US 20140120587A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0120587 A1 Haas et al. (43) Pub. Date: May 1, 2014

(54) ENZYMATIC AMINATION Publication Classification (51) Int. Cl. (75) Inventors: Thomas Haas, Muenster (DE); Jan CI2P 17/12 (2006.01) Christoph Pfeffer, Hanau (DE); Kurt CI2PI3/00 (2006.01) Faber, Graz (AT); Michael Fuchs, CI2P 17/04 (2006.01) Muelheim an der Ruhr (DE) (52) U.S. Cl. CPC ...... CI2P 17/12 (2013.01); C12P 17/04 (2013.01); C12P 13/001 (2013.01) (73) Assignee: Evonik Degussa GmbH, Essen (DE) USPC ...... 435/122; 435/126; 435/190: 435/128 (21) Appl. No.: 14/126,607 (57) ABSTRACT A method comprising the steps (a) contacting a hydrocarbon (22) PCT Filed: Jan. 10, 2012 comprising a hydroxyl group with a biological agent having oxygen-dependent and cofactor-dependent carbohydrate oxi (86). PCT No.: PCT/EP2012/050288 dase activity in the presence of oxygen and carbohydrate S371 (c)(1), oxidase cofactor, and (b) contacting the hydrocarbon pro (2), (4) Date: Dec. 16, 2013 duced in step a) with a biological agent having transaminase activity and a biological agent having cofactor-dependent (30) Foreign Application Priority Data amino acid dehydrogenase activity in the presence of amino acid dehydrogenase cofactor and the Substrate amino acid of Jun. 15, 2011 (EP) ...... 1117OO11.8 the amino acid dehydrogenase. Patent Application Publication May 1, 2014 Sheet 1 of 7 US 2014/O120587 A1

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ENZYMATIC AMINATION amines starting with compounds comprising a hydroxyl 0001. The present invention relates to a method compris group, preferably attached to an aromatic or aliphatic carbon ing the steps a) contacting a hydrocarbon comprising a ring, which compounds may be derived from renewable start hydroxy group with a biological agent having oxygen-depen ing materials rather than compounds obtained by way of dent and cofactor-dependent carbohydrate oxidase activity in cracking fossil resources. Another problem underlying the the presence of oxygen and carbohydrate oxidase cofactor, present invention is to provide an entirely enzymatic route and b) contacting the hydrocarbon produced in step a) with a towards the production of organic amines, preferably one biological agent having transaminase activity and a biological with a broad substrate specificity and/or improved yield and/ agent having cofactor-dependent amino acid dehydrogenase or product specificity compared to reactions described in the activity in the presence of amino acid dehydrogenase cofactor prior art and one that does not require the addition of hazard and the Substrate amino acid of the amino acid dehydroge ous reagents and/or harsh reaction conditions. nase, an aqueous mixture comprising a biological agent hav 0007 Another problem involves the provision of a suite of ing oxygen-dependent and cofactor-dependent carbohydrate reactions for the production of amines that has, in toto, a oxidase activity, a biological agent having transaminase redox balance of 0. activity, a biological agent having amino acid dehydrogenase 0008 Another problem underlying the present invention activity, oxygen, the Substrate amino acid of the amino acid is to provide a system that may be used to produce amines dehydrogenase, carbohydrate oxidase cofactor and amino starting with compounds comprising a hydroxyl group, pref acid dehydrogenase cofactor and preferably a biological erably attached to an aromatic or aliphatic carbon ring, with agent having formate or glucose dehydrogenase activity for out the need to Subject intermediates to purification and/or transaminating a hydrocarbon comprising a hydroxy group, extraction procedures or have a change of Solvent. and a use of Such an aqueous reaction mixture. 0009. The problem underlying the present invention is 0002 The era of non-renewable fossil fuels is coming to solved by the subject-matter of the attached independent and an end. While chemists have been able to rely on a virtually dependent claims. unlimited Supply of coal, petroleum and natural gas, the avail 0010. In a first aspect, the problem underlying the present ability of such resources, formed by bacteria, plancton, plant invention is solved by a method comprising the steps and animal matter buried in ocean sediments over millions of 0011 (a) contacting a hydrocarbon comprising a years, is expected to be limiting in the very near future. hydroxyl group with Moreover, burning fossil fuels has been linked with the 0012 a biological agent having oxygen-dependent and increase in atmospheric concentrations of CO and associated cofactor-dependent carbohydrate oxidase activity climate changes, most notably global warming. Therefore, 0013 in the presence of oxygen and carbohydrate oxi next generation processes for the production of chemicals dase cofactor, and conventionally derived from fossil resources will have to start 0.014 (b) contacting the hydrocarbon produced in step with renewable resources, i.e. materials that are easily and, in a) with terms of geological time scales, rapidly replenishable. 0015 a biological agent having transaminase activity 0003 Industrial biotechnology, i.e. the application of bio and catalysts such as enzymes or organisms as industrial cata 0016 a biological agent having cofactor-dependent lysts, offers alternatives to many conventional processes amino acid dehydrogenase activity using fossil resources as input. Not only are biocatalysts able 0017 in the presence of amino acid dehydrogenase to convert compounds made from renewable materials, often cofactor and the Substrate amino acid of the amino acid agricultural or process wastes that would otherwise have to be dehydrogenase. disposed of, but they do not require the use of toxic com 0018. In an embodiment of the first aspect, the hydrocar pounds and, last but not least, reduce greenhouse gas emis bon comprising a hydroxyl group comprises a 5 or 6 mem sions compared to conventional approaches. bered ring carrying at least one Substituent selected from the 0004 Amines, organic derivates of ammonia, represent of group comprising —(CH), OH, wherein X is 0 to 4. a class of industrially sought-after compounds, including 0019. In an embodiment of the first aspect, the hydrocar bulk chemicals such as aniline, often prepared by conversion bon comprising a hydroxyl group is selected from the group of compounds made from fossil carbon sources such as halo of compounds represented by formulae (I) or (II) genated alkanes or alkenes. The reactions used for the Syn thesis of amines depend on the use of extreme temperatures, toxic compounds Such as HCN and expensive or environmen (I) tally hazardous catalysts. Examples of industrially relevant 2N, 1N OH amines include aniline, a precursor to various industrial E A. chemicals, lysine, an essential amino acid, and di-(aminom ethyl)-furan (DAMF), a monomer used for the production of polymers. 0005. The prior art teaches a chemical route towards the (II) synthesis of DAMF and chemically related amines, involving the stepwise oxidation of HMF to DFF, followed by transfor mation to the corresponding dioxime and Subsequent nickel catalyzed reduction of the latter (El Hajj et al., 1987). A biotechnological route based on renewable materials such as sugars would be desirable. 0020 wherein up to two out of A, B, C, D, E and F are 0006. Therefore, the problem underlying the present atoms each and independently selected from the group invention is to provide a system that may be used to produce comprising N, S and O and the others are C, US 2014/O120587 A1 May 1, 2014

