Characterization and Substrate Specificity of a Recombinant Acetyl Xylan Esterase from Halalkalibacterium halodurans...

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Biotechnology

Background:

• Acetylxylan esterases (AXEs) are crucial for hydrolyzing acetyl groups in xylan.
• Current AXEs suffer from narrow substrate specificity, limiting their industrial utility.
• Engineering AXEs can enhance their performance for lignocellulose applications.

Purpose of the Study:

• To engineer a CE7 family acetylxylan esterase (HhAXE) from *Halobacillus halodurans* for improved industrial applications.
• To enhance substrate specificity and catalytic efficiency of HhAXE using computational strategies.
• To evaluate the potential of engineered HhAXE as a biocatalyst for lignocellulose valorization.

Main Methods:

• Site-directed mutagenesis guided by PROSS-based thermostabilization and machine learning.
• Enzyme purification and characterization of optimal activity and stability conditions.
• Assessing enzyme activity with various substrates and in the presence of metal ions and organic solvents.
• Molecular docking and dynamics simulations to understand structural modifications.

Main Results:

• The wild-type HhAXE showed optimal activity at pH 8.5 and 40 °C, with stability in alkaline conditions.
• Fe³⁺, Mn²⁺, and DMSO significantly enhanced the enzyme's activity.
• The engineered mutant L260R exhibited a 136.16% increase in activity towards ρNPA and improved affinity for longer-chain substrates.
• The mutant demonstrated significantly higher deacetylation of beechwood, arabinoxylan, and corncob.
• Computational simulations revealed structural basis for enhanced substrate binding and catalytic efficiency.

Conclusions:

• The engineered HhAXE-L260R represents a promising biocatalyst for lignocellulose valorization.
• Dual computational strategies are effective for optimizing AXE performance.
• The study provides a valuable computational approach for future enzyme engineering endeavors.