NL2013351B1 - Enzymatic conversion using hydrogen peroxide. - Google Patents

Abstract

The present invention relates to the use of a method for the enzymatic generation of H202, wherein the H202 produced is consumed in an enzymatic conversion reaction by a heme-dependent peroxygenase or heme-dependent peroxidase. In the process according to the invention H20 2 is produced by (a) enzymatically converting alcohol, using an enzyme with alcohol oxidase activity, and (b) enzymatically converting the product formed in coversion a) into the corresponding carboxylic acid and alcohol, using an enzyme with formaldehyde dismutase activity. Optionally, the corresponding carboxylic acid may be further converted using an enzyme with formate oxidase activity. The present invention also relates to carrier products to which enzymes with alcohol oxidase activity, with formaldehyde dismutase activity, with formate oxidase acivity and with heme-dependent peroxygenase or heme-dependent peroxidase activity are immobilized in any combination.

Description

P100169NL00

Enzymatic conversion using hydrogen peroxide

FIELD OF THE INVENTION

The invention relates to a new and efficient hydrogen peroxide (H2O2) generation system, especially for use in chemical conversions by heme proteins, wherein the H2O2 is generated from alcohol.

BACKGROUND OF THE INVENTION

Hydrogen peroxide can be used in enzymatic conversion reactions, in particular in oxyfunctionalization reactions. For this purpose H2O2 has been prepared in the art by various methods and from various starting materials.

SUMMARY OF THE INVENTION

Heme-dependent peroxygenases and heme-dependent peroxidases are versatile catalysts for specific oxyfunctionalization/oxidation reactions even on nonactivated hydrocarbons. Unlike the related cytochrome P450 monoxygenases, peroxygenases generate their catalytically active oxyferryl species with simple H2O2 instead of prohibitively expensive nicotinamide cofactors and molecular oxygen. This inherent advantage makes them attractive catalysts for preparative chemical synthesis. However, despite of the great potential of peroxygenases and peroxidases, their practical application is severely hampered by the poor operational stability caused by the rapid H2C>2-related oxidative inactivation. In this regard, gentle in situ generation of H2O2 from molecular oxygen could be a way of choice to maintain H2O2 at a low level not compromising enzyme activity or stability.

So far glucose oxidase (GOX) seems the most commonly used enzyme for in situ H2O2 formation, but the valuable glucose as co-substrate diminishes the cost-efficiency. A low yield of reducing equivalents coming from high molecular weight of glucose (one H2O2 per 180 g glucose co-substrate) represents a wasteful process. Other limitations of the glucose-based system are the restricted application in aqueous media as well as the necessary additional neutralization for the by-product, gluconic acid.

Another option is to use methanol as electron donor. Thereby, the cosubstrate consumption per mol H2O2 could be reduced to 32 g co-substrate. Moreover, methanol is cheap, readily available and can also act as a co-solvent that enhances the solubility of the hydrophobic substrates in aqueous reaction media. This approach, however, has an inherent limitation of generating as a byproduct formaldehyde, which is a known inactivator of enzymes.

Furthermore, as the availability of H2O2 is rate limiting for the enzymatic reactions which depend on the supply of H2O2, there is a need for a process with a higher availability of H2O2, but also for a process wherein the availability of H2O2 can easily be fine-tuned for the particular conversion process.

Hence, it is an aspect of the present invention to provide an improved process for the enzymatic in situ production of H2O2 from suitable co-substrates, such as alcohols.

The combined use of enzymes with alcohol oxidase and formaldehyde dismutase activities according to the present invention was found to deliver such higher availability of H2O2 with alcohol as an electron donor. A further improvement of the H2O2 producing system was attained by a trienzyme system i.e. the combined use of enzymes with alcohol oxidase, formaldehyde dismutase and formate oxidase activities.

It is a further aspect of the present invention to use the H2O2 produced by the combined use of enzymes with alcohol oxidase and formaldehyde dismutase activities and optionally also an enzyme with formate oxidase activity for specific enzymatic oxyfunctionalization reactions.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiments of the invention will now be described, by way of example only.

In view of the summary of the invention described above, one embodiment of the present invention relates to the use of a method for the enzymatic generation of H2O2, wherein a. a suitable alcohol is enzymatically converted into the corresponding aldehyde, using an enzyme with alcohol oxidase activity, and b. aldehyde formed in conversion a) is enzymatically converted into carboxylic acid and alcohol, using an enzyme with aldehyde dismutase activity wherein the H2O2 produced is consumed in an enzymatic conversion reaction by a heme-dependent peroxygenase or heme-dependent peroxidase.

It goes without saying that the alcohol produced in conversion step b) in turn can especially be used in a further conversion according to conversion step a).

In a further embodiment the invention relates to the use of H2O2 in an enzymatic conversion reaction by a heme-dependent peroxygenase or heme-dependent peroxidase, wherein the H2O2 is generated by a. enzymatically converting a suitable alcohol into the corresponding aldehyde, using an enzyme with alcohol oxidase activity thereby producing H2O2, and b. enzymatically converting the aldehyde formed in conversion a) into a carboxylic acid and an alcohol, using an enzyme with aldehyde dismutase activity.

In a further embodiment, the invention relates to the use of a method for the enzymatic generation of H2O2, wherein a. a suitable alcohol is enzymatically converted into the corresponding, using an enzyme with alcohol oxidase activity, b. aldehyde formed in conversion a) is enzymatically converted into the corresponding carboxylic acid and alcohol, using an enzyme with aldehyde dismutase activity, and c. carboxylic acid formed in conversion b) is enzymatically converted using an enzyme with formate oxidase activity wherein the H2O2 produced is consumed in an enzymatic conversion reaction by a heme-dependent peroxygenase or heme-dependent peroxidase.

