Fast automated online xylanase activity assay using HPAEC-PAD

Area of Science:

• Analytical Chemistry
• Biochemistry
• Enzymology

Background:

• Automated control is common in biochemical reactions but often neglected in chemical analysis.
• Traditional methods involve rudimentary in situ analysis or offline measurements of stored aliquots, risking sampling and storage errors.

Purpose of the Study:

• To implement automated reaction control, sampling, and analysis for chemical assays.
• To exemplify this by examining xylanase activities on xylotetraose and soluble xylan.

Main Methods:

• Automated reaction control, sampling, and analysis were performed within a temperature-controlled autosampler.
• Xylanase activity was measured using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).
• An adapted 13-minute HPAEC-PAD method with specific gradient elution was used for analyzing xylooligosaccharides.

Main Results:

• The automated system successfully monitored enzymatic hydrolysis of xylan.
• The detection limit for xylooligomers ranged from 0.35-1.83 mg/L.
• Specific activities were determined: 294 U/mg for Aspergillus niger EX, 1.69 U/mg for Bacillus stearothermophilus EX, and 0.36 U/mg for Bacillus subtilis EX.

Conclusions:

• The developed automated method enables precise and efficient analysis of enzyme activity.
• This approach minimizes errors associated with traditional sampling and storage.
• It provides a robust platform for kinetic studies of enzymatic reactions.