Gene cloning and characterization of a xylanase from a newly isolated Bacillus subtilis strain R5

A novel mesophilic strain, R5, was isolated from Osaka, Japan. The growth temperature of the strain ranged from 10 to 40 degrees C with optimal growth at 30 degrees C. Cells of strain R5 were highly motile small rods. The full-length 16S rRNA sequence was 99% homologous to that of Bacillus subtilis strain 168. The optimum pH and NaCl concentration for growth of the strain were 7.0 and 3%, respectively. Based on the biochemical characteristics and 16S rRNA sequences R5 was identified as a strain of B. subtilis. The strain R5 produced protease, cellulase, amylase, lipase/esterase, xylanase and a biosurfactant extracellularly. The gene encoding xylanase was cloned and expressed in Escherichia coli. The gene encoded a protein consisting of 213 amino acids with a relative molecular mass of 23 kDa. The gene product was purified and examined for enzymatic characteristics. The recombinant enzyme exhibited highest activity at temperatures ranging from 40 to 50 degrees C and at pH 6.0. The enzyme activity was enhanced in the presence of metal cations. The V(max) and K(m) values of the recombinant enzyme towards xylan from beech-wood were 5550 nkat/mg and 4.5 mg/mL, respectively.