# Patent

## Title
NADH oxidase from Lactobacillus

## Abstract

The present invention is directed to a new NADH oxidase from Lactobacillus, nucleic acids encoding the NADH oxidase, methods of producing the NADH oxidase, as well as there use in producing improved NADH oxidase enzymes and for producing chrial enantiomer-enriched organic compounds, such as alcohols and/or amino acids.

## Background
This application is a Divisional of U.S. application Ser. No. 10/219,288, filed on Aug. 16, 2002, now U.S. Pat. No. 6,987,013, issued on Jan. 17, 2006.


FIELD OF THE INVENTION
The present invention relates to an NADH oxidase from Lactobacillus, as well as the nucleic acids coding for this enzyme and vehicles containing these nucleic acids. In particular the invention relates to an oxidase from Lactobacillus brevis (DSM 20054).
BACKGROUND OF THE INVENTION
Dehydrogenases, classified under E.C. 1.1, are enzymes that reversibly catalyse the oxidation or reduction of certain compounds such as for example alcohols and ketones. Some of the known dehydrogenases are cofactor-dependent, in other words they require further molecules to balance the electron transfer processes. NADH and NADPH for example are known to be such cofactors.
NADH is formed in the oxidation of, for example, alcohols to ketones according to the following reaction scheme. In order to avoid having to add stoichiometric amounts of expensive NAD+, the formation of NAD+ can be initiated by a second enzyme system (Enzyme Catalysis in Organic Synthesis, Ed.: K. Drauz, H. Waldmann, VCH, 1st Edition, p. 721; reaction scheme 1).




Systems known previously described using lactate dehydrogenase and pyruvate with the formation of lactate, or glutamate dehydrogenase with ketoglutarate and ammonium with the formation of glutamate for regenerating NAD+. The disadvantage of these systems is that auxiliary substrates, such as pyruvate or ketoglutarate, have to be used and products, such as lactate or glutamate, are formed, which for preparative utilization have to be removed from the product. In addition, equilibrium reactions go to completion only with great technical difficulty.
Alternatively, a second enzyme system may be an NADH oxidase that accepts atmospheric oxygen as oxidised cosubstrate and at the same time generates water or hydrogen peroxide with the formation of NAD+. In this respect, NADH oxidases are advantageous in that the reaction catalyzed by them is irreversible, O2 is used as regeneration substrate, and H2O or H2O2 are formed as product. Enzymes of both groups (H2O-forming and H2O2-forming enzymes) are, in principle, known in the biochemical literature (for example, for H2O-forming enzymes see Lopez de Felipe, F. et al., J. Bacteriol. Vol. 180 (1998), 3804-08; for H2O2-forming enzymes see Nishiyama, Y. et al. J. Bacteriol. Vol. 183 (2001), 2431-2438 and the literature cited therein). H2O2-forming enzymes are less suitable as a regeneration enzyme since peroxide is known to be harmful to enzymes and should be decomposed as far as possible in situ, which requires further, disadvantageous but not impossible process steps.
