Expression And Characterization Of Novel α-galactosidases

α-Galactosidase(EC3.2.1.22) is one type of glycoside hydrolases which widely existed in nature, and are usually classi?ed into glycoside hydrolase families 4, 27, 36, 57 and 110 based on their a mino acid sequence similarity. These enzymes can hydrolyze α-1,6-galactosidic linkages from the non-reducing end of various polysaccharides. Some α-galactosidases not only have hydrolysis activity but also have transglycosylation activity. α-Galactosidases have great potential for application in various industries, including the food, feed, sugar-producing, chemical and medical industries. To date, the researches on α-galactosidases from Mesorhizobium sp. and Pontibacter sp. have not been reported. There are few reports on α-galactosidase showing alkaline and double pH optima and on glycoside hydrolases family 27 α-galactosidase showing transglycosylation activity.In this study, we selected three genes agaAJB07, agaAHJG4 and agaAHJ8 from three bacterial strains Mesorhizobium sp., Streptomyces sp. and Pontibacter sp., respectively. They encode α-galactosidase AgaAJB07(GH36), AgaAHJG4(GH36) and AgaAHJ8(GH27), respectively. The three genes mentioned above were inserted into pEASY-E2 vector, then expressed successfully in Escherichia coli BL21(DE3).Using histidine-tagged to purified recombinant enzyme(Aga AJB07, AgaAHJG4 and AgaAHJ8), and subjected to biochemical characterizations, transglycosylation activity and acceptor speci?city assays.(1) Purified recombinant AgaAJB07 was optimal activity at pH 6.5 and 45 °C. The enzyme was stable at pH5.0-10.0 and 50 °C for more than 60 min. The enzyme was also stable when trypsin and proteinase K were added to the reaction mixture. Furthermore, AgaAJB07 was activated by 0.5 mmol/L ZnSO4 and 30 mmol/L PbAc. These favorable properties make AgaAJB07 a potential candidate for applications in the aquaculture.(2) Purified recombinant AgaAHJG4 was an alkaline α-galactosidase with double optimal pH of 8.0 and 9.5. AgaAHJG4 was apparently optimal at 35 °C,and retained 19.3% of the maximum activity at 10 °C and 35.3% of the maximum activity at 20 °C. The enzyme activity was stable at pH 7.5-10.0 for more than 60 min. Because there is a potential relationship between the desiccation tolerance of seed and the activity of alkaline α-galactosidase when germination of seed begins, AgaAHJG4 might be used to improve the seed desiccation tolerance.(3) Purified recombinant AgaAHJ8 showed optimal activity at pH 5.5 and 50 °C. The enzyme activity was stable at pH 4.0-8.0 for more than 60 min. Proteinase K, trypsin, NaCl, Zn2+ and Pb2+ have little effect on its activity. AgaAHJ8 is valuable for the basic research on the mechanism of tolerance to salt, Zn2+, and Pb2+.(4) The result of TLC and MS assays indicated that AgaAJB07, AgaAHJG4 and AgaAHJ8 had the transglycosylation activity. When 40 mmol/L p-nitrophenyl-α-D-galactopyranoside was used as the donor, AgaAJB07 had transglycosylation activities toward 400 mmol/L D-xylose, D-glucose, D-galactose, L-sorbose, D-fructose, D-mannose, α-lactose, mannitol, sorbitol, ethanol, 2-propanol, and glycerol, while rAgaAHJG4 had transglycosylation activities toward 400 mmol/L D-galactose, D-fructose, α-lactose, sucrose, raffinose, and stachyose, and AgaAHJ8 had transglycosylation activities toward 400 mmol/L sucrose, raffinose, and isoamyl alcohol. Self-condensation and hydrolysis activities were detected with AgaAJB07 when 40 mmol/L p-nitrophenyl-α-D-galactopyranoside or 400 mmol/L melibiose was used as a substrate. For AgaAHJG4, self-condensation and hydrolysis activities were detected when 40 mmol/L p-nitrophenyl-α-D-galactopyranoside, 400 mmol/L melibiose or 400 mmol/L stachyose was used as a substrate. For AgaAHJ8, no self-condensation activity was observed.In summary, two GH36 enzymes and one GH27 enzyme were expressed in E. coli and purified. Novel biochemical properties of the three enzymes were revealed: AgaAJB07(GH36) from Mesorhizobium sp. showed tolerance to Zn2+ and Pb2+, and had broad transglycosylation acceptors; AgaAHJG4(GH36) from Streptomyces sp. showed optimal activity at pH 8.0 and 9.5 as well as low-temperature activity; AgaAHJ8(GH27) from Pontibacter sp. showed tolerance to Na+, Zn2+, and Pb2+, and showed transglycosylation activity. The three enzymes might be valueable for basic research and potential industrial applications.