Fast automated online xylanase activity assay using HPAEC-PAD
In contrast to biochemical reactions, which are often carried out under automatic control and maintained overnight, the automation of chemical analysis is usually neglected. Samples are either analyzed in a rudimentary fashion using in situ techniques, or aliquots are withdrawn and stored to facilitate more precise offline measurements, which can result in sampling and storage errors. Therefore, in this study, we implemented automated reaction control, sampling, and analysis. As an example, the activities of xylanases on xylotetraose and soluble xylan were examined using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The reaction was performed in HPLC vials inside a temperature-controlled Dionex™ AS-AP autosampler. It was started automatically when the autosampler pipetted substrate and enzyme solution into the reaction vial. Afterwards, samples from the reaction vial were injected repeatedly for 60 min onto a CarboPac™ PA100 column for analysis. Due to the rapidity of the reaction, the analytical method and the gradient elution of 200 mM sodium hydroxide solution and 100 mM sodium hydroxide with 500 mM sodium acetate were adapted to allow for an overall separation time of 13 min and a detection limit of 0.35–1.83 mg/L (depending on the xylooligomer). This analytical method was applied to measure the soluble short-chain products (xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and longer xylooligomers) that arise during enzymatic hydrolysis. Based on that, the activities of three endoxylanases (EX) were determined as 294 U/mg for EX from Aspergillus niger, 1.69 U/mg for EX from Bacillus stearothermophilus, and 0.36 U/mg for EX from Bacillus subtilis.
KeywordsAutomatic activity assay Xylanase HPAEC-PAD Enzyme
This work was performed as part of the Cluster of Excellence “Tailor-Made Fuels from Biomass,” which is funded by the Excellence Initiative of the German federal and state governments to promote science and research at German universities. The scientific activities of the Bioeconomy Science Center were financially supported by the Ministry of Innovation, Science, and Research within the framework of the NRW Strategieprojekt BioSC (no. 313/323-400-002 13). Specials thanks go to Priya Philip of the Institute for Biochemical Engineering of RWTH Aachen University for supplying the RAMOS fed-batch technique.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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