IMPROVED PROCEDURES FOR PREPARATION AND CHARACTERIZATION OF MYROTHECIUM CELLULASE: PART 2. PURIFICATION PROCEDURES

Abstract

Two methods are described for purification of the cellulase of Myrothecium verrucaria from concentrated culture filtrates. The steps of method I are (1) fractionation with ammonium sulphate, (2) elution through Sephadex G25, (3) elution through Sephadex G75, (4) precipitation with polymethacrylic acid, and (5) elution through Amberlite CG50 with citrate buffer containing a gradient of urea concentration. The steps of method II are precipitation by saturated ammonium sulphate, (2) and (4) as above, elution through DEAE-cellulose with phosphate buffer containing 7 M urea, followed by (2) and (3) as above. The two methods gave final products with identical specific activities toward carboxymethyl cellulose; the increase in specific activity was approximately 12-fold. Starch-gel electrophoresis at pH 6.8 showed no major indications of heterogeneity. The purified enzyme was unstable but could be stored frozen in dilute salt solution.Enzyme passed through DEAE-cellulose without an inhibitor of cellulase activity was contaminated by DEAE-substituted oligoglucosides and subject to proteolysis. Urea could be replaced by cellobiose as an inhibitor but the latter gave enzyme contaminated by products of transfer reactions.Binding of urea by the resin is shown to influence significantly the resolution attainable in chromatographic fractionations on Amberlite CG-50 with buffers containing gradients of urea concentration.Procedures for dialysis and desalting of cellulases and a compact reaction vessel for pH stats are described.