THEREOF

The invention relates to novel polypeptides having high NADPH oxidase activity; in particular high NADH oxidase activity and high NADPH oxidase activity. The invention also relates to nucleotides encoding these polypeptides; vectors and host cells comprising these nucleotides; and use of these polypeptides in the biocatalysis of chemical oxidation reactions.

Oxidoreductases constitute an important group of biocatalysts facilitating stereoselective reactions, for example, the reduction of ketones or the oxidation of alcohols and amines. Oxidoreductases act on the substrate by the transfer of electrons from or to a cofactor. The nicotinamide-based nucleotides NAD(H) and NADP(H) belong to the most widely used cofactors. As nicotinamide cofactors are very expensive, regeneration of cofactors is a necessity for the economical feasibility of a biocatalytic process. Regeneration of the reduced cofactors may be achieved in a number of ways, for example, using (engineered) formate dehydrogenase, glucose dehydrogenase, or phosphite dehydrogenase. However, no universal regeneration system for the oxidized forms NAD ⁺ and NADP ⁺ is known.

Coupled substrate or coupled enzyme systems constitute two possibilities for NAD(P) ⁺ recycling. In these set-ups the cofactor is regenerated via the reduction of a carbonyl group of a cosubstrate, catalyzed either by the production enzyme itself (coupled substrate) or by an additionally added dehydrogenase (coupled enzyme). However, carbonyl reductions by dehydrogenases in coupled enzyme or coupled substrate set-ups normally provide little thermodynamic driving force for the mostly energetically unfavorable biocatalytic oxidation. This makes it necessary to supply the carbonyl compound in excess to achieve high substrate conversion rates.

Glutamate dehydrogenase can be used to regenerate NADP ⁺ in the reduction of a-Ketoglutarate to L-glutamate. However this method has the problem of requiring NH ₃ for the reductive amination, and also suffers from problems with substrate stability and a more complicated down-stream processing due to the formation of glutamate.

Electrochemistry can also be used for the regeneration of cofactors but the productivity of the majority of electroenzymatic processes is rather low.

A further NAD(P) ⁺ regeneration method is the application of soluble I bacterial NAD(P)H oxidases which use molecular oxygen as oxidant. This method has the advantage of being cheap and enabling straight-forward downstream processing because only hydrogen peroxide or water are formed as byproducts. Moreover, the high redox potential of oxygen results in a high thermodynamic driving force. The electron and hydrogen transfer from NADH to oxygen is catalyzed by bacterial NAD(P)H oxidases via a four electron transfer producing water or a two electron transfer producing hydrogen peroxide.

A system that is capable of effectively regenerating both NADH and NADPH is highly desirable. Such a versatile system could be applied to regeneration of either NADH or NADPH in an oxidoreductase catalysed reduction. Accordingly, only one cofactor regenerating enzyme, rather than two, need be kept in the chemical toolbox. Most NAD(P)H oxidases are NADH specific or at least prefer NADH over NADPH. NAD ⁺ regeneration has been performed with a water forming NADH oxidase from Lactobacillus brevis (L. brevis) [Geuke et al. Enzyme Microb. Technol. 2003, 32, 205-21 1 ] but the enzyme is specific for NADH and therefore cannot be used for NADP ⁺ regeneration. The only water-forming NAD(P)H oxidase that accepts NADPH in addition to NADH is found in Lactobacillus sanfranciscensis (L. sanfranciscensis) (Z-sNOX) and this has been heterologously expressed in Escherichia coli (E. coli). While K _m values for NADH and NADPH are almost the same for both cofactors, maximum velocity (v _max ) with NADPH reaches only around 30% of maximum velocity with NADH [Riebel et al. Adv. Synth. Catal. 2003, 345, 707-712].

The present inventors have found new NAD(P)H oxidases which have high NADPH regeneration activity, in particular both high NADH and high NADPH regeneration activity.

Accordingly the present invention provides a polypeptide having NADPH oxidase activity and comprising an amino acid sequence having at least 40% sequence identity to SEQ ID No.1 .

NADPH oxidase activity is the activity of the polypeptide in catalyzing reaction of NADPH and oxygen to produce NADP ⁺ and water. Similarly, NADH oxidase activity is the activity of the polypeptide in catalyzing reaction of NADH and oxygen to produce NAD ⁺ and water.

Activity may be measured in any way conventional in the art. A preferred technique for analyzing the NAD(P)H oxidase activity is by measuring the decrease in absorption of visible light, or UV, due to the oxidation of NADH or NADPH, by addition of a crude lysate of NAD(P)H oxidase diluted to a specific concentration. Absorption of light may be measured at, for example, 340 nM (e=6220 M ^"1 cm ^"1 ). The apparent maximum rate, v _max , is calculated from this via the Michaelis Menten equation, as illustrated in Example 4. As used herein apparent v _max values of an enzyme are given for a cell free extract containing this enzyme.

Sequence identity or similarity is herein defined as a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. Usually, sequence identities or similarities are compared over the whole length of the sequences, but may however also be compared only for a part of the sequences aligning with each other. In the art, "identity" or "similarity" also means the degree of sequence relatedness between polypeptide sequences or polynucleotide sequences, as the case may be, as determined by the match between such sequences. Preferred methods to determine identity or similarity are designed to give the largest match between the sequences tested. In context of this invention a preferred computer program method to determine identity and similarity between two polypeptide or polynucleotide sequences includes BLASTP and BLASTN, respectively (Altschul, S. F. et al., J. Mol. Biol. 1990, 215, 403-410, publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894). Preferred parameters for polypeptide sequence comparison using BLASTP are gap open 10.0, gap extend 0.5, Blosum 62 matrix. Preferred parameters for nucleotide sequence comparison using BLASTN are gap open 10.0, gap extend 0.5, DNA full matrix (DNA identity matrix).

SEQ ID No. 1 is the amino acid sequence of wild type NADH oxidase 2 obtainable from Streptococcus mutans (S. mutans) (SmNOX2). The invention comprises mutants of SEQ ID No. 1 . Methods for making mutations are known to the person of skill in the art, examples being random mutagenesis (for example with the aid of PCR or by means of UV irradiation) and site directed mutagenesis.

The present invention further provides a nucleic acid sequence which encodes a polypeptide as defined above.

