Method for producing an aqueous solution containing alkali salts of glycolic acid and lactic acid

Abstract

The present invention relates to a method for preparing an aqueous solution containing the alkali salts of glycolic acid and lactic acid by treating, in vitro, the alkali salts of xylonic acid and / or arabinonic acid in an aqueous solution with an enzyme system comprising a dehydratase, an aldolase, glycolaldehyde dehydrogenase, lactate dehydrogenase and NAD+ as a cofactor, and then isolating the enzyme system.

Description

[Technical Field]

[0001] The present invention relates to a method for preparing an aqueous solution containing the alkali salts of glycolic acid and lactic acid. Such solutions are used in the cosmetics industry as a base for skin peeling products.

[0002] The present invention involves carbon-economical enzymatic processes for producing chemicals from renewable carbon sources. Specifically, the present invention relates to the atom-economical production of alkaline glycolates and alkaline lactates by an enzymatic process in which the C1 oxidation products of the pentoses D-xylose and L-arabinose, D-xylonate and L-arabonate, are converted to the C2 building blocks glycolic acid (glycolate) and the C3 building block lactic acid (lactate).

[0003] In this document, D-xylose is used interchangeably with xylose, L-arabinose is used interchangeably with arabinose, L-arabonate is used interchangeably with arabonate and arabonic acid, D-xylonate is used interchangeably with xylonate and xylonic acid, glycolate is used interchangeably with glycolic acid, lactate is used interchangeably with lactic acid, and glucose is used interchangeably with D-glucose. [Background technology]

[0004] Glycolic acid occurs naturally, for example in sugarcane and sugarbeet, but its synthesis is currently achieved primarily from fossil fuels due to the low concentration of the substance in these renewable raw materials. As synthetically produced glycolic acid often contains formaldehyde residues, which is particularly problematic when used in cosmetics (e.g., skin scrubs (Sharad, 2013)), and alternative synthesis methods are needed.

[0005] A chemoenzymatic method for producing glycolic acid involves the conversion of glycolonitrile, synthesized from formaldehyde and hydrogen cyanide, to glycolic acid via nitrilase (Panova et al., 2008; Ben-Bassat et al., 2008). This method cannot be considered sustainable due to the toxic and energy-intensive chemicals used, which are generally derived from fossil sources. Another method is the conversion of ethylene glycol to glycolic acid by microbial whole-cell biocatalysis (Gao et al., 2014). In this second example, the starting material is also typically produced by an energy-intensive chemical process. A chemical method for producing glycolic acid that utilizes renewable raw materials as starting materials is the conversion of bio-oil-derived glyoxal to glycolic acid in the presence of zeolite as a catalyst (Dapsens et al., 2014). Bio-oil is obtained by pyrolysis of biomass, a high-energy process that destroys the original composition of the biomass and preserves the synthetic potential of the raw materials.

[0006] To develop sustainable methods for the production of chemicals from biomass, less energy-intensive methods that utilize various existing biomass materials are needed. Apart from efficient and gentle methods for depolymerizing renewable feedstocks into short-chain sugars, efficient technologies for further converting these carbohydrate intermediates into chemical products are also needed. A particular challenge in this context is the efficient and as complete conversion as possible of the mixture of substances produced after depolymerization into valuable material streams. Straw, wood, and other plant-based feedstocks generally contain hemicellulose and cellulose, from which monosaccharides, primarily the pentoses xylose and arabinose, the hexoses glucose, mannose, and galactose, as well as acids derived from these sugars (e.g., glucuronic acid), can be released in various compositions. Methods for the efficient separation of C6 and C5 sugars would enable these types of material streams to be processed separately (e.g., WO 2011 / 014894). For example, corn husks contain 10% arabinose, 16% xylose, 64% glucose, 4% galactose, and 2% mannose in terms of total sugars (Hromadkova & Ebringerova, 1995), while wheat straw contains 5% arabinose, 30% xylose, 56% glucose, 1% galactose, and 2% mannose (Collins et al., 2014). These percentages demonstrate that the ratio of arabinose to xylose differs significantly between these two biomass types (corn husks: 1:1.6, wheat straw: 1:6).