In a further embodiment, the present invention relates to a method for the conversion of a substrate by an enzymatic reaction selected from the group consisting of hydroxylation, epoxidation, N-oxidation, sulfoxidation, O- and N-dealkylation, chlorination, bromination, polymerization, decolorization, and one-electron oxidation, wherein a. a suitable alcohol is enzymatically converted into the corresponding aldehyde, using an enzyme with alcohol oxidase activity, and b. aldehyde formed in conversion a) is enzymatically converted into the corresponding carboxylic acid and alcohol, using an enzyme with aldehyde dismutase activity, wherein c. H2O2 produced in conversion (a) is used for the conversion of the substrate by a heme-dependent peroxygenase or heme-dependent peroxidase.

In a further embodiment, the present invention relates to a method for the conversion of a substrate by an enzymatic reaction selected from the group consisting of hydroxylation, epoxidation, N-oxidation, sulfoxidation, O- and N-dealkylation, chlorination, bromination, polymerization, decolorization, and one-electron oxidation, as described above, wherein furthermore the carboxylic acid formed in conversion b) is enzymatically converted using an enzyme with formate oxidase activity and thereby producing H2O2. A suitable alcohol for use according to the method of the invention can be selected from the group consisting of methanol, ethanol, 1-propanol, 1-butanol, 1-pentanol, 1- hexanol, 2-chloroethanol, 3-chloro-1-propanol, 4-chloro-l-butanol, 2-mercaptoethanol, 2- methoxyethanol, 2-methyl-1-butanol, 2-butanol, 2-propen-l-ol, 2-cyanoethanol, 1-octanol, 2-octanol, 1,3-butylene glycol, 1,4-butynediol, 2-propanediol, 3-phenyl-l-propanol, benzyl alcohol, decanol, dodecanol, erythritol, isoamyl alcohol and tetradecanol.

More in particular, a suitable alcohol for use according to the method of the invention can be selected from the group consisting of alcohols having one to five carbon atoms, and in particular non-branched alcohols. More preferably, the alcohols comprise methanol, ethanol, 1-propanol, 1-butanol and 1-pentanol. It is especially preferred to use methanol or ethanol as the electron donor. Combinations of two or more different alcohols may also be used.

With the bi-enzyme system of enzymes with alcohol oxidase activity and with formaldehyde dismutase activity and methanol as co-substrate, toxic formaldehyde is transformed into a useful product, which theoretically doubles the electron yield (2 mol H2O2 from 32 g co-substrate) as compared to the use of alcohol oxidase alone.

With the tri-enzyme system of the enzymes with alcohol oxidase, formaldehyde dismutase and formate oxidase activities and methanol as co-substrate, the carbon end product is carbon dioxide, and the electron yield from 32 g co-substrate can be as high as 3 mol H2O2.

With “variant” of a polynucleic acid encoding an enzyme used according to the present invention is meant a polynucleic acid encoding an enzyme with similar functionality naturally found in the same or a different organism.

With “mutant” of a polynucleic acid encoding an enzyme used according to the present invention is meant any event (mutation) that changes the genetic structure thereof, while the enzyme encoded by the polynucleic acid remains functionally active. Such mutation may be a natural or a man-made mutation of the parent structure. Such mutation may encompass the replacement of a single nucleic acid base, the replacement of a number of nucleic acid bases, or the removal or insertion of one or more nucleic acid bases. The mutation of the polynucleic acid may or may not alter the structure of the enzyme encoded by it. The amino acid sequence of the enzyme encoded by a thus altered polynucleic acid preferably has at least 50% identity with the parent sequence, more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and most preferably at least 91%, 92%, 93%, 94%, 95%.

In the context of the present invention, with an “enzyme with alcohol oxidase activity” is meant an oxidoreductase enzyme or variant or mutant thereof capable to oxidize an alcohol and thereby producing H2O2 and an aldehyde. Suitable alcohol oxidases are especially classified as EC 1.1.3.13.

Such alcohol oxidases and the genes encoding these enzymes can suitably be derived from organisms such as Pichiapastoris, Pichia methanolica, Pichiafinlandica, Pichia naganishii, Pichia philodendra, Pichia trehalophila, Pichia anomala, Pichia acacia, Pichia alni, Pichia americana, Pichia amethionina, Pichia amylophila, Pichia angophorae, Pichia angusta, Pichia antillensis, Pichia harkeri, Pichia besseyi, Pichia bimundalis, Pichia bispora, Pichia bovis, Pichia burtonii, Pichia cactophila, Pichia canadensis, Pichia capsulate, Pichia caribaea, Pichia castillae, Pichia chambardii, Pichia ciferrii, Pichia delftensis, Pichia deserticola, Pichia dryadoides, Pichia etchellsii, Pichia euphorbiae , Pichia euphorbiiphila, Pichia fabianii, Pichia faecalis , Pichia farinose, Pichia fermentans, Pichia fluxuum, Pichia galaeiformis, Pichia glucozyma, Pichia guilliermondii, Pichia hampshirensis, Pichia haplophila, Pichia heedii, Pichia heimii, Pichia henricii, Pichia holstii, Pichia inositovora, Pichia jadinii, Pichia japonica, Pichia kluyveri, Pichia kodamae, Pichia lynferdii, Pichia maganishii, Pichia media, Pichia membranifaciens, Pichia methylivoria, Pichia mexicana, Pichia meyerae, Pichia minuta, Pichia mississippiensis, Pichia nakasei, Pichia nakazawae, Pichia norvegensis, Pichia ofunaensis, Pichia ohmeri, Pichia onychis, Pichia opuntiae, Pichia petersonii, Pichia philogaea, Pichia pijperi, Pichia pini, Pichia populi, Pichia pseudocactophila, Pichia anprcimm Pichia rnhm/lpns:i<i Pichia rhni!am>n\i\ Pichia \alicaria Pichia scolvti Pichia segobiensis, Pichia silvicola, Pichia spartinae, Pichia stipites, Pichia strasburgensis, Pichia subpelliculosa, Pichia sydowiorum, Pichia tannicola, Pichia thermotolerans, Pichia toletana, Pichia triangularis, Pichia veronae, Pichia wickerhamii, Pichia xylose, Candida boidinii, Candida cariosiliqnicola, Candida methanolovescens, Candida pignaliae, Candida sithepensis, Candida sonorensis, Candida tropicalis, Candida aaseri, Candida albicans, Candida amapae, Candida, anatomiae, Candida ancudensis, Candida antillancae, Candida apicola, Candida apis, Candida atlantica, Candida atmosphaerica, Candida auringiensis, Candida austromarina, Candida azyma, Candida beechii, Candida berate, Candida berthetii, Candida blankii, Candida boleticola, Candida bombi, Candida bombicola, Candida buinensis, Candida butyri, Candida cantarellii, Candida caseinolytica, Candida castellani, Candida castellii, Candida castrensis, Candida catenulate, Candida chilensis, Candida chiropterorum, Candida chodatii, Candida ciferrii, Candida claussenii, Candida coipomoensis, Candida conglobate, Candida cylindracea,

