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EP-4739771-A1 - NAD(P)H OXIDASE

EP4739771A1EP 4739771 A1EP4739771 A1EP 4739771A1EP-4739771-A1

Abstract

The present invention relates to a method for oxidizing NADH to NAD+ and/or NADPH to NADP+ comprising the step of incubating a protein having NADH and/or NADPH oxidase activity with NADH and/or NADPH in an aqueous solution, said protein comprising an amino acid sequence selected from the group consisting of i) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 1, ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 2 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 2, wherein the stringent conditions preferably comprise washing at 65°C, and at a salt concentration of 0.1 to 2x SSC.

Inventors

  • STAUNIG, Nicole
  • DUPONT, MARIA
  • Koch, Dennis

Assignees

  • Annikki GmbH

Dates

Publication Date
20260513
Application Date
20240708

Claims (13)

  1. 1. A method for oxidizing NADH to NAD+ and/or NADPH to NADP+ comprising the step of incubating a protein having NADH and/or NADPH oxidase activity with NADH and/or NADPH in an aqueous solution, said protein comprising an amino acid sequence selected from the group consisting of i) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 1, ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 2 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No . 2.
  2. 2. The method according to claim 1, wherein said protein comprises a polypeptide consisting of the amino acid sequence GX1GX2X3X4, wherein Xi is a non-polar amino acid residue, X2 is a non-polar amino acid residue, X3 is a non-polar amino acid residue and X4 is a non-polar amino acid residue.
  3. 3. The method according to claim 2, wherein Xi is glycine, and/or X2 is tyrosine, and/or X3 is isoleucine, and/or X4 is glycine or alanine .
  4. 4. The method according to any one of claims 1 to 3, wherein said protein comprises a polypeptide consisting of the amino acid sequence GX1GX2X3X4X5X6X7X8X9X10X11X12, wherein X5 is a peptide consisting of 6 to 12, preferably 8 to 10, more preferably 10, amino acid residues, Xe is a non-polar or a polar amino acid residue, X7 is a peptide consisting of 4 to 8, preferably 4 to 6, more preferably 6, amino acid residues, Xs is an acidic amino acid residue or a non-polar amino acid residue, Xg is a nonpolar amino acid residue or a basic amino acid residue, X10 is a non-polar amino acid residue or a polar amino acid residue, Xu is a peptide consisting of 2 to 6, preferably 2 to 4, more preferably 4, amino acid residues, and X12 is a polar amino acid residue or a basic amino acid residue.
  5. 5. The method according to claim 4, wherein X5 is a peptide consisting of amino acid sequence IELVEAFAES (SEQ ID No. 9) and/or X7 is a peptide consisting of amino acid sequence KQVTLV (SEQ ID No. 10) and/or Xu is a peptide consisting of amino acid sequence DRIL (SEQ ID No. 11) .
  6. 6. The method according to claim 4 or 5, wherein Xe is glycine or asparagine and/or Xg is aspartic acid or alanine and/or Xg is glycine or arginine and/or X10 is leucine or serine and/or X12 is asparagine or arginine.
  7. 7. The method according to any one of claims 4 to 6, wherein X4 is non-polar and Xe is a polar amino acid residue, preferably glycine and asparagine, respectively, Xg is a non-polar amino acid residue, preferably alanine, X10 is a polar amino acid residue, preferably serine, and Xg and X12 are a basic amino acid residue, preferably arginine.
  8. 8. The method according to any one of claims 4 to 6, wherein X4 is a non-polar amino acid residue, preferably alanine, Xe is a non-polar or a polar amino acid residue, preferably glycine and asparagine, respectively, Xg is a non-polar amino acid residue, preferably alanine, X10 is a polar amino acid residue, preferably serine, and Xg and X12 are a basic amino acid residue, preferably arginine .
  9. 9. The method according to any one of claims 4 to 6, wherein X4 and Xe are non-polar amino acid residue, preferably glycine, Xg is an acidic amino acid residue, preferably aspartic acid, Xg is a non-polar amino acid residue, preferably glycine, X10 is a nonpolar amino acid residue, preferably leucine, and X12 is a polar amino acid residue, preferably asparagine.
  10. 10. The method according to any one of claims 1 to 9, wherein the protein is provided in a host cell overexpressing and secreting the protein and/or a lysate and/or a homogenate of a host cell overexpressing intracellularly the protein.
  11. 11. The method according to any one of claims 1 to 10, wherein the aqueous solution comprises at least one organic co-solvent, preferably isopropanol.
  12. 12. The method according to claim 11, wherein the aqueous solution comprises 0.1 to 25% (v/v) , preferably 0.2 to 20% (v/v) , more preferably 0.5 to 15% (v/v) , of the at least one organic co-solvent .
  13. 13. The method according to any one of claims 1 to 12, wherein the method comprises a step of oxidising enzymatically a compound, where NADH is formed from NAD+ and/or NADPH is formed from NADP+ .

