Abstract
The ripening-related biological activity (Priem and Gross, 1992, Plant Physiol 98:399-401) of free N-glycans in fruit was recently reported. Thus, enzymes involved in N-glycan metabolism may be important in understanding the regulation of ripening. N-acetyl-β-D-glucosaminidase (EC 3.2.1.30) was purified to homogeneity and characterized from climacteric `Golden Delicious' apple (Malus domestica Borkh.) fruit tissue. This enzyme cleaves terminal N-acetylglucosaminyl residues from the non-reducing end of glycans. The enzyme was extracted in 25 mM Na-acetate (pH 5.0) containing 2% PVP, 0.1 mM EDTA, and 0.5 mM DTT. Activity was 0.02 μmol/min/gfw in the crude extract. Ammonium sulfate fractionation (30 to 70%), Mono-Q FPLC and Con A-Sepharose 4B resulted in a 4.7% yield and 923-fold purification. The enzyme had a Km for P-nitrophenyl-N-acetyl-β-D-glucosaminide of 136 μM. Optimal pH and substrate concentration were 6.0 and 0.5 mM, respectively. Activity was inhibited at substrate concentrations above 0.5 mM. The molecular weight of the enzyme was estimated to be 29 kD using SDS-PAGE.
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