Candida dendrica, Candida dendronema, Candida deserticola, Candida diddensiae,

Candida diversa, Candida drimydis, Candida dubliniensis, Candida edax, Candida entomophila, Candida ergastensis, Candida ernobii, Candida ethanolica, Candida euphorbiae, Candida euphorbiiphila, Candida fabianii, Candida famata, Candida famata var. famata, Candida famata var. flareri, Candida fennica, Candida fermenticarens, Candida fibrae, Candida firmetaria, Candida floricola, Candida fluviatilis, Candida freyschussii, Candida friedrichii, Candida fructus, Candida galacta, Candida geochares, Candida glabrata, Candida glaebosa, Candida glucosophila, Candida gropengiesseri, Candida guilliermondii, Candida guilliermondii var. guiltiermondii, Candida guilliermondii var. membranaefaciens, Candida haemulonii, Candida homilentoma, Candida humicola, Candida humilis, Candida hydrocarbofumarica, Candida incommunis, Candida inconspicua, Candida insectalens, Candida insectamans, Candida insectorum, Candida intermedia, Candida ishiwadae, Candida javanica, Candida karawaiewii, Candida kefyr, Candida krissii, Candida kruisii, Candida krusei, Candida lactis-condensi, Candida lambica, Candida langeroni, Candida laureliae, Candida lipolytica, Candida llanquihuensis, Candida lodderae, Candida lusitaniae, Candida lyxosophila, Candida magnolia, Candida maltose, Candida maris, Candida maritime, Candida melibiosi, Candida melibiosica, Candida membranifaciens, Candida mesenterica, Candida methanosorbosa, Candida milleri, Candida mogii, Candida montana, Candida multi vp.mmis Candida rrmsap. Candida napndpndra Candida natalp.nsis Candida nemodendra, Candida norvegensis, Candida norvegica, Candida odintsovae, Candida oleophila, Candida oregonensis, Candida ovalis, Candida palmioleophila, Candida paludigena, Candida paralipolytica, Candida parapsilosis, Candida parapsilosis var. obtuse, Candida pararugosa, Candida paratropicalis, Candida pelliculosa, Candida peltata, Candida petrohuensis, Candida pini, Candida populi, Candida pseudointermedia, Candida pseudolambica, Candida pseudotropicalis, Candida psychrophila, Candida pulcherrima, Candida quercitrusa, Candida quercuum, Candida railenensis, Candida reukaufii, Candida rhagii, Candida robusta, Candida rugopelliculosa, Candida rugosa, Candida saitoana, Candida sake, Candida salida, Candida salmanticensis, Candida santamariae, Candida santjacobensis, Candida savonica, Candida schatavii, Candida sequanensis, Candida shehatae, Candida shehatae var. Insectosa, Candida shehatae var. lignose, Candida shehatae var. shehatae, Candida silvae, Candida silvanorum, Candida silvatica, Candida silvicultrix, Candida solani, Candida sophiae-reginae, Candida sorbophila, Candida sorbosa, Candida sorboxylosa, Candida spandovensis, Candida stellate, Candida stellatoidea, Candida succiphila, Candida suecica, Candida tanzawaensis, Candida tapae, Candida techellsii, Candida tenuis, Candida torresii, Candida tsuchiyae, Candida utilis, Candida vaccinii, Candida valdiviana, Candida valida, Candida vanderwaltii, Candida variabilis, Candida vartiovaarae, Candida versatilis, Candida vinaria, ,Candida vini, Candida viswanathii, Candida vulgaris, Candida wickerhamii, Candida xestobii, Candida zeylanoides , Aspergillus ochraceus, Aspergillus terreus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus clavatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus oryzae, Aspergillus ustus, Aspergillus versicolor, Ogataea angusta, Ogataea glucozyma, Ogataea henricii, Ogataea minuta, Ogataea siamensis, Achatina achatina, Achatina fulica, Arion ater, Basidiomycota, Gloeophyllum trabeum, Helix aspersa, Kuraishia capsulate, Paecilomyces var iotii, Passalora fulva, Penicillium chrysogenum, Penicillium purpurascens, Phanerochaete chrysosporium, Phlebiopsis gigantean, Polyporus obtusus, Poria contigua, Radulodon casearius, Thermoascus aurantiacus, Comamonas sp., Hansenula sp.

More in particular, the alcohol oxidases and the genes encoding these enzymes can suitably be derived from Pichia pastoris, Pichia methanolica, Pichia fmlandica, Pichia naganishii, Pichia philodendra, Pichia trehalophila, Candida boidinii, Candida cariosiliqnicola, Candida methanolovescens, Candida pignaliae, Candida sithepensis, Candida sonorensis, Candida tropicalis, Aspergillus ochraceus and Aspergillus terreus.

With the expression “enzyme with aldehyde dismutase activity” is meant here an oxidoreductase enzyme or variant or mutant thereof capable to catalyze the following reversible chemical reaction: 2 aldehyde carboxylic acid + alcohol. Suitable formaldehyde dismutases are especially classified as EC 1.2.99.4.