The NADH oxidases from Enterococcus (and Streptococcus) faecalis (Schmidt, H. L. et al., Eur. J. Biochem. 156 (1986), 149-55), Leuconostoc mesenteroides (Koike, K. J. et al., Biochem. (Tokyo) 97 (1985), 1279-88), Streptococcus mutans (Higuchi, M. et al., Biosci. Biotechnol. Biochem. 58 (1994), 1603-07), Mycoplasma capricolum (Klomkes, M., Altdorf, R., Ohlenbusch, H. D., Biol. Chem. Hoppe Seyler 366 (1985), 963-9), Sulfolobus solfataricus (Arcari, P. et al., J. Biol. Chem. 275 (2000), 895-900), Thermus thermophilus (Erdmann, H. et al., J. Mol. Biol. 230 (1993), 1086-8) and from Thermus aquaticus (Cocco, D. et al., Eur. J. Biochem. 174 (1988), 267-71) have been biochemically characterised.
However, there remains a need in the art for NADH oxidase with high activity, stability, etc. that when coupled with a dehyrogenase can be used for NAD+ regeneration.
SUMMARY OF THE INVENTION
Therefore, an object of the present invention is to provide an NADH oxidase with high activity that is suitable when coupled with dehydrogenases for NAD+ regeneration. In particular the NADH oxidase should be able to be produced easily and should be available in sufficient amount for advantageous use on an industrial scale.
Another object of the invention is an isolated NADH oxidase obtained from Lactobacillus, preferably from Lactobacillus brevis, more preferably Lactobacillus brevis DSM 20054. In one embodiment of the present invention, the NADH oxidase has the amino acid sequence in SEQ ID NO:2.
Another object of the invention are those nucleic acid sequences that encode the NADH oxidase, as well as vectors and host cells containing the same. In one embodiment the nucleic acid sequence is SEQ ID NO:1.
Another object of the invention is for a process for producing a rec-NADH oxidase, by cultivating a host cell containing the nucleic acid, which encodes the rec-NADH oxidase and collecting the rec-NADH oxidase produced.
Another object of the present invention is a process for producing a rec-NADH oxidase by mutating a nucleic acid encoding the rec-NADH oxidase; screening the mutated nucleic acid obtained in an expression system, which allows protein encoded by the nucleic acid sequence to be expressed; detecting a mutated nucleic acid that encodes an rec-NADH oxidase with improved activity; and isolating the mutated nucleic acid that encodes an rec-NADH oxidase with improved activity. Therefore, another object of the present invention are those rec-NADH oxidases obtained by these processes, as well as, vectors, host cells, and uses therein.
Another object of the present invention is for a method of producing a chiral enantiomer-enriched organic compound by contacting/reacting the organic compound with the rec-NADH oxidase described herein.
Another object of the invention is a whole-cell catalyst containing the rec-NADH oxidase and/or a nucleic acid encoding the oxidase; which catalyst may also contain a dehydrogenase and/or a nucleic acid encoding the dehydrogenase.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the optimum pH of NADH oxidation.
FIG. 2 shows NADH oxidation at varying conditions, pH, time, and buffer conditions.
FIG. 3. shows the NADH oxidation at various temperature.
FIG. 4 shows the GC spectrum of a sample in which the R-alcohol had been almost completely oxidized.



