Abstract
N-Acetyl-β- d-glucosaminidase (EC 3.2.1.30) was purified to homogeneity and characterized from climacteric ‘Golden Delicious’ apple fruit cortical tissue. Ammonium sulphate fractionation (30–70%), Mono-Q anion-exchange and Con A-Sepharose 4B chromatography resulted in a 4.7% yield and 763-fold purification. The M r of the native enzyme was ca 236 000 using Superose 12 gel filtration, suggesting it was composed of eight similar subunits since only a single M r 29 000 silver-stained band was observed upon SDS-PAGE. The enzyme had a K m and V max for p-nitrophenyl- N-acetyl-β- d-glucosaminide of 0.23 mM and 8.48 nmol min −1, respectively, and for p-nitrophenyl- N-acetyl-β- d-galactosaminide of 1.49 mM and 10.58 nmol min −1, respectively. Estimates of V max/ K m for both substrates indicated that p-nitrophenyl- N-acetyl-β- d-glucosaminide was the most efficient and preferred substrate. The kinetics of mixed-substrate reactions and N-acetyl-β- d-glucosamine inhibition indicated the enzyme has only one active site which binds both substrates.
Published Version
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