Process for the preparation of an aqueous solution containing glycolic acid

Abstract

The invention relates to a process for the preparation of an aqueous solution containing glycolic acid, in which ethylene glycol, which is present in an aqueous solution, is oxidised by treatment with a first NAD-dependent oxidoreductase and NAD+ in vitro to form glycol aldehyde and this is oxidised in vitro with a further NAD-dependent oxidoreductase and NAD+ to form glycolic acid, and the reduced NADH created by these oxidations is oxidised again by means of an NADH oxidase or an alcohol dehydrogenase and is thus regenerated.

Description

[0001] Process for preparing an aqueous solution containing glycolic acid

[0002] The invention relates to a process for producing an aqueous solution containing glycolic acid.

[0003] Background of the invention

[0004] Although glycolic acid also occurs naturally, for example in cane and beet sugar, its synthesis is currently largely based on fossil fuels due to the low concentrations of the substance in these renewable raw materials. Since the resulting glycolic acid often still contains traces of formaldehyde, which is particularly problematic when used in cosmetic products (e.g., skin peels (Sharad, 2013)), there is a great need for alternative synthesis methods.

[0005] Chemo-enzymatic methods for glycolic acid production involve the conversion of glycolonitrile, synthesized from formaldehyde and hydrogen cyanide, to glycolic acid using nitrilases (Panova et al., 2008; Ben-Bassat et al., 2008). This process cannot be considered sustainable due to the toxic, energy-intensive chemicals used, which are generally derived from fossil sources.

[0006] One chemical process for producing glycolic acid using a renewable feedstock is the conversion of glyoxal derived from bio-oil to glycolic acid in the presence of zeolite as a catalyst (Dapsens et al., 2014). Bio-oil is produced by pyrolysis of biomass—an energy-intensive process that destroys the original composition of the biomass, leaving the synthetic potential of the feedstock unused.

[0007] Glycolic acid (or the corresponding alkali glycolate) can also be formed by reacting chloroacetic acid (produced by chlorination of acetic acid) with alkali, but this produces large amounts of alkali chlorides as a byproduct. In addition, glycolic acid purified by electrodialysis still contains traces of chloroacetic acid and dichloroacetic acid (a byproduct of chlorination) (EP 0784047 B1).

[0008] However, glycolic acid can also be obtained by oxidation of ethylene glycol (ethane-1,2-diol), the simplest dihydric alcohol. The oxidation can be achieved, for example, by oxygen in the presence of the bimetallic catalyst AuPt (supported by CeO2) (Tschirner et al., 2022).

[0009] Ethylene glycol is produced by the hydrolysis of ethylene oxide (at 200 °C), which in turn is obtained by the oxidation of ethylene (obtained from fossil fuels). The resulting byproducts (oligoethylene glycols) must be separated by distillation, which is an energy-intensive process. To avoid byproducts, Shell's OMEGA process (Only Mono Ethylene Glycol Advantage) can be used, for example. In this process, ethylene oxide is first reacted with CO2 to form ethylene carbonate, which is then hydrolyzed to ethylene glycol (using K1 and K2MOO4 as catalysts) while releasing CO2. Other processes include the Rh- or Co-catalyzed hydrohydroxymethylation of formaldehyde or the hydroformylation of formaldehyde to glycolaldehyde, which is subsequently reduced to ethylene glycol (Berger, 2016).

[0010] The processes mentioned for the production of ethylene glycol cannot be described as sustainable or environmentally friendly due to the raw materials, catalysts or reaction conditions used.

[0011] However, alternatives based on sustainable resources also exist. Certain microorganisms can ferment biomass to ethanol, which can be dehydrated to ethylene in the presence of phosphoric acid or aluminum oxide. This ethylene can then undergo the previously described steps (oxidation to ethylene oxide and subsequent hydrolysis) to produce ethylene glycol (Wong et al., 2023).

[0012] However, ethylene glycol can also be obtained by pyrolysis of sugars and the subsequent catalytic hydrogenation of the Ci-Cs oxygenates produced by pyrolysis, such as glycolaldehyde, glyoxal, acetol (hydroxyacetone) or pyruvaldehyde (methylglyoxal) (US 10077222 B2; Tulio, 2017).

[0013] The oxidation of ethylene glycol to glycolic acid via the intermediate glycolaldehyde can be accomplished biotechnologically. Among the microorganisms that catalyze the oxidation are Pichia naganishii AKU 4267 and Rhodotorula sp. 3Pr-126, which convert 100 g / l of ethylene glycol to 105 g / l and 110 g / l of glycolic acid, respectively, in 120 h (Kataoka et al., 2001).

[0014] Another microorganism capable of oxidizing ethylene glycol to glycolic acid is the yeast Yarrowia lipolytica IMUFRJ 50682, which is already known to produce various hydrolases for the depolymerization of poly(ethylene terephthalate). Resting cells of Y. lipolytica were able to oxidize 548.6 mM ethylene glycol (34.1 g / L; 1000 mM starting concentration) to 429.5 mM glycolic acid (32.7 g / L) within 72 h in a bioreactor, corresponding to a yield of 74% (based on the amount of substrate converted) (Carniel et al., 2023). The enzymes involved are described by Carniel et al. as similar to the membrane-bound dehydrogenases of acetic acid bacteria (Gluconobacter, Acetobacter). Zhang et al. (2016) and Hua et al. (2019) describe the conversion of ethylene glycol to glycolic acid by the industrially widely used acetic acid bacterium Gluconobacter oxydans. Thus, immobilized G.oxydons cells produced up to 66.9 g / L of glycolic acid (880 mM) from ethylene glycol in 72 h in an open bioreactor with C gassing (Hua et al., 2019). Zhang et al. were even able to produce 113.8 g / L of glycolic acid (1.50 M; molar yield 92.9%) after 45 h using a bioengineered G. oxydons strain overexpressing a membrane-bound alcohol dehydrogenase (mADH) (Zhang et al., 2016).

[0015] Although ethylene glycol is not a natural substrate for Escherichia coli, E. coli is able to oxidize ethylene glycol to glycolic acid when a mutant of propanediol oxidoreductase (encoded by / ucO) and glycolaldehyde dehydrogenase (encoded by aldA) are overexpressed (Boronat et al., 1983; Pandit et al., 2021).

[0016] WO 2020 / 168408 A1 describes the in vitro production of glycolic acid, whereby the glycolaldehyde is formed from a pentose 5-phosphate by aldol cleavage using deoxyribose phosphate aldolase (DERA) mutants, and the oxidation of glycolaldehyde to glycolic acid is also accomplished with aldA. In this way, 2.81 mM glycolic acid could be produced from 20 mM xylulose, 7.87 mM glycolic acid from 20 mM ribulose, and 0.9 mM glycolic acid from 20 mM ribose.

[0017] The first oxidation step (ethylene glycol to glycolaldehyde) can also be accomplished by a cytosolic medium-chain alcohol dehydrogenase (Gox0313) from Gluconobacter oxydans DSM2003. This can convert 500 mM ethylene glycol to 484.2 mM glycolaldehyde in 14 h (30 °C and pH 8.5) in vitro, whereby the regeneration of the cofactor NAD +with an NADH oxidase (NOX-2) from Lactobacillus brevis ATCC367. However, the second oxidation step (glycolaldehyde to glycolic acid) is not catalyzed by Gox0313 (Zhang et al., 2015).

[0018] An alternative to the dehydrogenases already mentioned are oxidases. Gao et al. (2014) describe resting cells of Burkholderia sp. EG13, which can oxidize 200 mM ethylene glycol (12.4 g / L) to glycolic acid (98.8% conversion) in 24 hours. The enzymes involved are unspecified oxidases that are subject to substrate inhibition at an ethylene glycol concentration of 250 mM and product inhibition at a glycolic acid concentration of 200 mM. To circumvent the inhibitory effect of glycolic acid, Gao et al. used an anion exchange resin to continuously remove glycolic acid from the reaction mixture. Thus, product concentrations of 793 mM (60.3 g / L) can be achieved in the fed-batch process.