DETAILED DESCRIPTION OF THE INVENTION
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of molecular biology. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
Since an NADH oxidase (NOX) from Lactobacillus species is now available, it is possible advantageously to achieve the aforementioned object. Most particularly preferred is the use of an NADH oxidase (NOX) from Lactobacillus brevis, preferably DSM 20054 (SEQ ID NO:2), which is encoded by the nucleic acid of SEQ ID NO:1 or Lactobacillus kefir (EP 91107067.0; DE 4014573).
The NADH oxidase is able to convert the NADH formed in the reaction into NAD+, according to the reaction scheme shown in the Background of the Invention section of this application. On account of the irreversibility of the reaction O2 to H2O, there is a complete conversion of the employed racemic alcohol to ketone due to the equilibrium pressure, a highly enantiomer-enriched unreacted optical antipode of the employed alcohol remaining. Examples of the use of the NADH oxidase (NOX) include, inter alia, the production of R-alcohols when coupled with an S-alcohol dehydrogenase, of S-alcohols when coupled with an R-alcohol dehydrogenase, or the production of D-amino acids when coupled with an L-amino acid dehydrogenase. The coupling with hydroxyacid dehydrogenases and all other NAD-dependent dehydrogenases is also possible in this context. As further important products there may also be mentioned the oxidation products of the dehydrogenase reaction, for example ketones, keto acids or aldehydes.
In another embodiment, the invention is concerned with nucleic acids coding for an NADH oxidase according to the invention.
In spite of the relative ease of cultivation of the Lactobacillus strains and easy accessibility of the enzyme via chromatographic methods, it has become possible by specifying the nucleic acids coding for an NADH oxidase (NOX) according to the invention to obtain, in a furthermore preferred way and manner, substances that enable the enzymes necessary for an enzymatic industrial process for the production of enantiomer-enriched compounds to be made available in sufficient amounts via recombinant techniques. It is possible with the nucleic acids to obtain the enzymes in high yields from rapidly growing host organisms. Also, the gene sequences according to the invention have to be employed for the production of improved mutants.
In another embodiment, the invention relates to plasmids or vectors containing one or more of the nucleic acids according to the invention.
As plasmids or vectors there may, in principle, be used all embodiments for this purpose available to the person skilled in the art. Such plasmids and vectors may be found in the article by Studier et al., Methods Enzymol. 1990, 185, 61-69 or in the brochures published by the companies Novagen, Promega, New England Biolabs, Clontech or Gibco BRL. Further preferred plasmids and vectors may be found in: DNA cloning: A Practical Approach. Volume I-III, edited by D. M. Glover, IRL Press Ltd., Oxford, Washington D.C., 1985, 1987; Denhardt, D. T. and Colasanti, J.: A survey of vectors for regulating expression of cloned DNA in E. coli. In: Rodriguez, R. L. and Denhardt, D. T (eds), Vectors, Butterworth, Stoneham, MA, 1987, pp 179-204; Gene expression technology. In: Goeddel, D. V. (eds), Methods in Enzymology, Volume 185, Academic Press, Inc., San Diego, 1990; Sambrook. J., Fritsch, E. F. and Maniatis, T. 1989. Molecular cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Preferable plasmids include: pKK-177-3H (Roche Biochemicals), pBTac (Roche Biochemicals), pKK-233 (Stratagene) or pET (Novagen). With the exception of the TOPO series, which has integrated a resistance to kanamycin, all other plasmids should contain a β-lactamase for ampicillin resistance.
The invention also provides microorganisms containing the nucleic acids according to the invention.
The microorganism in which the nucleic acids are cloned serves for the propagation and production of a sufficient amount of the recombinant enzyme. The processes for this are well known to the person skilled in the art (see below). Examples of such microorganisms include, for example, prokaryotes or eukaryotes, such as Pseudomonas, Streptomyces, Arthrobacter, Bacillus, Staphylococcus, E. coli, Candida, Hansenula, Pichia and baculoviruses. E. coli strains are preferably used. Most particularly preferred are: E. coli NM 522, JM109, JM105, RR1, DH5α, TOP 10- or HB101. Plasmids with which the gene construct containing the nucleic acid according to the invention is preferably cloned in the host organism are disclosed above.
The invention also includes nucleic acids that hybridize under stringent conditions with the single-strand nucleic acids according to the invention or with their complementary single-strand nucleic acids. The expression “stringent conditions” is understood to be the conditions described by Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), 1.101-1.104). Preferably a stringent hybridisation takes place according to the present invention if, after washing for one hour with 1×SSC and 0.1% SDS (sodium dodecylsulfonate) at 50° C., preferably at 55° C., more preferably at 62° C. and most preferably at 68° C., and more preferably for 1 hour with 0.2×SSC and 0.1% SDS at 50° C., preferably at 55° C., more preferably at 62° C. and most preferably at 68° C., a positive hybridization signal is still observed.
In another embodiment, the invention provides primers for producing the gene sequences according to the invention by amplification, for example, the polymerase chain reaction. The primers include the sense and antisense primers coding for the corresponding amino acid sequences. Suitable primers may in principle be obtained by methods known to the person skilled in the art. The discovery of primers according to the invention is carried out by comparison with known DNA sequences or by translation of the visually detected amino acid sequences into the codon of the organism under consideration (e.g. for Streptomyces: Wright et al., Gene 1992, 113, 55-65). Common features in the amino acid sequence of proteins of so-called superfamilies are also of use for this purpose (Firestine et al., Chemistry & Biology 1996, 3, 779-783). Further information relating to the above may be found in “Oligonucleotide synthesis: a practical approach”, edited by M. J. Gait, IRL Press Ltd, Oxford Washington D.C., 1984; PCR Protocols: A guide to methods and applications, edited by M. A. Innis, D. H. Gelfound, J. J. Sninsky and T. J. White. Academic Press, Inc., San Diego, 1990. The following primers are most preferred:










DIFAAGDSA AS-Primer
(SEQ ID NO:3)



 



MKVTVVGCT S-Primer
(SEQ ID NO:4)






In another embodiment, the present invention relates to a process for the production of improved rec-NADH oxidases and rec-NADH oxidases obtained thereby or nucleic acids coding therefor, starting from the nucleic acids according to the invention coding for an NADH oxidase (NOX),

    a) the nucleic acids are subjected to mutagenesis,
    b) the nucleic acids obtained from a) are cloned in a suitable vector and this is transferred to a suitable expression system, and
    c) the proteins formed having an improved activity and/or selectivity are detected and isolated. This process may be executed once or any desired number of times in succession.


The procedure for improving the enzymes according to the invention by mutagenesis methods has long been known to the person skilled in the art. As mutagenesis methods there may be used all methods for this purpose available to the person skilled in the art. In particular these include saturation mutagenesis, random mutagenesis, shuffling methods as well as site-directed mutagenesis (Eigen M. and Gardinger W. (1984) Evolutionary molecular engineering based on RNA replication. Pure & Appl. Chem. 56(8), 967-978; Chen & Arnold (1991) Enzyme engineering for nonaqueous solvents: random mutagenesis to enhance activity of subtilisin E in polar organic media. Bio/Technology 9, 1073-1077; Horwitz, M. and L. Loeb (1986) “Promoters Selected From Random DNA-Sequences” Proceedings Of The National Academy Of Sciences Of The United States Of America 83(19): 7405-7409; Dube, D. and L. Loeb (1989) “Mutants Generated By The Insertion Of Random Oligonucleotides Into The Active-Site Of The Beta-Lactamase Gene” Biochemistry 28(14): 5703-5707; Stemmer PC (1994). Rapid evolution of a protein in vitro by DNA shuffling. Nature. 370; 389-391 and Stemmer PC (1994) DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc Natl Acad Sci USA. 91; 10747-10751).
The new nucleic acid sequences that are obtained are cloned according to the methods specified hereinafter in a host organism (literature, see below) and the expressed enzymes are detected and then isolated using suitable screening methods (Roberts J., Stella V. J. and Decedue C. J. (1985) A colorimetric assay of pancreatic lipase: rapid detection of lipase and colipase separated by gel filtration. Lipids 20(1): 42-45; Pratt R. F., Faraci W. S. and Govardhan C. P. (1985) A direct spectrophotometric assay for D-alanine carboxypeptidases and for the esterase activity of beta-lactamases. Anal. Biochem. 144(1): 204-206; Brückner, H., R. Wittner, and H. Godel (1991) Fully automated high-performance liquid chromatographic separation of DL-amino acids derivatized with o-Phthaldialdehyde together with N-isopropyl-cysteine. Application to food samples).
The present invention also provides using NADH oxidase (NOX) according to the invention, optionally improved by mutation, for the production of chiral enantiomer-enriched organic compounds such as, for example, alcohols or amino acids. Such compounds are used in a similar manner as described in DE10037101, Enzyme Catalysis in Organic Synthesis, Ed.: K. Drauz, H. Waldmann, VCH, 1st Edition.
The nucleic acids according to the invention and improved still further that code for the NADH oxidase (NOX) as described herein, are preferably suitable for the production of whole-cell catalysts (DE10037115.9 as well as the literature cited therein). The invention provides a whole-cell catalyst containing a cloned gene for a dehydrogenase and a cloned gene for an NADH oxidase. Preferably the whole-cell catalyst according to the invention should contain an NADH oxidase (NOX) from Lactobacillus. More preferably, NADH oxidase is from Lactobacillus brevis, including Lactobacillus brevis DSM 20054 or Lactobacillus kefir DSM 20587. The production of such an organism is known to the person skilled in the art (PCT/EP00/08473; PCT/US00/08159; relevant literature, see below).
The advantage of such an organism is the simultaneous expression of both enzymes, whereby only a rec-organism still has to be used for the reaction. In order to match the expression of the enzymes with respect to their reaction rates, the coding nucleic acids may be carried on various plasmids having different copy numbers and/or promoters of different strengths may be used. In one embodiment, the enzymes are encoded on plasmids with similar copy numbers in a host cell; and/or under the control of promoters of similar strength. With enzyme systems matched in this way there is advantageously no accumulation of a possible inhibiting intermediate compound(s), and the reaction under consideration may proceed at an optimal overall rate. This is described in PCT/EP00/08473; and Gellissen et al., Appl. Microbiol. Biotechnol. 1996, 46, 46-54.
In a process for producing NADH oxidase from Lactobacillus advantageous expression of the NADH oxidase (NOX) the microorganism can be achieved by cultivating the microorganism under aerobic conditions.
