A new procedure using high-performance liquid chromatography for the rapid separation of cellulase proteins is described. The cellulase components of Trichoderma reesei are fractionated on a DEAE anion-exchange column using a phosphate buffer at pH 6.2. Activities of the individual components obtained from T. reesei QM6a, a wild strain, and several mutant strains have been determined_ Each system examined contained /&glucosidase, at least two exo-p-1.4 glucanases and five endo-s-l,4 glucanases with the endo-$-I,4 glucanases accounting for 20-36 o? and the exe-@-I,4 glucanases for 64-80% of the soluble protein.
1NTRODUCTION
The cellulases produced by microorganisms are complex mixtures of several types of enzymes wherein the major ones are endo-#?-1,4 glucanase, exe-p-l,4 glucanase and cellobiase. These are frequently associated with other glycanases, glycosidases and proteases all of which act individually and/or synergistically to bring about the degradation and hydrolysis of various polymers often associated with cellulosic materials.
In order to evaluate these microorganisms for cellulase activity it is necessary to examine a large number of culture filtrates which have been produced under different conditions. This evaluation is usually done by the filter paper assay method'. This procedure gives useful information about the activity of the enzyme preparation but no information about the levels of the individual components and their role in saccharification. This type of information can best be obtained by fractionating the enzyme system.
Cellulase proteins have been fractionated on carbohydrhte and polyacrylamide-gel columnsZ-J. The separations are achieved primarily by ion exchange, steric exclusion and affinity chromatography. Since these matrices are sensitive to changes in pH, ionic strength and pressure, the gradient eIution of components must be carried out slowly. A typical run may require 18 to 24 h. One way to decrease this time is to increase the mechanical stability of the support material, by king inorganic