PROCESS FOR REDUCING CARBON DIOXIDE TO METHANOL

Field of the invention

The invention relates to new processes and products useful in the reduction of carbon dioxide (CO2) to methanol.

Background to the invention

Accumulation of carbon dioxide in the environment (produced, for example, by combustion of fossil fuels) is a growing problem, and means for lowering carbon dioxide levels are desirable. Conversion of carbon dioxide into methanol is a promising approach, since it can not only recycle the greenhouse gas but also produce valuable chemicals. Today, methanol is one of the most important feedstock in the chemical industry. Most of the 32 million tons of annually produced methanol is used to manufacture a large variety of chemical products and materials, including basic chemicals such as formaldehyde, acetic acid, methyl ferf-butyl ether (MTBE), as well as various polymers, paints, adhesive, construction materials, etc. Methanol is also a feedstock for chloromethanes, methylamines, methyl metacrylate, and dimethyl terephthalate, among others.

A number of reduction processes (both enzymatic and non-enzymatic) for reducing carbon dioxide to methanol are described in the art.

The enzymatic reduction of carbon dioxide for the production of methanol has been studied extensively in recent years (US 644071 1 B1 ; R. Obert, B.C. Dave, J.. Am. Chem.l Soc, 1999. 121 (51 ) : 12192-12193; B. El-Zahab, D. Donnelly, P. Wang, Biotechnol. Bioeng., 2008. 99(3) : 508-514; Y. Lu, et al., Catalysis Today, 2006. 1 15(1 -4) : 263-268). The process described in the scientific and patent literature is based on the use of three enzymes: formate dehydrogenase (FDH) from Candida boidinii, formaldehyde dehydrogenase (FaldDH) from Pseudomonas putida or aldehyde dehydrogenase (AldDH) from Saccharomyces cerevisiae and alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae as catalysts, and NADH as hydrogen and electron donor. The NADH-mediated reduction of CO2 for production of methanol can be described as a multistep reaction process (Scheme 1):

NADH NAD NADH NAD NADH NAD

C0 ₂ ^= HCOOH ^= HCHO ^= CH ₃ OH

F _ate DH F _ald DH ADH

F _ate DH - formate dehydrogenase

F _a]d DH - formaldehyde dehydrogenase

ADH - alcohol dehydrogenase

Scheme 1. Enzymatic conversion of CO2 into methanol

Various procedures for carrying out the enzyme-catalyzed reactions have been described, including those using free or immobilized enzymes and different procedures for regeneration and reuse of NADH.

Electrochemical and bioelectrochemical reduction methods have also been described. For example, Kuwabata and coworkers (Kuwabata, S et al, J. Am. Chem. Soc, 1994. 1 16: 5431 -5443) describe the bioelectrochemical conversion of CO2 to MeOH using two enzymes, a formate dehydrogenase (FDH) from Pseudomonas oxalyticus and methanol dehydrogenase (MDH) from Methylophilus methylotrophus, and either pyrolloquinoline quinone (PQQ) or methyl viologen (MV) as electron mediators in an electrolytic process. Despite this however, there remains a need for new, simpler and more cost-effective means for carrying out the reduction process.

Summary of the invention

The present inventors have studied each step in the reported biocatalytic processes for conversion of CO2 to methanol using three enzymes, and have observed that none of the enzymes reported to be used for the reduction of formate to formaldehyde (i.e. FaldDH from Pseudomonas putida (EC 1 .2.1 .46) and AldDH from Saccharomyces cerevisiae (EC1 .2.1 .3)) showed this catalytic activity.

Further, the inventors have found that FDH enzyme from Candida boidinii (both the commercial enzyme and a recombinant enzyme produced by the inventors), exhibits two reduction activities: (1 ) the reduction of carbon dioxide to produce formate, and (2) the conversion of formate into formaldehyde.

Thus the inventors have found that reduction of carbon dioxide to methanol is catalysed by only two enzymes: a formate dehydrogenase (FDH) ; and an alcohol dehydrogenase (ADH) or methanol dehydrogenase (MDH). The finding has been proven experimentally by carrying out the reduction reaction using only these two enzymes and observing that methanol is formed at the same rate as with a three-enzyme system . This finding will allow the development of a simpler process for methanol production from CO2, and is also much more economical, since FaldDH is the most expensive enzyme in the conventional "three enzyme" process.

Accordingly, the present invention in one aspect provides:

an enzymatic process for reducing CO2 to methanol, the process comprising:

(a) exposing CO2 to at least one formate dehydrogenase (FDH) enzyme; and

(b) exposing formaldehyde produced from CO2 in (a) to at least one alcohol dehydrogenase (ADH) or methanol dehydrogenase (MDH) enzyme;

wherein the electron donor for reduction of CO2 to formaldehyde is NADH or NADPH ;

and

wherein the amount of FDH enzyme in (a) is greater than the amount of any formaldehyde dehydrogenase (FaldDH) and/or aldehyde dehydrogenase (AldDH) enzyme which may be present or wherein FaldDH and/or AldDH enzyme is substantially absent from (a).

The invention further provides:

a non-electrolytic enzymatic process for reducing CO2 to methanol, the process comprising:

(a) exposing CO2 to at least one formate dehydrogenase (FDH) enzyme; and

(b) exposing formaldehyde produced from CO2 in (a) to at least one alcohol dehydrogenase (ADH) or methanol dehydrogenase (MDH) enzyme;

wherein the amount of FDH enzyme in (a) is greater than the amount of any formaldehyde dehydrogenase (FaldDH) and/or aldehyde dehydrogenase (AldDH) enzyme which may be present or wherein FaldDH and/or AldDH enzyme is substantially absent from (a).

Further provided is:

- the use of a FDH enzyme to catalyse reduction of formate to formaldehyde; - the use of a dehydrogenase enzyme combination comprising at least one FDH enzyme and at least one ADH and/or MDH enzyme, to catalyse reduction of CO2 to methanol, wherein the amount of FDH enzyme in the combination is greater than the amount of FaldDH and/or AldDH enzyme which may be present or wherein FaldDH and/or AldDH enzyme is substantially absent;

- a kit comprising FDH enzyme and ADH and/or MDH enzyme for simultaneous or sequential use in the reduction of CO2 to methanol by a process of the invention; and

- a matrix comprising a dehydrogenase enzyme combination as defined herein.

Description of the Figures

Fig. 1a. SDS-polyacrylamide gel electrophoresis of dehydrogenases. M, standard molecular weight markers; 1 , formate dehydrogenase from C. boidinii; 2, alcohol dehydrogenase from S. cerevisiae; 3, alcohol dehydrogenase from equine liver; 4, aldehyde dehydrogenase from S.cerevisiae; 5, formaldehyde dehydrogenase from Ps. putida; 6, glucose dehydrogenase from Pseudomonas sp.

Fig 1b. SDS-polyacrylamide gel electrophoresis of overexpressed formate dehydrogenase.

Fig. 2. CO2 reduction to formic acid catalyzed by formate dehydrogenase from Candida boidinii. Reaction conditions: 5 ml total volume, 3 mM NADH, 10 mM glucose or lactic acid, 7 h room temperature, 5.1 mg FDH, 1 .1 mg glucose dehydrogenase (GDH) or 1 .32 mg lactate dehydrogenase (LDH), GC/MS analysis of the product.

Fig. 3. Effect of carbonic anhydrase (CA) and cofactor regeneration on CO2 reduction catalyzed by formate dehydrogenase from Candida boidinii. Reaction conditions: 2 ml total volume, phosphate buffer 0.1 M pH 7.3, 1 mg/ml CA, 1 mg/ml FDH, 3 mM NADH, 10 mM glucose, 0.185 mg/ml GDH, HPLC analysis of formic acid. For each set of bars showing results for a particular reaction time, the bars from left to right represent results for: no regeneration (CA) ; no regeneration ; regeneration (GDH, CA) ; and regeneration (GDH).

Fig. 4. Effect of cofactor (a) and FDH concentration (b) on production of formic acid. Reaction conditions: (a) 2 ml total volume, phosphate buffer 0.1 M pH 7.3, 1 mg/ml CA, 1 mg/ml FDH ; (b) 2 ml total volume, phosphate buffer 0.1 M pH 7.3, 1 mg/ml CA, 3 mM NADH, HPLC analysis of formic acid. For each set of bars in Fig 4(a) showing results for a particular reaction period, the bars from left to right represent results for: no regeneration (CA, 3mM NADH) ; and no regeneration (CA, 0.4mM NADH). For each set of bars in Fig 4(b) showing results for a particular reaction time, the bars from left to right represent results for: no regeneration (CA, 0.06ml FDH) ; and no regeneration (CA, 0.1 ml FDH).

Fig. 5. Effect of substrate concentration on CO2 reduction catalysed by FDH from Candida boidinii. The yield of formic acid was calculated based on the amount of glucose used for cofactor regeneration. Reaction conditions: 1 ml total volume, PBS buffer 0.1 M pH 7.0, 1 mg FDH, 3 mM NADH, 10 mM glucose, 0.13 mg GDH, HPLC analysis of formic acid. For each set of bars showing results for a particular reaction time, the bars from left to right represent results for: 24mM CO2; 30mM

Fig. 6. Response surface for formic acid production at (a) 0.7, (b) 1 .55 and (c) 2.4 mM NADH.

Fig. 7. Effect of glucose concentration on CO2 reduction catalysed by FDH from Candida boidinii. Reaction conditions: 1 ml total volume, phosphate buffer 0.1 M pH 8.0 (CO2 saturated), 0.7 mM NADH, 0.48 mg CA, 0.02 mg GDH, 0.61 mg FDH, HPLC analysis of formic acid. For each set of bars showing results for a particular glucose concentration, the bars from left to right represent results for: 2h; 21 h; and 48h.

Fig. 8. Effect of carbonic anhydrase concentration on CO2 reduction catalysed by FDH . Reaction conditions: 1 ml total volume, phosphate buffer 0.1 M pH 8.0 (CO2 saturated), 0.7 mM NADH, 10 mM glucose, 0.02 mg GDH, 0.61 mg FDH, HPLC analysis of formic acid. For each set of bars showing results for a particular carbonic anhydrase/C02 (%w/w), the bars from left to right represent results for: 2h; 21 h; and 48h.

Fig. 9. NaHC03 as potential substrate for formic acid production. Reaction conditions: 1 ml total volume, 10 mM NaHCOs, 0.7 mM NADH, 10 mM glucose, 0.02 mg GDH, 0.61 mg FDH, HPLC analysis of formic acid. For each set of bars showing results for a particular substrate, the bars from left to right represent results for: 2h; 21 h; and 48h.

Fig. 10. Effect of NaHC03 concentration on formic acid production. Reaction conditions: 1 ml total volume, different concentrations of NaHCO3, 0.7 mM NADH, 10 mM glucose, 0.02 mg GDH, 0.61 mg FDH, HPLC analysis of formic acid. For each set of bars showing results for a particular NaHC03 concentration, the bars from left to right represent results for: 2h; and 21 h.

Fig. 1 1. Reduction of formic acid catalysed by commercial FDH with/without cofactor regeneration. Reaction conditions: pH 7.0, 30 °C, 5 mM HCOOH, 3 mM NADH, 10 mM glucose, 0.142 mg GDH, 1 mg FDH, 1 ml total reaction volume, HPLC analysis of formic acid

Fig. 12. Formaldehyde reduction to methanol catalysed by alcohol dehydrogenase from S. cerevisiae. Reaction conditions: PBS buffer 0.1 M pH 7.5 (0.5 mM NaCL), 5 ml total volume, 100 mM formaldehyde, 3 mM NADH, 10 mM glucose or lactic acid, 15 h at room temperature, 2.8 mg ADH, 0.44 mg GDH or 1 mg LDH, GC/MS analysis of the product.

Fig. 13. Effect of cofactor regeneration on the enzymatic reduction of formaldehyde. Reaction conditions: 1 ml reaction volume, 30 °C, 3 mM NADH, 0.41 mg ADH, 60 mM methanol, PBS buffer 0.1 M (0.15 M NaCI) pH 7.0 (cofactor generation: 10 mM glucose, 0.12 mg GDH).

Fig. 14. Effect of methanol concentration on ADH efficiency for the reduction of formaldehyde. Reaction conditions: 1 ml total reaction volume, 134 mM formaldehyde, 3 mM NADH, 10 mM glucose, 0.12 mg GDH, 0.41 mg ADH, PBS buffer 0.1 M (0.1 5 mM NaCI) pH 7.0 and 30 °C. For each set of bars showing results for a particular reaction time, the bars from left to right represent results for: 50mM MeOH ; 55mM MeOH ; 60mM MeOH ; 70mM MeOH ; 100mM MeOH ; and 150mM MeOH.

Fig. 15. Methanol production from CO2 catalyzed by FDH (commercial) and ADH, with cofactor regeneration. Reaction conditions: 5 ml total reaction volume, CO2 saturated PBS buffer (0.15 M NaCI) pH 7.5, 3 mM NADH, 10 mM glucose, 2.5 mg ADH, 0.1 mg GDH, 5.1 mg FDH.

Description of the Sequences

SEQ ID NO:1 - amino acid sequence of a Candida boidinii FDH enzyme (UniProt 013437).

SEQ ID NO:2 - nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 , as found in Candida boidinii.

SEQ ID NO:3 - nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 and optimised for codon usage in E. coli and insertion of restriction sites.

SEQ ID NO: 4 - amino acid sequence of ADH1 of S. cerevisiae strain ATCC 204508 (UniProt P00330)

SEQ D NO: 5 - amino acid sequence of ADH2 of S. cerevisiae strain ATCC 204508 (UniProt P00331 ) SEQ ID NO: 6 - amino acid sequence of MDH of B. methanolicus (UniProt P31005)

SEQ ID NO: 7 - amino acid sequence of AldDH of S. cerevisiae (SwissProt P541 1 5)

SEQ ID NO: 8 - amino acid sequence of AldDH of S. cerevisiae (SwissProt P22281 )

SEQ ID NO: 9 - amino acid sequence of FaldDH of P putida (UniProt P46154)

Detailed description of the invention

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19- 854287-9) ; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-021 82-9) ; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1 995 (ISBN 1 -56081 -569-8).

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference in to the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.

This disclosure references various internet sites and sequence database entries (e.g. UniProtKB/Swiss-Prot, UniProtKB/TrEMBL, GenBank). The contents of the referenced internet sites and sequence database entries are incorporated herein by reference as of 18 December 2013. Thus: accession numbers for the GenBank database refer to database release no. 199.0; accession numbers for the UniProtKB/Swiss-Prot database refer to database release no. 2013_12; and accession numbers for the UniProtKB/TrEMBL database refer to database release no. 2013_12. References to EC (Enzyme Commission) numbers and CAS numbers are also as of 18 December 2013.

As described above, the present inventors have surprisingly found that a C. boidnii formate dehydrogenase enzyme (FDH) has activity which catalyses not only reduction of carbon dioxide (CO2) to formate, but also reduction of formate to formaldehyde. The inventors have therefore shown that a formate dehydrogenase (FDH) enzyme can be used to catalyse reduction of formate to formaldehyde. In one aspect therefore, the invention relates to a process for reducing formate to formaldehyde, using a formate dehydrogenase (FDH) enzyme as described herein.

Use of FDH in this way can occur as a stage in a new reduction process for reducing CO2 to methanol. FDH enzyme can be used to catalyse reduction of CO2 to formate, and reduction of formate to formaldehyde, and an alcohol dehydrogenase (ADH) or methanol dehydrogenase (MDH) enzyme can be used to catalyse reduction of the formaldehyde to methanol. Unlike the biocatalytic processes of the prior art, the present process does not require the presence of formaldehyde dehydrogenase enzyme (FaldDH) or aldehyde dehydrogenase (AldDH) to reduce formate. In the present processes, these enzymes may be substantially absent, or present in a lower amount than FDH enzyme. CO2 is reduced to formaldehyde by activity of FDH enzyme only. Thus the inventors have shown that the conventional "three enzyme" biocatalytic processes for serial reduction of CO2 to methanol can be carried out using only two enzymes: FDH ; and ADH or MDH.

