Original Research ARTICLE
Citrate as cost-efficient β nicotinamide adenine dinucleotide phosphate regenerating agent for characterization and screening purposes of NADPH-dependent enzyme
- 1IBG-1: Biotechnology, Forschungszentrum Jülich, Helmholtz-Gemeinschaft Deutscher Forschungszentren (HZ), Germany
- 2Department of Food, Environmental and Nutritional Sciences (DEFENS), University of Milan, Italy
The economically efficient utilization of NADPH or NADH-dependent enzymes requires the regeneration of consumed reduction equivalents. This cofactor regeneration is classically done via an additional substrate, and if necessary enzyme. We now demonstrate an easy-to-apply cofactor regeneration approach, which can especially be used in screening applications. Simply by applying citrate to a buffer or directly using citrate/-phosphate buffer NADPH can be regenerated by native enzymes of the TCA cycle, practically present in all aerobic living organisms. Apart from viable-culturable cells, this regeneration approach can also be applied with lyophilized cells and even crude cell extracts. This is exemplarily shown for the synthesis of 1 phenylethanol from acetophenone with several oxidoreductases. The mechanism of NADPH regeneration by TCA cycle enzymes was further investigated by a transient isotopic labeling experiment feeding [1,5-13C]citrate. This revealed that the regeneration mechanism can further be optimized by genetic modification of two competing internal citrate metabolization pathways, the glyoxylate shunt and the glutamate dehydrogenase.
Keywords: citrate oxidation1, oxidoreductase screening2, nicotinamide cofactor3, reduction equivalent regeneration4, NADPH regeneration5, cofactor regeneration6
Received: 29 Mar 2018;
Accepted: 28 Nov 2018.
Edited by:Roland Wohlgemuth, Lodz University of Technology, Poland
Reviewed by:Luis P. Fonseca, Technical University of Lisbon, Portugal
Rajni Hatti Kaul, Lund University, Sweden
Copyright: © 2018 Oeggl, Neumann, Gätgens, Romano, Noack and Rother. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Mr. Reinhard Oeggl, Forschungszentrum Jülich, Helmholtz-Gemeinschaft Deutscher Forschungszentren (HZ), IBG-1: Biotechnology, Jülich, Germany, email@example.com
Dr. Dörte Rother, Forschungszentrum Jülich, Helmholtz-Gemeinschaft Deutscher Forschungszentren (HZ), IBG-1: Biotechnology, Jülich, Germany, firstname.lastname@example.org