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

Chong Sha1, Beenish Sadaqat1, Hongcheng Wang1

  • 1Biofuels Institute, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, China.

International Journal of Biological Macromolecules
|April 27, 2020
PubMed
Summary

We engineered a hyperthermophilic xylanase (TnexlnB) from Thermotoga neapolitana for improved biomass conversion. Directed evolution enhanced its optimal temperature to 85°C and stability, making it ideal for industrial applications.

Keywords:
Endo-xylanaseHeat-shock induction, site-directed mutation

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Area of Science:

  • Biotechnology
  • Enzymology
  • Biomass Utilization

Background:

  • Hyperthermophilic xylanases are crucial for converting xylan to sugar during biomass utilization.
  • Developing thermostable xylanases is essential to meet industrial demands.
  • Directed evolution offers an effective strategy for enzyme improvement.

Purpose of the Study:

  • To clone and express xylanase B (TnexlnB) from Thermotoga neapolitana.
  • To improve the thermostability and activity of TnexlnB through site-directed mutation.
  • To characterize the wild-type and mutant enzymes for biomass degradation applications.

Main Methods:

  • Gene cloning and soluble expression of TnexlnB in E. coli using a pHsh vector.
  • Site-directed mutagenesis targeting the terminal helices of TnexlnB.
  • Enzyme activity assays at various temperatures and pH conditions.
  • Determination of kinetic parameters (Km and Vmax) and thermal stability.

Main Results:

  • TnexlnB exhibited optimal activity at 75°C and pH 6.0, with activity enhanced by Mg2+, Ba2+, and Ca2+ ions.
  • Site-directed mutation increased the optimal temperature to 85°C.
  • The best mutant showed significantly improved catalytic efficiency (lower Km, higher Vmax) and retained >80% activity after 1 hour at 90°C.

Conclusions:

  • Engineered TnexlnB demonstrates enhanced thermostability and activity, suitable for high-temperature biomass degradation.
  • The improved enzyme holds potential for efficient and cost-effective biofuel production.
  • This study highlights the efficacy of directed evolution in creating robust industrial enzymes.