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

• Published in: Biochimica et biophysica acta Vol. 1425; no. 2; pp. 300 - 310
• Main Authors: Amutha, Boominathan, Khire, Jayant M., Khan, M.Islam
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 23.10.1998
• Subjects: Acetylglucosaminidase - antagonists & inhibitors; Acetylglucosaminidase - chemistry; Acetylglucosaminidase - isolation & purification; Amino Acids - analysis; Bacillus - enzymology; Bacillus - genetics; Electrophoresis, Polyacrylamide Gel; Fermentation; Hydrogen-Ion Concentration; Hydrolysis; Novel substrate specificity; Retaining β(1–4) N-acetylglucosaminidase; Substrate Specificity; Temperature; Thermostability; dns-, 5-dimethylaminonaphthalene-1-sulfonyl; pNP-α- d-GlcNAc, p-nitrophenyl-(α-2-acetamido-2-deoxy- d-glucopyranoside); Retaining β(1–4) N-acetylglucosaminidase; Novel substrate specificity; GalNAc, N-acetylgalactosamine; Me-Umb-β- d-GlcNAc-6-SO 4, 4-methyl-umbelliferyl- N-acetyl-β- d-glucosamine-6-sulfate; Chitotetraose, N,N′,N″,N‴-tetraacetyl chitotetraose; NBS, N-bromosuccinimide; NAGase, exo- N-acetyl-β- d-glucosaminidase; Chitobiose, N,N′-diacetyl chitobiose; pNP-β- d-Glc, p-nitrophenyl-(β- d-glucopyranoside); Thermostability; pNP-β- d-Xyl, p-nitrophenyl-(β- d-xylopyranoside); GlcNAc, N-acetylglucosamine; pNP-β- d-GlcNAc, p-nitrophenyl-(β-2-acetamido-2-deoxy- d-glucopyranoside); phenyl-β- d-GlcNAc, phenyl-(β-2-acetamido-2-deoxy- d-glucopyranoside); pNP-1-thio-β- d-GlcNAc, p-nitrophenyl-(1-thio-β-2-acetamido-2-deoxy- d-glucopyranoside); pNP-β- d-GalNAc, p-nitrophenyl-(β-2-acetamido-2-deoxy- d-galactopyranoside); DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); Me-Umb-β- d-GlcNAc, 4-methyl-umbelliferyl- N-acetyl-β- d-glucosamine; Chitotriose, N,N′,N″-triacetyl chitotriose; Me-Umb-β- d-GalNAc, 4-methyl-umbelliferyl- N-acetyl-β- d-galactosamine
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/S0304-4165(98)00081-6

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.