Engineering an enzymatic regeneration system for NAD(P)H oxidation

Abstract

a b s t r a c tA recently proposed coenzyme regeneration system employing laccase and a number of various redox mediators for the oxidation of NAD(P)H was studied in detail by kinetic characterization of individual reaction steps. Reaction engineering by modeling was used to optimize the employed enzyme, coenzyme as well as redox mediator concentrations. Glucose dehydrogenase from Bacillus sp. served as a convenient model of synthetic enzymes that depend either on NAD + or NADP + . The suitability of laccase from Trametes pubescens in combination with acetosyringone or syringaldazine as redox mediator was tested for the regeneration (oxidation) of both coenzymes. In a first step, pH profiles and catalytic constants of laccase for the redox mediators were determined. Then, second-order rate constants for the oxidation of NAD(P)H by the redox mediators were measured. In a third step, the rate equation for the entire enzymatic process was derived and used to build a MATLAB model. After verifying the agreement of predicted vs. experimental data, the model was used to calculate different scenarios employing varying concentrations of regeneration system components. The modeled processes were experimentally tested and the results compared to the predictions. It was found that the regeneration of NADH to its oxidized form was performed very efficiently, but that an excess of laccase activity leads to a high concentration of the oxidized form of the redox mediator -a phenoxy radical -which initiates coupling (dimerization or polymerization) and enzyme deactivation.

Biocatalytic Oxidation of Alcohols

“…The so-called laccase mediator systems (LMSs) comprise combinations of laccases and chemical redox dyes to aerobically regenerate NAD(P) + from NAD(P)H. Compared to the aforementioned NADH oxidases, LMSs excel by their indifference with respect to the cofactor (NADP + or NAD + ) regenerated, since the NAD(P)H oxidation step is performed by an unselective chemical mediator (Scheme 3) [65][66][67][68][69][70][71]. Quite often, the reduced mediator itself reacts with molecular oxygen, making co-catalysis by laccase superfluous [72][73][74][75][76][77].…”

Immobilization Screening and Characterization of an Alcohol Dehydrogenase and its Application to the Multi-Enzymatic Selective Oxidation of 1,-Omega-Diols

“…For this reason, BsADH requires an in situ recycling of NAD + when exploited in applied biocatalysis. Several enzymatic and chemoenzymatic recycling systems have been proposed for this type of biotransformations using laccases (Pham et al, 2015) and NADH oxidases (Nowak et al, 2015) among others.…”

Primjena matematičkog modeliranja u razvoju enzimskih kaskadnih reakcija

“…Najveći broj sustava s dva enzima za koje su razvijeni kinetički modeli predstavljaju reakcije katalizirane oksidoreduktazama koje zahtijevaju regeneraciju koenzima. [24][25][26][27][28][29] Takve reakcije se često primjenjuju za proizvodnju kiralnih produkata, 28,[30][31][32] u oksidacijama kataliziranim oksidoreduktazama, 26,29 itd. U reakcijama oksidacije mnogo je teže uspostaviti učinkovit sustav regeneracije i, posljedično, postići industrijsku primjenjivost.…”