Such formaldehyde dismutases and the genes encoding these enzymes can suitably be derived from organisms such as Pseudomonas putida, Pseudomonas abietaniphila, Pseudomonas agarici, Pseudomonas agarolyticus, Pseudomonas alcaliphila, Pseudomonas alginovora, Pseudomonas andersonii, Pseudomonas antarctica, Pseudomonas asplenii, Pseudomonas azelaica, Pseudomonas batumici, Pseudomonas borealis, Pseudomonas brassicacearum, Pseudomonas chloritidismutans, Pseudomonas cremoricolorata, Pseudomonas diterpeniphila, Pseudomonas fdiscindens, Pseudomonas frederiksbergensis, Pseudomonas gingeri, Pseudomonas graminis, Pseudomonas grimontii, Pseudomonas halodenitrificans, Pseudomonas halophila, Pseudomonas hibiscicola, Pseudomonas hydrogenovora, Pseudomonas indica, Pseudomonas japonica, Pseudomonas jessenii, Pseudomonas kilonensis, Pseudomonas koreensis, Pseudomonas lini, Pseudomonas lurida, Pseudomonas lutea, Pseudomonas marginata, Pseudomonas meridiana, Pseudomonas mesoacidophila, Pseudomonas pachastrellae, Pseudomonas palleroniana, Pseudomonas parafulva, Pseudomonas pavonanceae, Pseudomonas proteolyica, Pseudomonas psychrophila, Pseudomonas psychrotolerans, Pseudomonas pudica, Pseudomonas rathonis, Pseudomonas reactans, Pseudomonas rhizosphaerae, Pseudomonas salmononii; P.thermaerum, Pseudomonas thermocarboxydovorans, Pseudomonas thermotolerans, Pseudomonas thivervalensis, Pseudomonas umsongensis, Pseudomonas vancouverensis, Pseudomonas wisconsinensis, Pseudomonas xanthomarina, Pseudomonas xiamenensis, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas anguilliseptica, Pseudomonas citronellolis, Pseudomonas flavescens, Pseudomonas jinjuensis, Pseudomonas mendocina, Pseudomonas nitroreducens,

Pseudomonas oleovorans, Pseudomonas pseudoalcaligenes, Pseudomonas resinovorans, Pseudomonas straminae, Pseudomonas aurantiaca, Pseudomonas chlororaphis,

Pseudomonas lundensis, Pseudomonas taetrolens, Pseudomonas azotoformans,

Pseudomonas brenneri, Pseudomonas cedrina, Pseudomonas congelans, Pseudomonas rnrmantn Pseudomonas eostantinii Pseudomonas e xtremorientalis Pseudomonas fluorescens, Pseudomonas fiilgida, Pseudomonas gessardii, Pseudomonas libanensis, Pseudomonas mandelii, Pseudomonas marginal! s, Pseudomonas mediterranea, Pseudomonas migulae, Pseudomonas mucidolens, Pseudomonas orientalis, Pseudomonas poae, Pseudomonas rhodesiae, Pseudomonas synxantha, Pseudomonas tolaasii, Pseudomonas trivialis, Pseudomonas veronii, Pseudomonas fulva, Pseudomonas monteilii, Pseudomonas mosselii, Pseudomonas oryzihabitans, Pseudomonas plecoglossicida, Pseudomonas denitrificans, Pseudomonas pertucinogena, Pseudomonas balearica, Pseudomonas luteola, Pseudomonas stutzeri, Pseudomonas avellanae, Pseudomonas cannabina, Pseudomonas caricapapyae, Pseudomonas cichorii, Pseudomonas coronafaciens, Pseudomonas fuscovaginae, Pseudomonas tremae, Pseudomonas viridiflava, Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus carnosus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hyicus-intermedius, Staphylococcus lugdunensis, Staphylococcus saprophyticus, Staphylococcus sciuri, Staphylococcus simulans, Staphylococcus warneri, Homo sapiens.

More in particular, the formaldehyde dismutases and the genes encoding these enzymes can suitably be derived from Pseudomonas putida and Staphylococcus aureus.

With the expression “ enzyme with formate oxidase activity” is meant here an enzyme or variant or mutant thereof capable of catalyzing the oxidation of formate to yield carbon dioxide and hydrogen peroxide. Suitable formate oxidases are especially classified as EC 1.2.3.1.

Such formate oxidases and the genes encoding these enzymes can suitably be derived from organisms such as Aspergillus nomius, Aspergillus oryzae, Aspergillus aculeatus, Aspergillus alliaceus, Aspergillus caesiellus, Aspergillus caespitosus, Aspergillus candidus, Aspergillus carneus, Aspergillus clavatus, Aspergillus deflectus, Aspergillus egyptiacus, Aspergillus flscherianus, Aspergillus flavus, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus ibericus, Aspergillus lentulus, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus, Aspergillus parasiticus , Aspergillus penicilloides, Aspergillus restrictus, Aspergillus sojae, Aspergillus sydowii, Aspergillus tamari, Aspergillus terreus, Aspergillus ustus, Aspergillus versicolor, Debaryomyces vanrijiae, Debaryomyces artagaveytiae, Debaryomyces carsonii, Debaryomyces castellii, Debaryomyces coudertii, Debaryomyces etchellsii, Debaryomyces vlnhulnrisi I)t>hnr\>nm\>rt>K hnnsipnii Tieharvamvc.es kursannvii Deharvnmvces Maeckeri.

Debaryomyces marama, Debaryomyces macquariensis, Debaryomyces melissophilus, Debaryomyces mrakii, mycophilus, Debaryomyces nepalensis, Debaryomyces occidentalis, Debaryomyces oviformis, Debaryomyces polymorphus, Debaryomyces prosopidis, Debaryomyces pseudopolymorphus, Debaryomyces psychrosporus, Debaryomyces robertsiae, Debaryomyces singareniensis, Debaryomyces udenii, Debaryomyces vietnamensis, Debaryomyces vindobonensis, Debaryomyces yamadae, Zygosaccharomyces rouxii, Trichophyton tonsurans, Pseudomonas entomophila, Pseudomonas putida.