(0021 wherein R is selected from the group comprising 0041. In an embodiment of the first aspect, one or more, H. halogen, Substituted or unsubstituted alkyl, alkenyl, preferably all, of the biological agents selected from the alkynyl, -(CH2), CRO. —(CH), NH, -(CH.) group comprising the biological agent having oxygen-depen NO. (CH), O R. (CH), CHSH, dent and cofactor-dependent carbohydrate oxidase activity, —(CH), COOR, the biological agent having transaminase activity, the biologi (0022 wherein X is 0 to 20, preferably 0 to 4, more cal agent having formate or glucose dehydrogenase activity preferably 1 to 2. and the biological agent having cofactor-dependent alanine (0023) and wherein R is selected from the group com dehydrogenase activity are associated with a viable cell. prising H and substituted or unsubstituted alkyl, 0042. In a second aspect, the problem underlying the cycloalkyl, aryland heteroaryl. present invention is solved by a use of a mixture comprising 0024. In an embodiment of the first aspect, up to two out of a biological agent having oxygen-dependent and cofactor A, B, C, D, E and Fare atoms each and independently selected dependent carbohydrate oxidase activity, a biological agent from the group comprising N and O and the others are C, having transaminase activity, a biological agent having amino I0025 wherein R is selected from the group comprising acid dehydrogenase activity, oxygen, the Substrate amino acid (CH), CRO, —(CH), NH, -(CH), O of the amino acid dehydrogenase, carbohydrate oxidase R" and -(CH), COOR, cofactor and amino acid dehydrogenase cofactor and prefer 0026 wherein X is 0 to 4, more preferably 1 to 2. ably a biological agent having formate or glucose dehydro 10027) and wherein R is selected from the group com genase activity for transaminating a hydrocarbon comprising prising H and alkyl comprising 1 to 4 carbon atoms. a hydroxyl group, wherein one or more than one, preferably 0028. In an embodiment of the first aspect, A, B, C, D, E all biological agents are or comprise a heterologous polypep and F are all C atoms. tide having the activity of interest. 0029. In an embodiment of the first aspect, the hydrocar 0043. In a third aspect, the problem underlying the present bon comprising a hydroxyl group is a cycloalkanol, prefer invention is solved by an aqueous reaction mixture compris ably cyclohexanol. ing a hydrocarbon comprising a hydroxyl group, a biological 0030. In an embodiment of the first aspect, the oxygen agent having oxygen-dependent and cofactor-dependent car dependent and cofactor-dependent carbohydrate oxidase bohydrate oxidase activity, oxygen, carbohydrate oxidase activity is an activity provided by an enzyme selected from cofactor, a biological agent having transaminase activity, a the group comprising the M1 variant of galactose oxidase biological agent having amino acid dehydrogenase, amino from Fusarium NRRL 2903, pyranose oxidase from Phan acid dehydrogenase cofactor, the substrate amino acid of the erochaete chrysosporium, hexose oxidase from Chondrus amino acid dehydrogenase and preferably a biological agent crispus and homologues thereof. having formate or glucose dehydrogenase activity, wherein 0031. In an embodiment of the first aspect, the amino acid one or more than one, preferably all biological agents are or dehydrogenase is alanine dehydrogenase. comprise a heterologous polypeptide having the activity of 0032. In an embodiment of the first aspect, the transami interest. nase is selected from the group comprising the co-transami 0044. In a preferred embodiment of the third aspect, each nases from Vibrio fluvialis and Paracoccus denitrificans and of the biological agents specified is a whole cell biocatalyst homologues thereof. comprising one or more heterologous active polypeptides 0033. In an embodiment of the first aspect, step b) is car having the activities of interest, preferably one whole cell ried out in the presence of a biological agent having formate biocatalyst comprising more than one heterologous active dehydrogenase activity. polypeptides has all the activities of interest. 0034. In an embodiment of the first aspect, step b) is car ried out in the presence of a biological agent having glucose 0045. Further embodiments of the second and third aspect dehydrogenase activity. of the present invention comprise the embodiments of the first 0035. In an embodiment of the first aspect, the redox factor aspect of the present invention. produced by the biological agent having amino acid dehydro 0046. The present invention is based on the surprising genase activity is consumed by the biological agent having finding that a sequence of reactions exists that may be used to formate or glucose dehydrogenase activity. catalyse the oxidation and Subsequent transamination of a 0036. In an embodiment of the first aspect, step a) is car hydrocarbon comprising a hydroxyl group, wherein said ried out in the presence of an H-O-degrading activity. hydroxyl group is converted to an amine group, based entirely 0037. In an embodiment of the first aspect, the HO on biotechnological catalysts. Moreover, the present inven degrading activity is provided by a biological agent selected tors have Surprisingly found that all of these reactions are from the group comprising catalase and horse radish peroxi fully compatible with each other and may be carried out dase/ABTS and homologues thereof. simultaneously in the same reaction vessel, using a uniform 0038. In an embodiment of the first aspect, steps a) and b) buffer system. Moreover, the present inventors have surpris are carried out simultaneously in the same reaction mixture. ingly found a suitable sequence of reactions that may be 0039. In an embodiment of the first aspect, at least one carried out without an intoto addition or removal of electrons. component essential for the transaminase activity, preferably 0047. The inventive process may be used to convert a the biological agent having transaminase activity, is added to broad range of compounds. In a preferred embodiment, the the reaction mixture following addition of the components term "hydrocarbon comprising a hydroxyl group', as used essential for oxygen-dependent and cofactor-dependent car herein, refers to any organic compound comprising two car bohydrate oxidase activity. bonatoms linked via a C-C bond and comprising at least one 0040. In an embodiment of the first aspect, the level of hydroxyl group, the latter preferably attached to a carbon oxygen pressure is 2 to 7 bar, preferably 3 to 5 bar. atom. In a preferred embodiment, the carbon chain of Such a US 2014/O120587 A1 May 1, 2014

hydrocarbon is selected from the group comprising cyclic and ment, transaminase activity is provided by a transaminase or linear alkanes, alkenes, alkynes, alkyl or alkenyl aryls and homologues thereof. In another preferred embodiment, cata alkyl or alkenyl heteroaryls. lase activity is provided by a catalase or a homologue thereof. 0048. In a preferred embodiment, the hydrocarbon com In another preferred embodiment, amino acid, preferably ala prising a hydroxyl group is selected from the group of com nine, dehydrogenase activity is provided by an amino acid pounds represented by formula (III): dehydrogenase, preferably alanine dehydrogenase, or a homologue thereof. In another preferred embodiment, for mate dehydrogenase activity is provided by a formate dehy (III) drogenase or a homologue thereof. In another preferred embodiment, glucose dehydrogenase activity is provided by a glucose dehydrogenase or a homologue thereof. In another preferred embodiment, the biological agent is a polypeptide, preferably an isolated polypeptide, or a whole-cell biocata lyst. The biological agent, regardless of its nature, may be I0049 wherein R and R are each and independently immobilised. any chemical group and preferably part of an aromatic ring. 0057. In a preferred embodiment, the term “presence of a cofactor, as used herein, means that a cofactor required for 0050. In a preferred embodiment, the hydrocarbon com an activity or enhanced activity of an activity and/or enzyme prising a hydroxyl group is selected from the group of com is present, either free in Solution and/or tightly associated pounds represented by formula (IV): with said activity and/or enzyme. 0058. It is clear that reaction conditions compatible with the activities involved have to be chosen, more specifically (IV) pH, buffer, buffer concentration and the like. In a preferred embodiment, the concentration of buffer, preferably sodium phosphate, is more than 25 mM, preferably 25 to 250 mM, more preferably 40 to 220 mM, most preferably 90 to 160 mM, and the pH is 5 to 9, preferably 6.5 to 8.5. 0059. In a preferred embodiment, the term “activity”, as 10051 wherein each of R, R and R is each and inde used herein, refers to the ability to catalyse a chemical or pendently selected from the group comprising H. halo biological reaction. In other words, a chemical or biological gen, Substituted or unsubstituted alkyl, alkenyl, alkynyl, reaction reaches a state of equilibrium more rapidly if a Suit (CH), CRO, -(CH2), NH, -(CH2), NO, able activity is present. In a preferred embodiment, the activ -(CH2), OR. -(CH2), CH2SH, -(CH2), ity is displayed by a catalytically active polypeptide or cell COOR, comprising Such a polypeptide. 0052 wherein X is 0 to 20, preferably 0 to 4, 0060. In a most preferred embodiment, the biological I0053 and wherein R is selected from the group com agent is a biological cell. The person skilled in the art is prising H and substituted or unsubstituted alkyl, familiar with a wide range of biological cells that may be used cycloalkyl, aryland heteroaryl, to display a biological activity of interest. For example, the 0054 and wherein up to one or two carbonatomes of the cell may be a prokaryotic cell, preferably a bacterial cell such aromatic ring may each and independently be replaced as an Escherichia, Corynebacterium or Pseudomonas cell, by anatom selected from the group comprising N, O and most preferably an E. coli cell. The biological cell may also be S. a eukaryotic cell, preferably a unicellular eukaryotic cell, 0055. In a most preferred embodiment, the hydrocarbon most preferred a fungal biological cell, for example Candida comprising a hydroxyl group is HMF. tropicalis, Saccharomyces cerevisiae or Pichia pastoris. The 0056. In a preferred embodiment, the term “biological person skilled in the art is also familiar with a wide range of agent, as used herein, refers to any organism or molecule of techniques that may be used to genetically alter cells such that biological origin that has the activity specified. In a more they express a biological or other activity of interest. preferred embodiment, the biological agent is a purified or 0061 Step a) of the present invention involves the use of a isolated active biological macromolecule, most preferably a biological agent having carbohydrate oxidase activity. In a peptide or polypeptide, wherein the term "isolated’, as used preferred embodiment, the term “carbohydrate oxidase activ herein, refers to, in a particularly preferred embodiment, a ity', as used herein, refers to an enzymatic activity that is able biological macromolecule that has been Subjected to an iso to oxidise a carbohydrate, preferably a carbohydrate capable lation or purification procedure and is present at a purity of forming, at least transiently, a five- or six-membered ring higher than it is in its endogenous environment Such as the structure comprising at least one hydroxyl group, wherein at inside of the respective wild type cell. In another more pre least one hydroxyl group is oxidised, preferably such that it is ferred embodiment, the biological agent is a whole cell bio replaced by a carbonyl group. In a preferred embodiment, the catalyst comprising or being associated with Such a biological term "hydrocarbon produced in step a)', as used herein, macromolecule. For example, the biological macromolecule means that the hydrocarbon subjected to contact with a bio may be located inside the cell or be attached to a cellular logical agent having carbohydrate oxidase activity under the membrane, for example at the inside of the membrane in conditions specified with respect to step a) have the opportu contact with the cytosol or at the outside of the membrane in nity to first interact with the agent having carbohydrate oxi contact with the environment. In a preferred embodiment, dase activity, subsequently the hydrocarbon modified by way carbohydrate oxidase activity is provided by a carbohydrate of interaction with the carbohydrate oxidase activity may oxidase or a homologue thereof. In another preferred embodi proceed to react with the reagents specified with respect to US 2014/O120587 A1 May 1, 2014

