Siswa Setyahadi

Constitutive High Level Expression of an Endoxylanase Gene from the Newly Isolated Bacillus subtilis AQ1 in Escherichia coli

A xylanolytic bacterium was isolated from the sediment of an aquarium. Based on the 16S rDNA sequence as well as morphological and biochemical properties the isolate was identified and denoted as Bacillus subtilis (B. subtilis) AQ1 strain. An endoxylanase-encoding gene along with its indigenous promoter was PCR amplified and after cloning expressed in E. coli. In E. coli the recombinant enzyme was found in the extracellular, in the cytoplasmic, and in the periplasmic fraction. The specific activity of the extracellular AQ1 recombinant endoxylanase after 24-hour fermentation was very high, namely, 2173.6 ± 51.4 and 2745.3 ± 11u2009U/mg in LB and LB-xylan medium, respectively. This activity was clearly exceeding that of the native B. subtilis AQ1 endoxylanase and that of 95% homologous recombinant one from B. subtilis DB104. The result shows that the original AQ1 endoxylanase promoter and the signal peptide gave a very high constitutive extracellular expression in E. coli and hence made the production in E. coli feasible.