Description

NAD(P)H OXIDASE TECHNICAL FIELD The present invention relates to the field of enzymes, in particular enzymes capable to oxidase NADH and/or NADPH. BACKGROUND ART Biocatalytic redox processes are widely used to replace expensive chemical synthesis, the processes can be performed in shorter time using milder reaction conditions (ambient pressure and temperature ) and no extensive amounts of waste are formed. Redox reactions like the stereoselective reduction of keto groups or the regioselective oxidation of hydroxy groups are of special interest for pharma and food industries and are mostly performed by oxidoreductases , accompanied by the interconversion of the nicotinamide cofactors NAD(P)+ (oxidized form) and NAD(P)H (reduced form) . These redox reactions require a stoichiometric amount of NAD(P)+ or NAD(P)H for the conversion to be completed, however, the addition of such cofactors in industrial processes is very expensive and not cost-efficient. Introducing an additional enzyme to the reaction mixture, which converts the used cofactor back to the oxidation state (NAD(P)+ or NAD(P)H) necessary for the oxidoreductase-catalyzed redox reaction (either by oxidation or reduction) , can greatly reduce the amount of expensive nicotinamide cofactor. This method is known as cofactor regeneration and is widely used in industrial enzymatic processes (Chenault et al., 1988; Wang et al . , 2017) . There are enzymatic methods known in the art for regeneration of the reduced nicotinamide cofactor NAD(P)H - e.g., glucose and glucose dehydrogenase (GDH) , formate and formate dehydrogenase (FDH) or a secondary alcohol (like isopropanol) and alcohol dehydrogenase (ADH) . For the regeneration of the oxidized nicotinamide cofactor NAD(P)+ systems like pyruvate and lactate dehydrogenase (only for NAD+ regeneration) , acetaldehyde and alcohol dehydrogenase or a-ketoglutarate and glutamate dehydrogenase can be employed (Chenault et al., 1988) . All these methods require the addition of a second substrate to the reaction mixture, which makes it more expensive, because further processing steps (such as removal of the coproducts or reutilization of the substrate used for cofactor regeneration, if possible) are necessary. In complex reaction cascades with multiple biocatalytic steps each additional (co) substrate can complicate the downstream processing, serve as alternative substrate for the cascade enzymes, act as a noncompetitive inhibitor or denature the proteins. NAD(P)H oxidases, a type of oxidoreductases , allow the oxidation of NAD(P)H to NAD(P)+ without concomitant use of an additional substrate - oxygen is used as substrate while only generating water or hydrogen peroxide (water- or hydrogen per- oxide-forming NAD(P)H oxidases, respectively) . As the oxidizing compound hydrogen peroxide can denature or degrade enzymes, water-forming NAD(P)H oxidases are preferred in industrial applications. Examples for such enzymes are the NADH oxidase from Lactobacillus brevis (Geueke et al, 2003; EP 1 285 962 Al; US 7,435,574 B2) , Pyrococcus furiosus (Ward et al., 2001) , Borrelia burgdorferi (Riebel et al., 2002) and Streptococcus mutans (Higuchi et al., 1993; Matsumoto et al., 1996; Higuchi et al., 1999) , as well as the NAD(P)H oxidase from Lactobacillus sanfranciscensis (Riebel et al., 2003) . The drawback of these enzymes is their low total turnover numbers (TTN) due to the instability in oxidative process conditions. Recently, engineered versions of the NADH oxidase from Streptococcus mutans were described, which are more stable showing no loss of activity and thus can be employed for cofactor regeneration in redox processes without requiring the use of antioxidant agents like dithiothreitol (EP 2 527 436 Al; US 9, 315, 782 B2; US 9, 376, 667 B2) . However, the prior art does not reveal the performance of the NADH oxidase from Streptococcus mutans under process conditions, like in presence of organic solvents which have denaturing effects (Klibanov, 1997) . Thus, there is still a need for alternative enzymes for cofactor regeneration in oxidative process conditions. One important class of pharmaceuticals are bile acids and their derivatives. Cholic acid, e.g. can be used as starting material for the chemical synthesis of ursodeoxycholic acid (for treatment of gallstones) involving two main steps - the dehydroxylation at C12 and the epimerization of the hydroxy group at C7. For most of the reactions enzymatic alternatives using highly specific hydroxysteroid dehydrogenases are described literature, only the reduction of the ketone moiety at C12 to a methylene group has to be done chemically via a Wolf f-Kishner reduction (Tonin and Arends, 2018) . For the oxidation reactions required in both main steps NAD(P)H oxidases can be used for cofactor regeneration (Tonin et al., 2019) . US 2017/218417 discloses a method for the epimerization of chenodeoxycholic acid into ursodeoxycholic acid wherein the regeneration of NAD+ is achiev