The invention is thus also concerned with a process for reducing CO2 to methanol. In the process CO2 is exposed to at least one FDH enzyme (and reduced to formaldehyde via formate) (step (a)). The formaldehyde produced by the activity of FDH is exposed to at least one ADH or MDH enzyme to produce methanol (step (b)).

As explained above, reduction of CO2 to formaldehyde does not require FaldDH or AldDH enzyme. Accordingly, FaldDH and/or AldDH may be substantially absent from this reduction stage, or if present, may be present in a lower amount than FDH, as described herein.

Reduction of CO? to methanol

As used herein, reduction of CO2 to methanol describes a reduction process in which CO2 is reduced to formate (HCOOH), formate is reduced to formaldehyde (CH2O), and formaldehyde is reduced to methanol (CH3OH). The process, which may be referred to as a serial reduction, may be represented as shown below:

C0 ₂ → HCOOH→ CH2O→ CH3OH

For the avoidance of doubt, reference to a serial reduction does not necessarily imply separation in time of the three reduction reactions shown. Two or more of these reduction reactions (e.g. steps (a) and (b) above) may be occurring simultaneously in the overall conversion process.

Formate as used herein may be represented by formula HCOOH, and may alternatively be referred to as formic acid or methanoic acid. Formic acid may dissociate in solution. Therefore formate may also refer to the dissociated form, the formate ion, HCOO-.

Formaldehyde as used herein may be represented by formula CH2O, and may alternatively be referred to as formaline.

Methanol used herein may be represented by formula CH3OH, and may alternatively be referred to, for example, as methyl alcohol.

In addition to enzyme and substrate, the present reduction reactions also require an electron donor which can act as a source of reducing power.

An electron donor comprises a species which is able to provide (donate) electrons for the reduction reaction (also referred to as a reducing agent). Any suitable electron donor may be used.

In one aspect, the electron donor used in reduction by FDH according to the invention (reduction of CO2 to formate and/or formate to formaldehyde) comprises NADH or NADPH as described herein. NADH is the reduced form of the coenzyme NAD ⁺ (nicotinamide adenine dinucleotide), which itself acts as an electron acceptor (oxidising agent). NADPH is the reduced form of the coenzyme NADP ⁺ (nicotinamide adenine dinucleotide phosphate), which itself acts as an electron acceptor (oxidising agent). NADH and NADPH are available commercially from, e.g. Sigma Aldrich.

NADH or NADPH may also be the electron donor for reduction of formaldehyde to methanol.

Non-electrolytic processes

One or more steps in the reduction processes described herein may be non-electrolytic. In one aspect, the entire CO2 to methanol reduction process may be non-electrolytic. An electrolytic reduction process generates electrons electrochemically, typically by electrolysis. Such a process generally requires, for example, electrodes and electrical current as well as a suitable electrolyte. A non-electrolytic reduction process thus refers to a process which does not comprise electrolysis and which uses a non-electrochemical method to provide or regenerate electrons. Electron providers for use in the present process are described further herein.

Exposure of substrate and enzyme

The present process comprises exposing CO2 to one or more FDH enzymes to produce formaldehyde, and exposing the formaldehyde product to one or more ADH or MDH enzymes.

Exposing an enzyme substrate (e.g. CO2 or formaldehyde) to an enzyme generally means that the enzyme substrate is brought into close enough contact with the enzyme for the enzyme catalysed reaction with the substrate to take place under the given reaction conditions. Without wishing to be bound by theory it is believed that in the present process, exposure of CO2 to FDH will produce formate, exposure of formate to FDH will produce formaldehyde, and exposure of the formaldehyde to ADH or MDH will produce methanol. Exposure of substrate to enzyme typically may comprise contact between substrate and enzyme.

Exposure of substrate to enzyme can occur by any suitable means, e.g. by admixing of substrate and enzyme. In one example, CO2 may be provided in a C02-saturated buffer. CO2 may be bubbled through a solution or an immobilisation matrix as described herein, or may be provided as a solid.

In one example, CO2 may be provided in the process in the form of another substance which will be converted to CO2 which in turn will be exposed to FDH enzyme.

Enzymes

Dehydrogenase enzymes

The present process makes use of a number of dehydrogenase enzymes. A dehydrogenase enzyme generally has activity which catalyzes the removal of hydrogen or electrons from a substrate and the transfer of the hydrogen/electrons to an acceptor in an oxidation-reduction reaction. A dehydrogenase enzyme may be an oxidoreductase enzyme. Oxidoreductases have activity which catalyzes the transfer of electrons from one molecule (an electron donor) to another molecule (an electron acceptor ) and are generally classified in EC 1 in the EC number classification of enzymes. Oxidoreductases catalyze reactions similar to the following, A- + B→ A + B- where A is the reductant (electron donor) and B is the oxidant (electron acceptor), and may be oxidases or dehydrogenases. Dehydrogenases usually use NADP ⁺ , NAD ⁺ or a flavin enzyme as cofactor, which can act as electron acceptors (e.g. NAD ⁺ or NADP ⁺ ) or donors (e.g. NADH or NADPH) in the oxidoreductase reaction.

An enzyme which uses NAD ⁺ /NADH or NADP ⁺ /NADPH as cofactor for accepting or donating electrons may be referred to herein as a NAD-dependent or NADP-dependent enzyme.

Formate dehydrogenase enzyme (FDH)

In step (a) of the present process, formate dehydrogenase (FDH) enzyme is exposed to CO2.

FDH enzymes generally have activity which catalyses oxidation of formate (HCOO) to CO2, donating the electrons to a second substrate. An FDH for use herein also has activity which catalyses the reverse reaction, reducing CO2 to formate, and further catalyses reduction of formate to formaldehyde.

In one aspect, an FDH enzyme for use herein may comprise a NAD-dependent or NADP-dependent FDH enzyme. Examples of FDH enzymes include those for which the second substrate is NAD ⁺ (formate:NAD+ oxidoreductases) in class EC 1 .2.1 .2, and those for which the second substrate is a cytochrome (formate:ferricytochrome-b1 oxidoreductases) in class EC 1 .2.2.1 . Other FDH enzymes include those in EC 1 .2.1 .43 (NADP+-dependent FDH) and EC1 .2.2.3 (formate dehydrogenase (cytochrome c- 553)). Examples of NAD-independent FDH enzymes include those in EC 1 .2.7.5 and EC 1 .2.99.6 with molibden or wolfram prosthetic groups,

In one aspect, an FDH enzyme for use herein comprises a formate: NAD+ oxidoreductase, in EC 1 .2.1 .2 (CAS registry No. 9028-85-7).

FDH oxidative activity in oxidising formate to CO2, or reduction activity in reducing C02 to formate or formate to formaldehyde, may be assayed using any suitable assay.

For example, oxidation activity may be determined by incubating enzyme, NAD ⁺ and formate substrate in a suitable buffer (e.g. phosphate buffer) at a suitable pH (e.g. pH 7.0) and temperature (e.g. 37Ό for a suitable time (e.g. 5 minutes), and monitoring formation of NADH spectrophotometrically. Enzyme activity may be expressed in μιηοΙΝΑΟΗ/ιτιίη. Typically, enzyme activity may be determined from the initial reaction rate. An assay such as that used in the present Examples may be used.

Similarly, FDH reduction activity in reducing CO2 may be determined by incubating enzyme, NADH and C02-saturated buffer (e.g. PBS) under anaerobic conditions, at a suitable pH (e.g. pH7 and temperature (e.g. 35 °C), for a suitable time (e.g. 5 minutes), and monitoring consumption of NADH spectrophotometrically. Enzyme activity may be expressed in molNADH/min. An assay such as that used in the present Examples may be used.

Another assay for determining FDH reduction of CO2 to formate comprises incubating suitable concentrations of FDH, NADH and C02-saturated buffer (e.g. PBS), together with a suitable means for regenerating NADH (e.g glucose and GDH or lactate and LDH) under anaerobic conditions at a suitable pH (e.g. pH7.5) and temperature (e.g. 30°C) for a suitable time (e.g. up to 48h). Samples are removed anaerobically at given time intervals, and FDH enzyme inactivated, e.g. by addition of HCI. The concentration of formic acid in the samples may be determined by GC-MS and/or H PLC (e.g. as described in the present Examples), and enzyme activity expressed as rate of production of formic acid. A suitable method is described in the present Examples.

Reduction activity of FDH in reducing formate to formaldehyde may be determined by an assay which comprises incubating suitable concentrations of FDH , NADH, formate and buffer (e.g. phosphate buffer) together with a suitable means for regenerating NADH (e.g glucose and GDH or lactate and LDH) under anaerobic conditions at a suitable pH (e.g. pH7.3) and temperature (e.g. 30 Ό) for a suitable time (e.g. up to 29h). Samples are removed anaerobically at given time intervals. Typically the enzyme is inactivated, for example by reducing the pH. The samples can be analysed to determine concentration of formic acid (e.g. by HPPLC as described herein) and concentration of formaldehyde (e.g. by the Purpald assay, as described herein). Enzyme activity can be expressed as rate of consumption of formic acid and rate of production of formaldehyde. A suitable method is described in the present Examples.

An assay described above may be carried out in a similar way using another electron donor as appropriate, e.g. NAD(P)+/NADPH.

An FDH for use herein may be derived from any suitable species, such as any suitable microorganism, for example, a yeast or bacterial strain. Examples of such enzymes are available in the BRENDA database (www.brenda-enzymes.orQ). Examples of FDH enzymes in EC 1 .2.1 .2 include enzymes having an amino acid sequence as in UniProtKB/SwissProt Accession Nos P33160, 008375, P07658, B6VPZ9, Q845T0, Q0891 1 or 013437, or functional variants thereof.

In one aspect an FDH as referred to herein comprises a NAD-dependent FDH of a Candida species, for example of C. boidinii (UniProtKB/SwissProt 013437), or a functional variant thereof (e.g. a species homolog). A suitable FDH may comprise an amino acid sequence as in UniProt 013437, or as in SEQ ID NO:1 herein or a functional variant thereof. An FDH may comprise an amino acid sequence encoded by SEQ ID NO:2 or SEQ ID NO: 3 or a functional variant thereof. Sequence variants and species homologs are as described elsewhere herein. FDH may be the FDH used in the present Examples.

Suitable FDH enzyme may be obtained from naturally expressing cells, or may be expressed recombinantly in a suitable host strain, e.g. E. coli, using methods known in the art, or described elsewhere herein. Enzyme may also be commercially available. For example, C. boidinii FDH is available from Sigma-Aldrich Inc.

Alcohol dehydrogenase enzymes (ADH) and methanol dehydrogenase enzymes (MDH)

Reduction of formaldehyde to methanol in the present process comprises exposing an alcohol dehydrogenase (ADH) or methanol dehydrogenase (MDH) enzyme to formaldehyde.

Alcohol dehydrogenase (ADH) enzyme

ADH enzymes generally have activity which catalyses oxidation of an alcohol to an aldehyde or ketone. An ADH for use herein also catalyses the reverse reduction reaction, reducing aldehyde or ketone to alcohol. In particular, an ADH for use herein has activity which catalyses reduction of formaldehyde to methanol.

In one aspect, an ADH enzyme for use herein may comprise a NAD-dependent or NADP-dependent ADH enzyme.

ADH reduction activity in reducing formaldehyde may be determined using any suitable assay. For example, reduction activity may be determined by incubating enzyme, NADH and formaldehyde in a suitable buffer (e.g. PBS) under anaerobic conditions, at a suitable pH (e.g. pH7.5) and temperature (e.g. 37°C), for a suitable time (e.g. 5 minutes), and monitoring consumption of NADH spectrophotometrically at 340 nm . Enzyme activity may be expressed in pmolNADH/min. An assay such as that used in the present Examples may be used.

Another assay for determining ADH reduction of formaldehyde comprises incubating suitable concentrations of ADH, NADH, formaldehyde and buffer (e.g. PBS), together with a suitable means for regenerating NADH (e.g glucose and GDH or lactate and LDH) under anaerobic conditions at a suitable pH (e.g. pH7.5) and temperature (e.g. room temperature) for a suitable time (e.g. up to 15h). Samples can be removed anaerobically at given time intervals, and ADH enzyme inactivated, e.g. by addition of HCI. The concentration of methanol in the samples may be determined by GC-MS (e.g. as described in the present Examples), and enzyme activity expressed as rate of production of methanol. A suitable method is described in the present Examples.

An assay described above may be carried out in a similar way using another electron donor as appropriate, e.g NAD(P)+/NADPH.

An ADH for use herein may use NAD ⁺ /NADH as a cofactor for accepting/donating electrons (a NAD- dependent ADH or alcohol:NAD ⁺ oxidoreductase). Such enzymes include, for example, those in EC 1 .1 .1 .1 (CAS registry no. 9031 -72-5). Other ADH enzymes include those in class EC 1 .1 .2.8 (cytochrome C dependent) and those in EC 1 .1 .2.B3 (PQQ dependent). An ADH for use in the invention may be derived from any suitable species, such as any suitable microorganism, for example, a yeast or bacterial strain. Examples of such enzymes are available in the BRENDA database (www.brenda-enzymes.orQ).

Examples of ADH enzymes in EC 1 .1 .1 .1 include enzymes having an amino acid sequence as in GenBank Accession No AAA3441 1 , UniProtKB/SwissProt P00330 or UniProtKB/SwissProt P00331 or functional variants thereof.

In one aspect an ADH for use herein comprises a NAD-dependent ADH of a Saccharomyces species, for example S. cerevisiae. Examples include an enzyme having an amino acid sequence as in UniProtKB/SwissProt P00330 (ADH1 of S. cerevisiae strain ATCC 204508) or UniProtKB/SwissProt P00331 (ADH2 of S. cerevisiae strain ATCC 204508)), or functional variants (e.g. species homolog) thereof. A suitable ADH may comprise an amino acid sequence as in UniProtKB/SwissProt P00330 or P00331 , or as in SEQ ID NO:4 or 5 herein or a functional variant thereof. Sequence variants and species homologs are described elsewhere herein. ADH may be the ADH used in the present Examples.

Suitable enzyme may be obtained from a naturally expressing strain, or may be expressed recombinantly in a suitable host strain, e.g. E. coli, using methods known in the art, or described elsewhere herein. Enzyme may also be commercially available. For example, S. cerevisiae ADH is available from Sigma-Aldrich Inc.

Methanol dehydrogenase (MDH) enzyme

MDH enzymes generally have activity which catalyses oxidation of methanol to formaldehyde. An MDH for use herein also catalyses the reverse reduction reaction, reducing formaldehyde to methanol.

In one aspect, an MDH enzyme for use herein may comprise a NAD-dependent or NADP-dependent MDH enzyme.

MDH activity in reducing formaldehyde may be determined using any suitable assay. For example, an assay for reduction activity of ADH described herein may be used, substituting MDH for ADH.

An MDH for use herein may use NAD ⁺ /NADH as a cofactor for accepting/donating electrons (a NAD- dependent MDH or methanol:NAD ⁺ oxidoreductase). Examples of such enzymes may be found for example in EC 1 .1 .1 .1 , EC 1 .1 .99.37, or EC 1 .1 .1 .244 (CAS registry no. 74506-37-9). MDH enzymes may also include those in EC 1 .1 .2. B2 (NAD/NADP-dependent) and in EC 1 .1 .2.7 (methanol dehydrogenase quinohemoprotein dependent).

An MDH for use in the invention may be derived from any suitable species, such as any suitable microorganism, for example, a yeast or bacterial strain. Examples of such enzymes are available in the BREN DA database (www.brenda-enzymes.orQ). Examples of MDH in EC 1 .1 .99.37 include enzymes having an amino acid sequence as in UniProtKB/SwissProt Accession No A42952, GenBank Accession No. EIJ77618, or functional variants thereof.