It has been found that Lactobacillus grows significantly better in a standard medium under aerobic conditions than under the otherwise conventional anaerobic conditions. It is preferred to use a Lactobacillus brevis, preferably Lactobacillus brevis DSM 20054, or Lactobacillus kefir DSM 20587. According to the invention those conditions considered aerobic are those in which the microorganism, when cultivated in flasks as a shaken culture or when cultivated in a fermenter, is grown in the presence of measurable concentrations of oxygen. The expression “measurable oxygen concentration” is understood to mean that the dissolved oxygen is 1% of the saturation value for oxygen, the oxygen being measured under otherwise normal conditions with an oxygen electrode.
The nucleic acids according to the invention can be used for the production of rec-NADH oxidase (NOX). Recombinant techniques known in the art (see below) can be used to produce the enzymes described herein in an amount sufficient for an industrial process from host cells carrying the nucleic acids encoding the enzyme. The production of the rec-enzymes according to the invention is carried out by genetic engineering processes as described in, for example, Sambrook et al. 1989, Molecular cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Balbas P & Bolivar F. 1990; Design and construction of expression plasmid vectors in E. coli, Methods Enzymology 185, 14-37; Vectors: A Survey of Molecular Cloning Vectors and Their Uses. R. L. Rodriguez & D. T. Denhardt, Eds: 205-225). With regard to the general procedure (PCR and fusion PCR, inverse PCR, cloning, expression etc.), reference may be made to the following literature and the references cited therein: Riley J, Butler R. Finniear R. Jenner D, Powell S, Anand R. Smith J C, Markham A F (1990). A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. Nucl Acids Res. 18, 8186; Triglia T, Peterson M G, Kemp D J (1988). A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Res. 16, 8186; Sambrook J, Fritsch EF, Maniatis T (1989). Molecular Cloning. Cold Spring Harbour Laboratory Press; Vectors: A Survey of Molecular Cloning Vectors and Their Uses. R. L. Rodriguez & D. T. Denhardt, II.
The enzymes described herein may be used in the free form as homogeneously purified compounds, or as enzyme produced by recombinant technology. Furthermore the enzyme may also be employed as a constituent of an intact host organism or in conjunction with the macerated cell mass of the host organism purified to an arbitrarily high degree. It is also possible to use the enzymes in immobilised form (Bhavender P. Sharma, Lorraine F. Bailey and Ralph A. Messing, “Immobilisierte Biomaterialiern—Techniken and Anwendungen”, Angew. Chem. 1982, 94, 836-852). The immobilisation is preferably carried out by lyophilisation (Dordick et al. J. Am. Chem. Soc. 194, 116, 5009-5010; Okahata et al. Tetrahedron Lett. 1997, 38, 1971-1974; Adlercreutz et al. Biocatalysis 1992, 6, 291-305). It is most particularly preferred to carry out the lyophilisation in the presence of surfactants such as aerosol OT, polyvinylpyrrolidone, polyethylene glycol (PEG) or Brij 52 (diethyleneglycolmonocetyl ether) (Goto et al. Biotechnol. Techniques 1997, 11, 375-378). The use as CLECs is also possible (St Clair et al. Angew Chem Int Ed Engl 2000 January, 39(2), 380-383).
The expression “optically enriched (enantiomer-enriched) compounds” within the scope of the invention is understood to mean the presence of an optical antipode in a mixture with the other antipode, in an amount greater than 50 mole %.
Lactobacillus brevis DSM 20054 and Lactobacillus kefir DSM 20587 have been filed at the German Collection for Microorganisms and Cell Cultures (DSMZ-Deutsche Sammlung von Mikroorganismen und Zelkulturen GmbH, Mascheroder Weg 1b, D-38124, Braunschweig, Germany) under the corresponding number and is accessible to the public.
The expression “nucleic acids” includes all types of single-strand or double-strand DNA, as well as, RNA or mixtures thereof.
The expression “improved rec-enzymes” as used herein means those that enzymes that have a modified substrate spectrum, are more active and/or more selective, or are more stable under the employed reaction conditions. For example, improved rec-enzymes may be those that are at least 1% (including at least 2, 3, 4, 5, 10, 15, 20% etc.) more active, more stable, and/or more selective than an enzyme which has not been modified, e.g., improved relative to the enzyme containing the amino acid sequence in SEQ ID NO:2.
Included within the present invention are protein sequences and nucleic acid sequences that exhibit an homology (exclusive of the natural degeneration) greater than 80%, preferably greater than 90%, 91%, 92%, 93% or 94%, more preferably greater than 95% or 96%, and particularly preferably greater than 97%, 98% or 99% with respect to one of these sequences, provided that the mode of action and/or purpose of such a sequence is retained, e.g., enzymatic activity. The expression “homology” (or identity) as used herein may be defined by the equation H (%)=[1−V/X]×100, where H denotes homology, X is the total number of nucleotide bases/amino acids of the comparison sequence, and V is the number of different nucleotide bases/amino acids of the sequence under consideration, referred to the comparison sequence. In any case the term “nucleic acids coding for amino acid sequences” covers all sequences that appear possible according to the degeneration of the genetic code.
Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.
EXAMPLES