More in particular the formate oxidases and the genes encoding these enzymes can suitably be derived from Aspergillus nomius, Aspergillus oryzae, Debaryomyces vanrijiae, Zygosaccharomyces rouxii, Trichophyton tonsurans, Pseudomonas entomophila and Pseudomonas putida.

German patent publication DE 3541582 relates to an enzymatic production process of formic acid by conversion of methanol into formaldehyde using an alcohol oxidase and subsequent enzymatic conversion of formaldehyde into formic acid using formaldehyde dismutase, wherein the H2O2 produced during this process is converted to water using catalase. Hence, in this document H2O2 produced is not used in the conversion of any substrate.

The hydroxylation, epoxidation, N-oxidation, sulfoxidation, O- and N-dealkylation, chlorination, bromination, polymerization, decolorization, and one-electron oxidation reactions according to the present inventions can suitably be catalysed by peroxygenases (E.C. 1.11.2.X) and peroxidases (E.C. 1.11.1 .X).

Suitable substrates for conversion by peroxygenases (E.C. 1.11.2.X) and peroxidases (E.C. l.ll.l.X) are alkanes (such as pentane, hexane, heptane, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, cyclopentane and cyclohexane), alkenes, alkynes, aromatic rings (such as benzene, naphthalene, toluene, phenanthrene, pyrene and p-nitrophenol), heterocycles (such as pyridine, dibenzofuran), sulphides (such as thioanisole), alcohols (such as benzyl alcohol, vanillyl alcohol, veratryl alcohol), aldehydes (such as benzaldehyde), various ethers (resulting in O-dealkylation).

Suitable reactions according to the present invention wherein the enzymatically generated H2O2 is enzymatically reacted with a substrate are reactions catalyzed by for example enzymes classified as E.C. 1.11.2.1 (also indicated as unspecific nernvvcxena se 1 F T 1 1 1 9 9 (mvelnnemvirlaiel FT 1119 4 (f'attv aeiH nerrwvoenpQet E C. 1.11.1.7 (peroxidase), E.C. 1.11.1.10 (chloride peroxidase) and E C. 1.11.1.18 (bromide peroxidase).

Suitable so-called unspecified oxygenases (E.C. 1.11.2.1) and genes encoding these enzymes can suitable be derived from organisms such as Agrocybe aegerita, Agrocybe acericola, Agrocybe amara, Agrocybe arvalis, Agrocybe cylindracea, Agrocybe dura, Agrocybe erebia, Agrocybe farinacea, Agrocybe firma, Agrocybe molesta, Agrocybe paludosa, Agrocybe parasitica, Agrocybe pediades, Agrocybe praecox, Agrocybe putaminum, Agrocybe re tiger a, Agrocybe semiorbicularis, Agrocybe sororia, Agrocybe vervacti, Coprinellus radians, Coprinellus amphithallus, Coprinellus angulatus, Coprinellus aureogranulatus, Coprinellus bipellis, Coprinellus bisporiger, Coprinellus bisporus, Coprinellus callinus, Coprinellus congregates, Coprinellus curtus, Coprinellus deliquescens, Coprinellus deminutus, Coprinellus dilectus, Coprinellus disseminatus, Coprinellus domesticus, Coprinellus ellisii, Coprinellus ephemerus, Coprinellus flocculosus, Coprinellus heptemerus, Coprinellus heterosetulosus, Coprinellus hiascens, Coprinellus impatiens, Coprinellus marculentus, Coprinellus mitrinodulisporus, Coprinellus pellucidus, Coprinellus plagioporus, Coprinellus pyrrhanthes, Coprinellus radians, Coprinellus sassii, Coprinellus sclerocystidiosus, Coprinellus subdisseminatus, Coprinellus subimpatiens, Coprinellus subpurpureus, Coprinellus truncorum, Coprinellus velatopruinatus, Coprinellus verrucispermus, Coprinellus xanthothrix, Coprinopsis cinerea, Marasmius rotula, Sulfolobus tokodaii. More in particular, the unspecified oxygenases and genes encoding these enzymes can suitable be derived from Agrocybe aegerita, Coprinellus radians, Marasmius rotula and Sulfolobus tokodaii. Also variants and mutants thereof can suitably be used in the process according to the present invention.

Suitable fatty acid peroxygenases (E.C. 1.11.2.4) and the genes encoding these can suitably be derived from organisms such as Bacillus megaterium, Bacillus subtilis, Clostridium acetobutylicum, Fusarium oxysporum, Sphingomonas paucimobilis. Also variants and mutants thereof can suitably be used in the process according to the present invention.

Peroxidase (E.C. 1.11.1.7) and the genes encoding these can suitably be derived from organisms such as Acorus calamus, Aedes aegypti, Aggregatibacter actinomycetemcomitans, Allium sativum, Arabidopsis thaliana, Arachis hypogaea, Armoracia rusticana, Arthromyces ramosus, Arundo donax, Beta vulgaris, Bjerkandera nt/n\tn Rnst tannic Rraccira nanus: Rrassira nlprarpa Rrassira rnnn Rnhalns huhalis