step b). This does not necessitate that the reagents and activi a formate dehydrogenase cofactor is reduced. In a preferred ties specified with respect to step b) be absent as step a) embodiment, the formate dehydrogenase cofactor is NAD". OCCU.S. In a preferred embodiment, the term “glucose dehydrogenase 0062. The person skilled in the art knows how to prepare activity, as used herein, refers to an activity capable of oxi biological agents having adequate activities, in particular by dising glucose, wherein NADH or NADPH is produced. The introducing Suitable polypeptides into cellular hosts by use of formate or glucose dehydrogenase requires the pres means of genetic engineering using standard molecular biol ence of formate or glucose, respectively. ogy techniques. Moreover, the person skilled in the art knows 0068. In a preferred embodiment, the term “ CRO, as how to employ such cells as whole-cell catalysts or use them used herein, refers to an aldehyd substituent in case R is as a starting material to prepare more or less isolated polypep hydrogen or to a keto substituent if R is an organic substitu tides having the activity of interest. Finally, the person skilled ent, wherein C is covalently bound to two carbon atoms. in the art is familiar with ways to preserve such activities or 0069. It is a particular strength of the present invention that use it to catalyse chemical reactions, for example by addition the sequence of reactions devised to oxidise and transaminate of essential cofactors, buffer reagents and adjusting pH and an alcohol may be chosen such that it has a net redox balance temperature conditions in a Suitable manner. Finding Such of 0, i.e. it does in toto not require the addition or removal of conditions is a matter of routine experimentation. electrons and/or oxidised or reduced redox cofactors. This is 0063. In a preferred embodiment, the carbohydrate oxi accomplished by employing an amino acid dehydrogenase, dase activity is an oxygen-dependent carbohydrate oxidase preferably analanine dehydrogenase that releases, in an oxi activity, i.e. it depends on the presence of molecular oxygen dised form, the cofactor used by the formate dehydrogenase as an oxidant. The person skilled in the artis aware of methods to oxidase formate or by the glucose dehydrogenase to oxi that may be used to Supply oxygen in an aqueous Solution, for dise glucose. However, the oxidation and amination may also example by bubbling gaseous oxygen through the aqueous be carried out in the absence of enzymes other than those from Solution or by adding to the aqueous solution oxygen-releas the group comprising carbohydrate oxidase, transaminase ing agents. and amino acid dehydrogenase. 0064. The carbohydrate oxidase activity is, aside from 0070 The present inventors have found that it is advanta oxygen, also dependent on a cofactor. In a preferred embodi geous to carry out step a) in the presence of H-O-degrading ment of the present invention, the term “cofactor', as used activity. In a preferred embodiment, the term “HO-degrad herein, refers to a redox factor that is either tightly associated ing activity, as used herein, refers to any activity that reduces with the molecule having the activity of interest or transiently the amount of H2O present in a given aqueous Solution. The interacting with Such a molecule, preferably as a Substrate. In person skilled in the art is familiar with many catalysts, of a preferred embodiment, the carbohydrate oxidase uses its inorganic, organic or even biological origin, that may be used cofactor as an oxidant, i.e. to sink electrons released upon for providing H2O-degrading activity. oxidation of the Substrate’s hydroxyl group. In another pre 0071 Moreover, the present inventors have surprisingly ferred embodiment of the present invention, the carbohydrate found that the entire Suite of reactions presented, i.e. no less oxidase cofactor is copper or a flavine cofactor, more prefer than five independent chemical reactions, are entirely com ably FAD". In another preferred embodiment, the cofactor is patible with respect to each other, enabling the person skilled selected from the group comprising FAD", NAD" and in the art to carry out all the reactions simultaneously in the NADP. same reaction vessel without the need to add or mix compo 0065 Step b) of the present invention involves the use of a nents in a particular order or to purify an intermediate. biological agent having transaminase activity. In a preferred 0072. In a preferred embodiment, homologues of any embodiment, the term “transaminase activity, as used amino acid or nucleic acid sequences referred to in this appli herein, is an activity capable of catalysing the conversion of a cation explicitly, for example by name or accession number or carbonyl function to an amine function. In a more preferred indeed the term “homologue of, or implicitly, for example by embodiment, the term “transamination activity, as used functional characterisation, are within scope of the present herein, refers to an activity that is able to catalyse the conver invention. In a preferred embodiment, the term “homologue'. sion of a carbohydrate having a hydroxyl group to a carbo as used herein, comprises amino acid or nucleic acid hydrate having an amine group, with the amine function sequences, respectively, that are 60, 70, 75,80, 85,90,92,94, replacing the hydroxyl group. 95, 96, 97,98 or 99% identical to the reference amino acid or 0066 Step b) also requires participation of an amino acid nucleic acid sequence. In a preferred embodiment, the term dehydrogenase activity. In a preferred embodiment, the term “homologue', with regard to amino acid sequence, com “amino acid dehydrogenase activity, as used herein, refers to prises, preferably in addition to the above sequence identity, an activity that is able to catalyse the conversion of an acid amino acid sequences that comprise one or more conservative comprising an amine function, i.e. an amino acid, preferably amino acid changes with respect to the reference sequence. In alanine, to an acid, wherein the amino function is replaced by a preferred embodiment, the term “homologue' of an amino carbonyl function. In a most preferred embodiment, the acid sequence or nucleic acid sequence comprises, preferably amino acid dehydrogenase is analanine dehydrogenase catal in addition to the above sequence identity, active portions ysing the conversion of alanine to pyruvate. In a most pre and/or fragments of the amino acid sequence or nucleic acid ferred embodiment, the amino acid dehydrogenase is depen sequence, respectively, and fusion constructs comprising the dent on a cofactor. In a preferred embodiment, the amino acid sequences or active portions and/or fragments thereof. In a dehydrogenase cofactor is NADH. preferred embodiment, the term “active portion', as used 0067 Finally, step b) may also involve a formate or glu herein, refers to an amino acid sequence or a nucleic acid cose dehydrogenase activity. In a preferred embodiment, the sequence, which is less than the full length amino acid term “formate dehydrogenase activity”, as used herein, refers sequence or codes for less than the full length amino acid to an activity capable of converting formate to CO., wherein sequence, respectively, wherein the amino acid sequence or US 2014/O120587 A1 May 1, 2014 the amino acid sequence encoded, respectively retains at least 007.9 FIG. 4 shows the results of pilot experiments aiming Some of its essential biological activity, e.g. as a carbohydrate to optimise the oxygen pressure with respect to the oxidation oxidase, co-transaminase, catalase, formate or glucose dehy reaction catalysed by the M1 variant of galactose oxidase drogenase or amino acid dehydrogenase. In a preferred from Fusarium NRRL2903. embodiment, the term “retains at least some of its essential 0080 FIGS. 5a and 5b show mass spectrometry results biological activity, as used herein, means that the amino acid demonstrating formation of benzylamine following oxidation sequence in question has a biological activity exceeding and and amination of benzylalcohol. distinct from the background activity and the kinetic param I0081 FIG. 6 shows NMR data demonstrating formation of eters characterising said activity, more specifically k, and DAMF following oxidation and amination of HMF. K. are preferably within 3, more preferably 2, most prefer ably one order of magnitude of the values displayed by the EXAMPLE 1. reference molecule with respect to a specific Substrate. In a preferred embodiment, the term “homologue' of a nucleic Oxidation of Various Cyclic Compounds Comprising acid comprises nucleic acids the complementary Strand of a Hydroxyl Function by Carbohydrate Oxidases which hybridises, preferably under stringent conditions, to 0082. A range of cyclic compounds comprising a the reference nucleic acid. Stringency of hybridisation reac hydroxyl group (FIG. 2) in addition to HMF was subjected to tions is readily determinable by one of ordinary skilled in the carbohydrate oxidase oxidation as follows. A whole cell art, and in generally is an empirical calculation dependent on preparation of the corresponding oxidase (20 mg, lyophilised probe length, washing temperature and salt concentration. In dry weight) was resuspended in the corresponding phosphate general longer probes require higher temperatures for proper buffer 1 mL: standard conditions: galactose oxidase: 100 annealing, while shorter probes need lower temperatures. mM sodium phosphate, pH-7, CuSO4.5H2O (3 mg/mL); Hybridisation generally depends on the ability of denatured pyranose and hexose oxidase: 100 mM Sodium phosphate, DNA to reanneal to complementary strands are present in an pH=8, 1 mM FAD at 30° C. and 120 rpm for 20 min (hori environment below their melting temperature. The higher the Zontal position). Afterwards, horseradish peroxidase (Sigma, degree of desired homology between the probe and hybridis # P8125) (30 uL. 10 mg/mL), 2,2'-azino-bis(3-ethylbenzthia able sequence, the higher the relative temperature which may Zoline-6-sulfonic acid) (ABTS) (30 uL. 10 mg/mL) and the be used. As a result, higher relative temperatures would tend respective hydrocarbon, for example HMF (5 mg, 0.04 mmol) to make the reaction conditions more stringent, while lower were added and the reaction mixture was transferred to an temperature less so. For additional details and explanation of oxygen apparatus consisting of a plexiglass cylinder (27 cm stringency of hybridisation reactions, see Ausubel et al. length}x10 cm diameter). The apparatus was primed with (1995). In a preferred embodiment, the term “homologue' of oxygen (technical grade) for about 1 min and pressurized to 4 a nucleic acid sequence, as used herein, refers to any nucleic bar. The whole apparatus was shaken at room temperature, acid sequence that encodes the same amino acid sequence as 170 rpm, 20 h (vertical position of the MG5 vial). As for the reference nucleic acid sequence, in line with the degen catalase, 20 uL of the stock solution Catalase from Micro eracy of the genetic code. coccus lysodeikticus (ca. 170000 U/mL, Sigma Aldrich 0073. In a preferred embodiment of the present invention, #60634), Catalase from Corynebacterium glutamicum the term “associated with a viable cell’, as used herein, means (>500000 U/mL, Sigma Aldrich #02071) were used instead that the biological agent characterised as Such is a polypep of horseradish peroxidase and 2,2'-azino-bis(3-ethylbenz tide located in the inside of the cell or attached to the surface thiazoline-6-sulfonic acid) (ABTS). or to the membrane of a cell. In a more preferred embodiment, I0083 Various carbohydrate oxidases, more specifically the polypeptide is fused to a membrane protein. galactose oxidase from Fusarium NRRL 2903 (M1 variant) (Escalettes et al., 2008), pyranose oxidase from Phanerocha 0074. In a most preferred embodiment, the hydrocarbon ete chrysosporium and hexose oxidase from Chondrus cris comprising a hydroxyl group is HMF, and the carbohydrate pus, used in recombinant form in E. coli BL21 (DE3) as lyo oxidase is preferably the M1 variant of galactose oxidase philized whole cells and obtained as described in Example 7. from Fusarium NRRL 2903 (Escalettes & Turner, 2008) or could be shown to convert the hydroxy groups of a range of the pyranose oxidase from Phanerochaete chrysosporium or cyclic substrates, including HMF, benzyl alcohol and ortho the hexose oxidase from Chondrus crispus or a homologue of hydroxybenzyl alcohol, to the corresponding carbonyl any of them, and the transaminase is Vibrioflurialis or Para coccus denitrificans transaminase or a homologue thereof. groups. 0075. The present invention is further illustrated by the EXAMPLE 2 following figures and non-limiting examples from which fur ther features, embodiments, aspects and advantages of the Dependency of the Oxidation Step on pH and Buffer present invention may be taken. Concentration 0076 FIG. 1 depicts a scheme illustrating the inventive I0084. The reaction was carried out in aqueous solution in Suite of reactions. phosphate buffer using the M1 variant of galactose oxidase from Fusarium NRRL 2903 understandard conditions, i.e. 40 0077 FIG. 2 depicts the range of components subjected to mmol HMF. 10 mg whole-cell preparation of galactose oxi oxidation, as described in Example 1, and Subsequent tran dase, ABTS (30 uL. 10 mg/mL), horse radish peroxidase (30 samination, as described in Example 5, in addition to HMF. uL. 10 mg/mL) and 100 mM sodium phosphate buffer pH 7. 0078 FIG.3 shows the results of pilot experiments aiming The oxygen pressure was 4 bar. The optimum pH was 7.0 for to optimise pH and buffer concentration with respect to the galactose oxidase (Tab. 2, FIG.3) As for catalase, 20LL of the oxidation reaction catalysed by the M1 variant of galactose stock Solution Catalase from Micrococcus lysodeikticus (ca. oxidase from Fusarium NRRL2903. 170000 U/mL, Sigma Aldrich #60634), Catalase from US 2014/O120587 A1 May 1, 2014

