Characterization of a novel exo-N-acetyl-beta-D-glucosaminidase from the thermotolerant Bacillus sp. NCIM 5120.

An exo-N-acetyl-beta-d-glucosaminidase from the thermotolerant Bacillus sp. NCIM 5120 was purified to homogeneity by chromatography on CM-cellulose, Sephacryl S-300 and phenyl-Sepharose. The enzyme has a Mr of 230000 as determined by size exclusion chromatography on Sephacryl S-300/Sephadex G-200 and exhibited a relative subunit Mr of 60000 on denaturing gel electrophoresis. It is a neutral protein with a pI of 6.79. The optimum pH and temperature for the enzyme activity are 6.0 and 70 degreesC, respectively. Determination of the reaction stereochemistry indicates that the enzyme is a retaining glycosidase with the beta anomer of GlcNAc formed as the initial product. Determination of the energy of activation with different leaving groups (p-nitrophenol and 4-methyl-umbelliferone) reveals that the enzyme exhibits a biphasic Arrhenius plot with two characteristic energy of activation with an inflection temperature of 50 degreesC. The activation energy at temperatures below the inflection point was found to be higher than that above the inflection point. The energy of activation for 4-Me-Umb-beta-d-GlcNAc was higher at temperatures below the inflection point than for pNP-beta-d-GlcNAc (60.3 and 43.2 kJ mol-1, respectively). It hydrolyzes specifically, terminally linked beta(1-4) GlcNAc residues from the non-reducing end of oligosaccharides. Comparative studies on the hydrolysis of chito-oligosaccharides by the exo-N-acetyl-beta-d-glucosaminidase indicates that chitobiose is the best substrate with a Km and kcat of 0.34 mM and 24 microoff min-1mg-1, respectively. It also exhibits strict substrate specificity with respect to the glycone substitution as well as anomeric linkage.