Expression of Streptomyces coelicolor [alpha]-Galactosidase Gene in Escherichia coli and Characterization

• Published in: Food science and technology research 2005-05, Vol.11 (2), p.207
• Main Authors: KONDOH, Kenji, MORISAKI, Kohei, KIM, Wook-Dong, M. KOTWAL, Sahebral, KANEKO, Satoshi, KOBAYASHI, Hideyuki
• Format: Article
• Language: English
• ISSN: 1344-6606; 1881-3984
• DOI: 10.3136/fstr.11.207

An α-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 64kDa. It was quite stable from pH 5.0 to 10.0 after treatment at 40°C for 60min, and was thermally stable up to 50°C. The enzyme acted on galacto-oligosaccharides, galactomanno-oligosaccharides and galactomannans as well as plant α-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°C. The catalytic domain showed the same specificity towards galacto-oligosaccharides, galactomanno-oligosaccharides and galactomannans as the enzyme containing the C-terminal region. These results indicated that the Cterminal region probably has an important role in stabilizing the enzyme.