Gene Cloning, Expression And Characterization Of α-galactosidase From Pedobacter Sp. MJ11

α-Galactosidase or melibiase (α-D-galactoside galactohydrolase; EC 3.2.1.22) is an exoglycosidase that catalytically removesα-linked terminal nonreducing galactose residues in different substrates.α-Galactosidase was widely distributed in animals, plants and microorganisms. And microorganism was the main sources of this enzyme for their various species, widely distribution and strongly adaption. Based on the amino acid sequence similarities,α-galactosidases have been classified into glycoside hydrolase (GH) families of 4, 27, 36 and 57. The GH-27 and GH-36 families contain the majority of knownα-galactosidases. Mostα-galactosidases of GH-27 are from eukaryote and those of GH-36 are from prokaryote.α-Galactosidases have a number of commercial applications, such as in the processing of food product, animal feed processing, medicine, chemical industry and so on. At present, there are two main problems in the study ofα-galactosidases: one is discovery of enzyme with good properties, another is high-level expression ofα-galactosidase to scale the production and solve problems in application.In this study, by using bioinformatical method, the amino acid sequence of microbialα-galactosidases from glycoside hydrolase (GH) familie 36 with molecular weight within the range 70-80 kDa were aligned and analyzed. Two conservative motif D-D-G-W and E-P-E-M were chosen to designed degenerated primers. Fragment ofα-galactosidase was cloned from Pedobacter sp. MJ11 by using degenerated PCR method, and the full length gene was obtained with TAIL-PCR cloning methods.Theα-galactosidase gene of Pedobacter sp. MJ11 was subject to heterogeneous expression in Escherichia coli BL21(DE3), function verification, purification and characterization.The gene consisted of 2166 nucleotides encoding a protein of 721 amino acids and a terminal codon. Theα-galactosidase gene was assigned to family 36 of the glycosyl hydrolases and the deduced amino acid sequence shares the highest identity of 54% with theα-galactosidase from Bacteroides thetaiotaomicron. All that indicated high novelty of theα-galactosidase genes.The expressed recombinant enzyme Aga-MJ11-H has a molecular weight of 80 kDa, which is agree to the calculated molecular weight of the mature enzyme from the deduced amino acid sequence of aga-MJ11, and was further identified by MS-based internal sequence analysis. The optimum of pH and temperature of the purified enzyme was 5.5 and 40°C, respectively, and the enzyme was stable in range of pH 4.0–10.0. The recombinant enzyme showed the hydrolytic activity for oligomeric substrates such as raffinose, stachyose and melibiose. The Km for pNPG and melibiose were determined as 2.94 and 22.37 mM, respectively. The purified enzyme also showed good protease tolerance to proteinase K, subtilisin A andα-chymotrypsin. All the features of the recombinant enzyme indicated it has potential applications in aquaculture. The enzyme studied in the present work may represent a member of GH-36α-galactosidases from genus Pedobacter which function was first identified.