High-Efficiency Secretion of β-Mannanase in Bacillus subtilis through Protein Synthesis and Secretion Optimization

The manno endo-1,4-mannosidase (beta-mannanase, EC. 3.2.1.78) catalyzes the random hydrolysis of internal (1 -> 4)-beta-mannosidic linkages in the mannan polymers. A codon optimized beta-mannanase gene from Bacillus licheniformis DSM13 was expressed in Bacillus subtilis. When four Sec-dependent and two Tat-dependent signal peptide sequences cloned from B. subtilis were placed upstream of the target gene, the highest activity of P-mannanase was observed using SPlipA,A as a signal peptide. Then a 1.25-fold activity beta-mannanase was obtained when another copy of groESL operon was inserted into the genome of host strain. Finally, five different promoters were separately used to enhance the synthesis of the target protein. The results showed that promoter P-mglv a modified maltose-inducible promoter, significantly elevated the production beta-mannanase. After 72 h of flask fermentation, the enzyme activity of P-mannanase in the supernatant when using locust bean gum as substrate reached 2207 U/mL. This work provided a promising beta-mannanase production strain in industrial application.