In one aspect an MDH as referred to herein comprises a NAD-dependent MDH of a Bacillus species, for example B. methanolicus (UniProtKB/SwissProt P31005) or a functional variant (e.g. species homolog) thereof. A suitable MDH may comprise an amino acid sequence as in UniProtKB/SwissProt P31 005, or as in SEQ ID NO:6 herein or a functional variant thereof (EC 1 .1 .1 .244). Sequence variants and species homologs are described elsewhere herein.

In one embodiment, an MDH for use in the invention does not comprise an MDH which uses PQQ as a co-enzyme. Examples of such enzymes include those in EC 1 .1 .2.7 (ethanokcytochrome c oxidoreductase). One specific example of this class of enzymes is the Methylophilus methylotophus MDH, UniProtKB/SwissProt Accession number P38539 .

Suitable MDH enzyme may be obtained from a naturally expressing strain, or may be expressed recombinantly in a suitable host strain, e.g. E. coli, using methods known in the art, or described elsewhere herein.

Aldehyde dehydrogenase enzymes (AldDH) and formaldehyde dehydrogenase (FaldDH) enzymes

According to the invention, reduction of formate to formaldehyde is catalysed by FDH. Unlike the prior art processes therefore, the present processes do not require the presence of FaldDH or AldDH enzyme.

Aldehyde dehydrogenase (AldDH) enzyme

AldDH enzymes generally have activity that catalyses the oxidation of aldehyde. An AldDH may for example use NAD ⁺ or NADP ⁺ as an electron acceptor. Examples of NAD-dependent and NAD(P)- dependent enzymes include those in in class EC 1 .2.1 .3 (aldehyde: NAD+ oxidoreductases or NAD- dependent aldehyde dehydrogenases; CAS registry no 9028-86-8) or those in class EC 1 .2.1 .5 (CAS no. 9028-88-0; aldehyde: NAD(P)+ oxidoreductase). For the purposes herein, AldDH enzyme in particular has activity which catalyses oxidation of formaldehyde.

AldDH activity in oxidising (form)aldehyde may be determined in any suitable assay. For example, oxidation activity may be determined by incubating enzyme, NAD ⁺ and (form)aldehyde substrate in a suitable buffer (e.g. PBS) at a suitable pH (e.g. pH 7.5) and temperature (e.g. 25 °C) for a suitable time (e.g. 5 minutes), and monitoring formation of NADH spectrophotometrically. Enzyme activity may be expressed in μιηοΙΝΑΟΗ/ιτιίη. Typically, enzyme activity may be determined from the initial reaction rate. An assay such as that used in the present Examples may be used.

In one aspect, AldDH as referred to herein does not have activity which catalyses the reduction of formate to formaldehyde. Typically, such an enzyme has substantially no detectable activity in an assay for such reduction activity. Activity which catalyses reduction of formate to formaldehyde may be assayed in any suitable way. For example, an assay may comprise incubating enzyme, NADH and formate substrate in a suitable buffer (e.g. PBS) at a suitable pH (e.g. pH 7.5) and temperature (e.g. 25°C) for a suitable time (e.g. 5 minutes), and monitoring consumption of NADH spectrophotometrically. Enzyme activity may be expressed in μιηοΙΝΑΟΗ/ιτιίη. Typically, enzyme activity may be determined from the initial reaction rate. An assay such as that used in the present Examples may be used.

An assay described above may be carried out in a similar way using another electron donor as appropriate, e.g. NAD(P)+/NADPH.

AldDH may be derived from any suitable species, such as any suitable microorganism, for example, a yeast or bacterial strain. Examples of such enzymes are available in the BRENDA database (www.brenda-enzymes.orQ). Examples of AldDH enzymes in class EC 1 .2.1 .3 include enzymes having an amino acid sequence as in UniProtKB/SwissProt Accession Nos P76217, P05091 , P30837, 035945, P47738, Q9CZS1 , Q9DBF1 , Q9FPK6, P1 1 884, P51 650, Q4F895, Q402C7, or in UniProtKB/TrEMBL (provisional) A6T782.

In one aspect AldDH as referred to herein comprises a NAD-dependent AldDH of Saccharomyces species, for example S. cerevisiae (e.g. UniProtKB/SwissProt P541 15 and P22281 ), or a functional variant (e.g. species homolog) thereof. The P541 1 5 and P22281 enzymes are in class EC 1 .2.1 .5. An AldDH may comprise an amino acid sequence as in UniProtKB/SwissProt P541 15 (SEQ ID NO:7), or as in UniProtKB/SwissProt P22281 (SEQ ID NO: 8) herein or a functional variant thereof. Sequence variants and species homologs are described elsewhere herein. AldDH may be the AldDH tested in the present Examples.

Suitable AldDH enzyme may be obtained from a naturally expressing strain, or may be expressed recombinantly in a suitable host strain, e.g. E. coli, using methods known in the art, or described elsewhere herein. Enzyme may also be commercially available. For example, S. cerevisiae AldDH is available from Sigma Aldrich Inc.

Formaldehyde dehydrogenase enzyme (FaldDH)

FaldDH enzymes generally have activity which catalyses the oxidation of formaldehyde to formate. A FaldDH as referred to herein may for example use NAD ⁺ or NADP ⁺ as an electron acceptor. Examples of NAD-dependent enzymes include those in class EC 1 .2.1 .46 (formaldehyde: NAD+ oxidoreductases or NAD-dependent formaldehyde dehydrogenases; CAS registry no 9028-84-6). Suitable enzymes may also be included in class EC 1 .1 .1 .284 (S-(hydroxymethyl)glutathione:NAD+ oxidoreductase or S-(hydroxymethyl)glutathione dehydrogenase; CAS registry no. 9028-84-6).

FaldDH activity in oxidising formaldehyde may be determined in any suitable assay. For example, oxidation activity may be determined by incubating enzyme, NAD ⁺ and formaldehyde substrate in a suitable buffer (e.g. PBS) at a suitable pH (e.g. pH 7.5) and temperature (e.g. 37°C) for a suitable time (e.g. 5 minutes), and monitoring formation of NADH spectrophotometrically. Enzyme activity may be expressed in μιηοΙΝΑΟΗ/ιτιίη. Typically, enzyme activity may be determined from the initial reaction rate. An assay such as that used in the present Examples may be used.

In one aspect, FaldDH as referred to herein does not have activity which catalyses the reduction of formate to formaldehyde. Typically, such an enzyme has substantially no detectable activity in an assay for such reduction activity. Activity which catalyses reduction of formate to formaldehyde may be assayed in any suitable way. For example, an assay may comprise incubating enzyme, NADH and formate substrate in a suitable buffer (e.g. PBS) at a suitable pH (e.g. pH 7.5) and temperature (e.g. 37°C) for a suitable time (e.g. 5 minutes), and monitoring consumption of NADH spectrophotometrically. Enzyme activity may be expressed in μιηοΙΝΑΟΗ/ιτιίη. Typically, enzyme activity may be determined from the initial reaction rate. An assay such as that used in the present Examples may be used.

An assay described above may be carried out in a similar way using another electron acceptor/donor as appropriate, e.g. NAD(P)+/NADPH.

FaldDH may be derived from any suitable species, such as any suitable microorganism, for example, a yeast or bacterial strain. Examples of such enzymes are available in the BRENDA database

(www.brenda-enzymes.orQ).

In one aspect FaldDH as referred to herein comprises a NAD-dependent FaldDH of Pseudomonas species, for example P. putida (UniProtKB/Swiss-Prot P46154; class EC 1 .2.1 .46) or a functional variant (e.g. species homolog) thereof. An FaldDH may comprise an amino acid sequence as in UniProtKB/Swiss-Prot P46154, or as in SEQ ID NO:9 herein or a functional variant thereof. Sequence variants and species homologs are described elsewhere herein. FaldDH may be the FaldDH tested in the present Examples.

Suitable FaldDH enzyme may be obtained from a naturally expressing strain, or may be expressed recombinantly in a suitable host strain, e.g. E. coli, using methods known in the art, or described elsewhere herein. Enzyme may also be commercially available. For example, P putida FaldDH is available from Sigma Aldrich Inc.

Electron donor and regeneration of the electron donor As explained above, the reduction reactions described herein require an electron donor or reducing agent. Any suitable electron donor may be used. In one aspect, the electron donor for one or more of the reduction reactions comprises NADH or NADPH. One or more enzymes used on the present processes, e.g. FDH, ADH and/or MDH may be NAD-dependent or NADP-dependent enzymes.

Since the electron donor becomes oxidised during the reduction process, the amount of donor may become rate limiting unless the donor is supplied continually or is regenerated. The reaction mix for the present process may also therefore comprise a suitable means for regenerating reducing agent (e.g. NADH or NADPH) which has been consumed (oxidised) in the reduction process.

Any suitable regeneration means may be used. Examples of systems for regeneration of NADH or NADPH include:

Lactate and lactate dehydrogenate (LDH) enzyme

In this system , lactate is converted to pyruvate as NAD ⁺ (or NADP ⁺ ) is reduced to NADH (WO 2007/022504A2) or NADPH.

Lactate dehydrogenase enzymes generally have activity which catalyzes the oxidation of lactate to pyruvate. LDH enzymes for use herein may use NAD ⁺ as an electron acceptor (NAD-dependent lactate dehydrogenases) and are thus able to regenerate NADH. Examples of such LDH enzymes include: those acting on L-lactate (EC 1 .1 .1 .27; CAS registry no. 9001 -60-9; S-lactate:NAD+ oxidoreductases) ; and those acting on R-lactate (EC 1 .1 .1 .28; CAS registry no. 9028-36-8; R- lactate:NAD+ oxidoreductases).

Methods for assaying oxidation of lactate by LDH are known in the art. For example, an assay is available from Worthington Biochemical Corporation.

Any suitable LDH enzyme may be used. Suitable LDH may be derived from any suitable species. Examples of such enzymes are available in the BRENDA database (www.brenda-enzymes.orQ). LDH may be that used in the present Examples.

LDH may be isolated from cells in which it is naturally expressed, or recombinantly expressed using known methods, or is available commercially. For example, rabbit muscle LDH is available from Sigma Aldrich Inc.

Glucose and glucose dehydrogenase (GDH) enzyme

A glucose/glucose dehydrogenase enzyme regeneration system is described in the art (e.g. "Glucose Dehydrogenase for the Regeneration of NADPH and NADH", Andrea Weckbecker and Werner Hummel, in Methods in Biotechnology, Vol17, 2005, pp225-238). In this system, glucose is converted to gluconic acid as NAD ⁺ is reduced to NADH (or NADP ⁺ is reduced to NADPH).

Glucose dehydrogenase enzymes generally have activity which catalyzes the oxidation of glucose. GDH enzymes for use herein may use NAD ⁺ as an electron acceptor and as thus able to regenerate the reduced NADH. Examples of such GDH enzymes include those in EC 1 .1 .1 .47; CAS registry no. 9028-53-9; beta-D-glucose:NAD(P)+ oxidoreductases or glucose 1 -dehydrogenases.

Methods for assaying oxidation of glucose by GDH are known in the art. For example an assay is available from Worthington Biochemical Corporation, or is described in Weckbecker and Hummel supra.

Any suitable GDH enzyme may be used. Suitable GDH may be derived from any suitable species. Examples of such enzymes are available in the BRENDA database (www.brenda-enzvmes.org). GDH may be that used in the present Examples. GDH may be isolated from cells in which it is naturally expressed, or recombinantly expressed using known methods, or is available commercially. For example, Pseudmonas sp GDH is available from Sigma Aldrich Inc.

Glutamate and glutamate dehydrogenase (GLDH) enzyme

In this system , glutamate is converted to 2-keto glutaric acid as NAD ⁺ is reduced to NADH (El-Zahab B et al, Biotechnol. Bioeng., 2008, 99(3) : 508-514) or as NADP ⁺ is reduced to NADPH.

Glutamate dehydrogenase enzymes generally have activity which catalyzes the oxidation of glutamate. GLDH enzymes for use herein may use NAD ⁺ as an electron acceptor and as thus able to regenerate the reduced NADH. Examples of such GLDH enzymes include those in EC 1 .4.1 .2 (CAS registry no. 9001 -46-1 , L-glutamate:NAD+ oxidoreductases (deaminating)) ; and in EC 1 .4.1 .3, (CAS registry no. 9029-12-3, L-glutamate:NAD(P)+ oxidoreductases (deaminating))

Methods for assaying oxidation of glutamate by GLDH are known in the art. For example, an assay is available from Calzyme Laboratories Inc.

Any suitable GLDH enzyme may be used. Suitable GLDH may be derived from any suitable species. Examples of such enzymes are available in the BRENDA database (www.brenda-enzymes.org).

GLDH may be isolated from cells in which it is naturally expressed, or recombinantly expressed using known methods, or is available commercially. For example, GLDH is available from Sigma Aldrich Inc.

Photochemical regeneration, e..g. Photosystem II (PSII) preparations

Photosystem I I (PSI I) preparations contain chlorophyll and 02-evolving complex, and can be extracted from green plants such as spinach. Extracted PSII preparation can be included in the reaction vessel and the reactions carried out in the presence of light, such that NADH can be regenerated and recycled automatically and continuously (US 6,440,71 1 B1 ). The PSI I preparations use the NAD ⁺ as an electron-acceptor in place of the acceptor quinone of green plants during exposure to light. As a result, the PSII preparation photo-oxidizes the water in the system to oxygen (O2), releasing hydrogen ions and electrons and thereby converting the NAD ⁺ back to NADH. Thus the PSII preparations provide photochemical regeneration.

Any other means of photochemical regeneration may be used.

Electrochemical regeneration

Reducing agent may be regenerated electrochemically. Suitable systems are known in the art.

Any of these regeneration means may be included in a reduction reaction according to the invention. In a preferred instance, the regeneration means comprises glucose and GDH.

Oxidised by-product of a regeneration system may be removed from the reaction by any suitable means. For example, pyruvate, produced by the lactate-LDH system may be removed by precipitation or crystallisation. Removal of the oxidised product prevents product build-up which might inhibit further reduction (e.g. of NAD ⁺ ) by the regeneration system and so inhibit regeneration of the reducing agent (e.g, NADH).

FaldDH and Aid DH enzymes

The inventors have shown that CO2 can be reduced to methanol without the use of FaldDH or AldDH. In particular, reduction of formate to formaldehyde does not require FaldDH or AldDH enzyme. Thus, FaldDH and/or AldDH may be substantially absent from reduction of formate to formaldehyde by FDH in step (a) of the process (or from step (a) of the process), or, if present, the amount of FDH enzyme is greater than the amount of any FaldDH and/or AldDH enzyme.

FDH amount is greater than the amount of FaldDH and/or AldDH to at least a detectable extent. FDH amount may be for example, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or at least 100% greater than the amount of any FaldDH and/or AldDH which may be present. FDH amount may be, for example, 2, 3, or 4 or more times greater than the amount of any FaldDH and/or AldDH which may be present.

Similarly, FaldDH and/or AldDH may be substantially absent in step (b) of the process, or, if present, the amount of ADH and/or MDH enzyme may be greater than the amount of any FaldDH and/or AldDH enzyme.

ADH and/or MDH amount may be greater than the amount of FaldDH and/or AldDH to at least a detectable extent. ADH and/or MDH amount may be for example, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or at least 100% greater than the amount of any FaldDH and/or AldDH which may be present. ADH and/or MDH amount may be, for example, 2, 3, or 4 or more times greater than the amount of any FaldDH and/or AldDH which may be present.

By substantially absent is meant that FaldDH and/or AldDH are not detectable in a suitable assay such as any of those described herein.

Enzyme amount may be initial enzyme amount - the amount of enzyme at the start of the reduction reaction. This may comprise, for example, the amount of enzyme added to the reaction mixture at time zero.

