Purification and characterization of thermostable beta-N-acetylhexosaminidase of Bacillus stearothermophilus CH-4 isolated from chitin-containing compost.
Thermostable exochitinase was purified to homogeneity from the culture fluid of Bacillus stearothermophilus CH-4, which was isolated from agricultural compost containing shrimp and crabs. The enzyme was a single polypeptide with a molecular mass of 74 kDa, and the N-terminal amino acid sequence was WDKVGVTDLI ISLNIPEADAVVVGMTLQLQALHLY. The enzyme specifically hydrolyzed C-4 beta-anomeric bonding of N-acetylchitooligosaccharides, as well as their p-nitrophenyl (pNP) derivatives. The enzyme also hydrolyzed pNP-beta-N-acetyl-D-galactosaminide (26% of the activity of pNP-beta-N-acetyl-D-glucosaminide). These results indicated that the enzyme is a beta-N-acetylhexosaminidase (EC 3.2.1.52). Kms for acetylchitooligosaccharides were 1 x 10(-4) to 6 x 10(-4) M, while those for the pNP derivatives were 4 x 10(-3) to 8 x 10(-3) M. The optimum temperature of the enzyme was 75 degrees C, and it retained 100 and 28% reactivity after heating at 60 and 80 degrees C, respectively. The enzyme exhibited 15 to 20% activity in a reaction mixture containing 80% organic solvents and maintained 91% of its original activity after exposure to 8 M urea. The optimum and stable pH was around 6.5. Fe2+, Zn2+, and Ca2+ activated the enzyme, but Hg2+ was inhibitory. N-Acetyl-D-glucosamine inhibited the enzyme competitively (Ki = 4.3 x 10(-4) M), whereas N-acetyl-D-galactosamine did not; in contrast, D-glucosamine and D-galactosamine activated it.
Sakai
K
K
Department of Applied Chemistry, Faculty of Engineering, Oita University, Japan.
Narihara
M
M
Kasama
Y
Y
Wakayama
M
M
Moriguchi
M
M
D000595:Amino Acid Sequence; D002688:Chitinases / Q000302:isolation & purification / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017421:Sequence Analysis; D013379:Substrate Specificity; D014866:Waste Products; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D014866:Waste Products; D002688:Chitinases; D001619:beta-N-Acetylhexosaminidases