[0019] A review article by Yamada & Isobe (2015) postulates an enzymatic process for the oxidation of ethylene glycol to glycolic acid using two microbial oxidases. The first step is catalyzed by enzymes from the class of alcohol oxidases (EC 1.1.3.13) (e.g., from Candida sp. or Pichia pastoris) or glycerol oxidases (e.g., from Aspergillus japonicus) to form hydrogen peroxide (H2O2). The final oxidation can be carried out by an aldehyde oxidase from Burkholderia sp. AIU 129 (Yamada & Isobe, 2015; Yamada et al., 2015). To optimize the process, the authors (Yamada et al., 2015) suggest investigating the influence of reaction conditions to increase the yield of glycolic acid.

[0020] An experimental study announced by Yamada & Isobe (2015) to optimize the reaction conditions for the conversion of ethylene glycol to glycolic acid using two microbial oxidases has not been published to date.

[0021] This is where the present invention comes in and aims to provide an efficient biocatalytic process for the production of glycolic acid.

[0022] Detailed description of the invention

[0023] The object of the invention for producing an aqueous solution of glycolic acid is achieved by treating ethylene glycol, which is dissolved in an aqueous solution, with a first NAD-dependent oxidoreductase and NAD + in vitro to glycolaldehyde and this with another NAD-dependent oxidoreductase and NAD + / n vitro to glycolic acid and the reduced NADH produced by these oxidations is re-oxidized and thus regenerated by means of an NADH oxidase or an alcohol dehydrogenase.

[0024] For the purposes of this description and claims, the term "in vitro" means that the process is not carried out by fermentation. The enzymes are therefore not contained in living cells and are not membrane-bound. Rather, they are preferably present as such in the aqueous solution.

[0025] In a preferred embodiment of the present invention, the first oxidoreductase is an alcohol dehydrogenase and / or the further oxidoreductase is an aldehyde dehydrogenase, in particular a glycolaldehyde dehydrogenase.

[0026] For the oxidation of the cofactor NADH and thus for the regeneration of NAD +Preferably, an alcohol dehydrogenase is used using a ketone or aldehyde, whereby in vitro NAD + and a secondary or primary alcohol is formed. For example, acetone, which is produced from 2-propanol by enzymatic cofactor regeneration in an enzymatically catalyzed reduction (see EP 2812439 B1 for examples), can be converted back to 2-propanol. This can then be recycled to the reduction reaction, forming a closed cycle that renders the heterogeneous catalytic gas-phase hydrogenation of acetone to 2-propanol (Al-Rabiah et al., 2022) obsolete.

[0027] For the oxidation of the cofactor NADH, it is best to use an NADH oxidase using air, preferably enriched with oxygen, or pure oxygen, which in vitro NAD + is formed.

[0028] Furthermore, applying overpressure (air or oxygen) during the reaction is preferred. Overpressure in this context means a pressure greater than ambient pressure (> 1 bar).

[0029] It has been shown that in the case of cofactor regeneration with an NADH oxidase, the overpressure not only improves the oxidation of the cofactor, but in the case of cofactor regeneration with an alcohol dehydrogenase, the evaporation losses of the cosubstrate used (preferably acetone) can also be minimized.

[0030] The overpressure is preferably more than 1.1 bar, even more preferably more than 1.2 bar, even more preferably more than 1.5 bar, even more preferably more than 2 bar, even more preferably more than 5 bar, and particularly preferably more than 7 bar. The upper limit of the overpressure does not exceed 20 bar, preferably not 10 bar, and particularly preferably not 7 bar.

[0031] By using NAD-dependent dehydrogenases for the oxidation of ethylene glycol to glycolic acid, the use of oxidases can be avoided, which have two major disadvantages: 1. the formation of hydrogen peroxide as a by-product of the oxidation, which is harmful to the enzymes used in higher concentrations and whose removal requires the addition of catalase, and 2. product inhibition, as described by Gao et al. (2014). Instead, enzymes are used that are dependent on the cofactor NAD. + which can be regenerated by using an O2-dependent NADH oxidase or an alcohol dehydrogenase with an aldehyde or ketone (preferably acetone) as a cosubstrate.

[0032] It has been shown that it is particularly advantageous if the alcohol dehydrogenase and the glycolaldehyde dehydrogenase are present in the reaction mixture as lysate of the corresponding cells that produce (express) them.

[0033] Preferred variants of the two processes according to the invention are schematically illustrated in the accompanying Figures 1 and 2. In further preferred embodiments of the process according to the invention, the concentration of ethylene glycol in the aqueous solution is 20-250 g / l.

[0034] The particularly preferred temperature range is between 20 and 40 °C.

[0035] The most preferred pH range of the reaction is between 6.0 and 9.0.

[0036] In another variant, the enzymes can be added to the reaction solution in a homogenate or powder form. The enzymes can be modified with a water-soluble polymer such as polyethylene glycol, immobilized in or on a solid matrix, or be part of a fusion protein. Furthermore, the enzymes can also be added to the reaction solution in lyophilized or spray-dried form.

[0037] In this context, homogenate refers to a physically and / or chemically treated cell suspension (e.g., treated by pressure, lysozyme, or ultrasound), whereby the cellular components are released from the cells. A lysate is obtained when the insoluble cellular components of the homogenate are removed, for example, by filtration or centrifugation (see Enzyme Production & Lysate Preparation for details).

[0038] The oxidoreductase used for the oxidation of ethylene glycol to glycolaldehyde is preferably an alcohol dehydrogenase (EC 1.1.1.1).

[0039] The oxidoreductase used for the oxidation of glycolaldehyde to glycolic acid preferably comes from group EC 1.2.1.21 (glycolaldehyde dehydrogenase).

[0040] The NADH oxidase used for cofactor regeneration can originate from one of the groups EC 1.6.3.3 (NADH oxidase (HjOj-forming)) or EC 1.6.3.4 (NADH oxidase (HjO-forming)), with the H2O-forming group being particularly preferred.

[0041] The oxidoreductase used for cofactor regeneration is preferably an alcohol dehydrogenase (EC 1.1.1.1).

[0042] The NAD-dependent oxidoreductase for the oxidation of ethylene glycol to glycolaldehyde preferably comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 1, 3 or 5 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 2, 4 or 6 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 2, 4 or 6.

[0043] SEQ ID No. 1:

[0044] MKAAVVEQFKKPLQVKEVEKPKISYGEVLVRIKACGVCHTDLHAAHGDWPVKPKLPLIPGHEGVGVIEEVGPGV THLKVGDRVGIPWLYSACGHCDYCLSGQETLCERQQNAGYSVDGGYAEYCRAAADYVVKIPDNLSFEEAAPIFC AGVTTYKALKVTGAKPGEWVAIYGIGGLGHVAVQYAKAMGLNVVAVDLGDEKLELAKQLGADLVVNPKHDD AAQWIKEKVGGVHATVVTAVSKAAFESAYKSIRRGACVLVGLPPEEIPIPIFDTVLNGVKIIGSIVGTRKDLQEAL QFAAEGKVKTIVEVQPLENINDVFDRMLKQINGRVVLKVD

[0045] SEQ ID No. 2:

