A water-forming NADH oxidase from Lactobacillus pentosus suitable for the regeneration of synthetic biomimetic cofactors

Abstract

The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox). Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13% FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyze the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H2O2 instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as by-product.

Efficient Bioconversion of Sucrose to High‐Value‐Added Glucaric Acid by In Vitro Metabolic Engineering

“…[15] Assays were conducted in 1.0 mL of the reaction system containing 200 mm Tris-HCl (pH 7.5), 50 mm disodium hydrogen phosphate, 100 mm sucrose, excess PGM (! [30] The NOX activity was analyzed in ar eaction system containing 200 mm Tris-HCl and 2mm NADH at 30 8C. The G6P was determined by the G6P dehydrogenase reaction, and the production of nicotinamide adenine dinucleotide phosphate (NADPH) was measured spectrophotometrically at 340 mm.T he activity of PGM was analyzed at 30 8Ci nar eaction solution including 200 mm Tris-HCl (pH 7.5) and 50 mm G1P.T he activity of MIPS was assayed at 30 8Ci n2 00 mm Tris-HCl (pH 7.5), 50 mm G6P as well as excess IPM.…”

Substrate scope of a dehydrogenase from Sphingomonas species A1 and its potential application in the synthesis of rare sugars and sugar derivatives

“…For product identification, enzyme assays were performed overnight with the addition of the cofactor recycling enzyme NADH oxidase (NOX) from Lactobacillus pentosus. Expression, purification and activation with FAD were performed as described previously (Nowak et al, 2015). Reaction mixtures contained 50 mM AbC, pH 7.9, 25 mM substrate, 5 mM NAD + and purified SpsADH.…”

Flavoenzyme‐mediated Regioselective Aromatic Hydroxylation with Coenzyme Biomimetics

“…NCBs are synthetic truncated versions of NAD(P)H with variable substituents (Figure ) . The biocatalytic applications of NCBs were demonstrated with dehydrogenases, ene reductases, azoreductases, cytochrome P450s, NADH oxidases, and two‐component flavoprotein monooxygenases (group E and F) . NCBs are attractive to study the impact of redox potential and mode of coenzyme binding in oxidoreductases in order to elucidate their mechanism, and to further apply these enzymes in large scale reactions …”