Purification and characterization of β-N-acetylglucos-aminidase from Grifola frondosa

Abstract

Using commercial API-ZYM screening kits, highly active α-glucosidase, β-glucosidase, and β-N-acetylglucosaminidase were found in Grifola frondosa, having potential for carbohydrate utilization. Of these, β-N-acetylglucosaminidase, which converts chitin to N-acetylglucosamine, was purified and characterized. The recovery was 24.5%, and the purified enzyme had a specific activity 0.67 U/mg protein. Chitinase activity was confirmed by zymogram analysis. The enzyme was also shown to be β-N-acetylglucosaminidase, as N-acetylglucosamine was the main hydrolysis product from colloidal chitin. Thus, the molecule was named NAG38, to indicate β-N-acetylglucosaminidase activity and a molecular weight of 38 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Enzymatic activity was optimal at pH 7.0 and 50 °C, with Km and Vmax values of 0.112 mM and 0.570 μmol/min/mg protein against p-nitrophenyl N-acetyl-β-D-glucosaminide. The bioactivity was inhibited by Hg2+, Ag+, Mg2+, Zn2+, Ca2+, and Mn2+, with residual enzyme bioactivity only 11.1% after incubation in Hg2+, but was not substantially inhibited by Ba2+, K+, and Na+.