74 times higher than it produced in the E.coli expression system.In this study,GMC was expressed in Bacillus subtilis and the enzyme activity of GMC was increased by molecular level optimization to 4.74 times higher than it previously expressed by E.coli expression system.This work offers the possibility of application of manganese catalase in industry.