Corynebacterium glutamicum (>500000 U/mL, Sigma Ald from Vibrio fluvialis (Vf-coTA) or Paracoccus denitrificans rich #02071) were used instead of horseradish peroxidase (pCR6) transaminases, see WO 2010/089171 for details of and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) transaminases (ABTS). I0089. The transaminases were used as lyophilized whole cell preparation (recombinantly in E. coli), alanine dehydro 0085. A similar suite of experiments was carried out and genase from Bacillus subtilis was purified prior to use and showed that the optimal pH value is 8.0 if pyranose oxidase is formate dehydrogenase was obtained from Codexis (#24.11, used. However, oxidation takes place over a wide pH range H624 11.01), see Example 7. from 5.0 to 9.0 in the case of both enzymes. Reaction Conditions were as Follows: 0090 Whole cell preparation of galactose oxidase from EXAMPLE 3 Fusarium NRRL 2903 (20 mg, lyophilised dry weight) was resuspended in sodium phosphate buffer (500 uL. 100 mM, Effect of Various Buffer Concentrations on the pH=7.0, 3 mg/mL CuSO4.5H2O) at 30° C. and 120 rpm, 30 Oxidation Steps min. Horseradish peroxidase (15uL. 10 mg/mL), ABTS (15 uL. 10 mg/mL) and HMF (5 mg, 0.04 mmol) were added and I0086 A range of experiments was carried out under stan the reaction mixture was placed into the oxygen apparatus dard conditions as specified in Example 1, except for the fact (vertical position, room temperature, 4 bar O. 170 rpm) for that various concentrations of buffer were used. It could be 20h. The reaction mixture was transferred to Eppendorf vials. shown that higher buffer strengths led to an increase in con A solution of whole cell preparation of ()-transaminase from version (FIG. 3). Vibrio fluvialis (20 mg, lyophilised dry weight) in P-buffer (500 uL. 100 mM, pH=7.0, 2 mM PLP, 200 mmol L-alanine, TABLE 2 140 mmol ammonium formate) was added, followed by for mate dehydrogenase (20 uL. Codexis) and purified alanine Optimisation of pH and buffer concentration using the M1 dehydrogenase from Bacillus subtilis (20 uL, 7.5 mg protein/ variant of galactose oxidase from Fusarium NRRL2903. mL). The reaction mixture was shaken at 30° C. and 120 rpm in a horizontal position for 20 h. The apparatus was primed Entry pH buffer strength mM yield .9% with oxygen for about 1 min and pressurized to 4 bar. The 1 S.O 50 38 reaction could be reproduced using as carbohydrate oxidase 2 6.O 50 32 3 7.0 50 34 pyranose and hexose oxidase. The reaction conditions were 4 8.O 50 31 identical except for the fact that the pH value was adjusted to 5 S.O 100 46 8, and 1 mM FAD was added rather than CuSO4.5H2O. 6 6.O 100 44 0091. This example illustrates that the transamination 7 7.0 100 42 reaction is fully compatible with the conditions of the oxida 8 8.O 100 47 tion reaction mixture. Irrespective of the transaminase used, 9 S.O 150 S4 10 6.O 150 65 HMF was converted to DAMF at a yield of more than 80% 11 7.0 150 69 after 20 h reaction time, more specifically 81% and 83% if 12 8.O 150 51 Vf-coTA or pCR6 transaminases were used. Other com pounds were oxidised and transaminated at a total combined yield as shown in Tab. 3. DFF formed during the oxidation step was quantitatively converted to DAMF. Remaining HMF EXAMPLE 4 did not inhibit the transamination, but was converted to the corresponding hydroxymethylamine. In an analogous fash Effect of Elevated Levels of Oxygen on the ion, HMF was converted by all transaminases to 5-(aminom Oxidation Step ethyl)furan-2-yl)methanol (AMFM). The rate of conversion and the identity of the products were determined using GC 0087 Pilot experiments were carried out to investigate the MS. dependency of the reaction on the oxygen pressure applied. More specifically, 40 mmol of two substrates, benzyl alcohol TABL33 and furfuryl alcohol, were subjected to oxidation by the galactose oxidase as specified in Example 1, horse radish A range of Substrates, see FIG. 2, were subjected to the sequential peroxidase (30 uL. 10 mg/mL) and ABTS (30 uL. 10 mg/mL) oxidation and transamination steps as described in examples 1 and 5. in 100 mM sodium phosphate buffer pH 7. The application of Substrate Rate of conversion 96 oxygen at elevated pressure was shown to be optimal at 4 bar 1 48 (FIG. 4). 2 56 3 51 EXAMPLE 5 4 Could not be extracted 5 13 6 33 One-Pot Transamination of Oxidation Products of 7 33 Various Cyclic Compounds Comprising a Hydroxyl Group in the Oxidation Reaction Mixture EXAMPLE 6 0088. Following incubation of the reaction mixture in Example 1 for 20h, the transamination reaction mix consist Simultaneous Oxidation by Carbohydrate Oxidases ing of transaminase, L-alanine, alanine dehydrogenase, and Transamination of Various Cyclic Compounds NADH and the corresponding recycling system (see above) Comprising a Hydroxyl Function was added and the reaction was shaken for another 20h. DFF 0092. The same experimental setup as outlined in was transaminated employing the recombinant transaminase Example 1 was added, except for the fact that HMF was used US 2014/O120587 A1 May 1, 2014

