The recombinant enzyme lichenase of size 30 kDa was over-expressed using E. coli cells and purified by immobilized metal ion affinity chromatography (IMAC) and size exclusion chromatography. The enzyme displayed high activity towards lichenan and β-glucan. The enzyme showed no activity towards carboxymethyl cellulose, laminarin, galactomannan or glucomannan. Surprisingly, affinity-gel electrophoresis on native-PAGE showed that the enzyme binds only glucomannan and not lichenan or β-glucan or other manno-configured substrates. The enzyme was thermally stable between the temperatures 60°C and 70°C. Presence of Cu(2+) ions at a concentration of 5 mM enhanced enzyme activity by 10% but higher concentrations of Cu(2+) (>25 mM) showed a sharp fall in the enzyme activity. Heavy metal ions Ni(2+), Co(2+) and Zn(2+) did not affect the activity of the enzyme at low concentrations (0-10 mM) but at higher concentrations (>10 mM), caused a decrease in the enzyme activity. The crystals of lichenase were produced and the 3-dimensional structure of native form of enzyme was previously solved at 1.50 Å. Lichenase displayed (β/α)(8)-fold a common fold among many glycoside hydrolase families. A cleft was identified that represented the probable location of active site.
Keywords: Cellulase; Clostridium thermocellum; Glycoside hydrolase; Lichenase.