This work is to clone the α-galactosidase gene from Thermoascus crustaceus JCM12803 and systematically study its enzymatic properties for obtaining high-quality α-galactosidase in industry. The gene sequence of α-galactosidase was cloned from T. crustaceus JCM12803 by RT-PCR and its enzymatic properties were systematically analyzed. As results revealed,the full-length of tcgal27A belonging to glycoside hydrolase 27 was 1 918 bp,contained 4 introns,the cDNA of tcgal27 was 1 419 bp,and encoded 472 amino acids. SignalP analysis indicated 24 residues in the N-terminal of tcgal27 might be signal peptides. Recombinant TCGal27A successfully expressed in Pichia pastoris had a high specific activity（336.5 U/mg）,and the broad substrate specificity,i.e.,presenting different degrees of degradation to melibiose（14.4 U/mg）,raffinose（9.1 U/mg）,gum tragon（3.6 U/mg）,konjaku flour（1.6 U/mg）,guar gum（1.3 U/mg）,stachyose（0.7 U/mg）. Using pNPG as the substrate,the Km and Vmax of TCGal27A were determined to be 1.6 mol/mL and 536.8 μmoL/（min·mg）,respectively. Like most fungal a-galactosidases,TCGal27A had an optimal acidic pH 4.5. Purified recombinant TCGal27A was thermophilic,exhibiting the maximum activity at 65℃,and the enzyme remained 86% of initial activity at 50℃ for 1 h. All above results imply that heat-tolerant protein TCGal27A with excellent enzymatic properties can be additive for feedstuff,and will be solid potential applicable value in digesting and improving the energy utilization ratio of soybean meal protein.
