Screening And Gene Cloning Study Of NADH Oxidase From Lactic Acid Bacteria

NADH oxidase (EC1.6.99.3, abbreviated as NOX) is a kind of oxidoreductases whichcan oxidise NADH to NAD+directly. Overseas researchs found that NADH oxidase existed inmany microbials, having an important role in microbial metabolism regulation. With the rapidgrowing of Oxidoreductase application in industry, the issue on the corresponding coenzymeregeneration has become hot topic. Because of the advantages such as the reactants oxygennot to be added, simple structure of the product, and no influence on the main productpurification, NOX shows great potential in the oxidized coenzyme NAD~+regeneration.In this paper, a strain of lactic acid bacteria was screened from kimchi which produceshigher activity of NADH oxidase, and was identified as Lactobacillus sp. LP-01. The genenox(Genbank:HQ441199) which encodes NADH oxidase was cloned from the genomic DNAof Lactobacillus sp. LP-01. And then the nox gene was cloned into pMD18-T and its geneticcharacter was analyzed. Furthermore, cloning and expression of the gene nox was studied forthe first time.Firstly,9strains of lactic acid bacteria were isolated from kimchi, and we obtaind onestrain which has the highest NADH oxidase activity. After the16S rDNA molecular analysis,the strain was proved to belong to Lactobacillus sp., and named Lactobacillus sp. LP-01.Secondly, the nox gene which encodes NADH oxidase was cloned from strainLactobacillus sp. LP-01. After the extraction of the genomic DNA PCR was runned with thedegenerate primers which were designed by comparison of the two known nox genes of twostrains of lactic acid bacteria(genbank accession number: AL935262.1and AF536177.1). Theamplified fragment was ligated to plasmid pMD18-T and transformed into E. coli DH5_α. Thesubseqently sequencing revealed the product(Genbank:HQ441199) was1353bp length,containing complete promoter and terminator, it encoded450amino acids with molecularweight of486,000Da. Amino acid sequence analysis found that the nox had4cysteines, andwe speculated it participated in disulfide bond formation and peptide chain folding. Therewere FAD-binding1(GCTHAGT) in N-terminal and FAD-binding2binding (TSDPDIYGAGDS) which was corresponded with the reports, that NOX catalyze the reaction withcoenzyme FAD participation. Moreover a NAD-binding binding domain (VIVIGGGYIGTELV) was also identifed, implying the combination of the substrate NADH during thereaction. Lastly, the expression of the gene nox(Genbank:HQ441199) was studied. we cloned thenox gene into expression vector pET-32a (+) with SalI and XhoI restriction sites, andtransformed into strain BL21(DE3) pLysS. whereafter, we fermented NOX enzyme with therecombination strain. By SDS-PAGE electrophoresis we found that recombinate NOX proteinwere existed in two forms of intracellular soluble protein and inclusion bodies, and theactivity of the NOX in intracellular soluble protein form of the cell was detected to be6.57U/g bacteria。...