PURIFICATION AND PROPERTIES OF N-ACETYL-BETA-D-GLUCOSAMINIDASE FROM HEVEA-BRASILIENSIS LATEX

Beta-N-Acetylglucosaminidase from Hevea brasiliensis latex was isolated by anion exchange and gel filtration chromatography. The purified enzyme showed a single protein band on native electrophoresis. It is a glycoprotein with a molecular mass of 92 kDa, consisting of two 46-kDa subunits and has a glycosidic content of 16%. It shows optimal activity at pH 6.0 and at 50-degrees-C; its amino acid composition has been established. It is inhibited by several monovalent or divalent ions. The enzyme hydrolyses p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-beta-D-GlcNAc) with apparent K(m) and V(m) values of 1-13 mM and 185 mM min-1 mg-1 of protein, respectively, at the optimum pH. p-Nitrophenyl-N-acetyl-beta-D-galactosaminide (pNP-beta-D-GalNAc) can also be used as a substrate but is less efficient. Glucosamine and galactosamine were competitive inhibitors with K(i) values of 2.2 mM and 7.5 mM, respectively, at pH 6.0. The results of kinetic studies suggest that two ionizable groups with pK values of about 4 and 6.5 may take part in the reaction, possibly in the substrate binding. The vacuolar location of the enzyme is in agreement with the idea that the latex might play a lysosomal role in intracellular digestion processes.