APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1994, p. 2911-2915, vol. 60, No. 8
Copyright @ 1994, American Society for Microbiology

Purification and Characterization of Thermostable
-N-Acetylhexosaminidase of Bacillus stearothemophilus
CH-4 Isolated from Chitin-Containing Compost


Department of Applied Chemistry, Faculty of Engineering, Oita University, Oita 870-11, Japan
Received 23 February 1994/Accepted 25 May 1994

Thermostable exochitinase was purified to homogeneity from the culture fluid of Bacillus stearothemophilus 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 WDKVGVTDLIISLNIPEADAVVVGMTLQLQALHLY. The enzyme specifically hydrolyzed C-4 -anomeric bonding of N- acetylchitooligosaccharides, as well as their p-nitrophenyl (pNP) derivatives. The enzyme also hydrolyzed pNP--N-acetyl-D-galactosaminide (26% of the activity of pNP--N-acetyl-D-glucosaminide). These results indicated that the enzyme is a P-N-acetylhexosaminidase (EC 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, and it retained 100 and 28% reactivity after heating at 60 and 80, 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.