A novel bifunctional endo-/exo-type cellulase from an anaerobic ruminal bacterium

Abstract

An anaerobic microorganism termed AN-C16-KBRB was isolated from the bovine rumen and demonstrated cellulolytic activity on a NB agar plate containing azo-carboxymethyl cellulose. The 16S rRNA gene of the strain was 98% similar to that of Clostridiaceae bacterium SK082 (AB298754) as the highest homology. A novel celEdx16 gene encoding a bifunctional endo-/exocellulase (CelEdx16) was cloned by the shotgun method from AN-C16-KBRB, and the enzyme was characterized. The celEdx16 gene had an open reading frame of 1,104-base pairs, which encoded 367 amino acids to yield a protein of molecular mass 40.4 kDa. The amino acid sequence was 53% identical to that of an endoglucanase from Clostridium thermocellum. CelEdx16 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific endocellulase and exocellulase activities of CelEdx16 were 15.9 and 3.6 × 10-2 U mg -1, respectively. The Michaelis-Menten constant (K m values) and the maximal reaction velocities (V max values) of CelEdx16 were 47.1 μM and 9.6 × 10-3 μmole min -1 when endocellulase activity was measured and 106.3 μM and 2.1 × 10-5 μmole min-1 when exocellulase activity was assessed. CelEdx16 was optimally active at pH 5.0 and 40°C.