Studies on Cellulase Produced by the Phytopathogens

Purification and Enzymatic Properties of Cellulase of Fusarium monilforme

Abstract

Cellulase [EC 3. 2. 1. 4] of a phytopathogenic fungus Fusarium moniliforme was purified by ammonium sulfate fractionation and column chromatography on Amberlite CG-50, DEAE-Sephadex A-25, and hydroxyapatite. The enzyme was finally separated into two fractions (Cellulases-I and -II). Cellulase-I was purified further by hydraxyapatite column chromatography, yielding cellulase-I'. Its activity was enhanced about 10-fold over that of the starting material, using sodium carboxymethylcellulose as a substrate. Cellulase-I' was homogeneous on disc gel electrophoresis and ultracentrifugal analyses. The optimum pH for cellulase-I' activity was found to be at. 4.5, and the enzyme was stable at pH 3-8. The optimum temperature for cellulase-I'activity was found to be 60°C, and heating at 60°C for 120min caused little loss of activity. Cellulase-I' was inhibited by Hg²⁺, N-bromosuccinimide and sodium picryl sulfate, while it was activated slightly by Co²⁺, Zn²⁺, hydroquinone, and ascorbic acid. Ca²⁺, Cu²⁺, Ba²⁺, and Cd²⁺ had no effect on the enzyme activity. EDTA and thiol agents such as PCMB and monoiodoacetic acid also had no effect on the enzymeactivity. The molecular weight of cellulase-I' was estimated to be about 25, 000 by Sephadex G-100 gel filtration. E₂₈₀^1%_1cm was calculated to be 15.0, and the ratio of E₂₈₀/E₂₆₀ of cellulase-I' was 1.74. The carbohydrate content of cellulase-I' was found to be about 26% as glucose, determined by the orcinol-sulfuric acid method. Cellulase-I' showed powerful activity toward insoluble celluloses such as Avicel and filter paper, whereas the enzyme was inactive toward cellobiose, salicine, sucrose, soluble starch, mannan, inulin, p-nitrophenyl α-D-glucopyranoside, and p-nitrophenyl β-D-glucopyranoside. Hence, the enzyme was practically free from β-glucosidase [EC 3. 2. 1. 21] activity.