Expression of Streptomyces coelicolor a-galactosidase gene in Escherichia coli and characterization

An alpha-galactosidase gene belonging to glycoside hydrolase family 27 from Streptomyces coelicolor was cloned and expressed in E. coli. The purified enzyme showed a single protein band on SDS-PAGE with a molecular mass of 64 kDa. It was quite stable from pH 5.0 to 10.0 after treatment at 40 degrees C for 60 min, and was thermally stable up to 50 degrees C. The enzyme acted on galacto-oligosaccharides, galactomanno-oligosaccharides and galactomannans as well as plant alpha-galactosidases. It consisted of an N-terminal catalytic domain (400 amino acid residues) and a C-terminal region (260 amino acid residues). The catalytic domain of the enzyme was constructed by deleting the C-terminal region from the enzyme and was found to be stable from pH 5.5 to 8.5 and up to 40 degrees C. The catalytic domain showed the same specificity towards galactooligosaccharides, galactomanno-oligosaccharides and galactomannans as the enzyme containing the C-terminal region. These results indicated that the C-terminal region probably has an important role in stabilizing the enzyme.