Targeted molecular engineering of a family 11 endoxylanase to decrease its sensitivity towards Triticum aestivum...

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• Bacillus subtilis endoxylanase XynA (BSXY) enhances arabinoxylan-containing materials in cereal industries.
• Wheat contains Triticum aestivum xylanase inhibitors (TAXI-I and TAXI-II) that reduce BSXY efficiency.

Purpose of the Study:

• To develop inhibitor-insensitive BSXY variants by site-directed mutagenesis.
• To overcome the negative impact of TAXI-I and TAXI-II on BSXY performance.

Main Methods:

• Utilized the structure of a TAXI-I/Aspergillus niger endoxylanase complex to guide mutagenesis.
• Employed site-directed mutagenesis to alter amino acids in BSXY, inducing steric hindrance or disrupting inhibitor interactions.
• Assessed catalytic performance and inhibitor sensitivity of engineered BSXY variants.

Main Results:

• The G12W mutation conferred inhibition insensitivity while maintaining catalytic activity.
• Other variants exhibited altered inhibitor sensitivities and enzyme activities.
• Gained insights into the binding modes of TAXI-I and TAXI-II with BSXY.

Conclusions:

• Site-directed mutagenesis is effective in creating BSXY variants resistant to wheat xylanase inhibitors.
• Engineered BSXY variants show potential for improved applications in cereal processing.
• Understanding inhibitor-enzyme interactions facilitates rational enzyme design.