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

Abstract A thermostable endoglucanase was purified to homogeneity from culture supernatants 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 isoelectric point of the enzyme was at pH 4.9. The temperature for maximum activity was 70 °C, with 85% of its maximum activity retained after 150 min of incubation at 50 °C, but was rapidly inactivated at 70 °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 °C. The enzyme was significantly inhibited by Hg 2+ , Cu 2+ , and Fe 2+ , and stimulated by Ca 2+ , Co 2+ , Mg 2+ , Mn 2+ , glycerol, DMSO, DTT, and EDTA. The enzyme also hydrolyzed filter paper, and Avicel ® 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.