Butia capitata, Camellia sinensis, Capra hircus, Capsicum annuum, Catharanthus roseus, Chromolaena odorata, Cicer arietinum, Citrus jambhiri, Coprinopsis cinerea, Cucumis melo, Cucumis melo var. inodorus, Cynara cardunculus, Elaeis guineensis, Elizabethkingia meningoseptica, Escherichia coli, Euphorbia characias, Fagopyrum esculentum, Fragaria vesca, Fragaria x ananassa, Glycine max, Gossypium hirsutum, Helianthus annuus, Homo sapiens, Hordeum vulgare, Ipomoea batatas, Ipomoea carnea, Jubaea chilensis, Landoltia punctata, Leptogium saturninum, Malus x domestica, Mentha arvensis, Momordica charantia, Mus musculus, Mycobacterium avium, Mycobacterium tuberculosis, Neurospora crassa, Nicotiana sylvestris, Nicotiana tabacum, Oryza sativa, Ovis aries, Pelargonium graveolens, Plasmodium falciparum, Pleurotus eryngii, Pleurotus ostreatus, Prunus persica, Pyrococcus furiosus, Raphanus sativus, Rattus norvegicus, Roystonea regia, Ruegeria pomeroyi DSS-3, Sabal minor, Sclerocarya birrea, Scutellaria baicalensis, Senecio squalidus, Sesbania rostrata, Solanum lycopersicum, Solanum melongena, Sorghum bicolor, Sphagnum magellanicum, Streptomyces thermoviolaceus, Sulfolobus acidocaldarius, Sus scrofa, Trachycarpus fortunei, Triticum aestivum, Vitis vinifera, Washingtonia fdifera, Yersinia pseudotuberculosis. Also variants and mutants thereof can suitably be used in the process according to the present invention.

Chloride peroxidase (E.C. 1.11.1.10) and the genes encoding these can suitably be derived from organisms such as Caldariomyces fumago, Aspergillus niger, Bazzania trilobata, Musa x paradisiaca, Streptomyces toyocaensis. Also variants and mutants thereof can suitably be used in the process according to the present invention.

Bromide peroxidase (E.C. 1.11.1.18) and the genes encoding these can suitably be derived from organisms such as Agrocybe aegerita, Ascophyllum nodosum, Corallina officinalis, Corallina pilulifera, Delisea pulchra, Ecklonia stolonifera, Fucus distichus, Gracilaria changii, Homo sapiens, Kappaphycus alvarezii, Laminaria hyperborea, Macrocystis pyrifera, Ochtodes secundiramea, Pseudomonas fluorescens, Pseudomonas putida, Saccharina latissima, Streptomyces aureofaciens, Streptomyces griseus, Streptomyces venezuelae, Synechococcus sp. Also variants and mutants thereof can suitably be used in the process according to the present invention.

Suitable substrates for conversion by heme-dependent oxygenases E.C. 1.11.2.1 are alkanes (such as propane, hexane and cyclohexane), alkenes, alkynes, aromatic rings (such as naphthalene, toluene, phenanthrene, pyrene and p-nitrophenol), heterocycles fsnrh as nvririinp Hihpnznfnranl various pthprs trpsnltirm in D-HpalEvlation'ï Snitahlp reactions catalysed by heme-dependent oxygenases E C. 1.11.2.1 include hydroxylation, epoxidation, A-oxidation, sulfooxidation, O- and A-dealkylation, bromination and one-electron oxidations. The heme-dependent oxygenases E C. 1.11.2.2 are able to oxidize phenols and have a moderate peroxygenase activity toward styrene in the absence of halides. The heme-dependent oxygenases E.C. 1.11.2.4 are able to hydroxylate saturated as well as unsaturated fatty acids.

According to the present invention the enzymes may be used for example in solution or immobilized to a carrier product. Immobilization means here associating the enzyme with an insoluble matrix. Suitable insoluble matrixes preferably are made of inert, insoluble materials, such as natural polymers (polysaccharides, proteins, carbon), synthetic polymers (polystyrene, polyacrylate, polymethacrylate, polyacrylamide, polyamides, vinyl, and allyl-polymers), inorganic natural minerals (bentonite, silica) or processed materials (porous or non-porous glass, metals, controlled-pore metal oxides). Depending on the nature of the insoluble matrix, the enzymes may be included in the matrix or may be bound to the surface of the product, and the enzymes may be reversibly or irreversibly immobilized. In the latter case, the enzymes may be covalently bound, entrapped, microcapsulated or cross-linked.

The respective enzymes used according to the present invention may be immobilized either each on a different carrier product or may be immobilized in any combination on one or two or more carrier products. In a specific embodiment of the present invention, the enzymes with alcohol oxidase activity, formaldehyde dismutase activity, optionally formate oxidase activity and with heme-dependent peroxygenase or heme-dependent peroxidase activity are all immobilized on the same carrier product. Accordingly a particular embodiment of the present invention relates to a carrier product to which enzymes with alcohol oxidase activity, with formaldehyde dismutase activity, optionally with formate oxidase activity as well as with heme-dependent peroxygenase or heme-dependent peroxidase activity are immobilized.

The process for the hydroxylation or epoxidation of a substrate according to the present invention may be carried out e.g. in a batch process or in a continuous process. A continuous process is particularly suitable when using the enzymes in immobilized form. Such continuous process may be carried out e.g. in a continous flow stirred tank, in a continuous flow packed bed, in a continuous flow membrane reactor, or in a continuous fluirli 7f>rl hf>rl rpartnr

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. For instance, in any process or step described herein wherein an enzyme may be used, in embodiments also a combination of different enzymes of the same class may be used. The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. The various aspects discussed in this patent can be combined in order to provide additional advantages. Furthermore, some of the features can form the basis for one or more divisional applications.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the enzymatic hydroxylation of ethyl benzene in the absence (

) and presence (

) of FDM; and Fig. 2 shows the enzymatic hydroxylation of ethyl benzene driven by AOX/FDM/FOX-mediated generation of H2O2 at pH 5.5 (

) or pH 6.0 (

). In both figures the x-axis indicates the time (t) in hours and the y-axis indicates the product concentration (C) in mM (millimole).

EXAMPLES

Materials

Enzymes

The unspecific peroxygenase from Agrocybe aegrita (AaeUPO, main isoform II) and Marasmius rotula (A/raUPO) were produced and purified as described by Ulrich et al., 2004 and Gröbe et al., 2011. The stock solution of AaeUPO used for all experiments contained 5.2 mg protein per mL (115 μΜ) with a specific activity of 98 U/mg related to the oxidation of veratryl alcohol. The A//0UPO solution contained 7.4 mg protein per mL (231 μΜ) and had a specific activity of 61 U/mg.