The amount of each of the above enzyme in a reaction mix for step (a) and/or step (b) may be determined using conventional methods. For example, detection could be mediated by Western blotting, using an antibody or antiserum raised against enzyme, or by mass spectrometry, detecting trypsin-generated peptides that are characteristic for enzyme. Enzyme activity may be determined according to an assay described herein.

PQQ-deoendent MDH

According to the invention, reduction of formate to formaldehyde is catalysed by the FDH enzyme. The reduction does not require the presence of ADH or MDH enzyme. In particular, the reduction does not require the presence of PQQ-dependent MDH enzyme.

Thus, in one aspect, MDH enzyme (e.g. PQQ-dependent MDH enzyme) may be substantially absent from the reduction of formate to formaldehyde by FDH in step (a) of the present process. MDH enzyme (e.g. PQQ-dependent MDH enzyme) may be substantially absent from step (a) of the present process.

PQQ-dependent MDH comprises an MDH which uses PQQ as a co-enzyme. Examples of such enzymes include those in EC 1 .1 .2.7 (,ethanol:cytochrome c oxidoreductase). One specific example of this class of enzymes is the Methylophilus methylotophus MDH (UniProtKB/Swiss-Prot. P38539).

By substantially absent is meant that the enzyme is not detectable in a suitable assay such as anyof those described herein. In one aspect, if MDH (e.g. PQQ-dependent MDH) is present, it is in a lower amount than FDH. FDH amount may be greater than the amount of MDH to at least a detectable extent. FDH amount may be for example, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or at least 100% greater than the amount of MDH. FDH amount may be, for example, 2, 3, or 4 or more times greater than the amount of any MDH which may be present. Enzyme amount may be initial enzyme amount - the amount of enzyme at the start of the reduction reaction. This may comprise, for example, the amount of enzyme added to the reaction mixture at time zero.

Amount of enzyme in a reaction mix may be determined using conventional methods. For example, detection could be mediated by Western blotting, using an antibody or antiserum raised against enzyme, or by mass spectrometry, detecting trypsin-generated peptides that are characteristic for enzyme. Enzyme activity may be determined according to an assay described herein.

Conditions for the reduction reactions

In general, the reduction reactions described herein are carried out in anaerobic conditions. Typically reagents and reactants are substantially free of oxygen. Oxygen can be excluded from the reaction by suitable means. For example, reagents and reactants may be flushed with nitrogen.

Any suitable conditions of pH and temperature may be used for the reduction reactions. For example, for reduction of CO2 to formate and then to formaldehyde by FDH, a temperature of about 30°C and pH of about 7 may be used. For reduction of formaldehyde by ADH, a temperature of about 20Ό and pH of about 7.5 may be used. For the overall reduction of CO2 to methanol process, temperature may be in the range of 20 -60 °C, preferably in the range of 20-30 °C, most preferably 25-30°C, e.g. about 30Ό. pH may be in the range of 5 - 8, most preferably 6 - 7.5, and preferably 7.

Reaction conditions used in any of the Examples may be used.

Any suitable reaction buffer may be used, for example, phosphate buffered saline (PBS).

Reagents and reactants may be used at any suitable amounts and as described further herein.

Enzyme amounts

The inventors have shown that in the present CO2 reduction process, FDH catalyses both reduction of CO2 to formate and reduction of formate to formaldehyde, whereas ADH (or MDH) catalyses only the reduction of formaldehyde to methanol. The ratio of FDH to ADH/MDH in the process may be adjusted accordingly. Thus, in one aspect of the CO2 reduction process, the amount of FDH enzyme in step (a) is greater than the amount of ADH and/or MDH enzyme in step (b).

In one aspect, FDH amount is greater to at least a detectable extent. FDH amount may be for example, at least 5, 1 0, 20, 30, 40, 50, 60, 70, 80, 90 or at least 1 00% greater than the amount of ADH and/or MDH enzyme. FDH amount may be, for example, 2, 3, or 4 or more times greater than the amount of ADH and/or MDH enzyme.

Enzyme amount may be initial enzyme amount - the amount of enzyme at the start of the reduction reaction. This may comprise, for example, the amount of enzyme added to the reaction mixture at time zero.

Enzyme amount may be determined by methods known in the art and described herein. For example, detection could be mediated by Western blotting, using an antibody or antiserum raised against enzyme, or by mass spectrometry, detecting trypsin-generated peptides that are characteristic for enzyme. Enzyme activity may be determined according to an assay described herein.

In one aspect, the amount of FDH enzyme in step (a) may be approximately 45-55%, for example, about 47, 49, 50, 51 or 53% w/w ratio to CO2 amount, such as about 50% w/w ratio to CO2 amount. In one aspect the process comprises a FDH concentration of approximately 50% w/w ratio to CO2 concentration, and is carried out at pH of about 7. CO2 concentration may be determined by any suitable means. For example, an assay based on titration with 0.1 M HCI (in the presence of phenolphthalein 0.5% in ethanol) of unreacted Ba(OH)2 0.05M (resulting from reaction of Ba(OH)2 with CO2) may be used. An example of such an assay is provided in the present Examples.

Carbonic anhydrase (CA) may be included in step (a) of the process. Typically, CA is provided at a concentration of up to 1 5% w/w ratio to CO2 concentration, for example, at 2, 4, 6, 8, 10, 12, 14% ratio.

Form of reaction components

Enzymes for use in the present processes may be provided as isolated enzyme preparations. Methods for preparing or obtaining such preparations are known in the art.

Steps (a) and (b) may occur in the same reaction mixture (e.g. in the same reaction vessel). In that case, reaction conditions specified for either step herein will be applicable also to the other step. For example, enzyme amounts specified for step (a) will also be the enzyme amounts in step (b). Use of a one-pot reaction may have a number of advantages. For example, it may prevent accumulation of formic acid, which may lower pH to an undesirable level, and in particular may prevent accumulation of formaldehyde which may react with enzymes and inactivate them.

Alternatively, steps (a) and (b) may occur in separate reaction mixtures (e.g. in separate reaction vessels). For example, the formaldehyde produced in step (a) by FDH catalysed reduction of CO2 and formate, may be channelled to a separate reaction vessel for reduction by ADH or MDH ; or FDH may be removed from the reaction mixture of step (a) before addition of ADH or MDH for reduction step (b).

Enzymes may be added to the reaction process simultaneously or sequentially. Thus, for example, FDH enzyme for use in step (a) and ADH or MDH enzymes for use in step (b) may be included together in the incubation at the start of the reaction, e.g. in the same reaction vessel. Alternatively, the enzymes may be added sequentially to the reaction process. Thus, for example, ADH or MDH may be included in the reaction at a later stage than FDH. ADH or MDH may be included in the same reaction vessel as FDH. Alternatively, ADH or MDH may be included in a separate reaction vessel to which the formaldehyde product of FDH-catalysed reduction has been directed, or may be added to the reaction mixture only after FDH has been removed.

Components in the present process may be provided (free) in solution and/or in immobilised form . Thus, for example, any of the enzymes for use in the process (e.g. FDH, ADH or MDH, LDH, GDH or GLDH), regeneration system components (e.g. PSI I), reducing agents (e.g. NADH), and/or other components may be provided in solution, in a suitable buffer such as those described herein. Alternatively, one or more of the components may be immobilised.

Any suitable immobilisation means may be used. Where PSI I is used as a regeneration means, any immobilisation means used is preferable transparent to allow light to access the PSI I.

Examples of immobilisation means include a micro-porous or nano-porous matrix such as those described in US 6,44071 1 B1 or WO 2007/022504 A2. Examples include sol-gel matrix.

Other examples include a silica matrix, optionally PEG-modified, such as those described in Wu, H et al, Chinese Chemical Letters, 2003, 14(4) : 423-425, or Wu, H et al, Abstracts of Papers of the American Chemical Society, 2004, 227: U1076-U1076. Further examples of immobilisation means include hydroxyapatite-polysaccharide capsules, such as those described in Zhang, L et al, J BiomaterialsSci. -Polymer Edition, 2009, 20(12) : 1661 -1674, or polystyrene particles such as those described in El-Zahab B et al, Biotechnol. Bioeng., 2008, 99(3) : 508-514. A solid immobilisation matrix may be provided as small particles or a powder which can be suspended in a medium in which the reaction takes place.

Enzyme combinations and kits

In one aspect, enzymes for use in the present process may be provided as a combination of one or more enzymes. Thus, in one aspect, the invention provides a dehydrogenase enzyme combination for use in the present process. A dehydrogenase enzyme combination may comprise, for example, one or more FDH enzymes and one or more ADH or MDH enzymes. The combination may additionally include, for example, one or more dehydrogenase enzymes for use in a regeneration system (e.g. LDH, GDH or GLDH). In one aspect, all dehydrogenase enzymes for use in the process may be included in the combination.

FaldDH and/or AldDH may be substantially absent from the enzyme combination. By substantially absent is meant that FaldDH and/or AldDH enzymes are not detectable in the incubation using a suitable detection method such as any described herein.

If FaldDH and/or AldDH is present, the amount of FDH in the combination is generally greater than the amount of FaldDH and/or AldDH enzyme. In one aspect, FDH amount is greater than the amount of any FaldDH and/or AldDH which may be present to at least a detectable extent. FDH amount may be for example, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or at least 100% greater than the amount of any FaldDH and/or AldDH which may be present. FDH amount may be, for example, 2, 3, or 4 or more times greater than the amount of any FaldDH and/or AldDH which may be present.

Similarly in one aspect, the amount of ADH and/or MDH in the combination may be greater than the amount of FaldDH and/or AldDH enzyme if present. In one aspect, ADH and/or MDH amount is greater than the amount of any FaldDH and/or AldDH which may be present to at least a detectable extent. ADH and/or MDH amount may be for example, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or at least 100% greater than the amount of any FaldDH and/or AldDH which may be present. ADH and/or MDH amount may be, for example, 2, 3, or 4 or more times greater than the amount of any FaldDH and/or AldDH which may be present.

Enzyme amount may be determined by known methods such as any of those described herein. For example, detection could be mediated by Western blotting, using an antibody or antiserum raised against enzyme, or by mass spectrometry, detecting trypsin-generated peptides that are characteristic for enzyme. Enzyme activity may be determined according to an assay described herein.

Enzymes in a combination may be provided in a single composition or formulation. Enzymes may be provided together in a suitable solution or immobilisation means, including any of those described herein.

Enzymes may be provided in separate formulations but as a combined product for use in the present processes. Thus in one aspect, the invention relates to a kit comprising at least one FDH enzyme, and at least one ADH or MDH enzyme, for simultaneous or sequential use in the reduction of CO2 to methanol by a process described herein. A kit may include one or more additional components for use in the process as described herein, for example, a suitable reducing agent (e.g. NADH) and/or a suitable regeneration means.

In general, FaldDH or AldDH is substantially absent from the kit, or if present, is provided at a lower amount than FDH. Where FaldDH and/or AldDH is present, the amount of FDH in the kit is generally greater than the amount of any FaldDH and/or AldDH enzyme. In one aspect, FDH amount is greater than the amount of any FaldDH and/or AldDH to at least a detectable extent. FDH amount may be for example, at least 5, 1 0, 20, 30, 40, 50, 60, 70, 80, 90 or at least 100% greater than the amount of any FaldDH and/or AldDH which may be present. FDH amount may be, for example, 2, 3, or 4 or more times greater than the amount of any FaldDH and/or AldDH which may be present. Similarly in one aspect, the amount of ADH and/or MDH in the kit may be greater than the amount of FaldDH and/or AldDH enzyme if present. In one aspect, ADH and/or MDH amount is greater than the amount of any FaldDH and/or AldDH to at least a detectable extent. ADH and/or MDH amount may be for example, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or at least 100% greater than the amount of any FaldDH and/or AldDH which may be present. ADH and/or MDH amount may be, for example, 2, 3, or 4 or more times greater than the amount of any FaldDH and/or AldDH which may be present.

Typically a kit includes instructions for using the kit components in a CO2 reduction process described herein.

Use of FDH

As described herein, FDH enzyme may be used to reduce formate to formaldehyde. Thus, in a further aspect the invention provides use of FDH enzyme for the reduction of formate to formaldehyde. Typically such use comprises exposing FDH enzyme to formate in the presence of a suitable reducing agent under suitable reaction conditions such that formaldehyde is produced.

Reaction conditions and particulars for reduction by FDH are as described elsewhere herein (e.g. in relation to step (a) of the CO2 reduction process).

The use of FDH according to the invention may form part of the process for reduction of CO2 to methanol described herein. Thus the use may additionally comprise using FDH to reduce CO2 to formate, and/or using ADH and/or MDH to reduce formaldehyde to methanol, as described herein.

Methanol product

Methanol product of the reduction process may be collected by any suitable means, for example, by distillation. For example, methods for recovery of methanol from a biodiesel process are described in David A. Chevront (2012) "Bioflexplant technology provides full methanol recovery." Biofuels International, January 2012, p64-65.

Further processing of methanol product

Methanol produced according to the present process may be used to manufacture a variety of chemical products and materials, including basic chemicals such as formaldehyde, acetic acid, methyl ferf-butyl ether (MTBE), as well as various polymers, paints, adhesive, and construction materials. Methanol may be used to produce formaldehyde, MTBE or acetic acid (1 1 %). Methanol is also a feedstock for chloromethanes, methylamines, methyl metacrylate, and dimethyl terephthalate, among others.

Thus the invention in one aspect provides a process for producing one or more of these products comprising process steps (a) and (b) as described herein.

Terms and definitions

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", can mean "including but not limited to", and are not intended to (and do not) exclude other moieties, additives, components, integers or steps. It will however also be understood that these terms encompass the meaning of and may in some instances be interpreted as meaning "consisting of".

An enzyme as referred to herein typically comprises a protein or peptide.

As used herein, "peptide" and "protein" can be used interchangeably and mean at least two covalently attached amino acids linked by a peptidyl bond. The term protein encompasses purified natural products, or products which may be produced partially or wholly using recombinant or synthetic techniques. The terms peptide and protein may refer to an aggregate of a protein such as a dimer or other multimer, a fusion protein, a protein variant, or derivative thereof. The term may also include modifications of the protein including any of those described herein.

Reference to an enzyme herein may include not only the enzyme per se as described, but also biologically active fragments of the enzyme, fusion proteins comprising the enzyme or biologically active fragments of the enzyme which fusion proteins retain enzyme activity, and derivatives of the enzyme, fragments or fusion proteins which retain enzyme activity. Thus, for example, reference to an FDH enzyme herein may include not only the FDH enzymes described herein including sequence variants as described), but also biologically active fragments of these FDH enzymes, fusion proteins comprising such FDH enzymes or fragments which retain FDH activity, or derivatives of the FDH enzymes, fragments or fusion proteins which retain FDH activity as described herein.

A derivative includes modified forms of the basic enzyme (or fragments or fusion proteins) described herein which retain enzyme activity. Modifications may include those made for improved isolation, purification, immobilisation or activity of an enzyme. Proteins may be modified for example, by glycosylation, acetylation, phosphorylation, PEGylation, ubiquitination, and so forth. A protein may comprise amino acids not encoded by a nucleic acid codon.

The term "biologically active fragment" as used herein generally means a fragment which retains a biological activity of a full length molecule functioning in vivo. For example, a biologically active fragment of an enzyme is typically capable of catalysing one or more of the reactions catalysed by the enzyme.

Similarly a functional variant of a molecule (e.g. of an enzyme) typically retains at least one biological activity of the molecule (e.g. enzyme).

For the avoidance of doubt, retention of enzyme activity does not require retention of the same level of activity as the enzyme per se. A biologically active fragment or fusion protein, or a derivative, or a functional variant, may display higher or lower activity than the reference enzyme. Typically, enough activity is retained to make the fragment, fusion protein, derivative or variant suitable for the given purpose. This may be, for example, retention of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99 % or more of the original activity of the reference enzyme. Activity which is retained may be substantially equivalent to that of the reference enzyme.

