Cloning and characterization of two distinct water-forming NADH oxidases from Lactobacillus pentosus for the regeneration of NAD

Abstract

Two uncharacterized nicotinamide adenine dinucleotide (NADH) oxidases (named as LpNox1, LpNox2) from Lactobacillus pentosus ATCC 8041 were cloned and overexpressed in Escherichia coli BL21 (DE3). The sequence analysis revealed that the two enzymes are water-forming Noxs with 64 % and 52 % identity to LbNox from Lactobacillus brevis DSM 20054. The optimal pH and temperature of the purified LpNox1 and LpNox2 were 7.0 and 8.0 and 35 and 40 °C, respectively, with K M of 99.0 μM (LpNox1) and 27.6 μM (LpNox2), and yielding catalytic efficiency k cat/K M of 1.0 and 0.2 μM(-1) s(-1), respectively. Heat inactivation studies revealed that the two enzymes are relatively instable. The application of LpNox1 for the regeneration of NAD(+) was demonstrated by coupling with a glycerol dehydrogenase-catalyzed oxidation of glycerol to 1,3-dihydroxyacetone. The characteristics of the LpNox1 could prove to be of interest in industrial application such as NAD(+) regeneration in dehydrogenase-catalyzed oxidations.

Characteristics of a water-forming NADH oxidase from Methanobrevibacter smithii, an archaeon in the human gut

“…Due to a conserved cysteine residue at position 42, NOX-ms should be an H 2 O-forming NOX. In order to determine the product of the NOX activity of NOX-ms, H 2 O 2 was quantified using the Amples Red and horseradish peroxidase method [ 17 ]. The results demonstrated that less than 1% of the theoretical yield of hydrogen peroxide could be detected.…”

Mycoplasma bovis NADH oxidase functions as both a NADH oxidizing and O2 reducing enzyme and an adhesin

“…The activities of several homologous proteins of NADH oxidase from Giardia lamblia 22 , Lactobacillus brevis 23 , Lactobacillus pentosus 24 , and Streptococcus pneumonia 14 , are successfully studied in the forms of recombinant proteins expressed by E. coli . In this study, our results by using rNOX expressed by E. coli firmly indicate that rNOX of M. bovis can oxidize NADH to NAD + and reduce O 2 to H 2 O 2 shown in Fig.…”

Broadening the Scope of Biocatalysis in Sustainable Organic Synthesis

“…[101] Alternatively, direct oxidation with molecular oxygen catalyzed by NAD(P)H oxidase( NOx) has been used (Scheme 10). [102] An elegant approach is to design cascade biotransformations that are selfsufficient in cofactor,t hrough hydrogen-borrowing mechanisms, in vitro or in vivo, with all of the enzymes co-expressed in as uitable host, thus minimizing waste generation. [103] Examples include combining an ADH with an amine dehydrogenase for conversion of an alcohol to an amine (see next section) and with aB aeyer-Villiger monooxygenase( BVMO) to convert an alcohol or ad iol into ac arboxylic acid ester or al actone, respectively.…”