A nucleic acid sequence according to the present invention can be cloned into a suitable vector and, after introduction into a suitable cell, the nucleic acid sequence can be expressed so as to produce a polypetide according to the present invention. Cloning and expression of a nucleic acid sequence are standard techniques known in the art. These are described, for example, in Sambrook, J., and Russell, D. Molecular Cloning, A Laboratory Manual, 3 ^rd ed., Cold Spring Harbour Laboratory, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY, 2001. Accordingly, the present invention further provides a vector which comprises a nucleic acid sequence as defined above.

A polypeptide according to the present invention can be produced by integrating the nucleic acid sequence encoding the polypeptide into the genome of a host cell and by (over)expressing it. Integration of the nucleic acid sequence can be effected using methods known to the person of skill in the art. It is also possible to over-express a polypeptide according to the present invention in the microorganism in which it occurs naturally, for example by placing a suitable promoter in front of the nucleic acid sequence according to the present invention in the genome of the microorganism; by integrating one or more copies of a nucleic acid sequence according to the present invention into the genome of the microorganism; or by overexpressing the nucleic acid sequence according to the invention in a suitable vector in its natural host.

The present invention further provides a host cell which comprises a nucleic acid sequence as defined above or a vector as defined above.

A further embodiment of the present invention is a method of generating NADP ⁺ by oxidation of NADPH comprising contacting NADPH with a polypeptide as defined above. Alternatively a method of generating NAD ⁺ by oxidation of NADH comprising contacting NADH with a polypeptide as defined above is provided.

SmNOX belongs to the large group of enzymes of the Rossmann-fold type. For these enzymes some conserved residues have been identified before which seem to contribute a major part to cofactor specificity determination. Especially an acidic residue, typically an aspartate at the C terminus of the second β-strand of the typical alternating α/β-regions is known to be important for NAD(H) binding, while NADP(H) specific dehydrogenases typically miss this acidic residue and instead show a basic residue at the position directly following the aspartate position.

Typically the polypeptide as defined above has NADH oxidase activity. Preferably NADH oxidase activity is measured in the same manner as NADPH activity. Preferably apparent maximum velocity v _max is used to measure both NADH and NADPH activity. The conditions for measuring v _max are preferably those described above.

Preferably the NADPH oxidase activity measured as apparent maximum rate v _max is at least 1.2 U/mg. More preferably v _max is at least 1 .6, 2.0, 2.4, 2.8, 3.2, 3.6; most preferably at least 4.0 U/mg. Preferably the NADH oxidase activity measured as apparent maximum rate v _max is at least 1.6 U/mg. More preferably v _max is at least 2.0, 2.4, 2.8, 3.2, 3.6, 4.0; most preferably at least 4.4 U/mg.

With U/mg is meant here the conversion of 1 μηηοΙ of substrate

[NAD(P)H] per minute per mg of total protein in the cell free extract.

It is particularly desirable to have a system where the NADPH and NADH oxidase activities are of comparable value. Such a system is very versatile because it could be applied to regeneration of either type of nicotinamide cofactor in an oxidoreductase catalysed oxidation of substrate with concurrent reduction of nicotinamide cofactor. Accordingly, only one cofactor regenerating enzyme, rather than two, needs to be kept in the chemical toolbox. Typically, the ratio of NADPH oxidase activity to NADH oxidase activity is from 1 :5 to 5:1. Preferably, the ratio of NADPH oxidase activity to NADH oxidase activity is from 1 :3 to 3:1 . Such activity can be measured by any standard technique, provided it is consistent for both types of oxidase activity. Typically the ratio of NADPH oxidase activity measured as apparent maximum rate v _max to NADH oxidase activity measured as apparent maximum rate v _max is from 1 :3 to 3:1 . Preferably the ratio is from 1 :2 to 2:1 . More preferably the ratio is from 1 :1 .5 to 1 .5:1 . Most preferably the ratio is from 1 :1.2 to 1 .2:1 .

Typically the polypeptide is one wherein the NADPH oxidase activity is water-producing NADPH oxidase activity and the NADH oxidase activity is water- producing NADH oxidase activity.

Water-producing NAD(P)H oxidases are desirable in order to avoid hydrogen peroxide production. Hydrogen peroxide acts destabilizing to many enzymes and therefore has to be destroyed by the addition of catalase. In addition hydrogen peroxide can cause by-product formation.

The polypeptide according to the present invention typically has at least 50% sequence identity with SEQ ID No. 1 . Preferably it has at least 60% sequence identity with SEQ ID No. 1 ; more preferably at least 70%, at least 80%, at least 90%, at least 95%, for example 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.8%. Typically the polypeptide of present invention comprises at least one mutation; for example 1 , 2, 3 or 4 mutations from SEQ ID No. 1. Preferably it comprises 2 or 3 mutations from SEQ ID No. 1 .

Typically the polypeptide comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID No.s 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 21 , 22, 23, 24, 25, 26, 27. Preferably it has at least 90% sequence identity with any one of SEQ ID No.s 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21 , 22, 23, 24, 25, 26 and 27. More preferably it has at least 95% sequence identity with any one of SEQ ID No.s 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21 , 22, 23, 24, 25, 26 and 27; still more preferably 97%, for example 98%, 99%, 99.5%, 99.7% or 99.8%. Most preferably the polypeptide described above comprises an amino acid sequence of any one of SEQ ID No.s 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21 , 22, 23, 24, 25, 26 and 27.

The difference between NADH and NADPH is the presence of a phosphate group. One way to improve the NADPH oxidase activity of an NADH oxidase active enzyme, is to increase the acceptance of this phosphate group.

Increasing acceptance of the phosphate group could be achieved by making mutations reducing steric hindrance, or increasing chemical compatibility, for example improving acid-base interaction, or polarity. The region of the enzyme of SEQ ID No. 1 found to have most influence on the binding of the phosphate group has been found to be in the position of amino acid residues 190 to 205. These amino acid residues are oriented such that they point into the phosphate binding region. Accordingly, replacing an amino acid at one of these positions with a more basic or less acidic amino acid than that of SEQ ID No. 1 is believed to increase the binding affinity of the polypeptide to the phosphate group and hence NADPH. Therefore, typically, in the polypeptide of the present invention at least one amino acid residue selected from an amino acid residue at any one of positions 190 to 205 of SEQ ID No.1 is replaced with either a more basic or less acidic amino acid residue. Preferred mutations are at positions 192, 193, 194, 199 and 200. Preferred mutations are D192A, D192N, V193R, V194H, A199R, G200K and combinations thereof. Particularly preferred are the combinations of mutations D192AA 193R and D194H/G200K.

