Effects of galactose and glucose on the hydrolysis reaction of a thermostable β-galactosidase from Caldicellulosiruptor saccharolyticus.

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. [ABSTRACT FROM AUTHOR]