Efficient xylan-to-sugar biotransformation using an engineered xylanase in hyperthermic environment.

Hyperthermophilic xylanases play a critical role in bioconversion from xylan to sugar in the process of biomass utilization. The discovery of new or improvement of existing xylanases based on directed evolution is expected to be an effective approach to meet the increasing demand of thermostable xylanases. In this work, a xylanase B gene (CTN_0623) from Thermotoga neapolitana (Tne) was cloned and xylanase B (herein named Tne xlnB) was solubly expressed in E. coli with a high amount using a heat shock vector pHsh. Tne xlnB showed the highest endo -β-1,4-xylan hydrolase activity at 75 °C and pH 6.0. Additionally, 1 mM Mg2+, Ba2+ and Ca2+ improved the activity of Tne xlnB by 31%, 37%, and 53%, respectively. The optimal temperature reached 85 °C by site-directed mutation at the last three helices of Tne xlnB. K m and V max towards birchwood xylan were determined for both wide type and the best mutant, as follow: 1.09 mg/mL, 191.76 U/mg and 0.29 mg/mL, 376.42 U/mg, respectively. Further characterization highlighted good thermal stability (>80% of enzymatic activity after 1 h at 90 °C) for the best mutant, which made this enzyme suitable for biomass degradation at high temperature. [ABSTRACT FROM AUTHOR]