Production, purification, and properties of an endoglucanase produced by the hyphomycete Chalara (Syn. Thielaviopsis) paradoxa CH32

The hyphomycete Chalara (syn. Thielaviopsis) paradoxa produces endoglucanas e activity during the late trophophase. The low molecular mass (35 kDa) end oglucanase purified from cultured broths works optimally at 37 degreesC; an d pH 5.0. The enzyme inactivates at pH below 3.0 and also at temperatures o f 50 degreesC;or higher, but it is stable at lower temperatures, including refrigeration temperature and freezing. The enzyme is inhibited by detergen ts, by EDTA, and by the divalent cations Hg2+ and Ag2+. It is also inhibite d to some extent by 10 mM Zn2+, Fe2+, and Mg2+, but it is stimulated by Mn2 +. Enzyme activity is not affected by reducing agents. In the presence of l ow concentrations of water miscible organic solvents (20%) endoglucanase ac tivity is inhibited by 7% (for methanol) to 50% (for acetonitrile), and it is totally inhibited at higher solvent concentrations (50%). Enzyme activit y is not affected by the water immiscible solvent ethyl acetate. Carboxy-me thylcellulose is the preferred substrate (K-m(app) = 8.3 g/L; V-max(app) = 1.1 muM/min). Hydrolysis of crystalline cellulosic substrates is very limit ed, but it is greatly enhanced by phosphoric acid swelling. The purified en zyme shows no activity toward disaccharides or aryl-glucosides. Its activit y is inhibited by cellobiose.