半乳糖苷酶.pdfVIP

Biotechnology Letters (2005) 27: 641–647 Springer 2005 DOI 10.1007/s10529-005-3660-2 Cloning and expression of the gene encoding Streptomyces coelicolor A3(2) a-galactosidase belonging to family 36 1 1 2 3 Kenji Kondoh , Kohei Morisaki , Wook-Dong Kim , Gwi-Gun Park , Satoshi Kaneko2 Hideyuki Kobayashi2,* 1Graduated school of Life and Environmental Science, University of Tsukuba, Ibaraki 305-0006, Japan 2Biological Function Division, National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan 3Departmant of Food Engineering and Biotechnology, Kyouguan University, Kyunggi-do 461-701, Korea *Author for correspondence (Fax: +81-29-838-7996; E-mail: hkobayas@nfri.affrc.go.jp) Received 16 December 2004; Revisions requested 10 January 2005; Revisions received 25 February 2005; Accepted 28 February 2005 Key words: family 36, a-galactosidase, glycoside hydrolase, oligosaccharides, Streptomyces coelicolor A3(2), substrate specificity Abstract The a-galactosidase gene of Streptomyces coelicolor A3(2) was cloned, expressed in Escherichia coli and characterized. It consisted of 1497 nucleotides encoding a protein of 499 amino acids with a predicted molecular weight of 57,385. The observed homology between the deduced amino acid sequences of the enzyme and a-galactosidase from Thermus thermophilus was over 40%. The a-galactosidase gene was assigned to family 36 of the glycosyl hydrolases. The enzyme purified from recombinant E. coli showed optimal activity at 40 C and pH 7. The enzyme hydrolyzed p -nitrophenyl-a-D-galactopyroside, raffinose, stachyose but not melibiose and galactomanno-oligosaccharides, indicating that this enzyme recognizes not only the galactose moi