Chloroperoxidase (CPO) from Galdariomyces fumago was purchased from Sigma-Aldrich and used as received. The enzyme concentration was determined spectrophotomerically to be 21 mg/mL (500 μΜ) (β4οο=91200 IVT'crn'1). CPO specific activity was -1500 U/mL, where one unit forms 1.0 mg of purpurogallin from pyrogallol in 20 seconds at pH 6.0 at 20°C.

Horseradish peroxidase (HRP) was purchased from Sigma-Aldrich with an activity of 950-2000 U/mg solid. One unit oxidizes 1 pmol of ABTS per minute at pH 5.0 at 25°C.

Alcohol oxidase (AOX) from Pichia patoris was purchased from Sigma-Aldrich as a solution with an activity of 1100 U/ml (27 U/mg protein) related to the oxidation of methanol. Alcohol oxidase from Candida boidinii was purchased from Sigma-Aldrich with an activity of 1.08 U/mg solid (13.43 U/mg protein).

Formaldehyde dismutase (FDM) from Pseudomonas putida was coexpressed with GroESL in E. coli BL21 Star (DE3) as described by Yanase et al (2002). Crude extracts were obtained by cell disruption of harvested cells using a Branson Sonifier 250. After centrifuging, the resultant supernatant was fractionated by precipitation with (NH^SCE at 60% to 80% saturation to get rid of the catalase. The precipitate obtained was then dissolved in phosphate buffer (pH 7.0) and desalted by ultrafiltration. The enzyme solution was shock-frozen with liquid nitrogen and freeze-dried overnight. FDM activity was determined to be 110 ± 10 U/mg according to Yanase et al, 2002. One unit of the enzyme activity was defined as the amount of the enzyme catalysing the formation of 1 pmol of formate in 1 minute.

Formate oxidase (FOX, isoform II) from Debaryomyces vanrijiae was cloned and expressed in E. coli BL21 Star (DE3) as described by Maeda et al (2008). After over-night cultivation in LB medium, the harvested cells was disrupted by ultrasonication and the cell-free extracts were used for protein purification using a HisTrap FF column. The target protein was eluted with an increasing gradient from 20 to 500 mM of imidazole. The pooled fractions containing the target protein were concentrated and dialyzed against 10 mM acetate buffer (pH 6.0). The FOX activity was assayed via the measurement of the amount of hydrogen peroxide generated during formate oxidation by using HRP and ABTS. One enzyme unit is defined as the amount of enzyme required to produce 1 pmol hydrogen peroxide per minute during the oxidation of formate at 30°C and pH 4.5.

Analytical procedures

Table 1. Details for GC and HPLC analysis

[a] Temperature profile: 90UC for 5 min, 20uC/min to 110 for 10 min, 40°C/min to 200 for 2 min.

[b] Temperature profile: 110°C for 15 min, 20°C/min to 170 for 3 min.

[c] Temperature profile: 170°C for 25 min. i [d] Temperature profile: 70°C for 8 min, 50°C/min to 100 for 5 min, 50°C/min to 200 for 2 min.

[e] Temperature profile: 70°C for 10 min, 50°C/min to 200 for 2 min.

[f] Eluent: heptane/ isopropanol = 98/2; flow rate = 1.0 mL/min, detection at 228 nm.

[g] gradient eluent: EfO/acetonitrile = 80/20 to 20/80 in 12 minutes; flow rate = 1.0 ) mL/min.

Example 1. Enzymatic oxygenations with AaeUPO (unspecific peroxvgenase from Agrocvbe aegrita)

Enzymatic oxygenation of alkanes (e.g. ethylbenzene, propylbenzen, 4-i chloro-ethylbenzene, tetralin, cyclohexane, cyclopentane) or alkenes (e.g. c/.ν-β-methylstyrene) was performed in 100 mM potassium phosphate buffer (pH 7.0) containing 10 mM alkane, 5 mM methanol, 50 nM AaeUPO, 60 nM alcohol oxidase and 0.2 g/L formaldehyde dismutase. The experiments were carried out in 1 ml scale (total volume) in 2 ml glass vials at 30°C under ambient atmosphere. After 2 hours, the reaction mixture was ) extracted with ethyl acetate or diisopropyl ether and the extracts were analysed by GC or HPLC. The results were shown in Table 1, Entry 1-8.

Conditions: [substrate]=10 mM, [methanol]=5 mM, [peroxygenase]=50 nM, [AOX]=60 nM, [FDM]=0.2 g/L in 100 mM phosphate buffer, r=30°C, 2 h.

Table 2. Peroxygenase-catalysed oxygenations driven by AOX/FDM-mediated H2O2 veneration

Example 2. Comparison of the bi-enzvme H7Q9 producing system with alcohol oxidase alone.

Figure 1 illustrates the ^aeUPO-catalysed hydroxylation of ethylbenzene driven by AOX/FDM-mediated H2O2 generation at the substrate concentration of 50 mM.

The presence of FDM had no effect on the initial rate with excess amount of methanol but improved the finial productivity. Addition of FDM resulted to full conversion of ethylbenzene after 3 days while in the absence of FDM the reaction retarded sharply and nearly ceased after 2 days.

We hypothesize that the dismutation of intermediate formaldehyde alleviated the enzyme inactivation and thus led to the higher conversion.

Conditions: [ethyl benzene]=50 mM, [methanol]=200 mM, [rifireUPO]=100 nM, [AOX]=60 nM, [FDM]=0.2 g/L in 100 mM phosphate buffer (pH 7.0), r=30°C. In all cases ee>98%.