[0046] ATGAAAGCTGCAGTTGTGGAACAATTTAAAAAGCCGTTACAAGTGAAAGAAGTGGAAAAACCTAAGATCT CATACGGGGAAGTATTAGTGCGCATCAAAGCGTGTGGGGTATGCCATACAGACTTGCATGCCGCACATGG CGACTGGCCTGTAAAGCCTAAACTGCCTCTCATTCCTGGCCATGAAGGCGTCGGTGTAATTGAAGAAGTAG GTCCTGGGGTAACACATTTAAAAGTTGGAGATCGCGTAGGTATCCCTTGGCTTTATTCGGCGTGCGGTCAT TGTGACTATTGCTTAAGCGGACAAGAAACATTATGCGAACGTCAACAAAACGCTGGCTATTCCGTCGATGG TGGTTATGCTGAATATTGCCGTGCTGCAGCCGATTATGTCGTAAAAATTCCTGATAACTTATCGTTTGAAGA AGCCGCTCCAATCTTTTGCGCTGGTGTAACAACATATAAAGCGCTCAAAGTAACAGGCGCAAAACCAGGTG AATGGGTAGCCATTTACGGTATCGGCGGGCTTGGACATGTCGCAGTCCAATACGCAAAGGCGATGGGGTT AAACGTCGTTGCTGTCGATTTAGGTGATGAAAAACTTGAGCTTGCTAAACAACTTGGTGCAGATCTTGTCG TCAATCCGAAACATGATGATGCAGCACAATGGATAAAAGAAAAAGTGGGCGGTGTGCATGCGACTGTCGT CACAGCTGTTTCAAAAGCCGCGTTCGAATCAGCCTACAAATCCATTCGTCGCGGTGGTGCTTGCGTACTCGT CGGATTACCGCCGGAAGAAATACCTATTCCAATTTTCGATACAGTATTAAATGGAGTAAAAATTATTGGTTC TATCGTTGGTACGCGCAAAGACTTACAAGAGGCACTTCAATTTGCAGCAGAAGGAAAAGTAAAAACAATTGTCGAAGTGCAACCGCTTGAAAACATTAACGACGTATTCGATCGTATGTTAAAAGGGCAAATTAACGGCCG CGTCGTGTTAAAAGTAGATTAA

[0047] SE ID Nr. 3:

[0048] MSGKMKAAVVHEFGKPLTIEELDIPTICPTQILVKM IACGVCHTDLHAASGDWPKKPHLPFIPGHEGVGTVVQV GSEVDWVKEGDVVGVPWLYSACGHCEHCLAGWETLCAKQEETGYSVNGCFAEYVVADPNYAHLPKGVDPVQV KVAPVLCAGLTVYKGLKMTDTRAGNWVAISGVGGLGQMAVQYAVAMGLNVVAVDIDDEKLATAKLGATYT VNARNTDPAAFMQEKVGGVHGGLITAVSTKAFSQAMGYVRAGGTLVLNGLPPGDFPISIFDVVM NAITIRGSI VGTRLDMIEALSFFAEGKVTSVTTTDRIDNINAIFDALKNGRVEGRVLDFRN SEQ. ID No. 4:

[0049] ATGTCCGGAAAAATGAAAGCCGCTGTGGTTCATGAATTTGGCAAACCACTGACCATTGAAGAACTGGACAT

[0050] TCCCACCATTAAACCCACCCAGATTTTGGTTAAAATGATTGCCTGCGGCGTGTGCCATACAGATTTGCATGC

[0051] TGCCAGCGGGGATTGGCCCAAAAACACATCTGCCGTTTATTCCCGGCCACGAAGGCGTTGGCACGGTTG

[0052] TGCAGGTTGGCAGCGAAGTAGACTGGGTAAAGGAAGGCGACGTTGTTGGCGTGCCTTGGCTCTATTCTGC

[0053] CTGCGGCCATTGCGAGCATTGTCTGGCTGGGTGGGAAACCCTTTGTGCGAAGCAAGAAGAAACCGGCTAT

[0054] TCCGTAAACGGCTGCTTTGCCGAATACGTGGTAGCAGACCCTAACTACATTGCTCATCTTCCCAAAGGCGTA

[0055] GACCCTGTAAAAGTTGCCCCCGTACTGTGCGCAGGCCTGACCGTTTATAAGGGCCTGAAAATGACAGACAC

[0056] GCGGGCAGGGAACTGGGTTGCCATTTCCGGCGTTGGGGGGTTAGGCCAGATGGCTGTGCAGTACGCCGT

[0057] AGCAATGGGGCTGAATGTTGTGGCGGTAGATATTGATGATGAAAAACTGGCAACCGCCAAAAAGCTTGGT

[0058] GCAACGTACACTGTTAACGCCAGAAATACAGATCCGGCAGCATTTATGCAGGAAAAAGTTGGTGGCGTAC

[0059] ACGGTGGATTGATCACCGCTGTTTCCACCAAAGCATTTTCTCAGGCTATGGGTTATGTGCGGGCAGGGGGC

[0060] ACTTTGGTGCTAAATGGGCTGCCGCCGGGTGATTTCCCAATTTCTATCTTTGACGTGGTCATGAATGCCATT

[0061] ACCATTCGCGGCTCCATAGTAGGCACACGGCTGGACATGATTGAAGCTCTTTCCTTCTTTGCGGAAGGGAA

[0062] GGTAACATCTGTCACCACAACGGATCGGATTGATAACATCAACGCAATTTTTGATGCGCTCAAGAACGGTC

[0063] GGGTGGAAGGCCGCGTGGTTCTGGACTTCCGCAACTGA

[0064] SEQ ID Nr. 5:

[0065] M NKTMKAAVARAFGKPLEIEEVEVPRPRAGELLVKIEACGVCHTDLHAVEGDWPVKPNPPFIPGHEGVGHVV

[0066] AVGEGVTHVKEGDRVGIPWLYSACGHCEHCLGGWETLCEQQQNAGYSVNGGFAEYALAAADYVGLLPKNVG

[0067] FVDIAPVLCAGVTVYKGLKMTDTRPGNWVVVSGIGGLGHMAVQYARAMGLNVAAVDIDDDKLDFAKRLGAE

[0068] VVVNAKATDPAAYLKKEIGGAHGALITAVSPKAFEQALGMVRRGGTVALNGLPPGDFPLSIFDMVLNGVTVRG

[0069] SIVGSRLDLQESLQFAEEGKVRATVATEKLENINSVFDRMRRGQIEGRIVLDMAA

[0070] SEQ ID Nr. 6:

[0071] ATGAACAAGACCATGAAGGCCGCGGTGGCCCGCGCGTTCGGCAAACCCCTGGAAATCGAAGAAGTCGAG

[0072] GTGCCGCGCCCGCGCGCGGGCGAACTGTTGGTGAAGATCGAGGCCTGCGGCGTCTGCCACACCGATCTGC

[0073] ACGCGGTCGAGGGCGACTGGCCGGTCAAGCCCAATCCCCCTTTCATCCCGGGCCACGAAGGCGTGGGCCA

[0074] CGTGGTGGCGGTGGGCGAAGGCGTGACGCACGTCAAGGAAGGCGACCGCGTCGGCATCCCCTGGCTGTA

[0075] TTCCGCCTGTGGCCATTGCGAGCATTGCCTGGGCGGCTGGGAGACGCTGTGCGAACAGCAGCAGAACGCC

[0076] GGCTACTCCGTCAACGGCGGCTTCGCCGAGTACGCGCTGGCCGCGGCCGACTACGTGGGCCTGCTGCCGA

[0077] AGAATGTCGGCTTCGTCGACATCGCCCCGGTACTGTGCGCCGGCGTCACGGTCTACAAAGGCCTGAAGAT

[0078] GACGGACACACGGCCAGGCAACTGGGTCGTGGTCTCCGGCATCGGCGGCCTGGGCCACATGGCGGTGCA GTATGCGCGCGCCATGGGCTTGAACGTGGCGGCCGTGGACATCGACGACGACAAACTGGATTTCGCCAAG CGTCTCGGCGCCGAGGTGGTCGTGAATGCCAAGGCCACCGATCCAGCGGCTTACCTGAAGAAGGAAATCG GCGGCGCCCACGGCGCGCTGATCACGGCGGTTTCGCCCAAGGCCTTCGAGCAGGCGCTGGGCATGGTGC GTCGCGGCGGCACGGTGGCGCTCAACGGCCTGCCGCCGGGCGACTTCCCGCTGTCGATCTTCGACATGGT GCTCAACGGCGTGACGGTGCGCGGTTCGATCGTGGGTTCGCGCCTGGACTTGCAGGAATCGCTGCAGTTC GCCGAGGAAGGCAAGGTGCGCGCGACCGTGGCGACCGAGAAGCTGGAGAACATCAACAGCGTGTTCGAC CGCATGCGCCGCGGCCAGATCGAAGGCCGCATCGTGCTGGACATGGCGGCGTAA

[0079] The NAD-dependent oxidoreductase for the oxidation of ethylene glycol to glycolaldehyde preferably comprises or consists of an amino acid sequence having an identity to SEQ ID No. 1, 3, or 5 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%. Particularly preferably, the NAD-dependent oxidoreductase according to the invention for the oxidation of ethylene glycol to glycolaldehyde comprises or consists of the amino acid sequence SEQ ID No. 1, 3, or 5.

