Efficient Expression And Characterization Of β-Mannanase And α-Galactosidase Applying In Feed

As feed additive with beneficial effects on the environment,enzymes are widely used in animal feed.β-mannanase and α-galactosidase have attracted significant attention for their abilities to increase feed efficiency.But most of the previously reported β-mannanases and α-galactosidase hardly show satisfactory results in practical applications due to their low yield,poor acid stability and susceptibility to digestive proteases.Therefore it is of great value to screen a noval feeding enzyme resistant to acid and proteases.The β-mannanase was isolated from the fermentation broth of a Bacillus sub tilis strain,which simutaneously overproduces proteases.The β-mannanase gene was cloned from the genome of the B.subtilis strain,and expressed in Escherichia coli Rosseta(DE3)and Pichia Pastoris GS115 respectively.The purified recombinant β-mannanase exhibited optimal activity at 50℃ and pH 5.5.It’s enzymatic activities was enhanced by the metal ions Zn2+、Co2+、Cu2+and Fe3+.In addition,the β-mannanase showed high acid stability and resistance to trypsin digestion.After one hour incubation in the acidic buffer(pH 2.0)and a succession of two hour treatment with porcine pancreatic protease(20U/ml),the remaining activities of the β-mannanase still retained more than 70%and 60%of the highest activity.The β-mannanase gene can be functionally expressed in E.coli and P.Pastoris.But the expression level of the β-mannanase in P.pastoris was higher.The activity of theβ-mannanase was 427U/ml efficacy after 72h induction with methanol under the shaking flask condition.Then high density fermentation was performed in 5 L fermentor,and the recombinant enzyme activity reached 8261.21 U/ml,which satisfies the requirements of large-scale production.In addition,the α-galactosidase gene from the Aspergillus niger was cloned and expressed in P.pastoris GS115 succesfully.But the activity of the α-galactosidase was only 12 U/ml after 72h induction with methanol under the shaking flask condition.Thus,further research is required to improve the expression levels of A.niger α-galactosidase,including testing other expression hosts.Our results show that it was important to select an appropriate expression host for improving its functional expressiong its functional expression.