Example 3, Enzymatic epoxidation of c/v-P-methylstyrene with MroUPO (unspecific peroxygenase from Marasmius rotula)

Enzymatic epoxidation of cv.v-P-methylstyrene was performed in 100 mM potassium phosphate buffer (pH 6.0) containing 10 mM 67.ν-β-ηιethyl styrene, 5 mM methanol, 50 nM MroUPO, 60 nM alcohol oxidase and 0.2 g/L formaldehyde dismutase. The experiments were carried out in 1 ml scale (total volume) in 2 ml glass vials at 30°C under ambient atmosphere. After 2 hours, the reaction mixture was extracted with ethyl acetate, and the extracts were analvsed hv GO The results were shown in Table Ί F.ntrv Q

Example 4. Enzymatic sulfoxidation of thioanisole with CPO (Chloroperoxidase from Caldariomyces fumago)

Enzymatic sulfoxidation of thioanisole was performed in 100 mM potassium phosphate buffer (pH 6.0) containing 10 mM thioanisole, 5 mM methanol, 50 nM CPO, 60 nM alcohol oxidase and 0.2 g/L formaldehyde dismutase. The experiments were carried out in 1 ml scale (total volume) in 2 ml glass vials at 30°C under ambient atmosphere. After 2 hours, the reaction mixture was extracted with ethyl acetate and the extracts were analysed by GC. The results were shown in Table 2, Entry 10.

Example 5, Tri-enzymatic in situ generation of

Enzymatic hydroxylation of ethylbenzene was performed in 100 mM potassium phosphate buffer (pH 5.5 or pH 6.0) containing 15 mM ethylbenzene, 5 mM methanol, 50 nM AaeUPQ, 60 nM alcohol oxidase, 0.4 g/L formaldehyde dismutase and 0.4 g/L formate oxidase. The experiments were carried out in 1 ml scale (total volume) in 2 ml glass vials at 30°C under ambient atmosphere. At intervals, aliquots were extracted with ethyl acetate and the extracts were analysed by GC.

The results at the two pH values are shown in Figure 2. CITED LITERATURE:

Ulrich et al.. 2004, R. Ullrich, J. Nuske, K. Scheibner, J. Spantzel, M. Hofrichter (2004) “Novel Haloperoxidase from the Agaric Basidiomycete Agrocybe aegerita Oxidizes Aryl Alcohols and Aldehydes”, Appl. Environ. Microbiol., 70, 4575-4581.

Gröbe et al.. 2011, G. Gröbe, R. Ullrich, M. J. Pecyna, D. Kapturska, S. Friedrich, M. Hofrichter, K. Scheibner (2011) “High-yield production of aromatic peroxygenase by the agaric fungus Marasmius rotuld\ AMB Express, 1,31-41.

Yanase et al.. 2002, H. Yanase, K. Moriya, N. Mukai, Y. Kawata, K. Okamoto, N. Kato (2002) “Effects of GroESL coexpression on the folding of nicotinoprotein formaldehyde dismutase from Pseudomonaspmtida F61”, Biosci. Biotechnol. Biochem., 66, 85-91.

Maeda et al.. 2008, Y. Maeda, M. Oki, Y. Fujii, A. Hatanaka, M. Hojo, K. Hirano, H. Uchida (2008) “doing and expression of three formate oxidase genes from Debaryomyces vanrijiae MH201”, Biosci. Biotechnol. Biochem., 72, 1999-2004.

German patent publication DE 3541582

Claims (14)

The use of H 2 O 2 in the enzymatic conversion reaction by a heme-dependent peroxygenase or heme-dependent peroxidase, the H 2 O 2 being prepared by: a) enzymatically converting a suitable alcohol into a corresponding aldehyde by means of an enzyme having the action of alcohol oxidase producing H 2 O 2, and b) enzymatically converting the aldehyde produced in conversion a) to 1 a carboxylic acid and an alcohol by means of an enzyme with the action of aldehyde dismutase. The use according to claim 1, wherein the suitable alcohol contains one to five carbon atoms. The use according to claim 2, wherein the alcohol is non-branched. The use according to any one of claims 1 to 3, wherein the carboxylic acid formed in conversion b) is further enzymatically converted by means of an enzyme with the action of format oxidase, thereby producing H2O2. The use according to claim 4, wherein the alcohol is methanol or ethanol. The use according to any of claims 1 to 5, wherein the conversion by an enzymatic conversion reaction by means of a heme-dependent peroxygenase or heme-dependent peroxidase is a reaction selected from the group consisting of hydroxylation, epoxidation, N-oxidation, sulfoxidation, O- and N-dealkylation, chlorination, bromination, polymerization, decolouration and one-electron oxidation. 7. A method for converting a substrate by an enzymatic conversion reaction selected from the group consisting of hydroxylation, epoxidation, N-oxidation, sulfoxidation, O- and N-dealkylation, chlorination, bromination, polymerization, decolouration and one-electron oxidation, by a) enzymatically converting a suitable alcohol into a corresponding aldehyde by means of an enzyme with the action of alcohol oxidase producing H 2 O 2, and b) enzymatically converting the aldehyde produced in conversion a) to a carboxylic acid and a alcohol by means of an enzyme with the action of aldehyde dismutase, wherein c) H 2 O 2 formed in conversion a) is used for the conversion of the substrate by a heme-dependent peroxygenase or heme-dependent peroxidase. A method according to claim 7, wherein the suitable alcohol contains one to five carbon atoms. A method according to claim 8, wherein the alcohol is non-branched. A method according to any of claims 7 to 9, wherein the carboxylic acid formed in conversion b) is further enzymatically converted by means of an enzyme with the action of format oxidase, thereby producing H 2 O 2. A method according to claim 10, wherein the alcohol is methanol or ethanol. A carrier to which at least two of the enzymes selected from the group consisting of an enzyme with the action of alcohol oxidase, an enzyme with the action of formaldehyde dismutase, an enzyme with the action of format oxidase and an enzyme with the action of heme -dependent 1 peroxygenase or heme-dependent peroxidase. A carrier according to claim 12, to which enzymes with the action of alcohol oxidase, enzymes with the action of formaldehyde dismutase, and enzyme with the action of heme-dependent peroxygenase or heme-dependent peroxidase are immobilized. A carrier according to claim 13, in addition to which an enzyme is immobilized with the action of format oxidase.