Purification and characterization of an endo-1,4-{u03B2}-D-glucanase and two exo-1,4-{u03B2}-D-glucanases from the cellulase system of Trichoderma reesei (1979)

An endo-1,4-β-D-glucanase (E.C. 3.2.1.4) and two exo-1,4-β-D-glucanases (exo-cellobiohydrolases I (D) and II, E.C. 3.2.1.91) have been purified to electrophoretic homogeneity from the extracellular culture filtrate of the imperfect fungus Trichoderma reesei QM 9414 grown on microcrystalline cellulose (Avicel). These three glycoprotein enzymes are the principal components of the cellulase system and constitute <95% of the extracellular protein produced by this organism when grown on cellulose or when incubated in the presence of sophorose (Q-β-D-glucopyranosyl (1->2) α-D-glucopyranose). The glucanases have been characterized with respect to a number of structural and enzymic properties. Neutral carbohydrate, predominantly mannose with some glucose, contributes 5, 21.2 and 14.1 percent of the weight of cellobiohydrolase I (D), cellobiohydrolase II and the endoglucanase, respectively. All three enzymes have a high proportion of acidic and hydroxylated amino acids, but much less of basic amino acids. Sedimentation equilibrium studies yielded the following molecular weights for the glucanases: cellobiohydrolase I (D) 53,220±1479; cellobiohydrolase II 54682±2683; endoglucanase 45,215±1483. Cellobiohydrolase I (D) is the most acidic (pH<sub>I</sub> <3.8) of the three enzymes, whereas the endoglucanase and cellobiohydrolase II have isoelectric points of 4.7 and 5.6, respectively. All three lose activity when exposed to alkaline conditions, and the extent of alkali lability is directly related to carbohydrate content. This instability, as well as the low amino sugar content of the enzymes, indicates that the carbohydrate may be linked to the polypeptide via O-glycosyl bonds to serine or threonine residues. Among the three enzymes, the endoglucanase is most resistant to thermal inactivation retaining 70% of its initial activity on swollen cellulose after a 20 min preincubation at 70°. The optimum pH for activity is ca.4.9 for the endoglucanase and cellobiohydrolase II, whereas cellobiohydrolase I (D) has a broader pH range for activity, between pH 5.2 to 5.6. The mechanism of action of each glucanase was investigated by quantitative high performance liquid chromatographic analysis of the products arising from various cellulosic substrates as a result of enzymic action. Cellobiohydrolases I (D) and II produce predominantly (>90%) cellobiose from either oligosaccharides or cellulose. Cellobiohydrolase I (D) is unique among the three glucanases in its ability to cleave cellotriose. The endoglucanase reduces the viscosity of carboxymethylcellulose solutions with a specific activity of 116 (expressed as the change in specific fluidity/min/mg protein). This enzyme also possesses significant transglycosylation activity. When the three glucanases are combined in the proportion 60:25:15, cellobiohydrolase I (D): cellobiohydrolase II: endoglucanase (w/w) respectively, the resulting mixture exhibits activity identical to that of the crude enzyme preparation, with either swollen or crystalline cellulose as the substrate. All three glucanases are essential for the degradation of crystalline cellulose. Cellobiohydrolase I (D) crossreacts immunologically with cellobiohydrolases I forms A, B and C, but not with cellobiohydrolase II, justifying the designation of CBH I and II as isozymes rather than forms of the same enzyme. An enriched β-glucosidase preparation from the culture filtrate of Trichoderma reesei QM 9414 formed a single precipitin line when allowed to react with antiserum to the β-glucosidase previously purified from a commercial T. viride cellulase preparation. The absence of any spurs of nonidentity indicates that these two enzymes are structurally very similar. Lack of crossreactivity is observed between cellobiohydrolase I (D) and the endoglucanase purified during this investigation, but the latter crossreacts weakly with antiserum to cellobiohydrolase II.