Gene cloning and characterization of a xylanase from a newly isolated Bacillus subtilis strain R5(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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A novel mesophilic strain, R5, was isolated from Osaka, Japan. The growth temperature of the strain ranged from 10 to 40℃ with optimal growth at 30℃. 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 23kDa. The gene product was purified and examined for enzymatic characteristics. The recombinant enzyme exhibited highest activity at temperatures ranging from 40 to 50℃ 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 beechwood were 5550nkat/mg and 4.5mg/mL, respectively.
社団法人日本生物工学会の論文