Enzyme activities for each of the enzymes are described elsewhere herein. Typically, enzyme activity which is retained comprises reduction activity as described herein.

Where an enzyme is described as having or not having a particular activity, generally, such an enzyme has/does not have activity which is detectable according to a suitable assay for that activity, examples of which are provided herein.

All references to "detectable" or "detected" are as within the limits of detection of the given assay or detection method.

Amount of enzyme may be defined in terms of enzyme activity or in terms of weight of enzyme (of known activity). Where enzymes or other components are in a single volume it will be appreciated that concentration may be regarded as a reasonable indicator of weight. Enzyme activities and methods for determining activity are described herein. Methods for detection of enzyme are also described.

The term "isolated" as used herein means a biological component (such as a nucleic acid molecule or protein, e.g. enzyme) that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids, proteins and peptides.

The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its environment within a cell, such that the peptide is substantially separated from cellular components (nucleic acids, lipids, carbohydrates, and other polypeptides) that may accompany it. In another example, a purified peptide preparation is one in which the peptide is substantially-free from contaminants, such as those that might be present following chemical synthesis of the peptide.

In one example, an peptide of the disclosure is purified when at least 50% by weight of a sample is composed of the peptide, for example when at least 60%, 70%, 80%, 85%, 90%, 92%, 95%, 98%, or 99% or more of a sample is composed of the peptide. Examples of methods that can be used to purify a peptide, include, but are not limited to the methods disclosed in Sambrook et al. (Molecular Cloning : A Laboratory Manual, Cold Spring Harbor, N.Y., 1989, Ch. 17). Protein purity can be determined by, for example, high-pressure liquid chromatography, SDS PAGE, or other conventional methods.

EC number as referred to herein is the Enzyme Commission number.

CAS registry number refers to the Chemical Abstracts Service number.

Sequence homoloas and variants

As used herein a homolog or variant of a protein or nucleic acid sequence (e.g. a gene) refers to a protein or nucleic acid sequence that is similar in sequence and in function to the reference sequence. A species homolog refers to a similar sequence (e.g. gene and/or protein) occurring in a different species to the reference sequence.

For any nucleotide or amino acid sequence, homologous sequences may be identified by searching appropriate databases. For example, suitable databases include GenBank (available at www.ncbi.nlm.nih.gov/Genbank) and UniProt (available at h ttp ://www .ebi.ac.uk/uniprot/).

Where appropriate, databases can be searched for homologous sequences using computer programs employing various algorithms. Examples of such programs include, among others, FASTA or BLASTN for nucleotide sequences and FASTA, BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. FASTA is described in Pearson, W R and Lipman, D J, Proc. Natl., Acad. Sci, USA, 85, 2444 2448, 1 988. BLASTP, gapped BLAST, and PSI-BLAST are described in Altschul, S F, et al., Basic local alignment search tool, J. Mol. Biol., 21 5(3) : 403 410, 1 990, Altchul, S F and Gish, W, Methods in Enzymology, 266, 460 480, 1996, and Altschul, S F, et al., "Gapped BLAST and PSI- BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389 3402, 1997

In addition to identifying homologous sequences, programs such as those mentioned above typically provide an indication of the degree of homology (or identity) between sequences. Determining the degree of identity or homology that exists between two or more amino acid sequences or between two or more nucleotide sequences can also be conveniently performed using any of a variety of other algorithms and computer programs known in the art. Discussion and sources of appropriate programs may be found, for example, in Baxevanis, A., and Ouellette, B. F. F., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, S. and Krawetz, S. (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. Calculations of sequence homology or identity (the terms are used interchangeably herein) between sequences may be performed as follows.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and nonhomologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the percent identity between two amino acid sequences is determined using the Needleman et al. (1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. In one embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) are a BLOSUM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et al. (1989) CABIOS 4:1 1 -17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

As used herein, a homologous or variant amino acid sequence generally has at least 60%, 65%, 70%, 75%, 80%, 81 %. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the reference sequence. Thus, for example, a species homolog of the C. boidinii FDH enzyme generally has at least 60%, 65%, 70%, 75%, 80%, 81 %. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the C. boidinii sequence.

Variants include insertions, deletions, and substitutions, either conservative or non-conservative.

In terms of amino acids, small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Therefore by "conservative substitutions" is intended to include combinations such as G ly, Ala; Val, lie, Leu; Asp, Glu; Asn, G in; Ser, Thr; Lys, Arg; and Phe, Tyr. Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.

As used herein, a homologous or variant nucleic acid sequence generally has at least 60%, 65% 70%, 75%, 80%, 81 %. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the reference sequence. Thus, for example, a species homolog of the C. boidinii FDH nucleic acid coding sequence, or gene sequence generally has at least 60%, 65%, 70%, 75%, 80%, 81 %. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the C. boidinii sequence. A functional variant is one in which the changes made with respect to the reference sequence do not substantially alter protein (enzyme) activity. For example, a functional variant of C. boidinii FDH enzyme typically retains FDH activity as described herein, in particular FDH activity in catalysing reduction of CO2 to formate and formate to formaldehyde. In general as used herein (and unless otherwise specified), enzyme homologs and variants are functional. Enzyme activities and methods for determining activity are described herein.

A fragment of an enzyme as referred to herein typically refers to a contiguous stretch of at least 8, 10, 12, 14, 1 5, 1 8, 20, 22, 25, 30, 40, 50, 100, 200, 300, 400, 500, 600, or more amino acids of the enzyme.

A fragment of a nucleic acid (e.g. m RNA or cDNA) encoding an enzyme may comprise a contiguous stretch of at least 8, 10, 12, 14, 15, 1 8, 20, 22, 25, 30, 40, 50, 100, 200, 500, 800, 900, 1000 or more nucleotides of the encoding nucleic acid.

Sequence information for enzymes referred to herein

As already explained herein, sequence accession numbers below are with reference to UniProtKB/Swiss-Prot version dated 18 December 2013.

SEQ ID NO: 1 - amino acid sequence of C. boidinii FDH (UniProt 013437)

MKIVLVLYDAGKHAADEEKLYGCTENKLGIANWLKDQGHELITTSDKEGETSELDKHIPD ADI I ITTPFHPAYIT KERLDKAKNLKLVVVAGVGSDHIDLDYINQTGKKI SVLEVTGSNVVSVAEHVVMTMLVLVRNFVPAHEQI INHDW EVAAIAKDAYDIEGKTIATIGAGRIGYRVLERLLPFNPKELLYYDYQALPKEAEEKVGAR RVENIEELVAQADIV TVNAPLHAGTKGLINKELLSKFKKGAWLVNTARGAICVAEDVAAALESGQLRGYGGDVWF PQPAPKDHPWRDMRN KYGAGNAMTPHYSGTTLDAQTRYAEGTK ILESFFTGKFDYRPQDI ILLNGEYVTKAYGKHDKK

SEQ ID NO: 2 - nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 , as found in Candida boidinii.

atgaagatcgttttagtcttatatgatgctggtaagcacgctgctgatgaagaaaaa ttatatggttgtactgaa aataaattaggtattgctaattggttaaaagatcaaggtcatgaactaattactacttct gataaagaaggtgaa acaagtgaattggataaacatatcccagatgctgatattatcatcaccactcctttccat cctgcttatatcact aaggaaagacttgacaaggctaagaacttaaaattagtcgttgtcgctggtgttggttct gatcacattgattta gattatattaatcaaacaggtaagaaaatctcagtcttggaagttacaggttctaatgtt gtctctgttgctgaa cacgttgtcatgaccatgcttgtcttggttagaaatttcgttccagcacatgaacaaatt attaaccacgattgg gaggttgctgctatcgctaaggatgcttacgatatcgaaggtaaaactattgctaccatt ggtgctggtagaatt ggttacagagtcttggaaagattactcccttttaatccaaaagaattattatactacgat tatcaagctttacca aaagaagctgaagaaaaagttggtgctagaagagttgaaaatattgaagaattagttgct caagctgatatcgtt acagttaatgctccattacacgcaggtacaaaaggtttaattaataaggaattattatct aaatttaaaaaaggt gcttggttagtcaataccgcaagaggtgctatttgtgttgctgaagatgttgcagcagct ttagaatctggtcaa ttaagaggttacggtggtgatgtttggttcccacaaccagctccaaaggatcacccatgg agagatatgagaaat aaatatggtgctggtaatgccatgactcctcactactctggtactactttagatgctcaa acaagatacgctgaa ggtactaaaaatatcttggaatcattctttactggtaaatttgattacagaccacaagat attatcttattaaat ggtgaatacgttactaaagcttacggtaaacacgataagaaataa

SEQ ID NO: 3 -nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 and optimised for codon usage in E. coli and insertion of restriction sites

ccatgGCTAAAATCGTTCTGGTTCTGTACGACGCTGGTAAACACGCTGCTGACGAAG AAAAACTGTACGGTTGCA CCGAAAACAAACTGGGTATCGCTAACTGGCTGAAAGACCAGGGTCACGAACTGATCACCA CCTCTGACAAAGAAG GTGAAACCTCTGAACTGGACAAACACATCCCGGACGCTGACATCATCATCACCACCCCGT TCCACCCGGCTTACA TCACCAAAGAACGTCTGGACAAAGCTAAAAACCTGAAACTGGTTGTTGTTGCTGGTGTTG GTTCTGACCACATCG ACCTGGACTACATCAACCAGACCGGTAAAAAAATCTCTGTTCTGGAAGTTACCGGTTCTA ACGTTGTTTCTGTTG CTGAACACGTTGTTATGACCATGCTGGTTCTGGTTCGTAACTTCGTTCCGGCTCACGAAC AGATCATCAACCACG ACTGGGAAGTTGCTGCTATCGCTAAAGACGCTTACGACATCGAAGGTAAAACCATCGCTA CCATCGGTGCTGGTC GTATCGGTTACCGTGTTCTGGAACGTCTGCTGCCGTTCAACCCGAAAGAACTGCTGTACT ACGACTACCAGGCTC TGCCGAAAGAAGCTGAAGAAAAAGTTGGTGCTCGTCGTGTTGAAAACATCGAAGAACTGG TTGCTCAGGCTGACA TCGTTACCGTTAACGCTCCGCTGCACGCTGGTACCAAAGGTCTGATCAACAAAGAACTGC TGTCTAAATTCAAAA AAGGTGCTTGGCTGGTTAACACCGCTCGTGGTGCTATCTGCGTTGCTGAAGACGTTGCTG CTGCTCTGGAATCTG GTCAGCTGCGTGGTTATGGCGGTGACGTGTGGTTCCCCCAGCCGGCTCCGAAAGACCACC CGTGGCGTGACATGC GTAACAAATACGGTGCTGGTAACGCTATGACCCCGCACTACTCTGGTACCACCCTGGACG CTCAGACCCGTTACG CTGAAGGTACCAAAAACATCCTGGAATCTTTCTTCACCGGTAAATTCGACTACCGTCCGC AGGACATCATCCTGC TGAACGGTGAATACGTTACCAAAGCTTACGGTAAACACGACAAAAAAtaactcgag

SEQ ID NO: 4 - amino acid sequence of ADH1 of S. cerevisiae strain ATCC 204508 (UniProt P00330)

MS IPETQKGVI YESHGKLEYKDIPVPKPKANELLINVKYSGVCHTDLHAWHGDWPLPVK LPLVGGHEGAGWVGMGENVKGWKIGDYAGIKWLNGSCMACEYCELGNESNCPHADLSGY THDGSFQQYATADAVQAAHIPQGTDLAQVAPILCAGITVYKALKSANLMAGHWVAI SGAA GGLGSLAVQYAKAMGYRVLGIDGGEGKEELFRS IGGEVFIDFTKEKDIVGAVLKATDGGA HGVINVSVSEAAIEASTRYVRANGTTVLVGMPAGAKCCSDVFNQVVKS I S IVGSYVGNRA DTREALDFFARGLVKSPIKVVGLSTLPEIYEKMEKGQIVGRYVVDTSK

SEQ D NO: 5 - amino acid sequence of ADH2 of S. cerevisiae strain ATCC 204508 (UniProt P00331 ) MS IPETQKAI IFYESNGKLEHKDIPVPKPKPNELLINVKYSGVCHTDLHAWHGDWPLPTK LPLVGGHEGAGWVGMGENVKGWKIGDYAGIKWLNGSCMACEYCELGNESNCPHADLSGY THDGSFQEYATADAVQAAHIPQGTDLAEVAPILCAGITVYKALKSANLRAGHWAAI SGAA GGLGSLAVQYAKAMGYRVLGIDGGPGKEELFTSLGGEVFIDFTKEKDIVSAWKATNGGA HGIINVSVSEAAIEASTRYCRANGTWLVGLPAGAKCSSDVFNHWKS IS IVGSYVGNRA DTREALDFFARGLVKSPIKVVGLSSLPEIYEKMEKGQIAGRYVVDTSK

SEQ ID NO: 6 - amino acid sequence of MDH of B. methanolicus (UniProt P31005)

MTNFFIPPASVIGRGAVKEVGTRLKQIGAKKALIVTDAFLHSTGLSEEVAKNIREAGLDV AIFPKAQPDPADTQVHEGVDVFKQENCDALVS IGGGSSHDTAKAIGLVAANGGRI DYQG VNSVEKPVVPVVAITTTAGTGSETTSLAVITDSARKVKMPVIDEKITPTVAIVDPELMVK KPAGLTIATGMDALSHAIEAYVAKGATPVTDAFAIQAMKLINEYLPKAVANGEDIEAREA MAYAQYMAGVAFNNGGLGLVHS I SHQVGGVYKLQHGICNSVNMPHVCAFNLIAKTERFAH IAELLGENVSGLSTAAAAERAIVALERYNKNFGIPSGYAEMGVKEEDIELLAKNAFEDVC TQSNPRVATVQDIAQI IKNAL SEQ ID NO: 7 - amino acid sequence of AldDH of S. cerevisiae (SwissProt P541 15)

MTKLHFDTAEPVKITLPNGLTYEQPTGLFINNKFMKAQDGKTYPVEDPSTENTVCEVSSA TTEDVEYAIECADRAFHDTEWATQDPRERGRLLSKLADELESQIDLVSS IEALDNGKTLA LARGDVTIAI CLRDAAAYADKVNGRTI TGDGYMNFTTLEPIGVCGQI IPWNFPIMMLA WKIAPALAMGNVCILKPAAVTPLNALYFASLCKKVGIPAGVVNIVPGPGRTVGAALTNDP RIRKLAFTGSTEVGKSVAVDSSESNLKKITLELGGKSAHLVFDDANIKKTLPNLVNGIFK NAGQICSSGSRIYVQEGIYDELLAAFKAYLETEIKVGNPFDKANFQGAITNRQQFDTIMN YIDIGKKEGAKILTGGEKVGDKGYFIRPTVFYDVNEDMRIVKEEIFGPVVTVAKFKTLEE GVEMANSSEFGLGSGIETESLSTGLKVAKMLKAGTVWINTYNDFDSRVPFGGVKQSGYGR EMGEEVYHAYTEVKAVRIKL

SEQ ID NO: 8 - amino acid sequence of AldDH of S. cerevisiae (SwissProt P22281 )

MLATRNLVPI IRAS IKWRIKLSALHYCMSDAETSEALLEDNSAYINNEKHNLFLEKIFSD YQPFKHDNRTQVSCSQHMRDYRPLLTLSSATRSVLFSLLASDMS I ILS I SPNTGILLCIG HLLASDIEDVVIVLSRGSPLVDLASTRIFKLAQNGTLRFAIKRTTFQELRFLRKSKDENV MEAATRGI ITIRQLYYENKVLPLRFTGNVATHIEENLEFEEQITWRTHVDSS IFPNTRCA YPSGYGPSAKIPCLSHKPNDILAYTGSTLVGRVVSKLAPEQVMKKVTLESGGKSTMAVFI QHDVTWAVENTQFGVFDRQGQCCIAQSGYTVHRSTLSQIVENNLEKDPSYVLHVDTESDI RGPFILKIHFES IPRRINSAKAENSKVLCGGPRENSVYLYPTLSATLTDECRIMKEEVFA PI ITILCVKTVDEAIQRGNNSKFGLAAYVTKENVHGI ILSTALKTVKLFI ICVHLASYQI PFGGNKNSGMGAELGKRALENYTEGNHVLPVSLVKETLAPNTETASPARWPIH

SEQ ID NO: 9 - amino acid sequence of FaldDH of P putida (UniProt P46154)

MSGNRGWYLGSGKVEVQKIDYPKMQDPRGKKIEHGVILKWSTNICGSDQHMVRGRTT A QVGLVLGHEITGEVIEKGRDVENLQIGDLVSVPFNVACGRCRSCKEMHTGVCLTVNPARA GGAYGYVDMGDWTGGQAEYLLVPYADFNLLKLPDRDKAMEKIRDLTCLSDILPTGYHGAV TAGVGPGSTVYVAGAGPVGLAAAASARLLGAAVVIVGDLNPARLAHAKAQGFEIADLSLD TPLHEQIAALLGEPEVDCAVDAVGFEARGHGHEGAKHEAPATVLNSLMQVTRVAGKIGIP GLYVTEDPGAVDAAAKIGSLS IRFGLGWAKSHSFHTGQTPVMKYNRALMQAIMWDRINIA EVVGVQVI SLDDAPRGYGEFDAGVPKKFVIDPHKTFSAA

EXAMPLES

The invention will now be described by way of specific Examples and with reference to the accompanying Figures, which are provided for illustrative purposes only and are not to be construed as limiting upon the teachings herein.