A particularly preferred embodiment of the present invention is a polypeptide having water-producing NADPH oxidase activity and water-producing NADH oxidase activity; and comprising an amino acid sequence having at least 40% sequence identity to SEQ ID No.1 , with the proviso that at least one amino acid residue selected from an amino acid residue at any one of positions 190 to 205 of SEQ ID No.1 is replaced with either a more basic or less acidic amino acid residue.

In the above embodiment, typically the ratio of water-producing NADPH oxidase activity measured as apparent maximum rate v _max to water-producing NADH oxidase activity measured as apparent maximum rate v _max is from 1 :3 to 3:1 . Preferably said polypeptide comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID No.s 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21 , 22, 23, 24, 25, 26 and 27. More preferably the NADPH oxidase activity measured as apparent maximum rate v _max is at least 1 .2 U/mg. Still more preferably the NADH oxidase activity measured as apparent maximum rate v _max is at least 1 .6 U/mg.

Vectors which are suitable for expressing a nucleic acid sequence according to the present invention are those normally used for cloning and expression, known to the person of skill in the art. Examples of suitable vectors for expression in £. co// ^' are pUC, pACYC, pET, pMS470A8, pQE, pBAD, pTrc, pRSET, pCold and pCYTEX. The skilled person can identify suitable promoters to be used. Suitable promoters include homologous and heterologous promoters. Heterologous promoters include both constitutive and inducible natural promoters as well as engineered promoters, which are well known to the person skilled in the art. Examples of constitutive and inducible promoters in Gram-negative microorganisms include, but are not limited to, tac, tet, trp-tet, Ipp, lac, Ipp-lac, laclq, T7, 75, 73, gal, trc, ara {PBAD),

Suitable host cells are those typically used in the art and known to the skilled person. These may be selected from, for example, gram-positive or gram- negative bacteria, yeast and fungi. Examples of suitable E. coli host cell strains include TOP10F', TOP10, DH 10B, DH5a H B101 , W31 10, BL21 (DE3)pLysS, BL21 Star(DE3) and BL21 (DE3) Gold. The choice of vector sometimes depends on the choice of a host cell; and the other way round.

A nucleic acid sequence according to the present invention can be amplified using, for example, cDNA, mRNA, genomic DNA or synthetic DNA as a template and the appropriate oligonucleotide primers by means of standard PCR amplification techniques. A nucleic acid sequence according to the present invention can also be obtained as synthetic DNA. The nucleic acid sequence obtained by amplification or synthetic DNA can be cloned into a suitable vector and can be characterized by DNA sequence analysis.

The nucleic acid sequences according to the present invention may, after isolation, be cloned into a suitable vector and expressed in a suitable host cell, using methods known in the art for production of a polypeptide according to the invention.

Typically the nucleic acid sequence of the present invention has at least 80% sequence identity to any one of SEQ ID No.s 1 1 , 12, 13, 14, 15, 16, 17, 18 and 19. Preferably it has at least 90% sequence identity with any one of SEQ ID No.s 1 1 , 12, 13, 14, 15, 16, 17, 18 or 19. More preferably it has at least 95% sequence identity with any one of SEQ ID No.s 1 1 , 12, 13, 14, 15, 16, 17, 18 or 19; still more preferably 97%, for example 98%, 99%, 99.5%, 99.7% or 99.8%. Most preferably, the nucleic acid sequence as described above has any one of SEQ ID No.s 1 1 , 12, 13, 14, 15, 16, 17, 18 or 19.

Stability of the polypeptide of the present invention is also an important factor. For example, stability in the presence of an organic solvent, to which enzymes are often sensitive. This can be assessed by measuring the activity of the polypeptide in the presence of a common organic solvent. Measurements can be made as described above. However, preferably maximum velocity v _max is used as the parameter for determining activity. Accordingly, the polypeptide of the present invention is typically one in which NADPH activity measured as v _max in the presence of an organic solvent is at least 10% of the NADPH activity in the absence of an organic solvent.

Preferably the activity is at least 20% of the activity in the absence of an organic solvent. More preferably it is at least 30%, for example at least 40%, 50%, 60%, 70%, 80% or 90% of the activity in the absence of an organic solvent. Any organic solvent may be used. A preferred reference solvent is 20% N- methylpyrrolidone.

Typically the polypeptide comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID No.s 28, 29, 30 and 31 .

Preferably it has at least 90% sequence identity with any one of SEQ ID No.s 28, 29, 30 and 31 . More preferably it has at least 95% sequence identity with any one of SEQ ID No.s 28, 29, 30 and 31 ; still more preferably 97%, for example 98% or 99%. Most preferably the polypeptide described above comprises an amino acid sequence of any one of SEQ ID No.s 28, 29, 30 and 31 .

Typically, in the method of the present invention the NADPH is formed in situ by reduction of NADP ⁺ , or the NADH is formed in situ by reduction of NAD ⁺ , during an oxidoreductase-catalysed oxidation of an alcohol or amine.

Preferably, the oxidation is of a C1-C12 primary or secondary alcohol, for example n- heptanol or 1 -phenylethanol.

By in situ is meant that the NADPH or NADH formation by the oxidoreductases-catalyzed oxidation of the alcohol or amine is taken place in the same reaction vessel as their NADPH or NADH oxidase-catalyzed regeneration. NADPH may be formed, for example, in a bioconversion reaction with two separate enzymes or with two separate cells; in a whole cell reaction (both enzymes in one and the same cell); and even in a hybrid enzyme containing both activities on a single (recombinant) polypeptide.

To confirm the activity of a polypeptide according to the present invention, the method of Example 4 may be carried out.

The present invention also provides a process for producing a ketone or aldehyde by oxidation of an alcohol, which process is carried out in the presence of a polypeptide as defined above.

