Purification and characterization of β-glucosidase produced by Trichoderma citrinoviride cultivated on microalga Chlorella vulgaris

Abstract

Microalga Chlorella vulgaris has a wide application as a source of biomass fuel, natural food coloring agent and dietary supplement. Its cell wall consists of multiple layers including cellulose and in order to utilize microalgae, the pre-treatment is necessary to breakdown rigid cell walls. β-glucosidase (EC 3.2.1.21) is a crucial enzyme to hydrolyze cellulose efficiently. In this study, β-glucosidase produced by Trichoderma citrinoviride cultivated on microalgae C. vulgaris was purified to homogeneity with a recovery of 8.5% and specific activity of 168.7 U/mg. The purified enzyme was obtained as a single band with the molecular mass of 110 kDa on SDS-PAGE. The optimum pH and temperature for enzyme activity and stability were 4.0 and 50°C, and 8.0 and 30°C, respectively. Metal ions (Mg2+, and Zn2+) activated the enzyme activity, whereas SDS inhibited moderately. Km, Vmax, Kcat, and Ki values of β-glucosidase were 0.96 mM, 300.42 μmol min−1 mg−1, 2.73 min−1 and 2.83 mM using p-nitrophenyl β-D-glucoside as a substrate, whereas they were 4.30 mM, 24.34 μmol min−1 mg−1, 0.022 min−1 and 0.53 mM using cellobiose, respectively.