Materials and methods

Materials

Carbonic anhydrase, CA (bovine erythrocytes; 31 52.7 U/mg solid), glucose dehydrogenase , GDH (Pseudomonas sp.; 276 U/mg), formaldehyde dehydrogenase, FaldDH (Pseudomonas putida), aldehyde dehydrogenase, AldDH (Saccharomyces cerevisiae; 314 U/mg solid), alcohol dehydrogenase, ADH (equine liver; 1 .33 U/mg solid; Saccharomyces cerevisiae 314 U/mg solid), NAD ⁺ and glucose were from Sigma Aldrich (NL). Formate dehydrogenase, FDH (Candida boidinii; 50 U/ml), lactate dehydrogenase from rabbit muscle, LDH (142 U/mg) and formic acid (99 %) was from Fluka. NADH was from Acros Organics. A plasmid containing a synthetic gene coding for FDH was obtained from Genscript. Vector plasmid pTrcHis2A was obtained from Life Technologies. Recombinant formate dehydrogenase (FDH) was produced at PRI. All other reagents were of analytical purity.

Methods

1. Protein determination

The Bradford method (M. M. Bradford, Anal. Biochem., 1976. 72: 248-254) was used to measure the protein concentration of enzyme preparations, using bovine serum albumin (BSA) as standard. The absorbance of the solutions was measured at 595 nm using a 96-well microplate (flat bottom) and a SAFIRE spectrophotometer (Tecan Benelux BVBA, Giessen, The Netherlands). The BSA standard curve was in the range of 0.1 - 1 .2 mg protein/ml. To have an accurate determination a new standard curve was performed before each experiment and the samples were made in duplicate.

2. Activity determination of formate dehydrogenase

2.1 - Oxidation of formic acid to carbon dioxide

The catalytic activity of FDH for the oxidation reaction was assayed with formate as substrate and NAD ⁺ as the electron acceptor. In a 3 ml plastic cuvette, 0.75 ml phosphate buffer 0.2 M, pH 7.0 and 0.75 ml of a 0.2 M sodium formate solution were added to 1 .1 ml deionized water. The solution was mixed by inversion and equilibrated about 10 minutes at 37 °C. The reaction started at the addition of 0.3 ml of 10.5 mM β-ΝΑϋ ⁺ solution and 0.1 ml enzyme solution (0.68 mg protein/ml; 1 U/ml). The formation of NADH was followed spectrophotometrically at 340 nm (£340 nm = 6.22 mlV ciTr ¹ ) for 5 minutes. The activity of the enzyme was determined based on the initial reaction rate determined from the slope of the linear part of the curve. One unit is the amount of enzyme which catalyzes the formation of 1 μιηοΙ of NADH per minute.

The effect of methanol, gluconic acid and glucose concentration on formate dehydrogenase activity has been studied in the range of 0 - 0.6 M for oxidation of formic acid. Other conditions were as above. The reactions were performed at ambient temperature. All experiments were performed in duplicate.

2.2 - Reduction of carbon dioxide to formic acid

The FDH-mediated CO2 reduction to formate was carried out anaerobically at 35 °C. In 1 ml anaerobic quartz cuvette, 0.98 ml PBS 10 mM (previously saturated with CO2 for 30 minutes) and 0.01 ml NADH 10 mM were added. The solution was saturated with CO2 for 5 minutes, then placed into the spectrophotometer and incubated for 10 minutes at 35 °C. The reaction started by the injection of 0.01 ml of formate dehydrogenase solution (10 U/ml), and the decrease in absorbance at 340 nm was measured for 5 minutes. One unit is the amount of enzyme that catalyzes the conversion of 1 μιηοΙ of NADH per minute. The amount of formate produced by reduction of CO2 was determined from the consumption of NADH. The experiments were carried out in duplicate.

3. Activity determination of formaldehyde dehydrogenase

Formaldehyde dehydrogenase activity was measured at 340 nm in 2 ml plastic cuvettes. The standard assay mixture contained 0.1 ml MQ water, 0.67 ml PBS buffer 10 mM (0.15 M NaCI) pH 7.5, 0.167 ml NAD ⁺ 5.7 mM and 0.033 ml formaldehyde 0.08 % (v/v). The solution was mixed by inversion and equilibrated about 5 minutes at 37 °C. Then reaction started at the addition of 0.01 ml enzyme solution (0.98 mg/ml; ~ 3 U/ml). The formation of NADH was followed spectrophotometrically at 340 nm (£340 nm = 6.22 mM- ¹ cnv ¹ ). The increase in absorbance was recorded for 5 minutes against a reference cuvette containing a similar concentration of substrate. One unit is the amount of enzyme which catalyzes the formation of 1 μιηοΙ of NADH per minute. The experiments were performed in duplicate. The activity of the formaldehyde dehydrogenase activity for the reverse reaction, namely the reduction of formate to formaldehyde, was determined under similar conditions, but using formate and NADH as substrates. One unit is the amount of enzyme that catalyzes the conversion of 1 μιηοΙ of NADH per minute.

4. Activity determination of alcohol dehydrogenase

4.1 - Kinetic studies

The kinetics of two different types of alcohol dehydrogenase, i.e. ADH from equine liver and Saccharomyces cerevisiae, respectively, was accomplished for the reduction of formaldehyde to methanol using NADH as electron donor.

In 1 ml anaerobic quartz cuvette, 0.97 ml PBS buffer 10 mM (150 mM NaCI) pH 7.5, 0.01 ml formaldehyde solution (range: 0.1 - 5 M for ADH form equine liver and 0.1 - 45 M for ADH from S. cerevisiae) and 0.01 ml NADH solution (range: 5 - 20 mM) were added. The cuvette was then placed into a spectrophotometer at 20Ό. The reaction started by the addition of 0.01 ml of alcohol dehydrogenase solution (1 mg solid /ml for ADH from equine liver and 1 .6 mg solid/ml for ADH from S. cerevisiae). The decrease in absorbance at 340 nm was measured for 5 minutes against a reference cuvette containing a similar concentration of NADH and formaldehyde. The Michaelis- Menten parameters were calculated from the double reciprocal plots for the reverse reaction of ADH. The experiments were performed in duplicate.

4.2 - Temperature effect on ADH activity

The influence of temperature on ADH activity for the reduction of formaldehyde to methanol for ADH from equine liver (15 - 70 °C) and ADH from S. cerevisiae (15 - 45 °C) has been investigated. In a 1 ml anaerobic quartz cuvette, 0.975 ml PBS buffer 10 mM (150 mM NaCI) pH 7.5, 0.01 ml of NADH solution 10 mM and 0.01 ml formaldehyde solution 1 M were added. The cuvette was then placed into a spectrophotometer and equilibrated at different temperatures. The reaction started by adding 0.005 ml of alcohol dehydrogenase solution (1 mg solid/ml). The decrease in absorbance at 340 nm was measured for 5 minutes against a reference cuvette containing a similar concentration of NADH and formaldehyde. One unit is the amount of enzyme which catalyzes the formation of 1 μιηοΙ of NADH per minute. The experiments were performed in duplicate. 5. Enzymatic reduction of CO? to formic acid

In a typical experiment, the reaction mixture contained 3 mM NADH and 5 mg FDH in PBS buffer 0.1 M (0.15 M NaCI) pH 7.5 saturated with CO2, in a total reaction volume of 5 ml. In the case of NADH regeneration, either 10 mM glucose and 1 .2 mg glucose dehydrogenase (GDH) (1 1 .1 mg/ml) or 10 mM lactate and 1 .32 mg lactate dehydrogenase (LDH) were added. The mixture was stirred and incubated at 30 °C for up to 48 h. Samples (300 μΙ) taken anaerobically at 2, 7 and 30 hours were analyzed by GC-MS and/or HPLC. The enzyme was inactivated by the addition of 0.05 ml HCI 2 M in each sample vials. The results are given as the concentration of formic acid produced, as determined from the area of the corresponding peak in the chromatogram . Control reactions were carried out in the same conditions without FDH.

The effect of glucose and CO2 concentration on the catalytic efficiency of FDH for CO2 reduction was determined in the range 15 - 60 mM for carbon dioxide and 10 - 150 mM for glucose, respectively.

In the case of the effect of carbonic anhydrase concentration, the reactions were carried out at fixed concentration of glucose (10 mM) and variable concentrations of CA (15 - 150 % (w/w), CA/CO2). The same protocol as described above was used.

Further optimization of the reaction was carried out using experimental design. A three level full factorial design was used. The three factors chosen were FDH concentration (A, 0.27 - 50.73 % FDH/CO2, w/w), NADH concentration (B, 0.12 - 2.98 mM) and pH (C, 4.48 - 9.52). Response variable was the product (i.e. formic acid) formation after 18 h. Analysis of variance was used to evaluate significance of factors, interactions among factors and presence of autocorrelation in the residuals of the regression analysis.

6. Reduction of formic acid to formaldehyde catalyzed by FDH

The reactions were performed at 30 Ό at a total reaction volume of 1 ml. The assay mixture contained 5 mM formic acid, 3 mM NADH , 0.142 mg GDH, 10 mM glucose and 1 mg FDH in 1 ml phosphate buffer 0.1 M pH 7.3 (O2 free). The NADH stock solution was flushed with nitrogen for 2 minutes before adding in the reaction vial. A typical experiment was as follows: in 1 ml reaction vial, 0.02 ml glucose 0.5 M, 0.02 ml GDH (2.13 mg/ml), 0.2 ml NADH 15 mM, 0.03 ml FDH (34 mg protein/ml) and phosphate buffer 0.1 M pH 7.3 (O2 free) were added. The total reaction volume was 1 ml. The reactions were initiated by the addition of 0.05 ml formic acid 0.1 M. The reaction medium was flushed with N2 for 1 min. The mixture was incubated at 30 °C for 29 hours. Samples (200 μΙ) taken anaerobically at different time intervals were analyzed by HPLC using an organic acid column to determine the formic acid, and by the Purpald assay, to determine the formaldehyde formed. The results are given as the concentration of formic acid consumed, as determined from the area of the corresponding peak in the chromatographic trace and as the concentration of formaldehyde produced, as estimated from the Purpald assay. Control reactions were carried out in the same conditions but (i) without enzyme and (ii) with thermally inactivated enzyme .

7. Reduction of formaldehyde to methanol catalyzed by ADH (GC-MS analysis and GO

The experiments were performed at room temperature in 20 ml glass vials with rubber septum. The reaction mixture contained 100 mM formaldehyde, 3 mM NADH, 2.5 mg ADH, 1 0 mM glucose or 10 mM lactic acid (in the case of cofactor regeneration) in PBS buffer 0.1 M (0.15 M NaCI) pH 7.5 in a total reaction volume of 5 ml. A typical experiment was as follows: to a solution of 3.9 ml PBS buffer 0.1 M (0.15 M NaCI) pH 7.5, 1 ml NADH 15 mM and 0.1 ml formaldehyde 5 M was added. The reaction started by the addition of 0.1 ml ADH (25 mg/ml). In the case of NADH regeneration, 0.1 ml formaldehyde 5 M, 1 ml NADH 15 mM, 0.1 ml GDH (4.4 mg/ml) or 0.1 ml LDH (10 mg/ml) and 0.1 ml lactic acid 0.5 M or 0.1 ml glucose 0.5 M were added to 3.6 ml PBS buffer 0.1 M (0.15 M NaCI) pH 7.5. The reaction started by the addition of 0.1 ml ADH (25 mg/ml). The mixture was stirred and incubated at room temperature for 15 h. The identity of the product was determined by GC/MS analysis. The results are given as the concentration of methanol, as determined from the area of the corresponding peak in the chromatogram. The control reaction was carried out in the same conditions without ADH.

To determine the effect of methanol concentration on the reduction activity of ADH, different concentrations of methanol in the range 0 - 200 mM were used. The mixture was incubated at 30 °C for 48 h. Samples (20 μΙ) taken (anaerobically) at different time intervals were diluted with 20 ul t-amyl alcohol (GC internal standard) and 160 μΙ ml PBS buffer pH 7.0 (0 ₂ free) and analyzed by GC. The results are given as the yield of methanol, as determined from the area of the corresponding peak in the GC chromatogram. The control reaction was carried out in the same conditions without enzymes.

8. Enzymatic conversion of CO? to methanol (GC/MS and GC analysis)

The experiments were performed at room temperature in 20 ml glass vials. The reaction mixture contained 3 mM NADH, 10 mM glucose, 0.1 mg GDH, 2.8 mg ADH and 5.1 mg FDH in a total volume of 5 ml PBS buffer 0.1 M pH 7.5 (CO2 saturated). The mixture was incubated at room temperature for 5 h and analyzed by GC/MS. The results are given as concentrations of methanol, as determined from the area of the corresponding peak in the GC/MS or GC chromatogram . The control reaction was carried out in the same conditions without enzymes. All reactions were performed in duplicate.

9. Analytical methods

9.1 - C0 ₂ determination

The amount of CO2 dissolved in buffer has been determined indirectly by titration with HCI 0.1 M (in the presence of phenolphthalein 0.5 % in ethanol) of unreacted Ba(OH)2 0.05 M (resulted from the reaction of Ba(OH)2 with CO2) (Scheme 2). In 10 ml glass vials, 2 ml CO2 saturated phosphate buffer, 4 ml Ba(OH)2 0.05 M and 4 drops of phenolphthalein were added. The unreacted Ba(OH)2 was titrated with HCI 0.1 N. The volume of acid was recorded and used for determination of CO2 present in the solution. The control reaction using 2 ml of phosphate buffer (O2 free) and 4 ml Ba(OH)2 0.05 M was performed.

Ba(OH) ₂ + C0 ₂ ^■ BaC0 ₃ + H ₂ 0

Ba(OH) ₂ + 2 HCI - BaCl ₂ + 2 H ₂ 0

Scheme 2. Determination of CO2 in aqueous solutions

9.2 - Formaldehyde assay by Purpald method

The analysis of formaldehyde using Purpald method was performed by the procedure described by Lee and Quesenberry, slightly modified (M. S. Quesenberry, Y. C. Lee, Analytical Biochem., 1996. 234: 50-55). The calibration of formaldehyde was carried out in the range of 1 .5 - 30 nmol. In 1 .5 ml glas vials, 300 μΙ of 34 mM Purpald (freshly prepared), dissolved in 2 M NaOH and 300 μΙ sample containing formaldehyde were added. The reaction mixture was incubated at room temperature for 30 minutes (for samples containing very low amount of formaldehyde, the reaction was incubated for 20 h at room temperature for color developing). The asorbance at 550 nm was measured using 96-well microplates. The volume of sample in each well was 200 μΙ. The reactions were performed in duplicate.

