Journal article
Cloning and characterization of a thermostable H2O-forming NADH oxidase from Lactobacillus rhamnosus
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 k(cat)/K(m) of 3.77×10(7) 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 H(2)O-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.
Language: | English |
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Year: | 2012 |
Pages: | 255-262 |
ISSN: | 18790909 and 01410229 |
Types: | Journal article |
DOI: | 10.1016/j.enzmictec.2012.01.009 |
Amino Acid Sequence Biotechnology Characterization Cloning, Molecular Cofactor regeneration Electrophoresis, Polyacrylamide Gel Enzyme Stability Escherichia coli H2O-forming NADH oxidase Hot Temperature Kinetics Lacticaseibacillus rhamnosus Lactobacillus rhamnosus Models, Molecular Molecular Sequence Data Multienzyme Complexes NAD NADH oxidase NADH, NADPH Oxidoreductases Recombinant Proteins Sequence Analysis, DNA Thermal stability Water