Purification and characterization of a thermostable and highly specific beta-N-acetyl-D-glucosaminidase from Aspergillus niger 419.
After a 10.5-fold purification, beta-N-acetyl-D-glucosaminidase (EC 3.2.1.52) produced by Aspergillus niger 419, showed the following main characteristics: maximum activity at 65 degrees C, pH 4.5; K(m) and kcat using p-nitrophenyl-N-acetyl-beta-D-glucosaminide as substrate, 0.2 mM and 0.93 x 10(4) min-1, respectively; Ea, 30.5 kJ/mol; molecular mass, 131,000 Da; pI 4.4. The activity after heating for 15 min at 70, 75 and 80 degrees C was 70, 28 and 13% of that found at 65 degrees C, respectively. The enzyme was active in reaction mixtures containing glycerol, ethanol, methanol, propan-2-ol, acetone or dioxan. The presence of Sr2+ or Ca2+ enhanced the activity, while it was inhibited by Cu2+ and Fe3+. The enzyme was highly specific for p-nitrophenyl N-acetyl-beta-D-glucosaminide and no activity was found when p-nitrophenyl derivatives of N-acetyl-beta-D-galactosaminide, beta-D-galactopyranoside and beta-D-N,N'-diacetylchitobiose were tested as substrates. Due to its thermostability, specificity and resistance to different organic solvents, the enzyme might be a potentially useful tool for the analysis and production of oligosaccharides.
Pera
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LM
PROIMI, Planta Piloto de Procesos Industriales Microbiologicos, San Miguel de Tucumán, Argentina.
Infante Majollí
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MV
Baigorí
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MD
D000118:Acetylglucosaminidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D001234:Aspergillus niger / Q000201:enzymology*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature
D000118:Acetylglucosaminidase