as a Substrate and all reagents required for the oxidation and liver rather than alanine dehydrogenase from Bacillus sub transamination were added right at the beginning, including tilies. Alternative transaminases, namely co-transaminase pyranose oxidase from Phanerochaete chrysosporium, from Alcaligenes denitrificans Y2k-2 (Ad-coTA) and co-tran horseradish peroxidase and ABTS, ()-transaminase from saminase from Pseudomonas putida KT2440 (Gene PP5182, Vibrio fluvialis, formate dehydrogenase and alanine dehydro pCR7) were used in experiments 4 and 5, respectively. In genase from Bacillus subtilis. 1 mM FAD was present and the addition, another amino acid dehydrogenase, glutamic dehy pH was 8. The reaction was allowed to proceed for 24 h. drogenase from Candida utilis, was used in experiment 5. 0093. The reaction was reproduced using as carbohydrate oxidase hexose oxidase under identical reaction conditions. 0096. The data presented illustrate that carrying out the inventive sequence of reactions in the same reaction vessel is EXAMPLE 7 optional. Moreover, alternative transaminases and amino acid dehydrogenases may be used. Finally, the presence of for Oxidation and Transamination by Alternative mate dehydrogenase is another optional feature. Transaminases, Amino Acid Dehydrogenases in the Absence/Presence of Formate Dehydrogenase, TABLE 4 Comprising Isolation of the Oxidation Product 0094. The oxidation reaction was performed as described Substrates used in Example 7. in Example 1, using compounds 1, 2 and 3, as depicted in Table 4, as Substrates. However, down stream reactions, more specifically the transamination, were not carried out simulta neously in the same reaction vessel, but the product of the try / 1 oxidation reaction was isolated by extraction using EtOAC (2x500LL), the conversion determined using GC-FID and the OH solvent was removed via a positive stream of air. The oxida tion product remaining in the vessel was Subsequently used for the transamination step, which was performed as described in example 5 of the application as filed, with modi fications and results as Summarised in Table 5. C 0095 Five experiments were conducted with each sub OH strate, wherein the first experiment (entries 1, 6 and 11) was each carried out under standard conditions, i.e. those described in the patent application, the second reaction was carried out in the absence of formate dehydrogenase (FDH) in c the presence of equimolar NADH cofactor, the third reaction was carried out using glutamic dehydrogenase from bovine TABLE 5

Sedential Oxidation - Transamination sequence with isolation of the intermediate.

Reaction conditions - Transamination Conv.9% Entry Substrate FDH AA-DH (OTA NADH Ox. Alc. Ald. Amine + Ala-DH Vf-coTA — >99 <1 <1 >99 - Ala-DH Vf-coTA 2 equiv. >99 <1 <1 >99 + BL-Glu-DH. Vf-coTA — >99 <1 <1 >99 + Ala-DH Ad-oTA — >99 <1 <1 >99 + CU-Glu-DH. pCR7 >99 <1 <1 >99 + Ala-DH Vf-coTA — >99 <1 <1 >998 - Ala-DH Vf-coTA 1 equiv. >99 <1 <1 >998 + BL-Glu-DH Vf-coTA — >99 <1 <1 >998 + Ala-DH Ad-oTA — >99 <1 <1 >998 + CU-Glu-DH pCR7 >99 <1 <1 >998 + Ala-DH Vf-coTA — >99 <1 5 95 - Ala-DH Vf-coTA 1 equiv. >99 <1 <1 >99 + BL-Glu-DH Vf-coTA — >99 <1 3 97 + Ala-DH Ad-oTA — >99 <1 1 89 1 5 + CU-Glu-DH. pCR7 >99 15 <1 85

Conversion was determined using GC FID (oxidation) and GC-MS (2nd step); + = FDH was added, - = no FDH was added; Ala-DH=alanine dehydrogenase from Bacilius subtiis, BL-Glu-DH = glutamic dehydrogenase from Bovine Liver, CU-Glu-DH = Glutamic Dehydrogenase from Candida utilis; Vf-coTA = ()-transaminase from Vibrio fluviais, Ad-coTA = ()-transaminase from Alcaiigenes denitrificans Y2k-2, pCR7 = ()-transaminase from Pseudomonas putida KT2440 (Gene PP5182); conversion of the oxidative reaction step; 'conversion towards the diamine is given, determined by derivatised GC-MS sample and HPLC-UV-analysis; 3conversion was determined by derivatised GC-MS-sample. US 2014/O120587 A1 May 1, 2014

EXAMPLE 8 TABLE 6-continued Use of Glucose Dehydrogenase in Lieu of Formate Substrates used in Example 6 and yields observed. Dehydrogenase and Further Substrates for the Oxidation/Transamination Reaction 0097. A range of substrates, see Table 6 was subjected to the oxidation and transamination reaction as described in CrC Example 6 except for the fact that glucose dehydrogenase (X-zymes, it B4A, 20 U), ammonium chloride (200 mM, 5 96% conv. equiv.) and glucose (140 mM, 2.8 equiv.) were used instead of FDH and ammonium formate. 0098. It could be demonstrated that additional substrates, Or OH notably with halogen Substituents, may be oxidised and tran C saminated using the inventive method. 68% conv.

TABLE 6 Substrates used in Example 6 and yields observed. Or OH F

OH 75% conv. Or {O OH >99% conv. O

OH 35% conv. c Cr >99% conv. 92% conv. O OH >97% conv. 82% conv. Derivat(2) HPLC: 1 OH (2) indicates text missing or illegible when filed OCOMe EXAMPLE 9 18% conv. Analytical Methods

OH (0099 Sample Preparation for HPLC-UV Measurements without Derivatisation. 0100. The reaction mixture was heated to 95° C. for 10 minto precipitate enzymes, diluted 1/1 with MeCN and cen trifuged (13000 rpm, 3 min). Samples of 100 uL were taken, orOMe diluted with 1 mL HO/MeCN 171 containing 0.1%TFA and 81% conv. subjected to HPLC-UV analysis using the following setup. Column: Phenomenex LUNA 5um C18(2) 100 A (250x4.60 mm); oven temp.: 30°C., flow: 0.25 mL/min, eluent A: HO OH (0.1%TFA), eluent B: MeCN (0.1%TFA); Gradient: 0-4 min 30% B, 4-30 min 30-100% B, 30-36 min 100% B, 36-46 min MeO O 30% B.

44% conv. Sample Derivatisation for GC-MS Measurements. 0101 Samples of 500 uL were treated with DMAP (500 uL. 10% in MeCH). Ethyl chloroformate (200LL) was added US 2014/O120587 A1 May 1, 2014

and the mixture was incubated at 60° C. and 300 rpm for 2 h. plate supplemented with 100 mg/ml Ampicillin and used to Extraction with dichloromethane (2x500 uL) and drying of grow E. coli Strain BL21 comprising the respective plasmid the combined organic phase over NaSO gave a sample was used to inoculate 50 ml of an over night culture Supple which was subjected to GC-MS analysis under following mented with the same amount of Ampicillin. Following incu conditions: Agilent 7890AGC system equipped with an Agi bation of said culture over night at 180 rpm and 30°C.4 ml of lent 975C mass-selective detector (El 70 eV) and a HP-5-MS the over night culture was used to inoculate 21 of the same column (30 mx0.25 mmx0.25 um film) using helium as car medium in a 51 flask. The flask was shaken at 37° C. and 120 rier gas at a flow of 0.55 mL/min. The following temperature rpm until an OD550 of 0.6 was reached, i.e. approximately program was used: 100°C., hold for 0.5 min, 10°C/min, to two to four hours. The expression of the respective enzyme 300° C. was subsequently induced by addition of 0.2 ml Anhydrotet racyclin (2 mg/ml), followed by incubation for three hours at FMOC Derivatisation for HPLC-UV Measurements. 30° C. and 180 rpm under vigorous shaking. Cells were then 0102 Sample preparation was performed to a standard spun down at 5000 rpm and 4°C. for 20 minutes. The super procedure involving formation of FMOC derivatives in a natant was disposed of the pellet was washed using physi glass HPLC vial, addition of borate buffer pH 9, addition of ological NaCl Solution, spun down and lyophilised. FMOC reagent and removing the FMOC reagent excess by 0105. In the case of alanine dehydrogenase, 4 g of the way of addition of EVA reagent, addition of dilution buffer. lyophilised cells were resuspended in phosphate buffer (50 The sample was subsequently subjected to HPLC analysis mM NaHPO, 300 mM. NaCl, 10 mM Imidazol) and lysed using ultrasonication (1 S pulse followed by incubation for 4 using the following conditions. Column: Phenomenex LUNA s, repeated for a total of 10 minutes). The cell lysate was spun 5um C18(2) 100 A (250x4.60mm); oven Temp. 30° C., flow: down twice (16000 rpm, 20 min, 4° C.), filtered using a 0.5 mL/min, eluent A: HO (0.1% TFA), eluent B: MeCN Syringe filter and applied to a His-trap column having a bed (0.1%TFA); Gradient: 0-2 min30% B, 2-15 min30-100% B, volume of 5 ml. Protein binding unspecifically was removed 15-25 min 100% B, 25-30 min 30% B. by applying phosphate buffer (50 mM NaH2PO4, 300 mM EXAMPLE 10 NaCl) comprising 20 mM imidazole. Protein was eluted using the same buffer, with a gradient from 50 mM to 80 mM Preparation of Enzymes imidazole being applied, followed by application of 60 ml of the same buffer comprising 150 mM imidazole Fractions 0103 Genes were optimized and synthesized by GeneArt comprising pure enzyme were pooled, dialyses against phos and delivered in a pMAL vector with amplicillin resistance. phate buffer to remove imidazole and concentrated via Protein-encoding insert was cut via Ndel and Xhol restriction Vivaspin tubes and centrifugation at 4000 rpm. enzymes (Fermentas) and ligated in a pET21a expression vector via T4 Ligase (Fermentas). If oxidases were used, REFERENCES enzymes were prepared using pET16b or pBT21 a plasmid constructs as follows: a fresh single colony from an LB agar 0106 T. El Hai, A. Masroua, J. C. Martin, and G. Decotes, plate supplemented with 100 mg/ml Ampicillin and used to Bull. Soc. Chim. Fr., 1987, 855-60 grow E. coli Strain BL21 comprising the respective plasmid 0107 F. M. Ausubel (1995), Current Protocols in Molecu was used to inoculate 50 ml of an over night culture Supple lar Biology. John Wiley & Sons, Inc. mented with the same amount of Ampicillin. Following incu 0.108 F. Escalettes; N.J. Turner, ChemBioChem 2008, 9, bation of said culture over night at 120 rpm and 30°C. 4 ml of 857-860 the over night culture was used to inoculate 600 ml of the 0109 R. Huang: W. Qi; R. Su; Z. He, Chem. Commun. same medium in a 31 flask. The flask was shaken at 37°C. and 2009, 46, 1115-1117. 120 rpm until an OD550 of 0.6 was reached, i.e. approxi 0110 D. Koszelewski; I. Lavandera; D. Clay; G. M. Gue mately two to four hours. The expression of the respective bitz: D. Rozzell; W. Kroutil, Angew. Chem. Int. Ed. 2008, enzyme was subsequently induced by addition of 0.6 ml 47, 9337-9340. IPTG (Isopropyl-b-D-thiogalactopyranosid, 238 mg/ml), fol 0111 P. D. Hanke, 2009, WO 2009/023174 A2, Chem. lowed by incubation over night at 20° C. and 120 rpm under Abst: 150:235,749. vigorous shaking. Cells were then spun down at 8000 rpm and (O112 F. Koopman; N. Wierckx, J. H. de Winde; H. J. 4° C. for 20 minutes. The supernatant was disposed of the Ruijssenaars, Bioresource Technol. 2010, 101, 6291-6296. pellet was washed using physiological NaCl Solution, spun 0113 M. P. J. van Deurzen: F. van Rantwijk; R. A. Shel down and lyophilised. don, J. Carbohydr. Chem. 1997, 16, 299-309. 0104. If other enzymes were used, constructs comprising 0114. T. H. de Koker; M. D. MoZuch: D. Cullen: J. the pASK-IBA35+ (IBA Biotagnology, Germany) plasmid Gaskell; P. J. Kersten, Appl. Environ. Microbiol. 2004, 70. backbone were used. A fresh single colony from an LB agar 5794-5800.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 6

