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

Börner, Tim LU ; Grey, Carl LU and Adlercreutz, Patrick LU (2016) In Biotechnology Journal 11(8). p.1025-1036
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... (More)

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.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Enzyme kinetics, Internal aldimine, Pyridoxamine, Size exclusion chromatography, Substrate-independent screening
in
Biotechnology Journal
volume
11
issue
8
pages
12 pages
publisher
John Wiley & Sons
external identifiers
  • Scopus:84977121545
  • Scopus:84979742008
  • WOS:000381071900005
ISSN
1860-6768
DOI
10.1002/biot.201500587
language
English
LU publication?
yes
id
ac0c46f3-10b4-4e5f-80b2-e0497dd7a1d2
date added to LUP
2016-07-18 11:53:17
date last changed
2017-01-15 04:40:18
@article{ac0c46f3-10b4-4e5f-80b2-e0497dd7a1d2,
  abstract     = {<p>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.</p>},
  author       = {Börner, Tim and Grey, Carl and Adlercreutz, Patrick},
  issn         = {1860-6768},
  keyword      = {Enzyme kinetics,Internal aldimine,Pyridoxamine,Size exclusion chromatography,Substrate-independent screening},
  language     = {eng},
  month        = {08},
  number       = {8},
  pages        = {1025--1036},
  publisher    = {John Wiley & Sons},
  series       = {Biotechnology Journal},
  title        = {Generic HPLC platform for automated enzyme reaction monitoring : Advancing the assay toolbox for transaminases and other PLP-dependent enzymes},
  url          = {http://dx.doi.org/10.1002/biot.201500587},
  volume       = {11},
  year         = {2016},
}