9.3 - HPLC analysis of formic acid

Formic acid was determined by HPLC using a Shodex RSpak KC-81 1 column (8.0 mm ID x 300 mm L) column thermostated at 50 °C using a Waters HPLC system equipped with UV detector and autosampler. The elution of the compounds were done isocratic using 3 mM H2SO4 in milli Q water with a flow rate of 1 ml/min and the sample injection volume of 10 μΙ. The eluting components were detected at 210 nm and the run time was 30 min (Method name: "ABE method", Hetty lab.) RT = 9.3 min

9.4 - GC/MS analysis of methanol

For analysis of methanol a Thermo Scientific Interscience gas chromatograph with AS 3000 autosampler coupled to a mass spectrometer DSQ I I was used. The column was an extended temperature range "etr" 50 m DB-WAXETR capillary column, internal diameter 0.32 mm, 1 μΜ film thickness from J&W Agilent. The multiplier voltage was 1272 V. Injection parameters were: 20 ml/min split flow with an injector temperature of 200 °C. Carrier gas was helium at constant pressure. A temperature gradient was used for elution as follows: 35 °C held for 6 min then ramped to 95 °C at 35 Ό/min no hold, then to 100 °C at 5 Ό/min no hold, then to 150 °C at 20 °C/min with 1 9 min hold. Injection volume was 1 μΙ (Method name: "method_08'). RT = 9.0 min

9.5 - GC/MS analysis of formic acid

For the detection of formic acid, the injection parameters of the previous method (9.4) were slightly modified: 10 ml/min split flow with an injector temperature of 50 °C. Carrier gas was helium at constant flow and splitless time of 2 min. The temperature gradient was the same as for methanol detection (method_07). RT = 1 8.2 min

9.6 - GC analysis of methanol

For analysis of methanol a Thermo Finnigan Isc. Focus GC with AS 3000 autosampler coupled to a FID detector was used. The column was 50 m DB-WAXETR capillary column, internal diameter 0.32 mm, 1 μΜ film thickness from J&W Agilent. The detector temperature was 210 °C. Injection parameters were: 200:1 split ratio with a split flow of 10 ml/min and injector temperature of 200 °C. Carrier gas was helium at constant flow (4 ml/min column flow). A temperature gradient was used for elution as follows: 1 00 °C no hold then ramped to 160 °C at 20 °C/min hold for 3 min. For accurate results 1 mM ferf-amylalcohol was used as internal standard. Injection volume was 1 μΙ. (Method name: " MeOH_Acid_ 1 M. MET, -) . RTMeOH = 1 .63 min; = 1 .85 min

10. Production of recombinant formate dehydrogenase

10.1 Candida bo/ ^' d/n// ^' formate DH synthetic gene

Relevant amino acid sequence (SEQ ID NO: 1 ) and nucleotide sequences (SEQ ID NO: 2 & 3) are shown in Sequence Information section herein.

10.2 - Cloning strategy:

Program for cloning formaldehyde dehydrogenase from Candida boidinii.

Synthetic gene:

The protein sequence of Candida boidinii formate dehydrogenase (SEQ ID N0.1 ) was reverse- translated to DNA optimized for expression in E. coli using the Java Codon Adaptation Tool (http://www.jcat.de/). The resulting DNA sequence (SEQ ID No.3) was ordered for synthesis from GenScript, and was delivered in cloning vector pUC57 as construct pUC-FDH . The entire lot of the lyophilised pUc vector containing the synthetic gene (4 was dissolved in 50 μΙ milliQ water (fresh). The plasmid was transformed by heatshock transformation of competent E.coli strain XL-1 blue, and the cells were plated on solid LB plates and selected on Ampicillin (100 μρ ηηΙ).

Vector The supplied vector plasmid, (20 μς) pTrcHis2A, was dissolved in 50 μΙ milliQ water (fresh). This plasmid was also transformed into competent E.coli strain XL-1 blue and plated on solid LB medium with Ampicillin. (100 μς/ιηΙ).

For both pUC-FDH and pTrcHis2A a single colony was cultured in 5 ml_ LB medium + ampicillin (100 μς/ιηΙ) on 200 rpm 37 ^S C (50 ml tube) overnight. From these cultures, 700 μΙ overnight, culture was taken out, 300 μΙ 50% glycerol (sterile) were added, well mixed and stored in -80 ^S C. The remainder was used for the isolation of plasmid DNA by Qiagen miniprep kit. After purification of the plasmid DNA, the insert was isolated from the plasmid by digestion with Ncol and Xhol restriction enzymes. The resulting mixture was separated on a 0.6% agarose gel and the appropriate fragments cut from the gel after separation :

Fragments from agarose gel were identified using the marker for estimation of the correct fragment sizes:

• Synthetic FDH : 1 1 50 bp

• pTrcHis2A: 4.4 kbp

DNA was purified from the gel fragments using the Qiagen Gel purification kit. After determination of the yield of the DNA fragments, concentrations were adjusted to 50 ng/μΙ.

The FDH 1 150 bp fragment was ligated into the Xhol/Ncol cut pTrcHis2A vector according to standard procedures, and the mixture transformed after 2 h incubation at room temperature to E.coli strain XL-1 blue. Transformed colonies were selected for on solid LB plates with Ampicillin 100 μg/ml. As a control for unwanted ligations, ligation without vector pTrcHis2A and ligation without insert were used. Colonies expressing the correct gene were identified by growing cultures in 5 ml LB with ampicillin 100 μg/ml overnight and isolating plasmid. Isolated plasmid was digested using Xhol and Ncol restriction enzymes and checked for fragments of 4.4 kbp and 1 1 50 bp by agarose gel analysis. Positive colonies were baptised pTrc-FDH.

10.3 Expression of FDH protein:

Strains E. coli XL-1 with pTrcHis2A and pTrc-FDH were grown in 10 ml LB with 1 % glucose and 100μg/ml ampicillin overnight at 37Ό and 250 rpm to make precultures. 100 ml 2xYT medium (16g/L Bacto Tryptone, 10g/L bacto Yeast Extract, 5g/L NaCI, pH7) was inoculated with 1 ml of the overnight cultures, and incubated at 37 ^S C and 250 rpm for about 2h. When OD600 reached 0.5, IPTG was added to the cultures to a final concentration of 1 mM. Cultures were further incubated at 37°C and 250 rpm . Cells were spun down at 3500 RPM for 15 min, the supernatant discarded and the pellet carefully dried. Pellets were stored at -20 ^S C.

For protein extraction, 2 ml of 0.1 M Sodium phosphate buffer, pH 7.5 was added to the pellet. The pellet was resuspended and 200 mg Zi02 beads, 100 μιη diameter added. The mix was shaken in Savant BIO101 Fastprep beadshaker at setting 6 for two x 1 0 sec. Preps were put on ice between shaking. Broken cells were treated with MSE Soniprep 150 Ultrasonic probe, 5 cycles, using the following settings: Amplitude 13, 10 s on/10 s off. Preps were centrifuged for 1 0 min 14000 rpm at 0 ^S C. The supernatant was used for enzyme assays as "negative control" (when derived from pTrcHis2A) or as "recombinant FDH".

11 Effect of methanol concentration on catalytic efficiency ofADH (GC analysis)

The experiments were performed at 30 Ό in 1 .5 ml glass vials. The reaction mixture contained 134 mM formaldehyde, 3 mM NADH, 0.4 mg ADH, 10 mM glucose, 0.12 mg GDH (in the case of cofactor regeneration) and different concentrations of methanol (0 - 200 mM) in PBS buffer 0.1 M (0.15 M NaCI) pH 7.0 in a total reaction volume of 1 ml. The NADH stock solution was flushed with nitrogen for 2 minutes each time the vial was opened. The mixture was incubated at 30 Ό 48 h. Samples (20 μΙ) taken (anaerobically) at different time intervals were diluted with 20 ul t-amyl alcohol (GC internal standard) and 160 μΙ ml PBS buffer pH 7.0 (O2 free) and analyzed by GC. The reaction vial was flushed with nitrogen for 1 minute each time the vial was opened. The results are given as the yield of methanol, as determined from the area of the corresponding peak in the GC chromatogram . The control reaction was carried out in the same conditions without enzymes.

Experiments and results

It is reported that the enzymatic production of methanol from carbon dioxide involves an initial reduction of CO2 to formate catalyzed by formate dehydrogenase (FDH), followed by reduction of formate to formaldehyde by formaldehyde dehydrogenase (FaldDH), and finally formaldehyde is reduced to methanol by alcohol dehydrogenase (ADH). In this process, reduced nicotinamide adenine dinucleotide (NADH) acts as cofactor for each dehydrogenase-catalyzed reduction. First screening tests in our lab using a cocktail of NADH and the three said enzymes, either free in solution or immobilized, showed the formation of methanol, with a low yield. The first step in the developing and optimization of this enzymatic pathway for the production of methanol from carbon dioxide was to show that each dehydrogenase could in fact catalyze the target reduction reaction.

1. Characterisation of enzymes

Formate dehydrogenase (FDH) from C. boidinii (commercial preparation), recombinant FDH produced in our lab by overexpression of the synthetic gene in E. coli, formaldehyde dehydrogenase (FaldDH) from Ps. putida, aldehyde dehydrogenase (AldDH) from S. cerevisiae, and two alcohol dehydrogenases (ADH), from S. cerevisiae and equine liver were tested to evaluate their ability to catalyze the direct and reverse reaction steps involved in methanol oxidation or carbon dioxide reduction respectively.

1.1 Purity of enzyme preparations

Since all enzymes are commercial preparations it was important to determine their purity by SDS- PAGE electrophoresis. The results are given in Fig. 1 a. All the studied enzymes had a high content of protein contamination, except the case of glucose dehydrogenase (lane 6) and alcohol dehydrogenase from Saccharomyces cerevisiae (lane 2). The recombinant FDH was very pure ( Fig 1 b).

The total protein content of the enzyme preparations is given in Table 1 . Alcohol dehydrogenase from Saccharomyces cerevisiae showed the highest amount of protein with a high purity demonstrated by SDS-PAGE experiment.

Table 1. Protein content of dehydrogenases determined by Bradford assay

^* The protein content is expressed in mg per ml of enzyme preparation Ί.2 Characterization of alcohol dehydrogenase

Characterization of the alcohol dehydrogenase activity towards formaldehyde reduction

Alcohol dehydrogenases are very well known industrial enzymes, extensively studied and applied in many industrial processes for the production of commodity chemicals. The use of alcohol dehydrogenase for the production of methanol from formaldehyde has been reported, but the reaction has not been studied in detail. Kinetic studies have been carried out for the reduction of formaldehyde to methanol using two aldehyde dehydrogenases, from S. cerevisiae and equine liver. Results are given in Table 2. The results show that ADH from yeast is the most efficient catalyst for the reduction of formaldehyde to methanol, with high maximum reaction rate and substrate inhibition occurring at relatively high formaldehyde concentration. The low temperature for maximum activity of yeast ADH is also in favor for the whole process of CO2 conversion to methanol, since the solubility of gases increases at lower temperatures. Further studies were carried out only with the yeast ADH.

Table 2. Kinetic parameters and properties of ADHs from S. cerevisiae and equine liver

a0.1 mM NADH, 160 mM formaldehyde, PBS buffer 10 mM, pH 7.5

b0.1 mM NADH, 10 mM formaldehyde, PBS buffer 10 mM, pH 7.5

Studies of potential inhibitors for alcohol dehydrogenase from S. cerevisiae

The effect of methanol, the main reaction product, gluconic acid, which is a by-product resulting in the NADH regeneration by glucose oxidation, and glucose on the activity of ADH from S. cerevisiae was studied. The results are summarized in Table 3. Methanol showed no inhibition at concentration levels below 100 mM. At increasing methanol concentration inhibition effects occur, and only 32 % activity is retained at a methanol concentration of 0.9 M. No inhibition of ADH was observed for either glucose and gluconic acid for concentration lower than 0.9 M, showing that glucose can be used for the regeneration of NADH.

Table 3. Inhibition studies of alcohol dehydrogenase from S. cerevisiae

1.3 Characterization of formaldehyde dehydrogenase

Initial velocity studies on the forward reaction of formaldehyde dehydrogenase from Pseudomonas putida were performed spectrophotometrically by following the formation of NADH at 340 nm . The measurements were carried out at 37 °C. The specific activity of FaldDH using formaldehyde as substrate was 2.3 mole/min/mg protein. No activity in the reverse reaction, namely the reduction of formate to formaldehyde, has been detected for this enzyme. Formaldehyde dehydrogenase from Pseudomonas putida is a unique enzyme that can catalyze NAD ⁺ -dependent oxidation of formaldehyde without the external addition of glutathione (S. Ogushi, M. Ando, D. Tsuru, (1984), J. Biochem. 96, 1 587-1591 ). Several studies have been reported on formaldehyde dehydrogenase from Pseudomonas putida unable to catalyze the reduction of formate to formaldehyde (L. Uotila, M. Koivusalo, J. Biol. Chem., 1974. 249, 7653-7663; M. Ando, T. Yoshimoto, S. Ogushi, K. Rikitake, S. Shibata, D. Tsuru, (J. Biochem., 1979. 85: 1 165-1 172).

No catalytic activity has been measured for the reduction of formate to formaldehyde in the presence of the aldehyde dehydrogenase from S. cerevisiae.

1.4 Characterization of formate dehydrogenase

Formate dehydrogenase (FDH) from Candida boidinii and the recombinant enzyme (FDH-R) were characterized to determine the oxidative activity, i.e. oxidation of formic acid to CO2. The results of the activity measurements are summarized in Table 4. The results show that the commercial FDH is approximately two times more active than the recombinant FDH for the oxidation of formic acid. The reduction activity of FDH for the conversion of CO2 into formic acid will be discussed later. Table 4. Specific activity of formate dehydrogenase towards formic acid oxidation to CO2

The effect of methanol, glucose and gluconic acid on the FDH activity was studied. No inhibition was observed for any of the compounds tested up to 0.6 mM (Table 5).

Table 5. Inhibition studies of formate dehydrogenase

2. Enzymatic reduction of CO2 to formic acid using formate dehydrogenase

2.1 Effect of cof actor regeneration system on catalytic efficiency (GC/MS analysis)

Two different cofactor regeneration systems were tested for enzymatic production of formic acid from CO2 catalyzed by FDH. One of the approach was the regeneration of NADH by production of pyruvic acid from lactic acid catalyzed by lactate dehydrogenase (LDH) from rabbit muscle. The second one was NADH regeneration by glucose oxidation to gluconic acid catalyzed by glucose dehydrogenase (GDH) from Pseudomonas sp.

The efficiency of both systems were compared with the experiment without cofactor regeneration (Scheme 3). The reactions were carried out at room temperature under anaerobic conditions. The product was identified and quantified by GC/MS analysis.

a) b) c)

Scheme 3. Schematic representation of CO2 reduction to formic acid catalyzed by FDH. a) no cofactor regeneration; b) cofactor regeneration by glucose oxidation catalized by GDH ; cofactor regeneration by lactic acid oxidation catalyzed by LDH.

Fig. 2 demonstrates the effect of cofactor regeneration on the production of formic acid after 7 h of incubation at room temperature. The reactions with NADH regeneration by GDH system produced much more formic acid than the regeneration system using LDH and the experiment without regeneration, while the control reaction without enzyme showed no product formation.

Overall, it appeared that the capability of the NADH regeneration system using glucose oxidation for formic acid production was almost 3-times higher than the reaction without cofactor regeneration. The first approach resulted in a formic acid concentration of 9.5 mM, which corresponds to a yield of 95 %, based on the amount of glucose introduced in the reaction.

