Secretory expression of β-mannanase in Saccharomyces cerevisiae and its high efficiency for hydrolysis of mannans to mannooligosaccharides.

Abstract

Degradation of mannans is a key process in the production of foods and prebiotics. β-Mannanase is the key enzyme that hydrolyzes 1,4-β-D-mannosidic linkages in mannans. Heterogeneous expression of β-mannanase in Pichia pastoris systems is widely used; however, Saccharomyces cerevisiae expression systems are more reliable and safer. We optimized β-mannanase gene from Aspergillus sulphureus and expressed it in five S. cerevisiae strains. Haploid and diploid strains, and strains with constitutive promoter TEF1 or inducible promoter GAL1, were tested for enzyme expression in synthetic auxotrophic or complex medium. Highest efficiency expression was observed for haploid strain BY4741 integrated with β-mannanase gene under constitutive promoter TEF1, cultured in complex medium. In fed-batch culture in a fermentor, enzyme activity reached ~ 24U/mL after 36h, and production efficiency reached 16U/mL/day. Optimal enzyme pH was 2.0-7.0, and optimal temperature was 60°C. In studies of β-mannanase kinetic parameters for two substrates, locust bean gum galactomannan (LBG) gave Km = 24.13mg/mL and Vmax = 715U/mg, while konjac glucomannan (KGM) gave Km = 33mg/mL and Vmax = 625U/mg. One-hour hydrolysis efficiency values were 57% for 1% LBG, 74% for 1% KGM, 39% for 10% LBG, and 53% for 10% KGM. HPLC analysis revealed that the major hydrolysis products were the oligosaccharides mannose, mannobiose, mannotriose, mannotetraose, mannopentaose, and mannohexaose. Our findings show that this β-mannanase has high efficiency for hydrolysis of mannans to mannooligosaccharides, a type of prebiotic, suggesting strong potential application in food industries.