Compositions and methods for using NADH oxidases
The present disclosure relates generally to bacterial NADH oxidases and, more particularly, to novel NADH oxidases obtained from Lactobacillus plantarum , and derivatives thereof that demonstrate enzymatic activity for NADH, NADPH, or both NADH and NADPH. The compositions comprising an NADH oxidase obtained from L. plantarum or derivatives thereof include: isolated enzymes; recombinantly produced enzymes and derivatives thereof, as well as catalytically active portions thereof; nucleic acids encoding an NADH oxidase obtained from L. plantarum , derivatives thereof, and portions thereof. The methods of the present invention include isolation of NADH oxidases obtained from L. plantarum , derivatives thereof, and portions thereof, and methods for enzymatic reactions comprising NADH oxidase obtained from L. plantarum , including the production of enantiomer-enriched organic compounds.
1. An isolated bacterial NADH oxidase, which is obtained from Lactobacillus plantarum , and regenerates NADP+ and NAD+, comprising
at least one amino acid mutation that facilitates enzymatic activity towards NADPH and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.
2. The isolated bacterial NADH oxidase of claim 1 , encoded by a nucleic acid sequence, which hybridizes under stringent conditions to the nucleic acid selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 18.
3. The isolated bacterial NADH oxidase of claim 1 , which comprises SEQ ID NO: 8.
4. The isolated bacterial NADH oxidase of claim 3 , encoded by a nucleic acid sequence comprising SEQ ID NO: 10.
5. The isolated bacterial NADH oxidase of claim 1 , which comprises SEQ ID NO: 16.
6. The isolated bacterial NADH oxidase of claim 5 , encoded by a nucleic acid sequence comprising SEQ ID NO: 18.
7. A method of producing an enantiomer-enriched organic compound, comprising:
reacting a substrate with a first enzyme selective for producing an enantiomer, wherein the first enzyme requires a oxidized nicotinamide-based cofactor for catalytic activity;
producing the enantiomer, its oxidized counterpart, and a reduced nicotinamide-based cofactor; and
oxidizing the reduced nicotinamide-based cofactor with a second enzyme selective for a nicotinamide-based cofactor,
wherein the second enzyme selective for a nicotinamide-based cofactor comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.
8. The method of producing an enantiomer-enriched organic compound of claim 7 , wherein the enantiomer comprises an L-nucleoside, and wherein the second enzyme selective for a nicotinamide-based cofactor comprises a NADH oxidase obtained from Lactobacillus plantarum.
9. The method of producing an enantiomer-enriched organic compound of claim 7 , wherein the second enzyme selective for a nicotinamide-based cofactor can oxidize NADPH and NADH.
10. The method of producing an enantiomer-enriched organic compound of claim 9 , wherein the second enzyme selective for a nicotinamide-based cofactor comprises SEQ ID NO: 8.
11. The method of producing an enantiomer-enriched organic compound of claim 9 , wherein the second enzyme selective for a nicotinamide-based cofactor comprises SEQ ID NO: 18.
12. The method of producing an enantiomer-enriched organic compound of claim 7 , wherein the second enzyme selective for a nicotinamide-based cofactor can catalyze more than 113,000 turnovers per active site.
13. The method of producing an enantiomer-enriched organic compound of claim 7 , wherein the second enzyme selective for a nicotinamide-based cofactor can catalyze more than 100,000 turnovers per active site in the absence of an externally added reducing agent.