Purification and characterization of b-1,4-glucosidase from Aspergillus glaucus

Abstract

A b-1,4-glucosidase (BG) was purified from fermentation liquor of Aspergillus glaucus by the following procedures: Ammonium sulfate precipitation, gel filtration on Sephadex G-100, and hydrophobic chromatography on Phenyl Sepharose Fast Flow. The specific activity on salicin was determined as 1747.4 U/mg. The molecular weight of this enzyme was determined as 23.5 and 92.5 kDa with SDS-PAGE and gel filtration on Sephadex G-100, respectively indicating that BG is a tetramer. The enzyme was stable at the pH ranging from 2.2 to 7.5, and its maximum activity was obtained at pH 3.6. The enzymatic activity gradually increased in the range from 40 to 60°C, but a sharp decrease occurred at 65°C. Enzymatic kinetics indicated that Michaelis-Menten contant (K_m) of the hydrolysis for salicin by BG was 2.58 mmol/L (pH 3.6, 60°C). Na⁺, K⁺, Ca²⁺, Mg²⁺, Ba²⁺, NO₃^– and SO₄^2- had no effects on BG activity. The enzyme activity was activated by Mn²⁺ and Fe²⁺, while it was strongly inhibited by Cu²⁺, Pb²⁺, Cd²⁺, SDS and EDTA, and slightly inhibited by Zn²⁺.

Key words: Aspergillus glaucus, b-1,4-glucosidase, purification, enzymatic properties.