Journal Highlight: Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP-dependent enzymes

Skip to Navigation

Ezine

  • Published: Sep 19, 2016
  • Author: separationsNOW
  • Channels: HPLC
thumbnail image: Journal Highlight: Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP-dependent enzymes
A generic method using an automated, standardizable HPLC platform was developed for the rapid and direct quantification of enzyme kinetics independent of the substrate, exemplifed by amine transaminase-catalyzed chiral amine synthesis.

Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP-dependent enzymes

Biotechnology Journal, 2016, 11, 1025-1036
Tim Börner, Carl Grey and Patrick Adlercreutz

Abstract: Methods for rapid and direct quantification of enzyme kinetics independent of the substrate stand in high demand for both fundamental research and bioprocess development. This study addresses the need for a generic method by developing an automated, standardizable HPLC platform monitoring reaction progress in near real-time. The method was applied to amine transaminase (ATA) catalyzed reactions intensifying process development for chiral amine synthesis. Autosampler-assisted pipetting facilitates integrated mixing and sampling under controlled temperature. Crude enzyme formulations in high and low substrate concentrations can be employed. Sequential, small (1 µL) sample injections and immediate detection after separation permits fast reaction monitoring with excellent sensitivity, accuracy and reproducibility. Due to its modular design, different chromatographic techniques, e.g. reverse phase and size exclusion chromatography (SEC) can be employed. A novel assay for pyridoxal 5'-phosphate-dependent enzymes is presented using SEC for direct monitoring of enzyme-bound and free reaction intermediates. Time-resolved changes of the different cofactor states, e.g. pyridoxal 5'-phosphate, pyridoxamine 5'-phosphate and the internal aldimine were traced in both half reactions. The combination of the automated HPLC platform with SEC offers a method for substrate-independent screening, which renders a missing piece in the assay and screening toolbox for ATAs and other PLP-dependent enzymes.

  • This paper is free to view for all users registered on separationsNOW.com until the end of October 2016.
    After this time, you can purchase it using Pay-Per-View on Wiley Online Library.

Follow us on Twitter!

Social Links

Share This Links

Bookmark and Share

Microsites

Suppliers Selection
Societies Selection

Banner Ad

Click here to see
all job opportunities

Copyright Information

Interested in spectroscopy? Visit our sister site spectroscopyNOW.com

Copyright © 2017 John Wiley & Sons, Inc. All Rights Reserved