[0080] Alternatively, the NAD-dependent oxidoreductase for the oxidation of ethylene glycol to glycolaldehyde preferably comprises or consists of an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 2, 4, or 6 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%. Particularly preferably, the nucleic acid encoding the NAD-dependent oxidoreductase according to the invention for the oxidation of ethylene glycol to glycolaldehyde comprises or consists of the nucleic acid sequence SEQ ID No. 2, 4, or 6.

[0081] The term "identity," as used herein, refers to the percentage of identical nucleotide or amino acid matches between at least two nucleotide or amino acid sequences aligned using a standardized algorithm. Such an algorithm can, in a standardized and reproducible manner, insert gaps into the compared sequences to optimize the alignment between two sequences, thus achieving a more meaningful comparison of the two sequences.

[0082] The percentage identity between sequences can be determined using one or more computer algorithms or programs known in the art or described herein. According to the invention, the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) provided by the National Center for Biotechnology Information (NCBI) is used to determine identity. The BLAST software suite includes various programs, including a tool called "BLAST 2 Sequences," which is used for the direct pairwise comparison of two nucleotide or amino acid sequences. "BLAST 2 Sequences" can also be accessed and used interactively via the NCBI World Wide Web. The blastn program (for nucleotide sequences) uses a word length (W) of 28, an expectation (E) of 0.05, M = 1, N = -2, and a comparison of both strands.For amino acid sequences, the blastp program uses as defaults a word length of 3 and an expectation (E) of 0.05 and the BLOSUM62 scoring matrix (Henikoff & Henikoff, 1989), alignments (B) of 50, expectation (E) of 0.05, M = 1, N = - 2.

[0083] Alternatively, the NAD-dependent oxidoreductase for the oxidation of ethylene glycol to glycolaldehyde preferably comprises or consists of an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 2, 4, or 6. As used herein, stringent conditions refer to conditions under which so-called specific hybrids, but not non-specific hybrids, are formed.

[0084] For example, hybridization can be performed by conventionally known methods, such as those described by J. Sambrook et al. in "Molecular Cloning, A Laboratory Manual," 2nd Ed., Cold Spring Harbor Laboratory (1989). Stringent conditions refer to washing at 65 °C and a salt concentration of 0.1x to 2x SSC (where 1xSSC is a mixture of 0.15 M sodium chloride / 0.015 M sodium citrate).

[0085] The enzymes used according to the invention for the oxidation of ethylene glycol to glycolaldehyde are preferably NAD-dependent alcohol dehydrogenases. Surprisingly, it has been shown that these enzymes are capable of oxidizing ethylene glycol in the presence of the cofactor NAD. + to glycolaldehyde. Accordingly, a further aspect of the present invention relates to the use of these enzymes for the oxidation of ethylene glycol to glycolaldehyde in the presence of the cofactor NAD+ .

[0086] A further preferred embodiment of the present invention is the use of an NAD-dependent oxidoreductase for the oxidation of ethylene glycol to glycolaldehyde, which preferably comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 1, 3 or 5 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 2, 4 or 6 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 2, 4 or 6.

[0087] According to a particularly preferred embodiment of the present invention, the NADH oxidase used for cofactor regeneration comprises an amino acid sequence which is selected from the group consisting of: i) an amino acid sequence which has an identity to SEQ ID No. 7, SEQ ID No. 9 or SEQ ID No. 11 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12 of at least 80%, and iii) an amino acid sequence which is encoded by a nucleic acid which binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12.

[0088] SEQ ID No. 7:

[0089] M KVVVVGCTHAGTAAVKTILNEHPDASVSVYERNDNVSFLSCGIALYVGGVVKDPAGLFYSSPEELASMGAKIN M EHNVKNIDNENKVVVIENLKTGETFEESYDKLVMTTGSWPIIPPIDGINSENILLCKNYNQANEIIKESKNAKKIV IVGGGYIGIELVEAFAESGKQVTLVDGLDRILNKYLDAEFTSVLEHDLQERGVTLALNQTVEKFVANESGAVTAVK TPVGEYEADLVILCVGFKPNTDLLKDKVEM LPNGAIVVDEYMRTSDEAIFAAGDSCAVHYNPTGGSAYIPLATN AVRMGALVGKNIVSPTVKYRGTQATSGLYLFGFNIGSTGLTENSAPHFGVEVRSVVEDNYRPEFMPTTEKVT M KLVYEVGTNRIVGGQIMSKYDVTQSANTLSLCVQNKMTIEDLAYVDFFFQPHFDRPWNYLNILAQAAVEQER KLAK

[0090] SEQ ID No. 8:

[0091] ATGAAAGTAGTAGTAGTAGGCTGTACACATGCAGGAACAGCGGCAGTTAAGACGATTTTAAATGAACATC CAGATGCATCAGTATCAGTATATGAGCGTAATGACAATGTCTCATTTCTATCTTGTGGGATTGCGTTGTATG TTGGTGGAGTTGTGAAAGATCCTGCAGGTTTGTTTTATTCAAGTCCAGAAGAACTTGCATCAATGGGCGCG AAAATTAACATGGAACACAATGTGAAAAATATAGATAATGAGAATAAGGTCGTAGTAATTGAGAATTTAAA AACAGGCGAAACATTTGAAGAAAGCTATGATAAGTTGGTAATGACAACTGGATCATGGCCAATTATTCCTC CAATTGATGGAATCAATAGTGAAAATATTCTTTTGTGTAAAAACTATAACCAAGCAAATGAAATTATTAAAG AATCAAAAAATGCTAAAAAGATTGTCATTGTTGGTGGTGGCTATATTGGAATTGAATTAGTTGAGGCATTT GCAGAATCTGGCAAGCAAGTGACGCTAGTTGATGGATTAGATCGTATTTTAAACAAATATTTAGATGCTGA

[0092] ATTCACTTCTGTTTTAGAGCATGATTTACAAGAAAGAGGCGTTACGCTAGCTTTAAACCAAACCGTCGAGAA

[0093] ATTGTTGCCAATGAATCAGGTGCTGTGACAGCTGTGAAAACACCAGTTGGAGAATATGAGGCTGATTTAG

[0094] TTATTTTATGTGTTGGATTTAAACCAAATACTGATTTGTTGAAGGATAAAGTAGAGATGTTGCCAAATGGTG

[0095] CCATCGTAGTGGATGAATATATGAGAACAAGCGATGAAGCGATTTTTGCTGCTGGCGATAGTTGCGCGGTT

[0096] CATTATAATCCAACTGGAGGCTCTGCGTATATTCCGTTAGCTACAAATGCAGTTAGAATGGGAGCTTTAGTT

[0097] GGGAAAATATTGTTTCTCCAACAGTTAAATATCGTGGCACGCCAAGCAACTTCTGGTTTATATTTTTGGT

[0098] TTTAATATAGGTCCAACCGGATTGACTGAAAATAGCGCTCCTCATTTTGGCGTAGAGGTTCGTTCAGTAGTT

[0099] GTAGAAGATAATTATCGTCCAGAGTTTATGCCGACAACAGAGAAAGTAACGATGAATTAGTTTATGAAGT

[0100] AGGAACGAATCGGATTGTTGGAGGTCAAATCATGTCAAAATATGATGTGACACAATCTGCCAATACGTTAT

[0101] CTTTATGTGTTCCAAATAAAATGACGATTGAGGATTTGGCTTATGTAGATTTCTTCTTCCAACCTCACTTTGA TCGTCCTTGGAACTATTTAATATTTTAGCGCAAGCTGTTGAGCAAGAGCGTAAACTAGCAAAATAA

