Mannosidase 3-mannosides, preparation

Results are expressed as fig of p-nitrophenol liberated per g of seed from 6 mM p-nitrophenyl a-D-mannoside in 1 hr at 37° and pH 5. Coarsely ground beans. Defatted sweet-almond meal. A commercial /3-D-glucosidase preparation purified from emulsin. a-D-Mannosidase activity is given per g of purified material. 

One unit of a-D-mannosidase liberates 1 /xg of p-nitrophenol from 6 mM p-nitro-phenyl a-D-mannoside in 1 hr at 37° and pH 5. Protein was determined by the method of Lowry and coworkers,59 with bovine albumin as the standard. Zn2+ was added to the preparation after Stage 3. 

Fig. 2.—Effect of Concentration of Substrate on the Rate of Hydrolysis4 of p-Nitro-phenyl a-D-Mannoside in Acetate Buffer, pH 3.5, at 37° by a Crude Preparation of Limpet a-D-Mannosidase in the Presence of , 0.1 mM ZnS04 and 0.1 M NaCl A, 0.1 M NaCl only and O, 0.1 mM ZnS04 only.

Proteins of a crude enzyme preparation obtained from the cultivation medium of the basidiomycete Phellinus abietis were separated by gel filtration and ion-exchange chromatography. The preparation contained a minimum of three enzymes capable of splitting a-D-mannosidic bonds a-D-mannosidase, exo-D-mannanase, and e/ido-D-mannanase, which were separated. Some properties of the D-mannanase complex of the crude enzyme preparation, and of a partially purified a-D-mannosidase, were examined. The D-mannanase complex exhibited two pH optima, its temperature optimum being 45 C. The pH optimum of purified a-D-mannosidase was at pH 5.0, the temperature optimum was at 60 °C the enzyme had a relatively high heat stability. The of a-D-mannosidase for 4-nitrophenyl a-D-mannopyranoside was 1.5xl0 M. Pure a-D-mannosidase did not split D-mannan. 

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