Abstract
A recombinant β-galactosidase from Caldicellulosiruptor saccharolyticus was purified with a specific activity of 211 U mg−1 by using heat treatment and His-trap affinity chromatography. The native enzyme was an 80-kDa trimer with a molecular mass of 240 kDa. Maximum activity was observed at pH 6.0 and 80ºC, and the half-life at 70ºC was 48 h. The enzyme exhibited hydrolytic activity for p-nitrophenyl-β-d-galactopyranoside (pNPGal), oNPGal, or lactose, whereas no activity for p-nitrophenyl-β-d-glucopyranoside (pNPGlu), oNPGlu, or cellobiose. The catalytic residues E150 and E311 of β-galactosidase from C. saccharolyticus were completely conserved in all aligned glycoside hydrolase family 42 β-galactosidases. The results indicated that the enzyme was a β-galactosidase. Galactose uncompetitively inhibited the enzyme. Glucose inhibition of the enzyme was the lowest among β-galactosidases. When 50 g l−1 galactose was added, the enzyme activity for pNPGal was reduced to 26%. When 400 g l−1 glucose instead of galactose was added, the activity was reduced to 82%. When adding galactose (200 g l−1), only 14% of the lactose was hydrolyzed after 180 min. In contrast, the addition of glucose (400 g l−1) did not affect lactose hydrolysis, and more than 99% of the lactose was hydrolyzed after 120 min.
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This study was carried out with the support of the 21C Frontier Project for Microbial Genomics, Ministry of Education, Science and Technology.
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Park, AR., Oh, DK. Effects of galactose and glucose on the hydrolysis reaction of a thermostable β-galactosidase from Caldicellulosiruptor saccharolyticus . Appl Microbiol Biotechnol 85, 1427–1435 (2010). https://doi.org/10.1007/s00253-009-2165-7
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DOI: https://doi.org/10.1007/s00253-009-2165-7