[0102] SE ID Nr. 9:

[0103] MSKIVIVGANHAGTAAINTILDNYGSENEVVVFDQNSNISFLGCGMALWIGKQISGPQGLFYADKESLEAKGAKI

[0104] YM ESPVTAIDYDAKRVTALVNGQEHVESYEKLILATGSTPILPPIKGAAIKEGSRDFEATLKNLQFVKLYQNAEDVI

[0105] NKLQDKSQNLNRIAVVGAGYIGVELAEAFKRLGKEVILIDVVDTCLAGYYDQDLSEMM RQNLEDHGIELAFGET

[0106] VKAIEGDGKVERIVTDKASHDVDMVILAVGFRPNTALGNAKLKTFRNGAFLVDKKQETSIPDVYAIGDCATVYD

[0107] NAINDTNYIALASNALRSGIVAGHNAAGHKLESLGVQGSNGISIFGLNMVSTGLTQEKAKRFGYNPEVTAFTDF

[0108] QKASFIEHDNYPVTLKIVYDKDSRLVLGAQMASKEDMSMGIHM FSLAIQEKVTIERLALLDYFFLPHFNQPYNY MTKAALKAK

[0109] SEQ ID Nr. 10:

[0110] ATGAGCAAAATTGTTATCGTGGGTGCAAATCATGCAGGCACCGCAGCAATTAATACCATTCTGGATAATTA

[0111] TGGCAGCGAAAATGAAGTGGTTGTGTTTGATCAGAATAGCAACATTAGCTTTCTGGGTTGTGGTATGGCAC

[0112] TGTGGATTGGTAAACAAATTAGCGGTCCGCAGGGTCTGTTTTATGCAGATAAAGAAAGCCTGGAAGCAAA

[0113] AGGTGCCAAAATCTATATGGAAAGTCCGGTTACCGCCATTGATTATGATGCAAAACGTGTTACCGCACTGG

[0114] TTAATGGTCAAGAACATGTTGAAAGCTACGAGAAACTGATTCTGGCAACCGGTAGCACCCCGATTCTGCCT

[0115] CCGATTAAAGGTGCAGCCATTAAAGAAGGTAGTCGCGATTTTGAAGCAACCCTGAAAAATCTGCAGTTCGT

[0116] GAAACTGTATCAGAATGCCGAAGATGTGATTAACAAACTGCAGGATAAAAGCCAGAATCTGAATCGTATT

[0117] GCAGTTGTTGGTGCAGGTTATATTGGTGTTGAACTGGCAGAAGCATTTAAACGTCTGGGTAAAGAAGTGA

[0118] TTCTGATTGACGTTGTTGATACCTGTCTGGCAGGTTATTATGATCAGGATCTGAGCGAAATGATGCGTCAG

[0119] AATCTGGAAGATCATGGTATCGAACTGGCATTTGGTGAAACCGTTAAAGCAATTGAAGGTGATGGTAAAG

[0120] TGGAACGTATTGTTACCGATAAAGCAAGCCATGATGTGGATATGGTTATTCTGGCAGTTGGTTTTCGTCCG AATACAGCACTGGGTAATGCAAAACTGAAAACCTTTCGTAATGGTGCCTTTCTGGTGGATAAAAAACAAGA

[0121] AACCAGCATCCCGGATGTTTATGCAATTGGTGATTGTGCAACCGTGTATGATAATGCCATTAACGACACCA

[0122] ACTATATTGCACTGGCAAGCAATGCACTGCGTAGCGGTATTGTTGCAGGTCATAATGCAGCCGGTCATAAA

[0123] CTGGAAAGTCTGGGTGTTCAGGGTAGCAATGGTATTTCAATTTTTGGCCTGAATATGGTTAGCACCGGTCT

[0124] GACCCAAGAAAAAGCCAAACGTTTTGGTTATAATCCGGAAGTTACCGCCTTTACCGATTTTCAGAAAGCCA

[0125] GCTTTATCGAGCATGATAACTATCCGGTTACGCTGAAAATTGTGTATGACAAAGATAGCCGTCTGGTTCTG

[0126] GGTGCACAGATGGCCAGCAAAGAAGATATGAGCATGGGTATTCACATGTTTAGCCTGGCCATTCAAGAGA

[0127] AAGTTACCATTGAACGTCTGGCCCTGCTGGATTATTTCTTTCTGCCGCATTTTAATCAGCCGTACAACTATAT GACCAAAGCAGCACTGAAAGCCAAATAA

[0128] SE ID Nr. 11:

[0129] M KVAVIGCTHAGTAAVKTILTENDDVEVVVFENDNISFLSCGIALYVGGVVKDVNGLFYSDPSELESLGATVYM

[0130] KHNVLSFDENTKVIQVENMETGEHFQESYDKLVIATGSWPIIPDLPGLDLENVMLCNFKHAQELIQTKQDKKR

[0131] VAVIGAGYIGIELVEAFAEDGKEVVLIDGADRVLPKYLDQEMTDLLEASLVDHGVQMQLGEFVESFLADDEGKV

[0132] RAVKTSKGEYECDMAVLCVGFQPNTELYKGKLE™ PNGAIIVDDYM HTSHPDIYACGDSCAVNYNPNDGHAYI

[0133] PLATNAVRMGSLVGKNIKADRVKYRGTQSTSGLKLFGWNIGSTGVTDNSASSFNLETRSVYVEDNYRPEFM PT

[0134] TEKVYMKLVYEVGTNRVVGGQLMSKYDITQSANTLSLAIQTKQTIEDLAYVDFFFQPHFDRPWNYLNILAQAAL SQEEELAQN

[0135] SEQ ID No. 12:

