Characterization of structure and enzymic activity of endo-l, 4-β-D-glucanases purified from Trichoderma viride

Abstract

Four electrophoretically distinct endo-1,4-β-D-glucanases have been isolated from a commercial Trichoderma viride enzyme preparation, Pancellase SS. These endoglucanases were distinguished by their affinity for microcrystalline cellulose; two were adsorbed onto a cellulose column at pH 5.0 (Endoglucanases III and IV) and two were not (Endoglucanases I and II). Endoglucanases II, III and IV have been purified subsequently by ion-exchange chromatography. Enzyme homogeneity has been established using polyacrylamide gel electrophoresis, slab gel eletrophoresis of sodium dodecyl sulfate-protein complexes and ultracentrifugation studies. With CM-cellulose as substrate Endoglucanases II, III and IV have specific viscosimetric activities of 1010, 60 and 250 specific fluidity change minute⁻¹ mg protein⁻¹, respectively, and from this substrate release reducing sugars at rates of 29.1, 28.2 and 9.16 µmole minute⁻¹ mg protein⁻¹, respectively. The specific activities of these endoglucanases are less when crystalline cellulosic substrates are employed. Endoglucanases II, III and IV have, respectively, E1%280 values of 11.97, 10.32 and 13.12, and molecular weights of 37,200, 52,000 and 49,000 as determined by sedimentation equilibrium. The three endoglucanases each contain mannose, galactose, glucose and glucosamine. Endoglucanase II contains significantly less carbohydrate (4.5% by weight) than Endoglucanases III and IV which contain 15.0% and 15.2%, respectively. Plots of viscosity decrease versus reducing sugar production indicate that Endoglucanases II and IV have similar modes of action on CM-cellulose which differ from that of Endoglucanase III. All three endoglucanases have similar pH optima of 4.0-4.5 and lose activity above pH 7.0. High pressure liquid chromatographic analysis permitted determination of enzymic rates of reaction with purified cellooligosaccharides. Endoglucanases II, III and IV display higher activities as the chain-length of cello oligosaccharide substrates progresses from two to six. Kinetic studies revealed differences in Endoglucanase III as compared to Endoglucanases II and IV; it is distinguished by low affinity for cellobiose (Km=162 compared to Km=1.03 and 1.26 mM) and its ten-fold higher activity with cellotriose (Vmax=2.49 compared to Vmax=0.339 and 0.279 µmole minute⁻¹mg protein⁻¹). In addition, Endoglucanase III differs from the other endoglucanases in its low activity on p-nitrophenyl glucoside, its preference for the third bond from the non-reducing end of cellopentaitol, and its significant transferase activity in the presence of large amounts of substrate-acceptor oligosaccharides. Results of high pressure liquid chromatographic analysis of the soluble sugars produced during endoglucanase reaction with cellulose was consistent with kinetic studies using oligosaccharide substrates. From phosphoric acid swollen cellulose, Endoglucanases II and IV yielded glucose, cellobiose and cellotriose as distinctive products, whereas Endoglucanase III action produced only glucose and cellobiose. Endoglucanases III and IV each produce substantial quantities of reducing sugars as well as short fibers from filter paper, whereas Endoglucanase II does not. These structural and catalytic properties may be used to describe more precisely the role of each component in the cellulase system.