In conclusion, regeneration of cofactor in the presence of GDH was used in further studies.

2.2 Effect of carbonic anhydrase on CO? reduction (HPLC analysis of formic acid)

Since the solubility of CO2 in aqueous systems is very low at room temperature and pressure, in order to further shift the reaction direction toward formic acid, carbonic anhydrase was added to the reaction mixture to increase the rate of hydration of carbon dioxide to form HCO3-, so that sequestered CO2 would be present in high concentration. The schematic representation of the transformation is presented in Scheme 4.

C0 ₂ HCOOH

acid

Scheme 4. Schematic representation of CO2 reduction to formic acid catalyzed by FDH with carbonic anhydrase and NADH regeneration

To examine the feasibility of GDH cofactor regeneration and carbon dioxide hydration, four reactions were conducted: with/without NADH regeneration and with/without carbonic anhydrase. The experiments were performed at 30Ό under anaerobic conditions, with 3 mM NADH and 1 mg protein/ml (FDH). The amount of formic acid produced was determined by H PLC at 21 0 nm using an organic acids column. The results are presented in Fig. 3. Without cofactor regeneration, the amount of formic acid decreases in time, due to the formation of NAD ⁺ which can determine the oxidation of the product in the presence of FDH.

The efficiency of cofactor regeneration has been demonstrated by the increasing of formic acid concentration in time, indication that NADH serves as a limiting reagent in the reaction. After 30 h of incubation at 30 Ό, 1 .63 mM of formic acid has been detected in the reaction mixture. The addition of carbonic anhydrase enhanced the product formation.

2.3 Effect of NADH and FDH concentration on CO? reduction (HPLC analysis of formic acid)

Figure 4 (a) and 4 (b) illustrate the enzymatic reduction of CO2 as a function of concentration of NADH and FDH respectively. The reactions were performed at 30°C under anaerobic conditions without NADH regeneration. The amount of formic acid produced was determined by HPLC at 21 0 nm using an organic acids column. Since NADH serves as a limiting reagent, it provides a relative measurement of the efficiency of the reaction and the yield of formic acid production. The results demonstrated that the amount of formic acid increased with the amount of NADH and FDH presented in the reaction.

2.4 Effect of CO? concentration on the catalytic efficiency (HPLC analysis of formic acid)

The solubility of CO2 in water has been improved using three different methods: increasing the surface of gas bubbling by using a glass frit with small pores, addition of carbonic anhydrase and CO2 bubbling at low temperature (ice). The concentration of C02 in the solution has been determined by titration using the method described in section 2.2.9.1 from the experimental section. The PBS buffer 0.1 M, pH 7.0 (0.1 5 M NaCI), boiled and cooled down under N2 atmosphere for complete removal of oxygen, was then bubbled with CO2 by different methods. The calculated concentrations of CO2 in the solutions are given in Table 5.

Table 5. Different methods for improving CO2 solvation in aqueous solution

The solvation of CO2 was greatly enhanced at low temperature reaching a maximum of 63 mM after 1 h of CO2 bubbling in cold solution. The addition of CA has no significant influence on gas solvation.

The CO2 concentration had no effect on formic acid production even after prolonged incubation time at 30 °C (Fig. 5). This could be explained by enzyme inactivation at high concentration of substrate. 2.5 Optimization of reaction parameters by experimental design

To optimise the reaction conditions to increase the conversion of CO2 to formic acid catalyzed by FDH from C. boidinii, an experimental design was applied. Three variables were considered as the most relevant to find the best conditions of the reduction. We chose pH, NADH concentration and enzyme concentration as the main factors and product (formic acid) formation after 18 h as response variable to evaluate the performance of FDH. The reactions were carried out in 1 .5 ml glass vials under the conditions described in the experimental section.

The effects of FDH concentration and pH on the formation of formic acid at 0.7, 1 .55 and 2.4 mM NADH are shown in Fig. 6 a), b) and c). At the levels evaluated in this experimental design the NADH concentration did not have significant effect compared to the FDH concentration and pH. The amount of formic acid increased with the amount of FDH added. A slow decrease in formic acid production occurred with the increasing of cofactor concentration, which suggests a slightly inactivation of the enzyme. The highest concentration of formic acid was 0.2978 mM when the concentration of FDH was 50.73 % (w/w) and pH 7.0 (data not shown).

2.6 Effect of glucose and carbonic anhydrase concentration on CO? reduction

The effect of glucose concentration used for cofactor regeneration on enzymatic CO2 reduction was investigated in the range of 10 - 150 mM using CA for CO2 hydration to bicarbonate. The results are given in Fig. 7. Glucose concentration had no effect on formic acid production even after prolonged incubation time of the reaction.

The solvation of CO2 in water involves the formation of several species including CO3 ² -, HCO3- and H2CO3 whose thermodynamic equilibrium and concentration distribution are subject to the effect of pH and other physicochemical properties of the solution. One question regarding the enzymatic reduction of CO2 was whether CO2 or its hydrated derivatives are transformed directly into formic acid. Even though it has been reported that C03 ^2_ and HCO3- had been supplied as substrates, there is currently a lack of knowledge regarding which species is directly involved in the biotransformation.

In our study the effect of CA concentration on enzymatic formic acid production has been investigated in the range of 15 - 150 % (w/w). The results are shown in Fig. 8. CA concentration had no effect on formic acid production even after prolonged incubation time of the reaction. These results demonstrate that HCO3- is not a substrate for FDH from C. boidinii.

This observation was confirmed in experiments using 10 mM NaHC03 instead of CO2 saturated buffer. The results shown in Fig. 9 demonstrate that CO2 is the prefered substrate for FDH from C. boidinii and undergoes a rather low afinity for bicarbonate.

At a high concentration of NaHCC (1 M) in the reaction mixture, the amount of formic acid produced increased slowly in time (Fig. 10). The explanation could be the amount of CO2 present in the bicarbonate solution, which serve as a substrate for FDH.

2.7 Reduction of CO? to formic acid using recombinant FDH-R

The recombinant formate dehydrogenase FDH-R was used for the conversion of CO2 into formic acid under the optimal conditions determined for FDH using the experimental design (pH 7, 50% w/w catalyst, 3 mM NADH) and the glucose - glucose dehydrogenase system for cofactor regeneration. After 24h incubation, 0.75 mM formic acid was obtained, corresponding to a formic acid yield of 25.2% (based on NADH). Comparable results (0.64 mM) were obtained when using FDH as catalyst, under the same conditions.

3. Reduction of formic acid to formaldehyde using formate dehydrogenase

3.1 Reduction of formic acid to formaldehyde with FDH

The reduction of formic acid to formaldehyde in the presence of NADH as electron donor seems to be the limiting step for the overall cascade reaction, due to the lack of commercial availability of active enzymes. In our studies, two different aldehyde dehydrogenases were tested, formaldehyde dehydrogenase from Ps. putida and aldehyde dehydrogenase from S. cerevisiae, which showed no activity for the desired transformation.

Surprisingly, the commercial formate dehydrogenase from C. boidinii showed the ability to catalyze the reduction of formate to formaldehyde. The reactions were performed at 30 °C with/without cofactor regeneration. The conversion of the substrate has been determined from HPLC analysis. The results are presented in Fig. 1 1 .

The results show that in the presence of FDH and glucose - GDH for cofactor regeneration, almost 10 % of the substrate was consumed after 30 h of reaction incubation. For the experiment without NADH regeneration the amount of formic acid decreased progressively reaching 25% conversion, which can be explained by the accumulation of NAD ⁺ in the reaction which increases the rate of formate oxidation in the presence of FDH.

3.2 Formate reduction to formaldehyde using FDH-R

The commercial FDH from C. boidinii was not a pure enzyme, but a complex mixture of proteins. To determine unambiguously if the formate reduction activity observed is an intrinsic activity of FDH and is not originating from another contaminating protein, we have tested the pure recombinant FDH-R in this reaction. The formaldehyde produced in the reaction was determined quantitatively by Purpald assay (colorimetric). Since glucose used for cofactor regeneration could disturb the colorimetric measurement by reacting with Purpald reagent, the reactions were performed without NADH regeneration. The catalytic efficiencies of commercial FDH for the reduction of formic acid compared to the recombinant FDH are given in Table 6.

Table 6. Catalytic efficiency of commercial and cloned FDH for the reduction of formic acid (Purpald determination of formaldehyde)

An absorbance background was also recorded for the control experiments. This can be explained by the possible reaction of Purpald reagent with sugars present in enzymatic preparations. For catalytic efficiency determination, the difference between sample absorbance and control absorbance (without substrate) was used. Surprisingly, the catalytic efficiency of the recombinant enzyme for the reduction of formic acid was almost 3.8-fold higher then commercial preparation

4. Reduction of formaldehyde to methanol

4.1 Effect of cofactor regeneration system on catalytic efficiency ofADH (GC/MS analysis)

In our study, two different cofactor regeneration systems were tested for enzymatic production of methanol from formaldehyde catalyzed by ADH from S. cerevisiae. One of the approach was the regeneration of NADH by production of pyruvic acid from lactic acid catalyzed by lactate dehydrogenase (LDH) from rabbit muscle. The second one was NADH regeneration by glucose oxidation to gluconic acid catalyzed by glucose dehydrogenase (GDH) from Pseudomonas sp. The efficiency of both systems were compared with the experiment without cofactor regeneration (Fig. 12). The reactions were carried out at room temperature under anaerobic conditions. The product was identified and quantified by GC/MS analysis.

Fig. 12 demonstrates the effect of cofactor regeneration on the production of formic acid after 15 h of incubation at room temperature. The reduction of formaldehyde was enhanced by NADH regeneration using GDH system , showing 38 % of substrate conversion within 15 h of reaction. In conclusion, regeneration of cofactor in the presence of GDH was used in further studies. The control reaction without enzyme showed no product formation.

4.2 Effect of methanol concentration on catalytic efficiency of ADH (GC analysis)

To examine the feasibility of GDH cofactor regeneration, the reduction of formaldehyde to methanol was conducted with/without NADH regeneration. The experiments were performed at 30 °C, 3 mM NADH 0.4 mg ADH, and 60 mM methanol. The reaction has been monitored by GC using a polar column and the percentage of methanol has been calculated based on the conversion of formaldehyde. The results are presented in Fig. 13. It can be seen that in the case of ADH-mediated reduction of formaldehyde using cofactor regeneration the product yield was about 55 % after 50 h of incubation.

To evaluate the effect of methanol concentration on the catalytic activity of ADH, the capacity of enzyme to catalyse the reduction of formaldehyde was investigated according to the assay described in section 1 1 of the Materials and Methods The methanol concentration in the reaction was in the range of 50 - 150 mM. The control reaction contained about 50 mM methanol derived from formaldehyde stock solution (a typical commercial grade formalin may contain 10-12 % methanol, as a stabilizer for limiting oxidation and polymerization). The standard reaction conditions were: 134 mM formaldehyde, 3 mM NADH, 10 mM glucose, 0.12 mg GDH, 0.41 mg ADH, PBS buffer 0.1 M (0.1 5 mM NaCI) pH 7.0 and 30 °C. The results are presented in Fig. 14.

Surprisingly, the methanol concentration had a favourable effect on the catalytic efficiency of ADH up to 70 mM in the reaction mixture at prolonged incubation time. The product yield was about 77.5 % after 50 h of incubation. The methanol concentration had no significant effect on enzyme activity in the first stages of the reaction.

5. Methanol production from C0 ₂

Since commercial FDH showed activity also for formic acid reduction, we evaluated the capacity of FDH and ADH to catalyze the conversion of CO2 to methanol. Overall, the process involved reduction of CO2 to formaldehyde catalyzed by commercial FDH from C. boidinii and recombinant FDH, respectively, followed by formaldehyde reduction to methanol catalyzed by ADH from S. cerevisiae. In the process, NADH cofactor was regenerated by glucose oxidation catalyzed by GDH. The experiments were performed as described in section 8 of the Materials and Methods. The reaction was monitored by GC/MS using a polar column and the yield of methanol has been calculated based on the concentration of glucose used for cofactor regeneration. The results for methanol production in solution system are shown in Fig. 15. The overall reaction process is shown in Scheme 5.

acid GDH

Scheme 5. Reduction of CO2 to methanol catalysed by FDH and ADH Surprisingly, enzymatic methanol production from CO2 is possible using two enzymes (FDH and ADH) instead of three (FDH, FaldDH, ADH), reported in literature R. Obert, B.C. Dave, J.. Am. Chem.l Soc, 1999. 121 (51 ) : 12192-12193; P.K. Addo, et al., Electrochemical and Solid State Letters, 201 1 . 14(4) : p. E9-E13). The concentration of methanol was 0.47 mM after 5 h of incubation at room temperature:

Result for free enzymes in solution (2 enzyme reaction, 3 mM NADH, Glucose/GDH, 5h)

FDH, ADH,

CH30H, CH3OH, μητιοΙ MeOH/ μητιοΙ MeOH/

mM μιηοΙ mg mg mg FDH mg ADH

0.47 2.35 5.1 2.8 0.46 0.84

For results confirmation, the experiments were repeated in duplicate and the samples were analyzed by GC. A new methanol calibration curve was performed using t-amyl alcohol as internal standard. The samples (100 μΙ) were diluted with 10 μΙ HCI 2M (for enzyme inactivation) and 10 μΙ t-amyl alcohol 10 mM. After 5 h of incubation, 0.9 mM methanol was found in the reaction mixture.

FDH-R was also tested for the production of methanol from CO2 using the two-enzyme approach described above. The reaction was carried out at the optimal conditions of pH and enzyme loading as determined by experimental design for the reduction of CO2 to formate. The rest of the conditions were similar to the experiment described above. Table 7 shows the progress of the reaction and the methanol yield for both FDH and FDH-R.

Table 7. Methanol production from CO2 catalyzed by FDH (commercial) and FDH-R (recombinant) in combination with ADH and with cofactor regeneration. Reaction conditions: 5 ml total reaction volume, CO2 saturated PBS buffer (0.15 M NaCI) pH 7.0, 3 mM NADH, 1 0 mM glucose, 2.5 mg ADH, 0.1 mg GDH, 5.1 mg FDH.

6. Comparison of 2 enzyme system with 3 enzyme system

The present CO2 reduction process using only two enzymes was compared with the three enzyme process described in the prior art. Results are shown below for enzymes free in solution and with listed reaction conditions. Comparison of reaction with 2 enzymes vs. 3 enzymes (3 mM NADH , Glucose/GDH, 5h) 2 enzyme process (free)

FDH, FaldDH , ADH,

CH30H, μιτιοΙ MeOH/ μιτιοΙ MeOH/ μιτιοΙ MeOH/ μιηοΙ mg mg mg mg FDH mg FaldDH mg ADH

1 .35 0.96 0 0.55 1 .41 - 2.46

3 enzyme process (free)

FDH, FaldDH , ADH,

CH3OH, μιτιοΙ MeOH/ μιτιοΙ MeOH/ μιτιοΙ MeOH/ μιηοΙ mg mg mg mg FDH mg FaldDH mg ADH

1 .2 0.96 0.2 0.55 1 .20 5.98 2.27

Conclusions

From the results obtained the inventors concluded the following:

1 . Methanol production is possible using two enzymes (FDH, ADH) instead of three (FDH, FaldDH and ADH) with cofactor regeneration; 0.9 mM was obtained after 5h of incubation, with NADH regeneration (GDH)

2. Efficient regeneration of cofactor was obtained using glucose oxidation catalysed by glucose dehydrogenase from Pseudomonas sp.

3. Enzyme concentration has a significant influence on enzymatic reduction of CO2 to formic acid.

4. Recombinant FDH proved to be a promising catalyst for formic acid reduction.

5. Methanol concentration had a favourable effect on ADH activity up to 70 mM in the reaction mixture.