Heterologous Expression And Enzymic Properties Of β-mannanase From Aspergillus Sulphureus

β-mannanase, which catalyzes the random cleavage of β-D-1,4-mannopyranosyl linkages within the main chain of mannans (e.g., galactomannans, glucomannans and galactoglucomannans), is a kind of hemicellulase. It can be produced by various resources in nature, and has good applications in food, textile, paper-making and animal feed.β-mannanase has better performance especially when it was used as feed additive because it can effectively digest high-fibre feeds, balance ecological environment of the gastrointestinal tract, promote the growth of animals and so on. However, this enzyme could lose its activity easily because of the acidic environment in the gastrointestinal tract,so it is necessary to obtain a, β-mannanase with good acid resistance.In this study, Aspergillus sulphureus which has the β-mannanase activity was isolated from fungus in our laboratory, and the β-mannanase encoding gene (man) was cloned by RT-PCR. Sequence analysis showed that the gene contained an ORF of 1146 bp, which encoded a protein of 383 amino acids with a calculated molecular weight of 41 kDa and a isoelectric point (pI) value of 3.91. The deduced amino acid sequence of the gene showed highest similarity to P-mannanase gene of Aspergillus terreus which belongs to the glycoside hydrolase family 5 and their similarities were 71.24 %. The mature peptide-encoding gene was expressed efficiently in E.coli Rosseta (DE3) and P.Pastoris GS115 respectively and the enzymatic properties of the purified P-mannanase were investigated.The results showed that the optimal temperature and pH for recombinant enzyme were 65 ℃ and 5.5. The enzyme activity was high when the enzyme was treated at 50-70 ℃.when it was treated at 65 ℃ for 60 min, almost no apparent change was observed in its activity. The enzyme was stable between pH 3.0-8.0 at 65 ℃ and the enzyme activity still remained 60 % when it was treated at pH 3.0 and at 65 ℃ for 1 hour. EDTA, Zn2+, Cu2+,Co2+ and Fe3+ had the possitive effect on its activity while Hg2+, Ag+, SDS and Tween 80 inhibited it activity. The kinetic parameters such as Km and Vmax of the β-mannanase towards locust bean gum were 3.79 mg/mL and 345.05 U/mg, respectively. The production of this β-mannanase in P.Pastoris GS115 was higher than that in E.coli Rosseta (DE3). The activity of the β-mannanase was 58 U/mL after 72 h induction with methanol under the shaking flask condition, and the recombinant enzyme activity reached 587 U/mL when the high density fermentation was performed in 5 L fermentor.