Figure 1 illustrates a general example of the present invention in a water-forming coupled enzyme reaction. An alcohol is oxidized to a ketone, catalysed by alcohol dehydrogenase (ADH). NAD(P) ⁺ cofactor is simultaneously reduced to NAD(P)H. The reduced cofactor is recycled to form NAD(P) ⁺ using NAD(P)H oxidase (NOX). Simultaneously, molecular oxygen is converted to water.

The invention is further illustrated by the following examples. It is not, however, intended to be limited thereto.

EXAMPLES Example 1 : Homology modeling of S. mutans NOX2

Homology modeling for S. mutans NOX2 was based on an x-ray structure of NADH oxidase from Streptococcus pyogenes (template:2bcOA.pdb, 2.00 A). Sequence identity between target and template is 77.5%. The homology model was created with the automated protein structure homology-modeling server SWISS- MODEL developed by the Protein Structure Bioinformatics group at the SIB - Swiss Institute of Bioinformatics and the Biozentrum University of Basel (version February 2008).

Example 1 shows the successful homology modeling of S. mutans

NOX2.

Example 2: Site directed mutagenesis of a synthetic S. mutans NOX2 gene

£. CO// TOP10F' was originally bought from Invitrogen (Carlsbad, CA, USA), E. coli BL21 -Gold (DE3) was from Stratagene (La Jolla, CA, USA). Materials for cloning were from Fermentas (St. Leon-Roth, Germany) if not stated otherwise. All other chemicals were purchased from Sigma-Aldrich, Fluka (St. Louis, MO, USA) or Roth (Karlsruhe, Germany) if not stated otherwise.

A synthetic S. mutans NOX2 gene was ordered at DNA2.0 (Menlo Park, CA, USA) and ligated into a Nde\/Hind\\\ cut pMS470A8 vector (Balzer 1992) downstream of the tac-promoter to give the vector pMSsNI Wt. Site directed

mutagenesis of the S. mutans NOX2 gene was performed following the Stratagene Quickchange Site-directed mutagenesis kit instruction (Stratagene, La Jolla, CA, USA). Complementary primers containing the desired mutations were used in polymerase chain reactions with the pMSsNIWt plasmid as template to produce mutated plasmids with staggered nicks. Primers used for site directed mutagenesis PCR are shown in Table 1 . Sequences are given for the forward primers binding to the coding strand, reverse primers have the reverse complementary sequence, mismatched bases are underlined.

Table 1 : Primers used in PCR for site directed mutagenesis of S. mutans N0X2

a) Numbering refers to S. mutans N0X2 sequence beginning with 1 for the starting methionine

b) reverse primers have reverse complementary sequence

c) mutated plasmid pMSsN1 M4 carrying the Val194→His mutation was used as template d) mutated plasmid pMSsN1 M5 carrying the Ala199→Arg mutation was used as template e) mutated plasmid pMSsN 1 M6 carrying the Gly200→Lys mutation was used as template

All amplifications were done with a Gene Amp PCR System 2700 (Applied Biosystems, Foster City, CA, USA). 50μΙ_ PCR reaction mixtures contained 28pM template plasmid, 0.2μΜ of each primer, 200μΜ of each dNTP and 5μΙ_ of 10x reaction buffer supplied with the polymerase. 2.5 units of Pfu turbo polymerase (Stratagene) were added to each tube. The amplification protocol comprised 30 seconds of initial denaturation at 95°C, 18 cycles of denaturation (30s ,95°C), annealing (1 min, 55°C) and extension (6 min, 68°C) and a final 7 minutes extension period at 68°C.

For digestion of unmutated template DNA 10U of Dpn\ restriction enzyme were added to each tube and the preparations were incubated for 1 h at 37°C. Competent £ co// TOP10F' cells were transformed by electroporation with 2μΙ_ of the reaction mixture. Finally plasmid DNA was extracted from transformants cultivated in LB medium with Gene JetTM Plasmid Miniprep Kit (Fermentas, St. Leon-Roth, Germany) and successful incorporation of the desired mutations was verified by dideoxy sequencing using primers pMSfw and pMSrv.

Sequencing primers:

pMSfw: 5'-GTGAGCGGATAACAATTTCACACA-3'

pMSrv: 5'-GTTTTATCAGACCGCTTCTGCG-3'

Example 2 demonstrates the successful site directed mutagenesis of S. mutans NOX2 gene.

Example 3: Preparation of cell free extracts containing S. mutans NOX2 variants with engineered cofactor binding site from shake flask cultivations

Electrocompetent £ coli BL21 (DE3) Gold cells were transformed with plasmid pMSsNIWt or one of 15 variants thereof with mutations in the gene section coding for the cofactor binding site as described in Example 2. Additionally a plasmid pMSsN2 was transformed into £ coli BL21 (DE3) giving a strain with overexpression of Lactobacillus sanfranciscensis NOX (LsNOX) for benchmarking activities. pMSsN2 is identical to pMSsNI except that it carries the gene coding for LsNOX (synthetic variant, ordered at DNA 2.0) instead of the SmNOX2. The gene coding for LsNOX (gene accession number AB035801 ) was codon pair optimised according to a procedure described in WO08000632 resulting in SEQ ID No. 54. The synthetic gene was ordered at DNA2.0 Inc. (Menlo Park, CA94025 US). The synthetic gene is coding for the amino acid sequence according to protein accession number BAB19268. Precultures of all resulting £. coli strains were cultivated in 50mL of LB media containing 100mg/L of Ampicillin (LB/Amp media) in baffled 300ml_ shake flasks at 37°C and 130 rpm overnight. For main cultures 250ml_ of LB/Amp-Medium in 1 L baffled flasks were inoculated with overnight culture to an OD of 0,05 and cultivated at 37°C and 130rpm. NOX production was induced by addition of 1 mM IPTG at an OD of 0.8. Cells were harvested after an overnight induction period at 25°C and 1 10rpm by centrifugation for 15 minutes at 5000rcf (Avanti J-20 XP, Beckman Coulter, Krefeld, Germany, rotor JA-10). Cell pellets were diluted in 50mM potassium phosphate buffer pH 7.0 to a final volume of 25 ml_. Cell breakage was achieved by ultrasonication with a Branson sonifier 250 (Branson ultrasonic corporation, Danbury, CT, USA) for 6 minutes at 50W with continuous cooling, pulsed with one 700ms pulse per second with a disruptor horn of 1 cm diameter. Cell free lysates were prepared by collecting the supernatant of centrifugation at 36000 rcf (rotor JA-25.50) for 45 minutes and concentrating it to half the volume via Vivaspin 20 centrifugal concentration tubes with 30 kD molecular weight cutoff (Sartorius,Gottingen, Germany). The protein content was determined with the bichinonic acid protein assay (BCA) kit (Thermo Scientific, Waltham, MA, USA) using BSA as standard. SDS/PAGE gel electrophoresis

