Purification and characterization of α-galactosidase from Aspergillus niger CCRC31494

• Main Authors: Chien-Ping Kuo, 郭建平
• Other Authors: Tsong-Rong Yan
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/95614059241767240006

碩士 === 大同大學 === 生物工程研究所 === 90 === An extracellular enzyme with high α-galactosidase activity was purified from Aspergillus niger CCRC31494 culture filtrate. The molecular mass of this enzyme was estimated to be 254 kDa on Sephacryl S-300HR gel filtration, and 64 kDa by SDS-PAGE. Therefore, purified α-galactosidase was considered to be a tetramer composed of four 64 kDa subunits. After treating the purified enzyme with Endoglycosidase H, we could observe a 55 kDa dragged band in SDS-PAGE, so we supposed that purified α-galactosidase has 14 % N-linked oligosaccharides. α-Galactosidase, optimally active at pH 4.0 and 60oC, was measured by p-nitrophenyl α-D-galactopyranoside. In pH 3.0~4.0, it could retain most of its activity. At the optimum temperature, this enzyme was unstable, but it was stable at 40oC up to 4 hours. Kinetic parameters of α-galactosidase toward mNP-α-Gal, oNP-α-Gal, and pNP-α-Gal were Km=5.01 mM, Km=1.79 mM, and Km=1.405 mM, respectively. Some natural substrates such as melibiose, raffinose, and stachyose could be hydrolyzed by α-galactosidase. This enzyme hydrolyzed melibiose more efficiently than that of raffinose and stachyose in 6 hours. So it can be used in sugar-making and food industry to remove these interfering substrates. In addition, we investigated that the activity of α-galactosidase was strongly inhibited by 1mM metal ions─Ag+, Hg2+,and 25mM saccharides─ galactose, melibiose.