[0007] Currently, biotechnology efforts are primarily focused on the conversion of biomass to chemical products through fermentation, i.e., methods aimed at converting substrates during the growth of microorganisms in a reactor. These fermentative whole-cell processes, despite all the technological advances in the field, such as metabolic engineering and heterologous pathway expression, are limited by the physiological constraints of cellular production (solvent, temperature, mass transport, and tolerance to high substrate and product concentrations, as well as by-products from other metabolic pathways within the cell) (Claassens et al., 2019). The energy demands of these intracellular processes, especially the metabolic (often CO2-releasing) steps, result in long processing times and significantly lower carbon yields than theoretically possible. Furthermore, because these processes generally utilize biosynthetic metabolic pathways that release CO2 from the substrate, even the theoretical carbon yield in the product is only a fraction of the carbon used. Depending on the metabolic pathway, 1–4 carbons are lost from C6 sugars and 1–3 carbons are lost from C5 sugars. Typical key data for such processes are summarized in the literature (Salusjarvi et al., 2019).

[0008] For example, fermentative production of glycolic acid from glucose via the glyoxylate shunt using metabolic engineering in recombinant Escherichia coli (E. coli) strains is known (WO2007 / 141316A2, WO2007 / 140816A1, WO2010 / 108909A1, WO2011 / 036213A2). In one example, in a fermentation process lasting 93 hours, fermentative production of 31.3 g / L of glycolic acid in the fermentation supernatant was achieved, resulting in a yield of 0.22 g of glycolic acid per gram of glucose (WO2007 / 141316A2). WO2011 / 036213A2 then describes a further optimized fermentation process of the system, obtaining 53.9 g / L of glycolic acid within 40 hours through adaptive process control, with a yield of 0.36 g of glycolic acid per gram of glucose.

[0009] Furthermore, the fermentative production of glycolic acid from pentoses, for example, by combining the ribulose-1-phosphate pathway with the glyoxylate shunt, has been described (Pereira et al., 2016). In this process, metabolic engineering of an Escherichia coli (E. coli) strain achieved a yield of 0.62 g of glycolic acid per gram of xylose. The glycolic acid end point concentration after 85 h was 41 g / L in the fermentation broth, representing a yield of 61% of the theoretical value (1.22 mol / mol). Generally, the upper limit of fermentative approaches to glycolic acid production is currently 65.5 g / L of glycolic acid end point and 90% of the theoretical yield when pure glucose is used as a substrate (Deng et al., 2018; Salusjarvi et al., 2019). (A 100% theoretical yield implies the loss of one carbon atom per sugar molecule.) In the case of pure xylose, the achieved values ​​are similar (44 g / l glycolic acid end point concentration, 87% of the theoretical yield (Pereira et al., 2016; Salusjarvi et al., 2019)). However, the achievable glycolic acid concentration in sugar mixtures (xylose + glucose) is an order of magnitude lower (Alkim et al., 2016; Salusjarvi et al., 2019). This also applies to similar systems realizing the fermentative conversion of sugars to ethylene glycol (Pereira et al., 2016; Salusjarvi et al., 2017; Uranukul et al., 2018; Salusjarvi et al., 2019).

[0010] In summary, it can be said that fermentation systems generally accept a loss of at least one carbon per sugar monomer and achieve low product end-point concentrations in the sugar mixture.

[0011] Cell-free conversion of metabolites was first demonstrated in 1897 by Eduard Buchner, who converted glucose to ethanol using cell lysate from Saccharomyces cerevisiae (Buchner, 1897). In 1985, Welch and Scopes presented a cell-free system for ethanol production (Welch & Scopes, 1985), but it was technically unusable due to lack of specificity.

[0012] Since then, several additional processes for producing chemicals from purified enzymes (enzyme isolates) have been described. For example, alcohol dehydrogenase has been used to produce high-quality chiral alcohols, and the cofactor NAD has been regenerated, for example, by adding glucose and glucose dehydrogenase (Goldberg et al., 2007). Generally, glucose or formate dehydrogenase (GDH or FDH) and alcohol dehydrogenase (ADH) are used for cofactor recycling (Schrittwieser et al., 2018).