1) Cultivation of Lactobacillus Brevis for Obtaining NADH Oxidase
Lactobacillus brevis DSM 20054 was cultivated in a standard medium for lactobacillae. In this connection it was found that the strain grew significantly better under the addition of oxygen than under the anaerobic conditions that are in fact normally used for lactobacillus strains. An enzyme test on the activity of an NADH oxidase showed that the strain particularly under these unusual cultivation conditions exhibits a high activity of an NADH oxidase (NOX).
For the enzyme purification and characterisation cells were therefore cultivated under the following conditions:
Medium: Pro 1 L: 10 g caseine peptone, tryptically digested; 10 g meat extract; 5 g yeast extract; 20 g glucose; 1 g Tween 80; 2 g K2HPO4; 5 g Na acetate; 2 g diammonium citrate; 0.2 g MgSO4×7H2O; 0.05 g MnSO4×H2O; pH=6.2-6.5.
Cultivation is carried out in a shaken flask (equipped with 2 baffles) at 30° C. for 2 days while stirring. The cell mass is then obtained by centrifugation and may be stored at −20° C. for a relatively long time.
2) Enzyme Isolation
a) Crude Extract Production
The cells were macerated by wet grinding with glass beads. For this, the moist bacterial mass (10 g) was suspended in 0.1 M sodium acetate buffer, pH 4.5 (30 ml) and macerated by grinding in a stirred cell glass bead mill (SCP disintegrator, Innomed-Konsult A B, Sweden) at 4000 rpm for 20 minutes. The macerated cells were centrifuged off and the supernatant was identified as crude extract.
The selected pH value of 4.5 has a decisive influence on the enzyme yield in the crude extract; under these conditions a crude extract preparation is obtained having a specific activity of 17.8 U/mg and a yield of 700 units, whereas when using a buffer with a pH of 7.4 the specific activity was only 2.6 U/mg and the yield was 580 units.
The enzyme activity was measured by means of the following photometric test (340 nm, 30° C. measurement temperature): 0.1 mM NADH; 100 mM potassium phosphate buffer pH 7.5; 1 mM DTT (=dithiothreitol); 10 μl enzyme solution per 1 ml (diluted if necessary). 1 unit NADH oxidase activity oxidises 1 μMole NADH/min.
b) Chromatographic Purification of the Enzyme
After the preparation of the crude extract in buffer with a pH of 4.5, the crude extract was immediately rebuffered and transferred to a Macro Q column (Biorad) equilibrated with 50 mM TEA pH 7.5+3 mM DTT+200 mM NaCl (=buffer A) (100 ml column volume, flow rate 4 ml/min, 10 ml fractions). The chromatography was carried out on an FPLC apparatus (Pharmacia) (pressure 0.1 MPa, room temperature). The proteins were eluted by means of an NaCl step gradient to 1 M NaCl (buffer B: buffer A+1 M NaCl), the following steps being used: 0-2 column volumes: 0% B (=200 mM NaCl), 2-5 column volumes: 40% B, 5-5.5 column volumes: 100% B. The enzyme activity and the protein content in the fractions were determined by means of the photometric test. The active fractions were combined and concentrated by evaporation in an Amicon stirred cell (YM10 membrane, 50 ml) while simultaneously rebuffering with hydroxylapatite equilibration buffer, see below.
This fraction pool was added to an hydroxylapatite ceramic type 20 μm column (Biorad) equilibrated with 10 mM Kpi buffer pH 6.7+3 mM DTT+200 mM NaCl (=buffer A) (15 ml column volume, flow rate 2 ml/min, 5 ml fractions; FPLC apparatus, pressure 0.1 MPa, room temperature). The proteins were eluted with a 3-step gradient to 500 mM KPi (buffer B: buffer A+500 mM Kpi) with the following stages: 0-3 column volumes 0% B, 3-7 column volumes 30% B, 7-9 column volumes 50% B, then rising to 100% B in 3 column volumes. The active fractions were combined and concentrated as described above, and rebuffered with the buffer required for the phenylsepharose.
This preparation was added, after the addition of ammonium sulfate (1.2 M final concentration in the sample), to a phenylsepharose High Sub column (Pharmacia) equilibrated with 50 mM TEA pH 7.5+3 mM DTT+1.2 M (NH4)2SO4 (=buffer A) (25 ml column volume, flow rate 1 ml/min, 3 ml fractions; FPLC apparatus, pressure 0.1 MPa, room temperature). The proteins were eluted using a 3-step gradient to 0 M ammonium sulfate (buffer B: buffer A without ammonium sulfate) with the following stages: 1 column volume 0% B, 1 column volume 20% B, 1 column volume 60% B, 2 column volumes 100% B). The active fractions were combined, concentrated by evaporation in an Amicon ultrafiltration cell, followed by the addition of further ammonium sulfate.
The last purification stage, which was carried out with the concentrated fraction from the phenylsepharose High Sub column, is a fine purification by chromatography on phenylsepharose 650C material (Tosohaas), equilibrated with 50 mM TEA pH 7.5+3 mM DTT+1.2 M (NH4)2SO4 (=buffer A). Chromatography conditions: column volume 2 ml, flow rate 1 ml/min; FPLC apparatus, pressure 0.1 MPa, room temperature, 2 ml fractions). The proteins are eluted with a 4-stage gradient to 0 M ammonium sulfate (buffer B: buffer A without ammonium sulfate) using the following stages: 10 column volumes 0% B, 5 column volumes 20% B, 5 column volumes 50% B, 5 column volumes 70% B, 10 column volumes 100% B).
The enzyme activity and protein yields of the various chromatography steps are summarised in Table 1.







