Secreting Expression of Bacillus subtilis β-Mannanase in Pichia pastoris

Objective:To construct an engineering Pichia pastoris with secretory over-expression of β-mannanase from Bacillus subtilis.Methods Results:A codon-optimized β-mannanase gene from B.subtilis MA139 cleaved by EcoRⅠ and XbaⅠ was fused downstream of an α-factor in the expression plasmid of pPICzαA,and transformed into E.coli.The resulting plasmid linearized with SacⅠ was transformed into P.pastoris X-33.The recombinant strain with high-level expression of β-mannanase was cultured in 10 liter fermenter.The β-mannanase activity of the supernatant reached 2100 U /mL after the yeast was induced by methanol for 72 h.The recombinant βmannanase showed optimum of activity at 40℃ and pH6.0.Conclusion:The B.subtilis β-mannanase was high-level expressed by P.pastoris.The biochemical characteristics of the β-mannanase suggest the enzyme has a prospective application in feed industry as an additive.