Constitutive High Level Expression of an Endoxylanase Gene from the Newly Isolated Bacillus subtilis AQ1 in Escherichia coli
Figure 3
Recombinant E. colis with and without xylanase activity and the comparison of properties of AQ1 and DB104 recombinant xylanase. (a) Recombinant E. colis on LB xylan, recombinant E.coli harbouring empty pGEM-T-easy vector (1), recombinant E.coli with vector pGEM containing AQ1xyn without native promoter (2), recombinant E.coli with vector pGEM containing AQ1xyn with native promoter (3), and recombinant E.coli with vector pGEM containing DB104xyn with native promoter (4). (b) The molecular mass of recombinant AQ1 endoxylanase in E. coli. For this analysis, the expression of the xylanase gene in cytoplasmic fraction with T7 promoter was used. (c) Effect of temperature on recombinant AQ1 endoxylanase and DB104 endoxylanase grown in LB and LB-xylan medium ( triangle was recombinant AQ1 xylanase, black one for that came from E. coli grown in LB-xylan, white one for that one grown in LB; square was DB104, black one for that one grown in LB-xylan, white one for that one grown in LB). For this temperature profile, enzymatic activity was measured in 50 mM sodium phosphate pH 7. (d) Effect of pH on recombinant AQ1 endoxylanase and DB104 endoxylanase from E. coli grown in LB and LB-xylan medium in different buffers. The reaction pHs were adjusted to 5–11 with the following buffers: 50 mM citrate buffer (pH 5,6), 50 mM phosphate buffer (pH 6–8), 50 mM Tris-HCl (pH 8–10), and 50 mM Tris-Glycine buffer (pH 10,11) ( triangle was recombinant AQ1 xylanase, black one for that came from E. coli grown in LB-xylan, white one for that one grown in LB; square was recombinant DB104 xylanase, black one for that one grown in LB-xylan, white one for that one grown in LB). (e) Thermostability of recombinant AQ1 endoxylanase and DB104 endoxylanase. Thermostability was determined by preincubating enzyme extract at 50, 55, and 60°C for designated time periods and then assaying the activity in pH 7.0 of 50 mM sodium phosphate at 50°C for 5 min as described in Materials and Methods. The given values in all activity assays are the means of triplicates, and the error bars indicate the standard deviation of these triplicates of independent experiment.
