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

Abstract

An exo- N-acetyl-β- 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 M r of 230 000 as determined by size exclusion chromatography on Sephacryl S-300/Sephadex G-200 and exhibited a relative subunit M r of 60 000 on denaturing gel electrophoresis. It is a neutral protein with a p I of 6.79. The optimum pH and temperature for the enzyme activity are 6.0 and 70°C, respectively. Determination of the reaction stereochemistry indicates that the enzyme is a retaining glycosidase with the β 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°C. 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-β- d-GlcNAc was higher at temperatures below the inflection point than for pNP-β- d-GlcNAc (60.3 and 43.2 kJ mol −1, respectively). It hydrolyzes specifically, terminally linked β(1–4) GlcNAc residues from the non-reducing end of oligosaccharides. Comparative studies on the hydrolysis of chito-oligosaccharides by the exo- N-acetyl-β- d-glucosaminidase indicates that chitobiose is the best substrate with a K m and k cat of 0.34 mM and 24 μmol min −1 mg −1, respectively. It also exhibits strict substrate specificity with respect to the glycone substitution as well as anomeric linkage.