(NuPAGE® Novex® 4-12% Bis-Tris Gels (1 .0 mm) from Invitrogen (Carlsbad, CA, USA) together with a NuPAGE MOPS SDS Running Buffer for Bis-Tris Gels) of the cell free extracts showed a strong protein band migrating to the expected position for 50kDa in each lane except in the lane with the cell free extract of an E. coli BL21 strain without plasmid. All strains were stored as glycerol stocks at -80°C. Cell free extracts were stored in aliquots at -20°C.

Example 3 demonstrates preparation of cell free extracts containing S. mutans NOX2 variants with engineered cofactor binding site from shake flask cultivations.

Example 4: Determination of catalytic constants for SmNOX2 variants with altered coenzyme specificity

Initial rate data of NAD(P)H oxidation were acquired measuring the decrease in NAD(P)H absorption at 340 nM (e=6220M ^"1 cm ^"1 ) in 50 mM potassium phosphate buffer, pH 7.0 at 25°C. Absorption measurements were performed on a Spectramax Plus 384 (Molecular Devices, Sunnyvale, CA, USA) in UV-star micro titer plates (Greiner, Kremsmijnster, Austria). The total reaction volume was 200μΙ_, reactions were started by addition of NAD(P)H. Apparent kinetic parameters were obtained from initial rate measurements at air saturation oxygen level with eight cofactor concentrations varying over a concentration range between 0 and 5 - 10 times the apparent K _m or, for variants were no saturation could be reached, to a maximum NAD(P)H concentration of 1 mM.. Enzymes were applied as crude lysates in dilutions chosen to give rates between 0,001 and 0,05 AAbs/min and a constant signal decrease for≥ 2 minutes. Appropriate controls containing crude lysate without overexpressed NOX verified that blank rates were insignificant for all conditions used. Results from initial rate measurements were fitted to the Michaelis Menten equation (1 ) using unweighted least-squares regression analysis performed with Sigmaplot program version 1 1 (Jandl).

v = v _max ^* A/(app _m +A) (1 )

v is the initial rate, v _max is the apparent maximum rate (U/mg total protein in cell free extract), A the cofactor concentration and app _m the apparent Michaelis constant for NAD(P)H. Apparent kinetic parameters for SmNOX2 wild type and mutants and LsNOX are summarized in table 2.

Table 2: Apparent kinetic values and calculated efficiencies for SmN0X2 mutants

NADH NADPH

v _max ^a) (U/mg) appK _m (μΜ) v _max /appK _m (U/(mg*mM)) v _max (U/mg) appK _m (μΜ) v _max /appK _m (U/(mg*mM)

SmN0X2 Wt (SEQ ID No. 1 ) 3.5 ± 0.1 6 ± 1 580 0.09 ^C

LsNOX 2.2 ± 0.1 9 ± 1 240 1.1 ± 0.1 5 ± 1 220

D192A ^b) (SEQ ID No. 2) 2.5 ± 0.1 18 ± 2 140 2.2 ± 0.1 140 ± 20 15

D192N (SEQ ID No. 3) 2.8 ± 0.1 19 ± 2 150 2.9 ± 0.1 190 ± 20 15

V193R (SEQ ID No. 4) 3.8 ± 0.4 9 ± 3 420 1.6 ± 0.1 140 ± 10 12

V194H (SEQ ID No. 5) 3.5 ± 0.1 1 1 ± 2 320 1.4 ± 0.1 150 ± 10 10

A199R (SEQ ID No. 6) 4.4 ± 0.1 9 ± 1 490 0.45 ^c)

G200K (SEQ ID No. 7) 3.4 ± 0.1 7 ±1 490 2.7 ± 0.2 120 ± 30 22

192A/193R (SEQ ID No. 8) 2.9 ± 0.1 23 ± 2 130 4.8 ± 0.2 5 ± 1 960

194H/199R (SEQ ID No. 9) 4.2 ± 0.1 12 ± 1 350 3.7 ± 0.1 71 ± 6 52

194H/200K (SEQ ID No. 10) 3.1 ± 0.1 7 ± 1 440 2.6 ± 0.1 12 ± 1 217

192A/193R/194H (SEQ ID No. 20) 2.0 ± 0.1 16 ± 2 130 4.2 ± 0.2 3 ±1 1400

192A/193R/194H/199R (SEQ ID No. 21 ) 2.3 ± 0.1 1 1 ± 0 210 6.3 ± 0 3 ± 0 2100

192A/193R/194H/200K (SEQ ID No. 22) 3.1 ± 0.4 8 ± 1 390 4.0 ±.0.3 3 ± 1 1300

193R/194H (SEQ ID No. 23) 4.4 ± 0.1 7 ± 2 630 4.6 ± 0.1 7 ± 1 660

193R/194H/199R (SEQ ID No. 24) 4.3 ± 0.1 7 ± 1 610 1.7 ± 0.1 4 ± 1 430

193R/194H/200K (SEQ ID No. 25) 4.6 ± 0.2 4 ± 1 1200 3.3 ± 0.2 2 ± 0 1650

a) Vmax and apparent K _m values were measured in cell free extracts at air-saturated oxygen levels

b) all indicated mutations confer to SmNOX2, numbering according to SmNOX2 amino acid sequence including the starting methionine c) as saturation with NADPH could not be achieved v _max /appK _m was calculated from the initial linear section of the Michaelis-Menten curve

Example 4, in Table 2 compares activity as v _ma x/app _m (U/mg mmol) for both NADPH oxidase activity and NADH oxidase activity of each of the prepared mutants against the wild type enzyme. Each of the mutants has a highly improved NADPH oxidase activity. Each of the mutants has a much lower ratio of NADH oxidase activity to NADPH oxidase activity.

