α-Galactosidase (α-D-galactoside, galactohydrolase; EC 3.2.1.22), also called melibiase, is an exoglycosidase that catalytically removesα-linked terminal non-reducing galactose residues in different substrates.α-Galactosidase has been widely applied in fields of medicine, food, chemical engineering, feed and so on. In feed industry, additive ofα-galactosidase in animal feed is the first choice to remove anti-nutritional factors in soybean meal. At present, the exploration of moreα-galactosidases with excellent properties was crucial to industrialization ofα-galactosidase in animal feed.In this study, Rhizopus sp. F78, Gibberella sp. F75, Streptomyces sp. S9 and S27, and Yersinia pestis biovar Microtus str. 91001 were selected as the research objects based on the evaluation of species-novelty andα-galactosidase production capacity. Some strains from Rhizopus and Gibberella are important in industries, but cloning and expression ofα-galactosidase genes from these genuses has not been reported yet. Streptomyces sp. S9 and S27 from the Huoyanshang mountain have special growth environment.By using bioinformatical methods, the amino acid sequence of microbialα-galactosidases from glycoside hydrolase (GH) family 36 were aligned and analyzed. GH-36α-galactosidases were classified according to the diversity of molecular weight. A set of degenerate primers was designed based on the conserved motifs. Gene fragments ofα-galactosidases from these tested strains were cloned by using degenerated PCR method, and the full length genes were obtained by combinating with other cloning methods. Thus the method of degenerate primer designing based on the motifs can amplify GH-36α-galactosidases availably.BLAST search was carried on to analysis the identities of amino acid sequence between obtained and reportedα-galactosidases. The highest identities ofα-galactosidases Aga-F78 (R. sp. F78), Aga-F75 (G. sp. F75), Aga-S9 (S. sp. S9), Aga-S27(S. sp. S27) and Aga-Y (Y. pestis biovar Microtus str. 91001) were 45, 69, 36, 55 and 57%, respectively, and the identities among these fiveα-galactosidases was 25-40%. All that indicated high novelty of theseα-galactosidase genes.The fiveα-galactosidase genes were subject to heterogeneous expression in Escherichia coli, function verification, purification and characterization. The optimum pH and temperature on activity of theα-galactosidases from fungi were 4.0-5.0 and 35-40°C, and that of bacteria were 7.0-7.5 and 50-60°C, respectively. The recombinantα-galactosidases from R. sp. F78 (Aga-F78-H), G. sp. F75 (Aga-F75-H) and S. sp. S27 (Aga-S27-H) had good thermostability; Aga-F78-H and Aga-F75-H had excellent resistance to some protease; the one from Y. pestis biovar Microtus str. 91001 (Aga-Y-H) had some cold-adapted enzyme properties; all of these properties have never been reported in otherα-galactosidases. Therefore, theseα-galactosidases may not only have practical application values in different fields, but also provide materials for the study of structure and function.In animal feed,α-galactosidase is commonly applied together with protease to increase the utilization of nutrient material. In addition, resistance to protease in gastrointestinal tract is very important forα-galactosidase to increase the efficacy by prolonging action time. Soybean meal hydrolysis test showed that application of fourα-galactosidases alone increased the content of galactose obviously. Combinating with Trypsin had little effect on Aga-F75-H, Aga-F78-H or Aga-S27-H in soybean meal hydrolysis test. Aga-F75-H increased the content of galactose 82.42 times without trypsin and more 36% together with trypsin. Collectively, Aga-F75-H, Aga-F78-H or Aga-S27-H, especially Aga-F75-H, are promising in industrial applications.