الفهرس 
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Abstract
Xylanases have potential applications in brewing industry, food and animal feed production, pharmaceutical industry, pulp biobleaching, bioconversion of waste, and biofuel production. However, the narrow temperature and pH ranges, the poor ionic strength resistance and low catalytic performance of most natural xylanases constrains their wide applicability under the harsh industrial processes. Therefore, improving xylanase functional properties is urgently needed. Protein engineering is a useful approach to get tailor-made xylanases. It includes rational and semi-rational design and directed evolution. The latter has drawn tremendous attention to drive progeny proteins with desirable trait(s).
Previously, Zhang et al. (2010) introduced 13 amino acids substitution in G. stearothermophilus xylanase XT6 wild type gene by several rounds of directed evolution followed by site-directed mutagenesis. The generated variant (FC06T) had an optimum activity at temperature 87 ºC and improved thermal tolerance by 52-fold. By directed evolution, we succeeded here to improve the catalytic activity of the Zhang’s variant. The gene of FC06T variant was chemically synthesized, inserted into expression plasmid pJ404 and mutated by EP-PCR kit. After mutagenesis, MEGAWHOP method (ligation-independent cloning method) was applied for subcloning and xylanase library was then expressed in E.coli DH5α.
The findings obtained can be summarized in the following:
1- A Library of 864 xylanase variants was expressed and screened.
2- Genes of variants with higher activity than the parent were sequenced.
3- Sequencing identified point mutations in four variants as follows; V161L mutation in CG4 variant, P209L mutation in CB10 variant, H378R and V144I mutations in DD3 variant and P18L mutation in FE8 variant.
4- The four mutant xylanases exhibited single protein bands equivalent to 45 kDa on SDS-PAGE after being purified by Ni-IMAC.
5- The highest specific activities were shown by the purified proteins of CG4 (2811U/mg) and CB10 (2957 U/mg) variants. Such findings and other parameters of purification scheme recommend these two variants for further characterization versus the parent FC06T variant.
6- The maximum activity of mutant V161L and P209L was at pH 7 and 85 ºC and 70 ºC, respectively. The thermal and alkaline tolerance of mutant V161L was markedly improved relative to the parent.
7- The two mutants and the parent xylanase displayed similar activity profile in the presence of different concentrations of metal ions suggesting that the point mutations reported here have no distinct effect on the metal binding site of the enzyme.
8- All xylanase variants showed a comparable inhibitory effect with PEG having nearly similar IC50 values.
9- The two mutants were more resistant to ethanol inhibition than the parent.
10- Substrate specificity of the two mutants was shifted from beechwood xylan to birchwood xylan. The potential of the two mutants to hydrolyze rice straw and sugarcane bagasse increased.
11- Both turnover number (kcat) and catalytic efficiency (kcat/kM) for the variants CB10 (P209L) and CG4 (V161L) toward birchwood xylan increased by 12.2 and 13 folds and by 5.7 and 6.5 folds, respectively.
12- The crystal structure of extracellular G. stearothermophilus xylanase XT6 shows that the mutations V161L and P209L are located on βα-loops. Conformational changes of the respective loops could potentiate the loop swinging, product release and consequently result in enhancement of the catalytic performance.