Example 5: Application of SmNOX2 mutants for cofactor recycling in alcohol-ketone conversions

The oxidation of n-heptanol to n-heptanone was chosen as model reaction for an alcohol to ketone conversion. Alcohol dehydrogenase from

Sphyngobium yanoikuyae (SyADH) was chosen as production enzyme in a coupled enzyme approach together with SmNOX2 for cofactor regeneration (see scheme 1 ). SyADH is known to be unselective and active with NADP ⁺ (preferred) and NAD ⁺ . Its preparation is described in Example 10.

Conversion experiments were set up in deep well plates that were sealed with aluminum seals and shaken at l OOOrpm at RT on a TITRAMAX 1000 shaker (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). The reaction mixture contained 500μΜ NAD(P) ⁺ and 10mM n-heptanol in 50mM potassium phosphate buffer, pH 7.0 in a total volume of 500μΙ_. SyADH and SmNOX2 (wild type or mutant 192A193R) were applied as crude E. coli lysates in amounts resulting in the initial activities given in table 2. After 20 hours 100μΙ_ of 50 mM n-butanol was added as internal standard for GC analysis and the mixture was extracted with 500μΙ_ ethyl acetate by mixing on a magnetic tumble stirrer Pacesetter VP710 (V&P Scientific, San Diego, CA, USA) for 2 minutes at 200 rpm. Substrate conversion was determined by GC-analysis on a Varian CP7503 gas chromatograph equipped with an FID detector (275°C) and a Chirasil-DEX CB column (25 m x 0.32 mm, 0.25 μηι film). H ₂ was used as carrier gas (2.4 mL/min). The following temperature program was used: 65°C - 4 min; 9°C/min to 1 10°C; 160°C - 4 min. Table 3: Results of conversion experiments with S. mutans NOX2 added for cofactor

a values given are concentrations detected by GC measurements of substrate and product after conversion, not all of the 10 mM applied could be recovered as substrate or product, no other major peaks were detectable in GC

b conversion was calculated as follows: product detected by GC/(substrate+product) detected by GC

d TTN (total turnover number) was calculated as follows: product detected by GC/0.5 mM cofactor applied

Results shown in table 3 confirm that conversion occurs with NAD ⁺ and NADP ⁺ to a high level and therefore that NADPH recycling with S. mutans NOX2 mutant 192A193R was made possible. Example 6: Preparation and cultivation of an expression library with random mutations in SmNOX2

A library of the SmNOX2 gene with random mutations was ordered at SeSaM Biotech. Average number of mutations per kb was stated to be 2.9 mutations by SeSaM Biotech (21 clones tested) and verified to be 2.6 mutations by the inventors (90 clones tested). 12 % of the clones had two mutations in 2 subsequent basepairs, 9% of the clones showed an insertion or deletion. SeSaM Biotech evaluated the total number of amino acids exchanged per clone (18 checked) to be 2.4. The library was based on the novel mutant SmNOX2 194H200K and was designed with a 5' Hind\\\ and 3' Nde\ restriction site. The library was digested with Hind\\\ and Nde\ and purified via Qiaquick PCR Purification Kit (Qiagen, Hilden, Germany). The mutated insert was ligated with a Hind\\\/Nde\ restricted and gel purified pMS470A8 vector. The ligation mixture contained 0.064 pmole of insert, 0.016 pmole of vector and 10U of T4 DNA ligase (Fermentas) and was kept at 22°C for two hours and 8°C for 1 hour. The ligation mixture was then heated to 65°C for 20 min and 3μί were transformed into

electrocompetent E. coli TOP10 F' cells. Transformants were picked into 60μί LB/Amp media in 384 well plates, grown overnight at 37°C and 60% of humidity and stored as 15% glycerol stocks at -80°C.

Cultivation of the expression library was done in 96 well plate format. Preculture plates with 150μΙ_ of LB/Amp media per well were inoculated from glycerol stock plates and cultivated at 37°C and 60% humidity for at least 12 hours. Main culture plates with V-shaped bottom contained 80μΙ_ of LB Amp media and were inoculated from the preculture plates. After 8 hours of growth at 37°C and 60% humidity SmNOX2 expression was induced by addition of 20μΙ_ of a 0.5mM IPTG solution in LB/Amp media. The plates were kept at 28°C and 60% humidity for 16 hours. Cells were harvested by 15 minutes of centrifugation at 2500g. Supernatant was decanted and the cell pellets were frozen at -20°C for at least two hours.

Example 6 demonstrates successful preparation of library of mutants from the novel SmNOX2 194H200K mutant enzyme. Example 7: Screening of SmNOX2 variants from a random mutagenesis library for enhanced NADPH/NADH oxidase activity

Screening assays were carried out in 96 well plates. After thawing the microtiter plates with frozen cell pellets cell lysis was accomplished by addition of 100μΙ_ lysis buffer (50 mM potassium phosphate buffer pH7, 1 mg/mL lysozyme), incubation for 10 minutes at room temperature and shaking with 1050 rpm on a Titramax 1000, and an additional 1 h incubation at 28° at 600rpm. Cell debris was separated by centrifugation at 2500g for 15 min at 4°C. The supernatant was diluted 1 +1 with 50mM potassium phosphate buffer pH 7 and used for screening assays.

Screening for enhanced NADPH/NADH oxidase activity ratio: 140μΙ_ of 50mM potassium phosphate buffer pH 7, were added to 10μΙ_ of diluted supernatant in two plates in parallel. Reactions were started by addition of 50μΙ_ of a 0.8mM NADH or NADPH solution. Initial rates of NADH and NADPH conversion were measured by detection of decrease in absorption at 340nm over three minutes. Activity with NADH and NADPH was compared for each well.

2800 clones were screened, 480 thereof were chosen for a re-screen and the best 40 thereof were measured in a re-re-screen. Cultivation and assay conditions for re-screen and re-re-screen were identical to the screening setup. In the re-re-screen for each variant four isolated colonies from streaks of the best 40 variants were cultivated and analyzed. From best variants plasmid DNA was isolated with Gene JetTM Plasmid Miniprep Kit (Fermentas, St. Leon-Roth, Germany) and sent for sequencing using primer pMSfw and pMSrv (sequences given in Example 2).

