Studies on Cellulase Produced by the Phytopathogens*1

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 hydroxyapatite column chromatography, yielding cellulase-T. 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 ultra-centrifugal 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 120 min 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 enzyme; activity. The molecular weight of cellulase-I′ was estimated to be about 25,000 by - Sephadex G-100 gel filtration.E2801cm1% was calculated to be 15.0, and the ratio of E280E260 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. Cellu-lase-F showed powerful activity toward insoluble celluloses such as Avicel and filter paper, whereas the enzyme was inactive toward cellobiose, salfcine, 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