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Cloning and characterization of a thermostable H2O-forming NADH oxidase from Lactobacillus rhamnosus

Paper ID Volume ID Publish Year Pages File Format Full-Text
17265 42655 2012 8 PDF Available
Title
Cloning and characterization of a thermostable H2O-forming NADH oxidase from Lactobacillus rhamnosus
Abstract

NADH oxidase (Nox) catalyzes the conversion of NADH to NAD+. A previously uncharacterized Nox gene (LrNox) was cloned from Lactobacillus rhamnosus and overexpressed in Escherichia coli BL21(DE3). Sequence analysis revealed an open reading frame of 1359 bp, capable of encoding a polypeptide of 453 amino acid residues. The molecular mass of the purified LrNox enzyme was estimated to be ∼50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 100 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had optimal activity at pH 5.6 and temperature 65 °C, and kcat/Km of 3.77 × 107 s−1 M−1, the highest ever reported. Heat inactivation studies revealed that LrNox had high thermostability, with a half-life of 120 min at 80 °C. Molecular dynamics simulation studies shed light on the factors contributing to the high activity of LrNox. Although the properties of Nox from several microorganisms have been reported, this is the first report on the characterization of a recombinant H2O-forming Nox with high activity and thermostability. The characteristics of the LrNox enzyme could prove to be of interest in industrial applications such as NAD+ regeneration.

► We characterize a novel NADH oxidase (LrNox) from Lactobacillus rhamnosus. ► The LrNox has kcat/Km of 3.77 × 107 s−1 M−1, the highest ever reported. ► LrNox has high thermostability with a half-life of 120 min at 80 °C. ► This is the first report on the characterization of a recombinant H2O-forming Nox with high activity and thermostability.

Keywords
Characterization; Cofactor regeneration; H2O-forming NADH oxidase; Lactobacillus rhamnosus; Thermal stability
First Page Preview
Cloning and characterization of a thermostable H2O-forming NADH oxidase from Lactobacillus rhamnosus
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 50, Issues 4–5, 5 April 2012, Pages 255–262
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering