Effects of galactose and glucose on the hydrolysis reaction of a thermostable beta-galactosidase from Caldicellulosiruptor saccharolyticus.
A recombinant beta-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 80 degrees C, and the half-life at 70 degrees C was 48 h. The enzyme exhibited hydrolytic activity for p-nitrophenyl-beta-D: -galactopyranoside (pNPGal), oNPGal, or lactose, whereas no activity for p-nitrophenyl-beta-D: -glucopyranoside (pNPGlu), oNPGlu, or cellobiose. The catalytic residues E150 and E311 of beta-galactosidase from C. saccharolyticus were completely conserved in all aligned glycoside hydrolase family 42 beta-galactosidases. The results indicated that the enzyme was a beta-galactosidase. Galactose uncompetitively inhibited the enzyme. Glucose inhibition of the enzyme was the lowest among beta-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.
Park
Ah-Reum
AR
Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea.
Oh
Deok-Kun
DK
D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002384:Catalysis; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005690:Galactose / Q000494:pharmacology*; D005947:Glucose / Q000494:pharmacology*; D006094:Gram-Positive Bacteria / Q000201:enzymology* / Q000235:genetics; D006207:Half-Life; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D007785:Lactose / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001616:beta-Galactosidase; D005947:Glucose; D007785:Lactose; D005690:Galactose