Example 7 describes the successful screening for NADH and NADPH oxidase activity of the enzymes from the library produced in Example 6. The best 40 enzymes are those with the closest NADPH/NADH oxidase activity ratio, and high NADPH and NADH oxidase activity.

Example 8: Screening of SmNOX2 variants from a random mutagenesis library for enhanced stability against N-methylpyrrolidon (NMP)

Screening assays were carried out in 96 well plates. After thawing the microtiter plates with frozen cell pellets cell lysis was accomplished by addition of 100μΙ_ lysis buffer (50 mM PPB, pH7, 1 mg/ml_ lysozyme), incubation for 10 minutes at room temperature and shaking with 1050 rpm on a Titramax 1000, and an additional 1 h incubation at 28°C at 600rpm. Cell debris was separated by centrifugation at 2500g for 15 min at 4°C. The supernatant was diluted 1 +1 with 50mM PPB, pH 7 and used for screening assays.

Screening for enhanced stability against NMP was performed as follows: In one plate 140μΙ_ of 20% NMP in 50mM PPB, pH 7, were added to 10μΙ_ of diluted supernatant. In a second plate 140μΙ_ of 50mM PPB, pH 7, without NMP were added to 10μΙ_ of diluted supernatant. The plates were shaken for 15 minutes at 20°C at 900rpm. After the incubation the NAD(P)H conversion was started by addition of 50μΙ_ of a 0.8mM NAD(P)H solution. Initial rates of NAD(P)H conversion were measured by detection of decrease in absorption at 340nm. Results of the two plates were compared for each well.

2800 clones were screened, 320 thereof were chosen for a re-screen and the best 20 thereof were measured in a re-re-screen. Cultivation and assay conditions for rescreen and re-re-screen were identical to the screening setup. In the re-re-screen for each variant four isolated colonies from streaks of the best 20 variants were cultivated and analyzed.

From best variants plasmid DNA was isolated with JetTM Plasmid Miniprep Kit (Fermentas, St. Leon-Roth, Germany) and sent for sequencing using primer pMSfw and pMSrv (sequences given in Example 2).

Example 8 describes the successful screening for stability against N- methylpyrrolidon (NMP) of the enzymes from the library produced in Example 6. The best 20 enzymes are the most stable in NMP and therefore expected to be most stable in an organic solvent in general.

Example 9: Cultivation of best SmNOX2 variants in 50mL shake flask cultures and measurement of activity with NADH and NADPH and of stability in NMP

Plasmid DNA isolated from the screened E. coli strains showing highest NADPH/NADH activity ratios or highest stability in NMP was transformed into BL21 -Gold (DE3) (Stratagene, La Jolla, CA, USA). Precultures of all resulting E. coli strains were cultivated in 5ml_ of LB/Amp-media containing 100mg/L of Ampicillin in plastic tubes at 37°C on a rotating wheel. For main cultures 50ml_ of LB/Amp-Medium in 300ml_ baffled flasks were inoculated to an OD of 0,05 and cultivated at 37°C and 130rpm. NOX production was induced by addition of 1 mM IPTG at an OD of 1 .1 - 1 .6. Cells were harvested after an overnight induction period at 25°C and 100rpm by centrifugation for 15 minutes at 3200rcf (Eppendorf centrifuge 581 OR) and 4°C. Cell pellets were frozen at -20°C. After thawing cell pellets were diluted in 50mM potassium phosphate buffer pH 7.0 to a final volume of 1 ml_. For cell breakage 400μΙ_ of this dilution were vortexed with a Turbomix top on a Vortex Genie 2 (Scientific Industries, Bohemia, NY, USA) with approximately 200mg of 0.3mm glass beads for 5 min at maximum speed. Another 600μΙ_ of 50mM potassium phosphate buffer pH 7.0 were added and cell free lysates were prepared by collecting the supernatant after centrifugation at 16100 rcf (Eppendorf Centrifuge 5415R) for 1 h.

The protein content was determined with the bichinonic acid protein assay (BCA) kit (Thermo Scientific, Waltham, MA, USA) using BSA as standard. Activity was assayed by initial rate measurement of NAD(P)H depletion at 340nm as described in Example 7 and 8 except that the final coenzyme concentration in the assay was 300μΜ instead of 200μΜ. Crude lysates were diluted 100 fold. Results for the two most interesting mutants, V194H/G200K/Y202C and V194H/G200K/Y202N, in comparison to mutant V194H/G200K concerning activity with NADH and NADPH are shown in Table 4. Results for the most interesting mutants regarding stability in NMP found in screening and mutant V194H/G200K are shown in Table 5 (NADH) and Table 6 (NADPH). Table 4: Activity with NADH and NADPH of best mutants from screening in comparison to starting point

Values are the average of two measurements; activity was measured at air-saturated oxygen level

Table 5: Activity after 15 min of incubation with and without 20%NMP for best mutants from screening in comparison to starting point, measurement with NADH

Values are the average of two measurements; activity was measured at air-saturation oxygen level

Table 6: Activity after 15 min of incubation with and without 20%NMP for best mutants from screening in comparison to starting point, measurement with NADPH

Values are the average of two measurements; activity was measured at air-saturation oxygen level Example 9 compares activity of mutant enzymes in the presence of NMP (an organic solvent). The results of Table 5 indicate that NADH oxidase activity of the V194H/G200K/E429K, V194H/G200K/K121 N/A421V and

V194H/G200K/F245Y/E367G mutants is particulary high in the presence of NMP. The results of Table 6 that these same 3 mutants have particularly high NADPH activity in the presence of NMP, as does the V194H/G200K/G363S mutant.