<21 Os SEQ ID NO 1 &211s LENGTH: 1935 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence

US 2014/O120587 A1 May 1, 2014 11

- Continued <223> OTHER INFORMATION: pyranose oxidase from Phanerochaete chrysosporium

<4 OOs, SEQUENCE: 2 catatgatgt toctagacac aacac cattc agagcagacg aaccatacga cqtatt cata 6 O gCaggaag.cg gaccalatagg agcaa.cattc gcaaaactat gcgtagacgc aaacctalaga 12 O gtatgcatgg tagaaatagg agcagcagac agctt cacaa goaalaccalat gaaaggagac 18O ccaaacgcac Caagaa.gc.gt acaatticgga C caggacaag taccalat acc aggataccac 24 O aaaaaaaacg aaatagaata ccaaaaagac atagacagat t cqtaaacgt aataaaagga 3OO gcactaagca catgcago at accaacaa.gc aacaaccaca tagcaac act agacccaagc 360 gtag taagca acagoctaga caaac catt cataagcc tag gaaaaaacco agcacaaaac 42O c catt.cgtaa acctaggagc agaag cagta acaagaggag taggaggaat gag cacacac 48O tgga catgcg caacaccaga attctitcgca cca.gcagact tcaacgcacc acacagagaa 54 O agaccaaaac taa.gcacaga cqcagcagaa gacgcaagaa tatggaaaga cct at acgca 6OO caa.gcaaaag aaataatagg aacaa.gcaca acagaatticg accacagcat aagacacaac 660 c tag tact aa gaaaatacaa cqacatatt c caaaaagaaa acgtaataag agaatticago 72 O c cactaccac tag catgcca cagactaa.ca gacccagact acgtagaatg gcacgcaa.ca 78O gacagaatac tagaagaact attcacagac ccagtaaaaa gaggaagatt cacac tact a 84 O acaaac caca gatgcacaaa act agtatt C aaa Cactaca gaccaggaga agaaaacgaa 9 OO gtag actacg cactagtaga agacic tacta ccacacatgc aaaac cc agg aaacc cagoa 96.O agcgtaaaaa aaatatacgc aagaa.gctac gtagtag cat gcggagcagt agcaa.ca.gca O2O caagtactag caaacago.ca cataccacca gacgacgtag taataccatt cocaggagga O8O gaaaaaggaa goggaggagg agaaagagac gcaacaatac Caacaccact aatgccalatg 14 O c taggaaaat a cataacaga acaac caatg acattctgcc aagtag tact agacago agc 2OO

Ctaatggaag tagtaagaaa cccaccatgg C caggactag actggtggala agaaaaagta 26 O gcaaga cacg tagaag catt cccaaacgac ccaataccaa taccatt cag agacccagaa 32O ccacaagtaa caataaaatt cacagaagaa cacccatggc acgtacaaat acacagagac 38O gcattcagct acggagcagt agcagaaaac atggacacaa gagtaatagt agact acaga 44 O ttctitcggat acacagaacc acaagaagica aacgaac tag tatto caa.ca acact acaga SOO gacgcatacg acatgccaca accaa.cattcaaatt cacaa tdagccalaga cqacagagca 560 agagcaagaa gaatgatgga cacatgtgc aac at agcac taaaaatagg aggatacct a 62O cCaggaag.cg aaccacaatt catgacacca ggact agcac tacacct agc aggaacaa.ca 68O agatgcggac tag acacaca aaaaa.cagta ggaaacacac actgcaaagt acaca acttic 74 O aacaacct at acgtaggagg aaacggagta atagaaacag gatt.cgcagc aaa.cccaa.ca 8OO Ctaacaagca tatgctacgc aataagagca agcaacgaca taatagcaaa atticggalaga 86 O

Cacagaggat aataacticga g 881

<210s, SEQ ID NO 3 &211s LENGTH: 1845 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Hexose oxidase from Chondrus crispus US 2014/O120587 A1 May 1, 2014 12

- Continued <4 OOs, SEQUENCE: 3 catatgatga cattcago at aaaaacaaga aaaaacacag tag acaaact aaaaaacgaa 6 O galactagacic tact agtaat aggaggagga ataac aggag Caggagtagc act acaa.gca 12 O acagcaa.gcg gactaaaaac aggactagta gaaatgcaag actitcgcaga aggaacaagc 18O agcagaag.ca caaaactagt acacggagga ataagat acc taaaaacatt cacgtagga 24 O gtag tagcag acacagtaca agaaagagca gtag tacaag gaatagc acc acacatacca 3OO agac catt co caatgctact accaatatac gacgacccaa gcago acatt cqacatgttc 360 agcgtaaaaa tag caatgga cct at acgac agact agcag gagtaac agg alacacaatac 42O gcaaactaca caataa.gcag agaagaagta Ctacaaagag alaccaaacct aaaaag.cgac 48O aalactactag gag caggagt atacctagac tacgtaalaca acgacgcaag act agtaata 54 O gaaaacataa aagaag caga caac tagga ggactaatgg caa.gc.cacgt aaaagtagta 6OO ggagtact ac acgacgacala C9gaagaata aacggagtaa alagcaaaaga cct actalaca 660 gacgaagaat t caaataaa agcaaaaatg gtaataalaca Caacaggacc atggg talaca 72 O aaaacactag gactagacga caaaaacgga gaaggagaag taataagacic aacaaaagga 78O gtacacctag tagtag acag cagcagacta agagtaccac alaccalacata citt cqacagc 84 O ggaataggag acggaagaat gat attcgta gtaccalagag aaaacaaaac at actitcgga 9 OO acaacagaca Cagact acac aggagactac aaa.cacccala gagtagalaca agcagacgta 96.O gactacctac taaaagcaat aaacaacaga tacccagaag cagaaataac aataaacgac O2O atagaa.gcaa gctgggcagg act aagacca Ctact aagcg gaaacggaag cagcgactac O8O aacggaggag gaa.gcaacac aggaaaagta agcgacgaaa gct tcgaaaa Cctaataaaa 14 O acagcagtag catacagcaa agacgaagca agcagaggag aagtagaaaa aag cataagc 2OO agcctaaaaa Cagcaa.gc.gc agaaaaaa.ca ctaagcc caa gccaagtaag cagaggaagc 26 O agcctaaaag taggagacga C9gactaata acactaa.gcg gaggaaaaat alacagacitac 32O agaaaaatgg cagcaggagc aatggaacta ataagaaaac tatt caaaga act attcaac 38O aaagacgtaa gagaagtaga cagcaaaaaa ctacaagtaa gcggaggaca Ctt Caccca 44 O acaaacgtag acgaaacaat galaattctac Caaaaaatag caatggacaa aggaatgc.ca SOO gcagcagacg cagcagacct agcaaacaga titcggaagca acacaag cag agtaataagc 560 tacgcagacg acgtagaa.gc agcaccagga cta acactag cagacacact aag cctacac 62O tacagogtaa acgaagaaat gacactaaac ctagtag act tcc tact aag aagaacaaac 68O tacgtact at tccacaaaga cqaactaatg atgaaaaaag acgcatt cqt agacgaaatg 74 O gcaagaataa tigaatggaa caagaagaa aaa.gcagcag cagcaaaa.ca act agacgaa 8OO acaatagoag aaag.ca.gcct agcataccta aaataataac to gag 845

