Production of free and substrate-bound cellulases of Cellulomonas flavigena Amtul Jamil Sami, M. Waheed Akhtar,* Nadeem Nawazish Malik and Bashir Ahmad Naz Division of Biochemistry, Institute of Chemistry, University of the Punjab, Lahore-I, Pakistan (Received 24 July 1987; revised 14 January 1988) Conditions for the production of extracellular carboxymethyl cellulase (CMCase) and avicelase activi- ties by a locally isolated Cellulomonas species identified as C. flavigena were optimized. The microbe produced maximal levels of free cellulases after 72 h of fermentation, when cultivated in the presence of 0.2% yeast extract, 0.5% Avicel, and 0.1% Tween 80 at 30°C. Conditions for the elution of substrate- bound cellulases from the residual Avicel were optimized. Higher proportions of the extracellular cellulases were bound to the substrate when higher concentrations of Avicel were used. When 2.0% Avicel was used as a carbon source, maximal levels of extracellular CMCase and avicelase produced were 13.2 and3 U m1-1 of the culture medium, respectively. Both the cellulases were most active at pH 6.5 and 50°C and were inhibited by ~-mercaptoethanol. Calcium chloride activated CMCase but had no effect on avicelase activity. Each of the enzymes lost activity when incubated at 70°C for 30 min. A comparative study of free and substrate-bound CMCase on gradient polyacrylamide gel electrophore- sis (PAGE) showed that free CMCase activity was composed of at least seven active fractions, while bound activity had four major bands of CMCase activity. Since a considerable level of protease was secreted by C. flavigena, the additional fractions in the free CMCase activity could be a result of proteolysis. Keywords: CMCase; avicelase; Cellulomonasflavigena Introduction Enzymes involved in the breakdown of cellulose to glucose fall into three basic groups: 1 endo-1,4-/3-D-glu- canase (1,4-/3-D-glucan 4-glucanohydrolase, EC 3.2.1.4), exo- 1,4-fl-D-glucanases (1,4-/3-D-glucan cello- biohydrolase, EC 3.2.1.91, and 1,4-/3-D-glucan gluco- hydrolase, EC 3.2.1.74), and fl-D-glucosidases (fl-D- glucoside glucohydrolase, EC 3.2.1.21). A large variety of fungal and bacterial species have been re- ported to produce one or more of these enzymes. A number of reviews dealing with the nature of cellulosic substrates, 2-5 production and properties of cellulases, and their action on cellulose hydrolysis 6-1° have been published. Amongst a variety of bacterial species reported for cellulolytic activity, Cellulomonas species have re- ceived considerable attention. Kim and Wimpeny ~ studied the growth and cellulolytic activities of C. flavigena. Grass-based lignocellulosic substrates have * To whom correspondence should be addressed been reported to enhance cellulase production by this organism. 12 A strain of C. uda was reported to be an efficient producer of crystalline cellulose hydrolysing activity when grown in the presence of crystalline cel- lulose. 13 Stoppok et al. 14 studied the formation, loca- tion, and regulation of endo-/3-1,4-glucanase and /3- glucosidases by C. uda in the presence of different carbon sources. Production of cellulases by C. uda was studied using printed newsprint as a carbon source. 15 Btguin and Eistn 16 reported the purification of three extracellular cellulases from the culture of Cellulomonas. One of these was found in the culture supernatant, while the other two were found to be bound to the cellulose added as a carbon source. Two extracellular endoglucanases were purified and par- tially characterized from culture supernatants of C. fermentans.17 Six major components exhibiting endo- glucanase activity were partially purified from culture filtrates of a Cellulomonas species. 18Langsford et al. 19 reported that the cellulase system of C. fimi was com- posed of only three enzymes, which had great affinity for, and were stabilized by binding to, an insoluble 626 Enzyme Microb. Technol., 1988, vol. 10, October © 1988 Butterworth Publishers