N- and C-terminal truncations of a GH10 xylanase significantly increase its activity and thermostability but decrease...

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• XynII from Volvariella volvacea exhibits high sodium dodecyl sulfate (SDS) resistance, indicating potential for industrial use under harsh conditions.
• The enzyme possesses a glycoside hydrolase family 10 (GH10) catalytic domain with unique N- and C-terminal extensions.

Purpose of the Study:

• To investigate the impact of N- and C-terminal deletions on XynII's enzymatic properties, including activity, substrate specificity, thermostability, and SDS resistance.
• To understand the role of terminal regions in XynII structure and function.

Main Methods:

• Construction and characterization of five XynII derivatives with varying N- and/or C-terminal deletions.
• Enzyme activity assays using beechwood xylan and various xylooligosaccharides.
• Thermostability and SDS resistance assessments (half-life measurements).
• Circular dichroism spectroscopy to analyze secondary structure changes.

Main Results:

• N- and C-terminal truncations significantly enhanced XynII's catalytic efficiency (kcat/Km) and thermostability.
• The XynIIΔNC4 mutant showed 2.53-fold higher catalytic efficiency and 3.0-fold greater thermostability compared to wild-type XynII.
• While activity against various substrates increased, SDS resistance was notably reduced in truncated mutants, with XynIIΔNC4 having a shorter half-life in SDS than wild-type XynII.

Conclusions:

• The N- and C-terminal segments of XynII contribute to its structural rigidity and SDS resistance.
• Terminal deletions enhance enzyme activity and thermostability by increasing structural flexibility.
• These findings provide a basis for engineering XynII for specific industrial applications requiring high activity and thermostability, potentially at the expense of SDS resistance.