<210s, SEQ ID NO 4 &211s LENGTH: 1935 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: pASK-IBA35+-AlaDH for E. coli

<4 OOs, SEQUENCE: 4 catatgatgg cagogcgcc gattggcagc gcgatt.ccgc gcaacaactg. g.gc.ggtgacc 6 O tgcgatagcg cgcagagcgg caacgaatgc aacaaag.cga ttgatggcaa Caaagat acc 12 O

US 2014/O120587 A1 May 1, 2014 18

- Continued gtcagggggg C9gagcct at ggaaaaacgc cagcaacgcg gcctttittac ggttcctggc 4500 cittittgctgg ccttittgctic acatgacccg aca 4533

1. A method, comprising the steps: R" is selected from the group consisting of H, and an alkyl (a) contacting a hydrocarbon comprising a hydroxyl group comprising 1 to 4 carbon atoms. with a first polypeptide having oxygen-dependent and 4. The method according to claim 3, cofactor-dependent carbohydrate oxidase activity or a wherein the hydrocarbon is a cycloalkanol. whole-cell biocatalyst comprising the first polypeptide 5. The method according to claim 1, in the presence of oxygen and a carbohydrate oxidase wherein the amino acid dehydrogenase is alanine dehydro cofactor, and genase. (b) contacting the hydrocarbon with a second polypeptide 6. The method according to claim 1, having transaminase activity or a whole-cell biocatalyst wherein the transaminase is selected from the group con comprising the second polypeptide and sisting of co-transaminase from Vibrio fluvialis, co-tran a third polypeptide having cofactor-dependent amino saminase from Paracoccus denitrificans, and a homo acid dehydrogenase activity or a whole-cell biocata logue thereof. lyst comprising the third polypeptide in the presence 7. The method according to claim 1, of an amino acid dehydrogenase cofactor and a Sub wherein the contacting b) is carried out in the presence of a strate amino acid for an amino acid dehydrogenase, fourth polypeptide having formate dehydrogenase activ wherein the hydrocarbon comprises a 5 or 6 membered ring ity or a whole-cell biocatalyst comprising the fourth carrying at least one substituent of—(CH), OH, and polypeptide. X is 0 to 4, and 8. The method according to claim 1, the first polypeptide is selected from the group consisting wherein the contacting b) is carried out in the presence of a of an M1 variant of galactose oxidase from Fusarium fifth polypeptide having glucose dehydrogenase activity NRRL 2903, pyranose oxidase from Phanerochaete or a whole-cell biocatalyst comprising the fifth polypep chrysosporium, hexose oxidase from Chondrus crispus, tide. and a homologue thereof. 9. (canceled) 2. The method according to claim 1, 10. The method according to claim 1, wherein the hydrocarbon of formula (I) or (II): wherein a redox factor produced by a sixth polypeptide having amino acid dehydrogenase activity or a whole cell biocatalyst comprising the sixth polypeptide is con (I) sumed by the fourth polypeptide or the whole-cell bio catalyst comprising the fourth polypeptide. 11. The method according to claim 1, wherein the contactinga) is carried out in the presence of a compound having an H-O-degrading activity. (II) 12. The method according to claim 11, wherein the compound is a seventh polypeptide compris ing catalase and horse radish peroxidase/ABTS, and a homologue thereof or a whole-cell biocatalyst comprising the seventh polypep tide. 13. The method according to claim 1, wherein up to two out of A, B, C, D, E, and F are atoms each wherein the contacting a) and the contacting b) are carried and independently selected from the group consisting of out simultaneously in the same reaction mixture. N. S., and O and others are C, 14. The method according to claim 12, the method further R is selected from the group consisting of H, halogen, comprising: Substituted or unsubstituted alkyl, alkenyl, alkynyl, adding at least one component essential for the transami (CH), CRO, -(CH2), NH, -(CH2), NO, nase activity to a reaction mixture following addition of —(CH), O R. —(CH), CHSH, and —(CH.) components essential for oxygen-dependent and cofac COOR, and X is 0 to 20, and tor-dependent carbohydrate oxidase activity. R" is selected from the group consisting of H, a substituted 15. The method according to claim 1, or unsubstituted alkyl, a substituted or unsubstituted wherein a level of oxygen pressure is from 2 to 7 bar. cycloalkyl, a Substituted or unsubstituted aryl, and a 16. The method according to claim 1, substituted or unsubstituted heteroaryl. wherein one or more of the first, second, and third polypep 3. The method according to claim 2, tides are associated with a viable cell. wherein up to two out of A, B, C, D, E and F are atoms each 17. A method for transaminating a hydrocarbon compris and independently selected from the group consisting of ing a hydroxyl group, the method comprising: N and O, and others are C, transaminating the hydrocarbon with a mixture comprising R is selected from the group consisting of -(CH2), a first polypeptide having oxygen-dependent and cofactor CRO, (CH), NH, (CH), O R', and dependent carbohydrate oxidase activity or a whole-cell —(CH), COOR, and x is 0 to 4, and biocatalyst comprising the first polypeptide, US 2014/O120587 A1 May 1, 2014

a second polypeptide having transaminase activity or a OXygen, whole-cell biocatalyst comprising the second polypep a carbohydrate oxidase cofactor, tide, a second polypeptide having transaminase activity or a a third polypeptide having amino acid dehydrogenase whole-cell biocatalyst comprising the second polypep activity or a whole-cell biocatalyst comprising the third tide, polypeptide, a third polypeptide having amino acid dehydrogenase activity or a whole-cell biocatalyst comprising the third OXygen, polypeptide, a Substrate amino acid for an amino acid dehydrogenase, an amino acid dehydrogenase cofactor, a carbohydrate oxidase cofactor, and a Substrate amino acid of the for an amino acid dehydro an amino acid dehydrogenase cofactor, genase wherein the hydrocarbon comprises a 5 or 6 membered ring wherein one or more of the first, second, and third polypep carrying at least one substituent of—(CH), OH, and tides or the whole-cell biocatalysts comprising the first, second, and third polypeptides are or comprise, respec X is 0 to 4, and tively, a heterologous polypeptide having a respective the first polypeptide is selected from the group consisting activity, of an M1 variant of galactose oxidase from Fusarium the hydrocarbon comprises a 5 or 6 membered ring carry NRRL 2903, pyranose oxidase from Phanerochaete ing at least one Substituent of (CH), OH, and X is 0 chrysosporium, hexose oxidase from Chondrus crispus, to 4, and and a homologue thereof. the first polypeptide is selected from the group consisting 18. An aqueous reaction mixture, comprising: of an M1 variant of galactose oxidase from Fusarium a hydrocarbon comprising a hydroxyl group, NRRL 2903, pyranose oxidase from Phanerochaete a first polypeptide having oxygen-dependent and cofactor chrysosporium, hexose oxidase from Chondrus crispus, dependent carbohydrate oxidase activity or a whole-cell and a homologue thereof. biocatalyst comprising the first polypeptide, k k k k k