A novel thermostable endoglucanase from the wood-decaying fungus Daldinia eschschokii (Ehrenb.: Fr.) Rehm

A thermostable endoglucanase was purified to homogeneity from culture supematants of the wood-decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm grown on 1.0% (w/v) carboxymethyl-cellulose using ammonium sulfate precipitation, ion-exchange, hydrophobic interaction, and gel filtration chromatography. The molecular weight of the enzyme was 46.4 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelecttic point of the enzyme was at pH 4.9. The temperature for maximum activity was 70 degrees C, with 85% of its maximum activity retained after 150 min of incubation at 50 degrees C, but was rapidly inactivated at 70 degrees C. The pH optimum of the enzyme activity was 6.0, and it was stable over a pH range of 4.0-7.0 at 50 degrees C. The enzyme was significantly inhibited by Hg2+, Cu2+, and Fe4+, and stimulated by Ca2+, Co2+, Mg2+, Mn2+, glycerol, DMSO, DTT, and EDTA. The enzyme also hydrolyzed filter paper, and Avicel (R) PH-101 at rates of 25.8%, and 7.3%, respectively when compared with carboxymethyl-cellulose. The enzyme did not hydrolyze soluble starch, oat spelt xylan, birch wood xylan, or locust bean gum. The enzyme catalyzed the hydrolysis of carboxymethyl-cellulose with a K-m of 1.74 mg/ml and a V-max of 0.63 U/min/mg protein. This enzyme was competitively inhibited by glucose and cellobiose with K-i values of 0.67 and 0.45 M, respectively. TLC showed that the endoglucanase produces cellotetraose, cellotriose, cellobiose, and a small amount of glucose. The deduced internal amino acid sequences of the D. eschscholzii endoglucanase showed similarity to the sequences of the glucosyl hydrolase family 5. (C) 2007 Elsevier Inc. All rights reserved.