Express Gene MEL1 Of α-galactosidase In E.coli And Yeast

α-galactosidase or melibiase is the enzyme of hydrolysising α -galactosis bonds, it is a exoglycosidase exist in nature widely.α -galactosidase can hydrolysis a -galactosis bond , namely it can hydrolysis galactosis complex of non-reductive ends binding α -bond, so it can hydrolysis oligosaccharides such as raffinose, stachyose, verbascose, et al. It also can hydrolysis heteropolysaccharide including α-galactosis bonds.Recent research to α-galactosidase indicates that the enzyme has widely application prospect in fields of feedstuff, refining sugar, foodstuff and pharmaceutical industry, et al. Soya bean cake is appled as the widest vegetal protein feedstuff at present time, but it includes a kind of anti-trophic factor of alpha-galactosis oligosaccharide such as raffinose, stachyose, verbascose etc, which affects absorbation and utilization of nutriments. Application of alpha-galactosidase can utilize this kind of oligosaccharide on the one hand, and increase utilization ratio of soya bean cake and eliminate flatulence by reducing anti-trophic factors at the same time. Experimentation indicates that addition of alpha-galactosidase can increase potent metabolism energy 8 percent and digestible protein 7 percent. Now the annual usage of legume feedstuff is ten million annually, eighty thousand tons legume was increasely utilized comparatively after using enzyme preparation widely. So it has great society and economy value to develop and propagate alpha-galactosidase as feedstuff additives.This research choose feasible expression system for alpha-galatosidase by comparing expression of alpha-galactosidase gene MEL1 in Saccharomyces cerevisiae, E. coli and pichia pastoris, which will set up for industrialized production of alpha-galactosidase. 1. To express MEL1 gene of alpha-galactosidase in Saccharomycescerevisiae.MEL1 gene was amplified in Saccharomyces cerevisiae AH109 with PCR, and Recombinated with ADHl constitutive promotor thus recombinant plasmid pGAD-GAL is constructed. The recombinant plasmid pGAD-GAL was transformed into yeast, and the blue yeast clonies was screened out on SD/Leu~ nutritional media with X-alpha-Gal. Blue colonies indicate the alpha -galactosidase was constitutively expression in Saccharomyces Cerevisiae, but expression quantity of alpha-galactosidase is low and secretive capability of protein is poor.2. To express MEL1 gene of alpha-galactosidase in E. coliMEL1 gene was amplified in Saccharomyces cerevisiae AH109 with PCR, and recombined into E coli expression vector pRSET. Recombinant plasmid pRSET-Gal was obtained and transformed into host E. coli BL21 (DE3) PlysS. Transformed strain was cultured in liquid LB, and induced with IPTG. It has a bright band about 50ku by SDS-PAGE analysis. X-a-Gal was added into lysed cultures, Blue reaction indicated alpha-galactosidase was expressed in E coli. Glycosylation was not necessary to keep activity of alpha-galactosidase. But it sometimes forms inclusion body after expression of alpha-galactosidase in this expression system, the process of protein purification and enzyme renaturation is complicated.3. To express MEL1 gene of alpha-galactosidase in pichia pastorisMEL1 gene was amplified in Saccharomyces cerevisiae AH109 with PCR, and cloned into integrated vector pGAPZ a A, constitutive and secreted expression plasmid pGAPZa-Gal was constructed. The lined recombinant plasmid pGAPZ a -Gal was transformed into pichia pastoris KM71 by electropotation, and the blue positive colonies was screened out on YPDS plate with X-a -Gal and lOOmg/ml Zeocin. There was a special 53ku band by SDS-PAGE from supernatant of yeast culture; Alpha-galactosidase enzyme...