[0136] ATGAAAGTTGCAGTAATCGGTTGTACCCATGCTGGACAGCTGCCGTTAAAACTATCTTAACAGAAAACGA

[0137] TGATGTAGAAGTTGTTGTTTTTGAACGCAATGATAATATCTCCTTCTTATCTTGTGGGATTGCCCTTTACGTA

[0138] GGTGGCGTGGTCAAAGACGTCAATGGCCTTTTCTATTCTGATCCAAGTGAGTTAGAATCTCTAGGCGCAAC

[0139] AGTTTATATGAAACACAATGTGCTATCGTTTGACGAAAACACTAAAGTTATCCAAGTAGAAAATATGGAGA

[0140] CTGGCGAACATTTCCAAGAATCATATGACAAGTTAGTCATTGCCACTGGTTCTTGGCCGATTATTCCTGATT

[0141] TACCAGGACTAGATCTAGAAAATGTCATGCTATGTAAAAACTTTAAACATGCCCAAGAATTAATCCAAACTA

[0142] AGCAAGATAAAAAACGAGTTGCTGTTATTGGTGCTGGTTATATTGGTATCGAGTTAGTTGAAGCCTTTGCT

[0143] GAAGACGGTAAAGAAGTTGTTTTAATCGATGGTGCTGACCGCGTCCTACCTAAGTATCTCGACCAGGAAAT

[0144] GACTGACTTATTAGAAGCTAGCTTGGTAGACCATGGCGTACAAATGCAATTAGGGGAATTTGTAGAATCAT

[0145] TCCTAGCCGACGATGAAGGTAAGGTACGTGCCGTTAAAACGTCTAAAGGTGAATATGAATGTGATATGGC

[0146] AGTCCTTTGTGTCGGCTTCCAACCTAACACAGAATTATATAAGGGCAAGTTAGAAACTATGCCGAATGGTG

[0147] CGATTATTGTAGATGACTATATGCACACCTCTCACCCAGATATCTATGCCTGTGGTGACTCATGTGCTGTCA

[0148] ACTATAATCCTAACGATGGCCATGCTTATATCCCGCTTGCCACTAATGCTGTCCGTATGGGTAGCTTAGTTG GTAAAAATATTAAAGCAGACCGCGTCAAATACCGGGGCACCCAGTCAACATCTGGTCTAAAATTATTCGGC TGGAATATTGGCTCAACTGGTGTTACCGACAATTCTGCTAGCAGCTTCAACCTTGAAACTCGTAGCGTCTAT GTTGAAGATAACTACCGCCCTGAATTCATGCCTACTACAGAAAAAGTTTATATGAAGTTAGTCTATGAAGTA GGCACAAATCGAGTTGTTGGCGGTCAGCTTATGTCTAAGTATGATATTACCCAATCAGCCAACACCCTATCA CTAGCTATCCAAACCAAGCAAACAATTGAAGATTTAGCCTATGTGGACTTCTTCTTCCAACCACACTTTGACC GCCCTTGGAACTATTTAAATATTTTAGCCCAAGCAGCACTAAGTCAAGAAGAAGAATTAGCTCAAAACTAA

[0149] The NADH oxidase selected from the group consisting of an amino acid sequence having an identity to SEQ ID No. 7 of at least 80%, an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 8 of at least 80%, and an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 8, is particularly well suited for the method according to the invention since it has a significantly higher stability compared to other NADH oxidases from the prior art.

[0150] The following examples describe preferred embodiments of the invention in more detail.

[0151] Materials

[0152] Glycolaldehyde dimer, ethylene glycol, IPTG (isopropyl-ß-D-thiogalactopyranoside), and alcohol oxidase (Candida boidinii; lyophilized powder, A6941-50UN) were purchased from Sigma-Aldrich. Sodium glycolate was purchased from AlfaAesar. Acetone, potassium dihydrogen phosphate, di-potassium hydrogen phosphate, and sodium dodecyl sulfate (SDS) were purchased from Carl Roth. NAD + , NADH disodium salt, zinc chloride and methanol were purchased from PanReac AppliChem (ITW Reagents) and triethanolamine was purchased from Chem-Lab NV.

[0153] Production of enzymes & preparation of lysates

[0154] General information on the expression of recombinant enzymes in E. coli

[0155] For recombinant enzyme production in an Escherichia coli strain, the gene to be expressed was first amplified by PCR using genomic DNA or its synthetic equivalent, adapted to the codon usage of E. coli, as a template, together with specific oligonucleotides carrying additional recognition sequences for restriction endonucleases. The gene fragment was then isolated from the reaction mixture. After nucleic acid digestion, the gene fragment encoding the target enzyme was ligated into the cleaved backbone of the expression vector pQE70-Kan. The ligation product was transformed into chemically competent E. coli cells (ToplOF), and the resulting colonies were used for plasmid isolation and restriction analysis. The results of the cloning step were verified by restriction enzyme digestion and DNA sequencing. The resulting construct carries the target gene under the IPTG-inducible T5 promoter.

[0156] For overexpression of the enzyme in E. coli, the resulting expression plasmid was transformed into the competent expression cells RB791. After 24 h of incubation at 37 °C, the resulting colonies were inoculated into LB medium for expression assays.

[0157] The next day, expression cultures with an optical density (OD550) of 0.02 were inoculated and shaken at 37°C until an OD550 of 0.3 was reached. The temperature was then lowered to 25°C, and the cultures were induced with 0.1 mM IPTG upon reaching an OD550 of 0.5. After 22 h, the cultures were harvested (separated from the medium by centrifugation in the form of a cell pellet) and analyzed for expression of the recombinant enzyme using SDS gel electrophoresis and an activity determination (use in a USE test or optical enzymatic assay).

[0158] Preparation of cell lysates using sonifier disruption

[0159] To prepare a cell suspension, the cell pellet prepared according to the above procedure was weighed into a suitable container, mixed with buffer, and dissolved while stirring. The mass fraction of biomass is typically 20%, with the remainder being buffer.

[0160] A Branson Sonifier 450 was used for cell disruption. The suspension was treated three times with 15 ultrasonic pulses each (device settings: Timer = 15; Duty Cycle = 50; Output Control = 3 - 5).

[0161] The resulting homogenate was centrifuged for 10 min at 4 °C and 16000 rpm (Eppendorf Centrifuge 5417R) to separate the insoluble cell fragments and obtain the lysate.

[0162] Table 1. Donor organisms and digestion conditions for the enzymes used in the examples.

[0163] Analytical methods

[0164] High Performance Liquid Chromatography (HPLC)

[0165] HPLC (high-performance liquid chromatography) was used to quantify ethylene glycol, glycolaldehyde, and glycolic acid. Detection was performed using a refractive index detector. A Phenomenex Rezex ROA-Organic Acid H+ (8%) column with a corresponding precolumn was used for the measurement and eluted isocratically with 1 mM sulfuric acid.

[0166] Determination of enzyme activities (optical-enzymatic assay)

[0167] Enzyme activities in the lysates were determined using a Shimadzu UV-1900 spectrophotometer. The formation or consumption of NADH was monitored at a wavelength of 340 nm via the change in absorbance. The measurements were performed with 0.2 mM cofactor (NAD +or NADH). For this purpose, 20 μl of a 10 mM stock solution of the cofactor was placed in a cuvette (Greiner bio-one semi-micro cuvette made of polystyrene), and the desired pH value was adjusted with 100 mM TEA-HCl buffer (870 μl). 10 μl of lysate (diluted or undiluted) and 100 μl of substrate solution were added to the cuvette, and the measurement was started immediately. The measurements were carried out at 25 °C as standard. The extinction coefficient of NAD(P)H at 340 nm (E = 6220 L mol 1 cm 1 ), the enzyme activity of the lysate can be determined in U / ml (based on the volume of the lysate) or U / g (based on the biomass used for production). 1 U represents 1 pmol of substrate turnover per minute (1 U = 1 pmol / min = 1.67-10' 8 cat).

[0168] The following examples describe preferred variants of the method according to the invention in more detail. The lysates used in these examples were prepared according to the methods described above.

[0169] Example 1

[0170] Oxidation of ethylene glycol to glycolic acid - Comparison of alcohol dehydrogenases with an alcohol oxidase from Candida boidinii

[0171] The following components were mixed in four 2 ml glass vials: 200 μl of a 500 mM TEA-HCl buffer (pH 8.5), 100 μl of an 820 mM ethylene glycol solution (final concentration 164 mM), 10 μl of glycolaldehyde dehydrogenase lysate, 10 μl of a 10 mM NAD + Solution (final concentration 0.2 mM), 2 U NADH oxidase I lysate, and alcohol oxidase or alcohol dehydrogenase (see Table 2 below for details). The alcohol oxidase solution also contained 5 μl of catalase lysate. It was made up to a total volume of 500 μl with deionized water.

[0172] The mixture was incubated for 20 h at 30 °C with continuous shaking (1200 rpm, Eppendorf Thermomixer).

