| NADH oxidase(NOX, EC 1.6.99.3) is a kind of oxidoreductases which catalyze the oxidation of NADH in the presense of oxygen. NADH oxidase widely exists in a variety of microbials and plays an important role in the regulation of microbial metabolism. It has showed great potencial industrial applications, because it can catalytze the regeneration of NAD+ with a relatively low cost. In this work, 3 genes encoding NADH oxidases were cloned from Lactobacillus rhamnosus. The properties of NADH oxidase encoded by the genes were investigated and the structure of the NADH oxidase was modified by site-direct mutagenesis.(1) After extraction of Lactobacillus rhamnosus genomic DNA, the 3 gene fragments encoding NADH oxidases including Nox(447)(Gene ID: 8420447), Nox(552)(Gene ID: 8420552) and Nox(603)(Gene ID: 8420603) were amplified with PCR, respectively. The amplified genes were ligated to the expression vector pET-28 a, and transformed into E.Coli BL21 for the expression.(2) Nox(447) was expressed and characterization of the Nox was performed. The optimum temperature, time and inducer concentration for the expression of Nox(447) was 15 oC, 4 h and 1.25 mM. The optimum concentration of the coenzyme FAD was 20 μM, the activity of Nox(447) declined with the increasing of FAD concentration. The optimum pH was 5.6, and the activity had been completely inactivated when the pH was 10. The optimal temperature for reaction was 45 oC, and the activity did not decrease significantly after incubation for 160 min at the optimal temperature. The half-life of Nox(447) was 3 h at 50 oC and the enzyme completely inactivated after 1 h at 55 oC. This indicated that the enzyme was unstable at high temperatures. The Km and Vmax were calculated as 345.47 μM and 69.23 U/mg.(3) There is no cysteine residue in the amino acid sequence of the Nox(447). Docking results showed that NADH interacted with the enzyme at HIS319, GLY298 and TYR341 of Nox(447) via hydrogen bonds. The mutants H319 A, G298 A, and Y341 A completely lost the activity. It indicated that these amino acid residues constituted the catalytic center of the enzyme.(4)The cloned Nox(603) had been modified by site-directed mutagenesis. Based on the structure of Nox(603), eight residues(Q18, A85, A218, A184, N186, N227, T346, S146) had been selected and substituted for polar cysteine. The analysis of circular dichroism spectra indicated that the secondary structure of the mutant enzymes did not change significantly, and the specific activity increased by 59.04%. Kinetic experiments showed that the Km of mutant enzymes decreased by 3.67 to 43.11 % compared to the wild-type enzyme, and the Vmax of mutant enzymes increased by 1.73 to 48.19 %. These results indicated that mutant enzymes had better substrate affinity and catalytic rate. The introduced Cys did not form a stable disulfide bond with the Cys in active site to resulte in the loss of the catalytic activity, and the mutant enzymes also showed advantages in kinetic experiments.(5) MOE 2014 was used for the substrate-docking analysis, and the scoring of molecular docking of mutant enzymes by scoring function decreased by 12.37 to 70.32 % compared to the wild-type enzyme. It indicated that the conformations of mutant enzymes after the structural modification combined with coenzyme and substrate more easily, the results were consistent with the kinetic experiments. |
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