[0013] In recent years, interest has shifted to processes that use highly selective conversions via enzymatic cascade reactions to obtain target chemicals in a single step (one-pot). EP 2700714 A1, US 8859247 B2, and EP 2204453 B1 describe a portfolio of cascade reactions in which glucose is converted to two molecules of pyruvate via a cascade of five enzymatic transformations. Starting from glucose, the cascade follows the non-phosphorylating Entner-Doudoroff pathway (sugar oxidation, dehydration, and aldol cleavage) to D-glyceraldehyde and pyruvate. D-glyceraldehyde is then further oxidized to D-glycerate, which is then dehydrated to pyruvate. This then serves as a platform for further conversion to a range of amino acids and alcohols. Thus, the enzymes in the cascade include glucose dehydrogenase, a promiscuous dihydroxy acid dehydratase (which catalyzes the dehydration of gluconate and D-glycerate), 2-keto-3-deoxygluconate aldolase, and an aldehyde dehydrogenase. The system can be minimized to three enzymes by using a similarly promiscuous dehydrogenase that accepts both glucose and D-glyceraldehyde as substrates.

[0014] A similar cascade exists for the pentose xylose, known as the Dahms pathway (Dahms, 1974). Here, xylose is first oxidized to 1,4-xylonolactone, which is cleaved to xylonate under the influence of lactonase. Dehydratase converts xylonate to 2-keto-3-deoxy-xylonate, which is then cleaved by aldolase to pyruvate and glycolaldehyde. This pathway can also convert arabinose in organisms such as Sulfolobus solfataricus (Kopp et al., 2020) through promiscuous enzymes in the pathway.

[0015] Pyruvate is a central intermediate in cellular metabolism, serving as a precursor for the amino acids alanine and acetyl-CoA, involved in the citric acid cycle. Reduction of pyruvate produces lactic acid and lactate, respectively, which have a wide range of applications. Lactic acid is used, for example, as an acidifier in the food industry; as a descaling agent, pH adjuster, or cleaning agent in the chemical industry; as an additive to anti-acne creams or moisturizers in the cosmetics industry; and as a precursor to acrylic acid or ethyl lactate (Wee et al., 2006). Furthermore, lactic acid (similar to glycolic acid) can be used in skin peels (chemical peels) (Smith, 1996). Difunctional lactic acid can also be polymerized to produce polylactide (PLA), a biodegradable and biocompatible plastic with applications in packaging, textiles, electronics, and medicine (Balla et al., 2021).

[0016] presented an in vitro variant of the Dahms pathway using purified enzyme isolates that converted xylose or xylonolactone (1 mM substrate concentration) to ethylene glycol (reduction of glycolaldehyde), glycolic acid (oxidation of glycolaldehyde), or lactate (reduction of pyruvate), respectively. The main focus of this study was the application of a spectrophotometric assay to evaluate the efficiency of the cascade based on NADH formation or consumption. For this purpose, 2 mM NAD was added as an external cofactor. Furthermore, this study also investigated the role of lactonase (from Caulobacter crescentus) in the Dahms pathway. Data suggest that spontaneous ring-opening of xylonolactone is rate-limiting, especially at pH 7. Only the addition of lactonase significantly accelerated the overall rate of the cascade, which could also be achieved by increasing the pH (Boer et al., 2019).

[0017] From WO 2014 / 162063 A1 it is known to obtain glycolic acid together with lactic acid from the pentose xylose via fermentation in a metabolically engineered eukaryotic system (S. cerevisiae). However, the achieved product concentrations are far below the theoretical yield. For example, co-fermentation of glucose (10 g / L) and xylose (20 g / L) over a 2-day fermentation period produced only 0.927 g / L of lactic acid and 0.696 g / L of glycolic acid, and even lower concentrations from pure sugar fermentation.

[0018] The present invention addresses this problem by providing a method for preparing an aqueous solution containing the alkali salts of both glycolic acid and lactic acid, which method exhibits high yields and is operable at higher concentrations. Summary of the Invention

[0019] The objective is to demonstrate in vitro that in aqueous solution, alkaline salts of xylonic acid and / or arabinonic acid are capable of catalyzing the synthesis of dehydratases, aldolases, glycolaldehyde dehydrogenases, lactate dehydrogenases, and NAD as a cofactor. + This is achieved by treating the protein with an enzyme system containing the compound, and then isolating the enzyme system.

