Purification and characterization of cellulases produced by two Bacillus strains

Cellulases produced by two Bacillus strains, CH43 and HR68, isolated from h ot springs in Zimbabwe, were purified to homogeneity from culture supernata nts. Both enzymes had molecular mass of 40 kDa and isoelectric point of 5.4 . The enzymes also resembled each other in N-terminal amino acid sequence w hich was Ala-Gly-Thr-Lys-Thr-Pro-Val-Ala-Lys-Asn-Gly-Gln, showing 100% homo logy with that of endoglucanases from Bacillus subtilis belonging to glycos ide hydrolase family five. The cellulases were optimally active in the pH r ange of 5-6.5. The optimum temperature was 65 and 70 degrees C for the endo glucanase of CH43 and HR68, respectively. The CH43 enzyme was stable at 50 degrees C in a pH range of 6-10, and HR68 at pH 6-8. Both the enzymes retai ned complete activity for at least 24 h at 50 degrees C. The enzymes showed highest activity with beta-glucan as substrate followed by carboxymethylce llulose. Significant activity was also observed with crystalline forms of c ellulose such as filter paper and Avicel, particularly for HR68 cellulase. For carboxymethycellulose, the CH43 and HR68 cellulases had a K-m of 1.5 an d 1.7 mg ml(-1), respectively, and V-max of 0.93 and 1.70 mmol glucose min( -1) mg protein(-1), respectively. The activity of the enzymes was not influ enced by most metal ions at 1 mM concentration, but was increased by about 38% by Co2+. The inhibition by Hg2+ and Mn2+ was higher for CH43 than for H R68 enzyme. Ag+ inhibited the CH43 activity but stimulated the HR68 activit y. The CH43 cellulase was inhibited by N-bromosuccinimide and iodoacetamide while HR68 was unaffected. (C) 2000 Elsevier Science B.V. All rights reser ved.