Directed evolution of a modified Geobacillus stearothermophilus xylanase for biotechnological applications

Abstract

Directed evolution using error-prone PCR (EP-PCR) was employed in the current study to enhance the catalytic efficiency of a thermostable Geobacillus stearothermophilus (G. stearothermophilus) xylanase XT6 parent. High-throughput screening identified two variants with enhanced activity. Sequencing analysis revealed the presence of a single amino acid substitution (P209L or V161L) in each variant. The maximum activity of mutant V161L and P209L was at 85 ºC and 70 ºC, respectively. Both mutants exhibited maximum activity at pH 7. The thermal and alkaline tolerance of mutant V161L only was markedly improved. The two mutants were more resistant to ethanol inhibition than the parent. 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. Both turnover number (kcat) and catalytic efficiency (kcat/kM) increased 12.2 and 5.7 folds for variant CB10 (P209L) and 13 and 6.5 folds for variant CG4 (V161L), respectively, toward birchwood xylan. Based on the previously published crystal structure of extracellular G. stearothermophilus xylanase XT6, V161L and P209L mutations are located on βα-loops. Conformational changes of the respective loops