TABLE 1







Purification of the NADH oxidase from Lactobacillus brevis DSM


20054 (OH-apatite = hydroxyapatite; PS = phenylsepharose)














Specific






Activity
Activity
Total Activity
Yield
Enrich-ment


Sample
[U/ml]
[U/mg]
[U]
[%]
Factor















Crude extract
35.1
17.8
702
100
1


Macro Q
77.3
10.4
270
38
0.6


OH-apatite
32.2
12.2
103
15
0.7


PS
44
58.6
87
12
3.3


PS 650C
98
116
98
14
6.5









An SDS-PAGE of the purified fractions according to Table 1 shows that the enzyme fraction after the last step still contains only 2 proteins, whose subunits have molecular weights of 38 and 50 kDa. These two protein bands can be individually N-terminally sequenced after blotting, and a comparison with sequences accessible in databanks shows that the 38 kDa band has very high similarities with known lactate dehydrogenases, while the 50 kDa band has very high similarities with NADH oxidases.
3) Biochemical Characteristic Data of the NADH Oxidase from L. Brevis 
The biochemical data were determined with an enzyme preparation purified by 4 chromatographic steps.
a) pH Dependence of the Oxidation
The pH optimum was measured in the range pH 4.0 to 10.0. Different buffers were used depending on the pH value. As a check on the influence of the buffer, when changing the buffer this pH value was measured twice in the different buffers (pH 7.0 and pH 8.5). 0.1 M citric acid/Na2PO4 was used for the range pH 4.0-7.0, 0.1 M TEA was used for the range 7.0-8.5, and 0.1 M glycine/NaOH was used for the range pH 8.5-10.0. FIG. 1 shows that the optimum of the NADH oxidation is at pH 8.0 to 8.5.
b) pH Stability of the NADH Oxidase
In order to determine the pH stability the NADH oxidase was incubated for 8 hours in the buffers listed under 3a), then the residual activity was measured in the standard test. FIG. 2 shows that the enzyme has a first sharp stability optimum at pH 5.0, is moderately stable in the range 5.5-7.5, and exhibits a second optimum at pH 8.5. At pH 8.5 the enzyme is still up to 100% stable after 8 hours in TEA/NaOH buffer as well as in glycine/NaOH buffer. The figure illustrates that the enzyme stable in the slightly alkaline range (pH 8-9) is also stable in a wider range. This range is useful for application purposes if the NADH oxidase for the oxidation is to be coupled with an NAD-dependent dehydrogenase, since the pH optimum of many dehydrogenases for the oxidation lies in a slightly alkaline range.
c) Temperature Optimum of the NADH Oxidase
Activity tests at various temperatures show that the NADH oxidase exhibits maximum activity at 40° C. (FIG. 3).
d) Temperature Stability of the NADH Oxidase
In order to determine the temperature stability enzyme samples were stored at 30° C. and 42° C., samples were taken at various times, and the residual activity was measured. The evaluation shows that the enzyme at 30° C. is in practice stable for ca. 400 minutes, following which it appears to deactivate according to a slow reaction kinetics (half-value time ca. 18 hours). At 42° C. the enzyme deactivates initially (up to ca. 120 minutes) relatively quickly, following which it appears to be stable at ca. 50% residual activity over a relatively long time (at least 16 hours).
e) Determination of the KM Value for NADH for the NADH Oxidase
For the substrate NADH the concentration was varied in the range from 0.0013 to 0.52 mM and the activity was measured photometrically. The evaluation shows that maximum activity is reached at 0.1 mM. Higher NADH concentrations (up to 0.5 mM) exhibit only a weak excess inhibition. The Km value is calculated to be 24 μM. This high affinity of the enzyme for NADH is very advantageous for applications in which NAD+ is to be regenerated by oxidation of NADH, since a prerequisite for this is as complete an oxidation of NADH as possible.
4) Protein Chemistry Characteristic Data of the NADH Oxidase from Lactobacillus Brevis 
a) N-Terminal Amino Acid Sequence
The results of several enrichments always revealed a protein whose subunits have a molecular weight according to SDS-PAGE of 50 kDa. The first 27 amino acids of the N-terminus were determined for this protein (automated Edman decomposition with an automated sequencer 4774 (Applied Biosystems) with online HPLC 120 A); the sequence is as follows: MKVTVVGCTHAGTFAIKQILAEEPDADXXVY (SEQ ID NO:5). In the sequence a conserved motif is underlined that acts as FAD-binding motif in NADH oxidases, and is a slight modification of the known ADP-binding site GXGXXG (SEQ ID NO:6). Databank searches (BLAST) already show for this protein fragment agreement with known NADH oxidases.
a) Determination of the Protein and Gene Sequences
The results of the databank searches show in some partial regions sequences that are present in virtually all NADH oxidases or in related NADH peroxidases. The amino acid sequence DIFAAGDSA (SEQ ID NO:3) is regarded as such a conserved region. An antisense codon is used as “C-terminal” primer for this sequence, and together with a primer that codes for the N-terminus MKVTVVGCT (SEQ ID NO:4), is used in a PCR. The PCR yields a 777 bp long fragment that is 59% identical to the NADH oxidase (NOX) from Enterococcus. A gene library is established by digesting genome DNA with 4 different restriction endonucleases (EcORV, Pvu II, Sca I and Stu I). Using specific primers derived from the cleavage sites of the restriction enzymes and sequences from the 777 bp fragment, further sequence fragments are then obtained by means of PCR, whereby the total sequence can be defined with the aid of overlapping DNA sequences and the stop codon.
The sequence for the NADH oxidase, (NOX) from L. brevis is reproduced in the sequence protocol (Seq. 1) given above.
5) Use of the NADH Oxidase from Lactobacillus Brevis for the Coenzyme Regeneration
A typical use of the NADH oxidase is the regeneration of NAD+. This may be necessary for example if NAD-dependent dehydrogenases are used for the oxidation, as has been shown in a general way hereinbefore (reaction scheme 1). This is demonstrated in the following example for the production of an enantiomer-pure alcohol corresponding to the following equations:

(R,S)-alcohol+NAD+−−>S-alcohol+ketone+NADH+H  (4)

NADH+H++1⁄202−−>NAD++H2O  (5)

with an R-specific alcohol dehydrogenase as catalyst for reaction (4), and the NADH oxidase for (5).

In this example an NAD mutant of the R alcohol dehydrogenase from Lactobacillus brevis (recADHG37D (NAD-mutant) DE10037101.9), coupled with the purified NADH oxidase from Lactobacillus brevis, was used as R-specific alcohol dehydrogenase. (R,S)-phenylethanol was used as alcohol, and the reaction was carried out as a batch reaction. The following reactants were used: 50 mM (R,S)-phenylethanol, 2 mM NADH, 50 mM TEA buffer pH 7.0 with 1 mM MgCl2 and with 5 mM DTT, 1 U recADHG37D and 2 U NADH oxidase.
Samples were taken after 0, 1, 2, 3, 4, 6, 9 and 24 hours and separated by gas chromatography (column: CP-Chirasil-DEX CB, length: 25 m, diameter: 25 μm (Chrompack). Temperature programme: 5 mins at 60° C., then 5° C./min up to 190° C.; column flow rate 1.3 ml/min; gas: helium. The acetophenone peak (product; retention time=16.9 min) as well as the enantiomers of phenylethanol (educts; retention times R-phenylethanol=20.8 min and S-phenylethanol=21.1 min) were detected. FIG. 4 shows the GC spectrum of a sample in which the R-alcohol had been almost completely oxidised, which is why a corresponding amount of the oxidation product acetophenone can be seen. As a control the same experiment was carried out, but without the addition of NADH oxidase. Gas chromatography analysis does not show any significant change in the two alcohols, and simply a small amount of acetophenone can be detected, which may possibly be attributed to a spontaneous oxidation of NADH by atmospheric oxygen.
The present application claims priority to German Application DE 101 40 088.8 filed on Aug. 16, 2002, the entire contents of which are incorporated herein by reference.
Obviously, numerous modifications and variations on the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

## Claims

1. A cell which expresses a recombinant NADH oxidase obtained from Lactobacillus, wherein the recombinant (rec) NADH oxidase comprises SEQ ID NO: 2.

2. The cell of claim 1, wherein the rec-NADI oxidase is obtained from Lactobacillus brevis.

3. The cell of claim 1, wherein the rec-NADH oxidase is obtained from Lactobacillus brevis DSM 20054.

4. The cell of claim 1, wherein the nucleic acid encoding the rec-NADH oxidase is carried on a plasmid.

5. The cell of claim 1, wherein the recombinant (rec) NADH oxidase is encoded by SEQ ID NO: 1.

6. A method of generating NAD+ from NADH, comprising culturing the cell of claim 1 under conditions in which NADH contained in the cell is converted to NAD+ by the action of the rec-NADH oxidase.

7. A method of generating NAD+ from NADH, comprising culturing the cell of claim 2 under conditions in which NADH contained in the cell is converted to NAD+ by the action of the rec-NADH oxidase.

8. A method of generating NAD+ NADH, comprising culturing the cell of claim 3 under conditions in which NADH contained in the cell is converted to NAD+ by the action of the rec-NADH oxidase.

9. A method of generating NAD+ from NADH, comprising culturing the cell of claim 5 under conditions in which NADH contained in the cell is converted to NAD+ by the action of the rec-NADH oxidase.