Example 10: Preparation of cell free extracts containing Sphingobium yanoikuyae short-chain dehydrogenase (SyADH)

SyADH was overexpressed from pEam_SyADH, a pEamTA vector carrying the SyADH gene (accession number EU427523, see Lavandera et al„

Organic Letters, 2008). Precultures of £. co// TOP10F' strains transformed with pEam_SyADH were cultivated in 50ml_ of LB media containing 100mg/L of Ampicillin (LB/Amp media) in baffled 300mL shake flasks at 37°C and 130 rpm overnight. For main culture 2x250mL of LB/Amp-Medium in 1 L baffled flasks were inoculated to an OD of 0,1 and cultivated at 37°C and 130rpm. SyADH production was induced by addition of 2mM IPTG at an OD of 1 .2. Cells were harvested after an overnight induction period at 25°C and 130rpm by centrifugation for 15 minutes at 5000rcf (Avanti J-20 XP, Beckman Coulter, Krefeld, Germany, rotor JA-10). Cell pellets from both flasks were pooled and diluted in 50mM potassium phosphate buffer pH 7.0 to a final volume of 25 mL. Cells were disrupted by passing them twice through a pre-cooled French press at a cell pressure of approximately 1300 bar. Cell free lysates were prepared by collecting the supernatant of centrifugation at 36000 rcf (rotor JA-25.50) for 45 minutes. SDS/PAGE gel electrophoresis (NuPAGE® Novex® 4-12% Bis-Tris Gels (1.0 mm) from Invitrogen (Carlsbad, CA, USA) together with a NuPAGE MOPS SDS Running Buffer for Bis-Tris Gels) of the cell free extracts showed strong protein bands migrating to the expected position at 28kDa. Cell free extracts were stored in aliquots at -20°C.

Example 10 demonstrates the successful preparation of cell free extracts containing Sphingobium yanoikuyae short-chain dehydrogenase (SyADH).

Example 1 1 : Production of R-ADH, S-ADH and SmNOX2 194H/200K (codon-pair optimized) £ coli strain TOP10 (Invitrogen, Carlsbad, CA, USA) was used for all cloning procedures. £ coli was also used for protein expression. For induction of gene expression L-arabinose was used at a final concentration of 0.02% (w/v).

The target gene according to SEQ ID No 10 (SmNOX2 194H/200K) was codon pair optimised according to a procedure described in WO08000632 resulting in SEQ ID No. 53 The genes of R-specific ADH of Lactobacillus brevis DSM 20054 (Gene accession no LBR544275) and S-specific ADH of Candida parapsilosis IFO 1396 (Gene accession no. AB010636) were codon-optimized by DNA2.0 Inc. (Menlo Park, CA94025 US) resulting in SEQ ID No 49 and SEQ ID No 51 , respectively coding for protein sequences in SEQ ID No 50 and SEQ ID No 52 respectively. attB sites were added to all genes upstream of the ribosomal binding site and downstream of the stop codon, respectively, to facilitate cloning using the Gateway technology (Invitrogen, Carlsbad, CA, USA). The synthetic genes were obtained from Geneart (Regensburg, Germany) (SmNOX2 194H/200K) and DNA2.0 Inc. (Menlo Park, CA94025 US) (S-ADH and R-ADH). The gene constructs were cloned into a Gateway adapted pBAD/A yc-HisC (Invitrogen, Carlsbad, CA, USA) derived expression vector using the Gateway technology (Invitrogen) via pDONRzeo (Invitrogen) as entry vector as described in the manufacturer's protocols (www.invitrogen.com) resulting in the three required pBAD-based expression vectors. Finally, chemically competent £. coli TOP10 cells were transformed with these expression vectors according to a standard procedure.

The resulting recombinant strains were cultivated in 200ml_ of complex media following standard procedures at 28°C and 180 rpm. Production of NOX and ADH was induced by addition of 0.02 % (w/v) L-arabinose. Cells were harvested by centrifugation and the supernatant was discarded. 1 ml of potassium phosphate buffer pH 7.5 was added to 0.5 g of wet cell pellet and cells were resuspended by vigorously vortexing. To achieve lysis, the cells were sonicated for 10 min according to a standard protocol. To remove cell debris, the lysates were centrifuged at 4 °C and 6,000 g for 20 min. The supernatants were transferred to a fresh tube and frozen at -20°C until further use.

Example 1 1 demonstrates the successful production of R-ADH, S- ADH and SmNOX2 194H/200K. Example 12: Application of SmNOX2 194H/200K for cofactor recycling in the production of acetophenone

The enantioselective oxidation of rac-1-phenylethanol to acetophenone was chosen as a further model reaction for an alcohol to ketone conversion. The NAD ⁺ -dependent alcohol dehydrogenase from Candida parapsilosis IFO 1396 (S-ADH) and the NADP ⁺ dependent R-specific alcohol dehydrogenase from Lactobacillus brevis were chosen as production enzymes in a coupled enzyme approach together with SmNOX2 194H/200K for cofactor regeneration.

Conversion experiments were carried out on 5ml scale capped glass vials and shaking at 240 rpm at 28°C on an IKA KS 130 basic shaker. The reaction mixtures contained 1 mM NAD(P) ⁺ and 50mM rac-1 -phenylethanol in 100mM potassium phosphate buffer, pH 7.5 in a total volume of 5ml_. 15ul of each ADH and SmNOX2 194H/200K were applied as cell free extracts prepared as described in Example 1 1. 500 uL samples were taken after 28 hours and the enzymatic conversion was stopped by the addition of 500 μΙ_ of acetonitrile. Subsequently, the stopped reaction samples were centrifuged by an Eppendorf Centrifuge 5415 R for 5 min at 4°C and 13,000 rpm. 800 μΙ_ of the supernatant were decanted for GC analysis. The substrate conversion was determined on an Agilent 5890 gas chromatograph equipped with a Gerstel PTV injector, an FID detector (250°C) and a Chiralsil-DEX CB column (25 m x 0.25 mm, 0.25 μηη film). Helium was used as carrier gas at constant pressure mode with a column pressure of 100 kPa. The following temperature program was used: 100°C for 5 min; 5°C/min to 200°C (total runtime: 25 min).

Table 7: Results of conversion experiments with S. mutans NOX2 194H/200K added for cofactor recycling in an oxidative ADH reaction

Conversion is calculated base on substrate decrease (product acetophenone is evaporating)

bTTN (total turnover number) was calculated as follows: substrate converted [mM]/1 mM cofactor applied

cee was calculated for the remaining enantiomer of 1 -phenylethanol

din these two experiments, additional SmNOX2 194H/200K (15ul) was added after 5 and 25 hours of reaction.

The results (shown in Table 7) confirm that conversion occurs with NAD ⁺ and NADP ⁺ to a high level and therefore that NADPH recycling with S. mutans NOX2 194H/200K was made possible.