[0173] For processing, 50 μl of the mixture was mixed with 150 μl ultrapure water and 200 μl MeOH and incubated at 60 °C for 20 min (1200 rpm, Eppendorf Thermomixer). After denaturation, the sample was centrifuged at max. 1 g for 10 min. 150 μl of the clear supernatant was transferred to HPLC vials and analyzed (HPLC, RI detection).

[0174] The results are shown in Table 2 below.

[0175] Table 2

[0176] The data in Table 2 show that the alcohol dehydrogenases lead to higher yields of glycolic acid compared to the alcohol oxidase from Candida boidinii (see Yamada & Isobe (2015)).

[0177] Example 2

[0178] Oxidation of ethylene glycol to glycolic acid - cofactor recycling with various NADH oxidases

[0179] The following components were mixed in three 2 ml glass vials: 200 μl of a 500 mM TEA-HCl buffer (pH 8.5), 163 μl of deionized water, 100 μl of an 810 mM ethylene glycol solution (final concentration 162 mM), 20 μl of alcohol dehydrogenase III lysate, 10 μl of glycolaldehyde dehydrogenase lysate, 5 μl of a 10 mM NAD + solution (final concentration 0.1 mM) and 2 U NADH oxidase lysate (see Table 3 below). The total volume of the mixture was 500 μL.

[0180] The mixture was incubated for 20 h at 30 °C with continuous shaking (1200 rpm, Eppendorf Thermomixer).

[0181] For processing, 50 μl of the mixture was mixed with 150 μl ultrapure water and 200 μl MeOH and incubated at 60 °C for 20 min (1200 rpm, Eppendorf Thermomixer). After denaturation, the sample was centrifuged at max. 1 g for 10 min. 150 μl of the clear supernatant was transferred to HPLC vials and analyzed (HPLC, RI detection).

[0182] The results are shown in Table 3 below.

[0183] Table 3 Example 3

[0184] Oxidation of ethylene glycol to glycolic acid - cofactor recycling using NADH oxidase

[0185] The reaction was carried out in a Multifors benchtop bioreactor (Infors AG). A glass reactor (volume 1.0 l) equipped with a stirrer, pH electrode, and C sensor was used as the vessel. pH was controlled by adding 5M NaOH.

[0186] Initially, 50 ml of an ethylene glycol solution (3.87 M), 173.6 ml of deionized water and 7.5 ml of a 2M TEA-HCl buffer (pH 8) were placed in the reactor and brought to 25 °C while stirring.

[0187] To start the reaction, 24 ml of alcohol dehydrogenase II lysate, 5 kll of NADH oxidase I, 5 kll of glycolaldehyde dehydrogenase lysate and 10 ml of 6 mM NAD + -solution (final concentration 0.2 mM). For the oxidation of NADH to NAD + Using NADH oxidase, air was constantly added to the reaction solution at a flow rate of 0.6 L / min via a sparger.

[0188] During operation, samples were continuously taken from the reactor solution and analyzed as follows: 50 μl of the mixture was first diluted with 150 μl of ultrapure water. Then, 40 μl of the diluted solution was added to 160 μl of ultrapure water and 200 μl of MeOH and incubated at 60 °C for 20 min (1200 rpm, Eppendorf Thermomixer). After denaturation, the sample was centrifuged for 10 min at max. 1 g. 150 μl of the clear supernatant was transferred to HPLC vials and analyzed (HPLC, RI detection).

[0189] After 17.5 h, 95% of the ethylene glycol (644 mM) was oxidized to glycolic acid. After completion of the reaction (42 h; 97% conversion), the pH of the reactor solution was adjusted to pH 4 with H2SO4, and the reactor contents were stirred for 1 h at 60 °C to deactivate the enzymes. The enzymes were first separated using a paper filter in a Büchner funnel, and the resulting cloudy solution was filtered again using a glass frit (P4), yielding a clear solution.

[0190] Example 4

[0191] Oxidation of ethylene glycol to glycolic acid - cofactor recycling using alcohol dehydrogenase and acetone

[0192] The following components were mixed in a 2 ml glass vial: 100 μl of a 1000 mM TEA-HCl buffer (pH 9), 204 μl of deionized water, 100 μl of an 813 mM ethylene glycol solution (final concentration 163 mM), 40 μl of alcohol dehydrogenase II lysate, 4 U of glycolaldehyde dehydrogenase lysate, 15 μl of a 10 mM NAD +solution (final concentration 0.3 mM) and 15 μl of acetone. The total volume of the mixture was 500 μl.

[0193] The mixture was incubated for 20 h at 25 °C with continuous shaking (1200 rpm, Eppendorf Thermomixer). 4 h after the start of the reaction, 20 μl of acetone were added.

[0194] For processing, 50 μl of the mixture was mixed with 150 μl ultrapure water and 200 μl MeOH and incubated at 60 °C for 20 min (1200 rpm, Eppendorf Thermomixer). After denaturation, the sample was centrifuged at max. 1 g for 10 min. 150 μl of the clear supernatant was transferred to HPLC vials and analyzed (HPLC, RI detection).

[0195] In this way, 50% of the ethylene glycol (163 mM) could be oxidized to glycolic acid.

[0196] literature

[0197] Sharad J (2013). Glycolic acid peel therapy - a current review. Clinical, Cosmetic and Investigational Dermatology, 6, 281-288. https: / / doi.org / 10.2147 / CCID.S34029

[0198] Panova, A., Mersinger, L. J., Liu, Q„ Foo, T., Roe, D. C., Spillan, W. L., Sigmund, A. E., Ben-Bassat, A., Wagner, L. W., O'Keefe, D., Wu, S., Petrillo, K., Payne, M. S., Breske, S. T., Gallagher, F. G., & DiCosimo, R. (2007). Chemoenzymatic Synthesis of Glycolic Acid. Advanced Synthesis & Catalysis, 349(8-9), 1462- 1474. https: / / doi.org / 10.1002 / adsc.200700061

[0199] Ben-Bassat, A., Walls, A. M., Plummer, M. A., Sigmund, A. E., Spillan, W., & DiCosimo, R. (2008). Optimization of Biocatalyst Specific Activity for Glycolic Acid Production. Advanced Synthesis & Catalysis, 350(11-12), 1761-1769. https: / / doi.org / 10.1002 / adsc.200800228

[0200] Dapsens, P.Y., Mondelli, C., Kusema, B.T., Verel, R., & Perez-Ramirez, J. (2014). A continuous process for glyoxal valorisation using tailored Lewis-acid zeolite catalysts. Green Chemistry, 16(3), 1176-1186. https: / / doi.org / 10.1039 / C3GC42353K

[0201] Tschirner, S., Weingart, E., Teevs, L., & Prüße, U. (2022). Oxidation of Monoethylene Glycol to Glycolic Acid with Gold-Based Catalyst and Glycolic Acid Isolation by Electrodialysis. Reactions, 3, 47-58. https: / / doi.org / 10.3390 / reactions3010004

[0202] Berger, A. (2016). Ethylenglycol, RD-05-01999 in Böckler, F., Dill, B., Eisenbrand, G., Faupel, F., Fugmann, B., Gämse, T., Matissek, R., Pohnert, G., Rühling, A., Schmidt, S., & Sprenger, G. (Ed.), RÖMPP [Online], Stuttgart, Georg Thieme Verlag. https: / / roempp.thieme.de / lexicon / RD-05-01999

[0203] Wong, M. K., Lock, S. S. M., Chan, Y. H., Yeoh, S. J., & Tan, I. S. (2023). Towards sustainable production of bio-based ethylene glycol: Progress, perspective and challenges in catalytic conversion and purification. Chemical Engineering Journal, 468, 143699. https: / / doi.Org / 10.1016 / j.cej.2023.143699

[0204] Tullo, A. H. (2017). Haldor Topsoe, Braskem investigate biobased ethylene glycol. Chemical and Engineering News, 95(46). Aufrufbar unter: https: / / cen.acs.org / articles / 95 / i46 / Haldor-Topsoe- Braskem-investigate-biobased.html

