Purification and Characterization of an Endo-cellulase from Acremonium cellulolyticus.

Abstract

An endo-cellulase from a commercial cellulase preparation of a filamentous fungus Acremonium cellulolyticus was separated and extensively purified to essential homogeneity by using consecutive column chromatographies of QAE-Toyopearl 550C, DEAE-Toyopearl 650S, Butyl-Toyopearl 650M and Bio-Gel P-100, and was designated as cellulase IV The purified enzyme was homoge-neous on both Native- and SDS-PAGE, and was completely free from β-glucosidase. The enzyme showed high specific activity for CMC and an extremely low specific activity for Avicel (a mi-crocrystalline cellulose powder), 106 and 0.08 U/mg of protein, respectively. The molecular mass of the enzyme was estimated to be about 38 kDa by SDS-PAGE. The enzyme was an acidic protein with a pI value of≤3.4. The N-terminal amino acid sequence from the 2nd up to the 27th residue of the purified cellulase was Gln-Ala-Ser-(Cys)-Phe-Glu-Trp-Phe-Gly-Ser-Asn-Glu-Ser-Gly-Ala-Glu-Phe-Gly-Ser-Gly-Asn-Ile-Pro-Gly-Val-Glu-. The optimum pH and temperature were 4.0 and 60-C, respectively. The enzyme was completely stable over the range of pH 5.0-12.0 at 4-C for 24 h and at temperatures below 50-C. Cellulase IV was characterized as a medium endo -type cellulase on the basis of its action on CMC and cellooligosaccharides. The enzyme was completely inhibited by 5 mM Hg2+, Fe3+ and KMnO4. Cellulase IV split various cellulosic substrates to pro-duce predominant cellobiose and a small amount of glucose as the final hydrolysis products.