Molecular cloning and characterization of multidomain xylanase from manure library

Abstract

The gene (manf-x10) encoding xylanase from an environmental genomic DNA library was cloned and expressed in Escherichia coli. The manf-x10 encoded a predicted protein of 467 amino acids residues with a molecular mass of 50.3 kD. Sequence analysis of manf-x10 gene revealed that the N-terminus had high homology to the catalytic domain of other bacterial xylanase enzymes. The optimal pH and temperature for xylanase activity were 7.0 and 40°C, respectively. In the presence of 1 mM solution of Co2+, Fe2+, Mg2+ and Zn2+, the relative xylanase activity was enhanced; however, it had almost no activity in the presence of 10 mM solution of Cu2+. The apparent Km and Vmax values obtained for the hydrolysis of rye arabinoxylan were 2.8 mg/ml and 49.5 μmol/min/mg, respectively. The C-terminus of the enzyme had high homology to a domain of unknown function found in several mannanase enzymes. Biochemical characterization of the C-terminus of the enzyme revealed a previously unrecognized carbohydrate binding module.