[0205] Kataoka, M., Sasaki, M., Hidalgo, A.-R. G. D., Nakano, M., & Shimizu, S. (2001). Glycolic acid production using ethylene glycol-oxidizing microorganisms. Bioscience, Biotechnology, and Biochemistry, 65(10), 2265-2270. https: / / doi.org / 10.1271 / bbb.65.2265 Carniel, A., Santos, A. G., Chinelatto, L. S., Castro, A. M., & Coelho, M. A. Z. (2023). Biotransformation of ethylene glycol to glycolic acid by Yarrowia lipolytica: A route for poly(ethylene terephthalate) (PET) upcycling. Biotechnology Journal, 18, e2200521. https: / / doi.org / 10.1002 / biot.202200521

[0206] Zhang, H., Shi, L, Mao, X., Lin, J., & Wei, D. (2016). Enhancement of cell growth and glycolic acid production by overexpression of membrane-bound alcohol dehydrogenase in Gluconobacter oxydans DSM 2003. Journal of Biotechnology, 237, 18-24. https: / / doi.org / 10.1016 / jjbiotec.2016.09.003

[0207] Hua, X., Du, G., & Xu, Y. (2019). Cost-practical of glycolic acid bioproduction by immobilized whole-cell catalysis accompanied with compressed oxygen supplied to enhance mass transfer. Bioresource Technology, 283, 326-331. https: / / doi.Org / 10.1016 / j.biortech.2019.03.094

[0208] Boronat, A., Caballero, E., & Aguilar, J. (1983). Experimental Evolution of a Metabolic Pathway for Ethylene Glycol Utilization by Escherichia coll. Journal of Bacteriology, 153(1), 134-139. https: / / doi.Org / 10.1128 / jb.153.l.134-139.1983

[0209] Pandit, A. V., Harrison, E., & Mahadevan, R. (2021). Engineering Escherichia coll for the utilization of ethylene glycol. Microbial Cell Factories, 20, 22. https: / / doi.org / 10.1186 / sl2934-021-01509-2

[0210] Zhang, X., Zhang, B., Lin, J., & Wei, D. (2015). Oxidation of ethylene glycol to glycolaldehyde using a highly selective alcohol dehydrogenase from Gluconobacter oxydans. Journal of Molecular Catalysis B: Enzymatic, 112, 69-75. https: / / doi.Org / 10.1016 / j.molcatb.2014.12.006

[0211] Gao, X., Ma, Z., Yang, L., & Ma, J. (2014). Enhanced Bioconversion of Ethylene Glycol to Glycolic Acid by a Newly Isolated Burkholderia sp. EG13. Applied Biochemistry and Biotechnology, 174, 1572-1580. https: / / doi.org / 10.1007 / sl2010-014-1114-9

[0212] Yamada, M., & Isobe, K. (2015). A Novel Microbial Aldehyde Oxidase Applicable to Production of Useful Raw Materials, Glycolic Acid and Glyoxylic Acid, from Ethylene Glycol. Fermentation Technology, 4(1), 1000116. https: / / doi.org / 10.4172 / 2167-7972.1000116

[0213] Yamada, M., Adachi, K., Ogawa, N., Kishino, 8., Ogawa, J., Kataoka, M., Shimizu, S., & Isobe, K. (2015). A new aldehyde oxidase catalyzing the conversion of glycolaldehyde to glycolate from Burkholderia sp. AIU 129. Journal of Bioscience and Bioengineering, 119(4), 410-415. https: / / doi.org / 10.1016 / jjbiosc.2014.09.005

[0214] Al-Rabiah, A. A., Boz, I., Akhmedov, V. M., Mostafa, M. M. M., & Bagabas, A. A. (2022). Highly Selective Gas-Phase Catalytic Hydrogenation of Acetone to Isopropyl Alcohol. Catalysts, 12(10), 1251. https: / / doi.org / 10.3390 / catall2101251 Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215(3), 403-410. https: / / doi.org / 10.1016 / S0022-2836(05)80360-2

[0215] Henikoff, S., & Henikoff, J. G. (1992). Amino acid substitution matrices from protein blocks. Proceedings of the National Academy of Sciences of the United States of America, 89(22), 10915-10919. https: / / doi.org / 10.1073 / pnas.89.22.10915

[0216] Sambrook, J., Fritsch, E. R., & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

[0217] Protein [Internet], Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Accession No. WP_033015595.1, alcohol dehydrogenase AdhP [Geobacillus stearothermophilus]. Verfügbar unter: https: / / www.ncbi.nlm.nih.gOv / protein / WP_033015595.l

[0218] Protein [Internet], Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Accession No. AEN03783.1, NAD+-dependent and Zn-containing alcohol dehydrogenase [Acetobacter pasteurianus NBRC 101655], Verfügbar unter: https: / / www.ncbi.nlm.nih.gOv / protein / AEN03783.l

[0219] Protein [Internet], Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Accession No. WP_006385522.1, alcohol dehydrogenase AdhP [Achromobacter xylosoxidans]. Verfügbar unter: https: / / www.ncbi.nlm.nih.gOv / protein / WP_006385522.l

[0220] Hidalgo, E., Chen, Y.-M., Lin, E. C. C., & Aguilar, J. (1991). Molecular cloning and DNA sequencing of the Escherichia coli K-12 aid gene encoding aldehyde dehydrogenase. Journal of Bacteriology, 173(19), 6118-6123. https: / / doi.org / 10.1128 / jb.173.19.6118-6123.1991

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Claims

Patent claims 1. A process for the preparation of an aqueous solution containing glycolic acid by treating ethylene glycol, which is present in an aqueous solution, with a first NAD-dependent oxidoreductase and NAD + / n vitro to glycolaldehyde and this with another NAD-dependent oxidoreductase and NAD + / n vitro to glycolic acid and the reduced NADH produced by these oxidations is re-oxidized and thus regenerated by means of an NADH oxidase or an alcohol dehydrogenase.

2. The method according to claim 1, characterized in that the first oxidoreductase is an alcohol dehydrogenase and / or the further oxidoreductase is an aldehyde dehydrogenase.

3. Process according to claim 2, characterized in that the aldehyde dehydrogenase is a glycolaldehyde dehydrogenase.

4. Method according to one of claims 1 to 3, characterized in that for the oxidation of the cofactor NADH an alcohol dehydrogenase is used using a ketone or aldehyde, whereby in vitro NAD + and a secondary or primary alcohol is formed.

5. Method according to one of claims 1 to 3, characterized in that for the oxidation of the cofactor NADH an NADH oxidase is used using oxygen, whereby in vitro NAD + is formed.

6. The method according to claim 5, characterized in that the NADH oxidase comprises an amino acid sequence which is selected from the group consisting of: i) an amino acid sequence which has an identity to SEQ ID No. 7, SEQ ID No. 9 or SEQ ID No. 11 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12 of at least 80%, and iii) an amino acid sequence which is encoded by a nucleic acid which binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No.

12.

7. Method according to one of claims 2 or 3, characterized in that the alcohol dehydrogenase and the glycolaldehyde dehydrogenase are present as lysate of the corresponding cells producing them.

8. The method according to any one of claims 1 to 7, characterized in that the NAD-dependent oxidoreductase for the oxidation of ethylene glycol to glycolaldehyde comprises an amino acid sequence which is selected from the group consisting of: i) an amino acid sequence which has an identity to SEQ ID No. 1, 3 or 5 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 2, 4 or 6 of at least 80%, and iii) an amino acid sequence which is encoded by a nucleic acid which binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 2, 4 or 6.

9. Use of an NAD-dependent oxidoreductase for the oxidation of ethylene glycol to glycolaldehyde, wherein the NAD-dependent oxidoreductase comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 1, 3 or 5 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 2, 4 or 6 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 2, 4 or 6.

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