[0020] Surprisingly, it has been shown that the objectives set out in the present invention can be achieved when the conversion is not carried out fermentatively, but rather when the enzyme is present intact in aqueous solution and therefore functions in vitro. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 shows the reaction scheme of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0022] The term "enzyme system" refers to the four enzymes: dehydratase, aldolase, glycolaldehyde dehydrogenase, lactate dehydrogenase, and the cofactor NAD +The term "enzyme system" refers to the entire system of enzymes. These four enzymes can be added, for example, to an aqueous solution together with their cofactors (enzyme isolates). However, it is also possible to suspend cells expressing a particular enzyme in an aqueous solution. In this case, the term "enzyme system" should be understood as a cell suspension. Furthermore, the term "enzyme system" should be understood as a homogenate that can be obtained from a cell suspension by, for example, exposing the cells to ultrasound to disrupt them. Furthermore, the term "enzyme system" should also be understood as a lysate obtained from a homogenate by separating solid cell components. Finally, the term "enzyme system" should also be understood as an enzyme present immobilized in or on a matrix.

[0023] The main advantage of the method according to the invention is that the conversion can be carried out under non-physiological conditions, such as high substrate concentrations, at which the fermentation process cannot function.

[0024] Another major advantage of the method according to the invention is the low NAD + This can be done at low concentrations because it is essentially regenerated during the process. The formation of glycolate from glycolaldehyde produces NAD while generating NADH. + whereas the formation of lactate from pyruvate consumes NADH and thereby NAD + Since NAD is a regenerating substance, endogenous regeneration is possible. + This is a crucial advantage due to the high cost of acetone and makes the process easier to implement on a large scale.

[0025] The reaction scheme is shown in the accompanying diagram, which also illustrates endogenous regeneration. Oxidation and reduction occur in NAD + However, it can be initiated by NADH as well, and therefore, for purposes of the claims and description, NAD + also refers to the corresponding NADH.

[0026] In a further preferred variant of the method according to the invention, the enzymes forming the enzyme system are present in a suspension, homogenate and / or lysate of the corresponding cells from which they are formed. When a suspension, lysate and / or homogenate is used, small amounts of cofactors present in the suspension, lysate and / or homogenate are used to provide the final oxidation and reduction steps of NAD. + It has been demonstrated that there is no need to add cofactors for the dehydrogenase separately, since this is already sufficient due to endogenous regeneration of the enzyme.

[0027] Thus, through endogenous cofactor regeneration, the system can function without the addition of organic co-substrates, which impairs energy and carbon efficiency and complicates processing. As a result, the method according to the invention uses all the same paired cofactors (NAD + We extend the core reactions of the Dahms pathway, i.e., dehydration and aldol cleavage, by incorporating a closed redox system that uses ATP (ATP / NADH) to oxidize glycolaldehyde to glycolic acid and reduce pyruvate to lactate.

[0028] In this context, a suspension refers to a suspension of resting cells. These cells are harvested (separated from the nutrient medium) after cultivation and suspended in an appropriate buffer system. In contrast to fermentation methods, which also function with whole cells, resting cells can no longer grow due to the removal of carbon sources and nutrients, but rather function solely to convert substrates (Lin & Tao, 2017). A homogenate in this context refers to a physically and / or chemically treated suspension (e.g., treated with pressure, lysozyme, or ultrasound) in which 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 for details).

[0029] According to a preferred embodiment of the process according to the invention, potassium salts are used as alkaline salts.

[0030] In a further preferred embodiment of the method according to the invention, the concentration of the alkali salt of xylonic acid and / or arabinonic acid in the aqueous solution is 50-300 g / l, 100-200 g / l, and finally 150-250 g / l.

[0031] Furthermore, the process according to the invention is preferably carried out at a temperature of 20 to 50°C, 25 to 40°C, even more preferably 30 to 40°C.

[0032] A particularly preferred pH range for the reaction is 7.5 to 8.5.

[0033] According to another preferred aspect of the invention, all reaction steps of the method, as well as the regeneration of cofactors, are carried out in a single reaction vessel (one-pot reaction), thereby avoiding the isolation of expensive intermediates. In this embodiment, all enzymes are used at the start of the reaction.

[0034] The present invention is further based on the surprising discovery that the yield of glycolate is higher when lactate is simultaneously formed, for example, by use of lactate dehydrogenase, compared to the sole production of glycolate (and vice versa, see Example 5 below).

[0035] Thus, the conversion of arabonate and / or xylonate via 4,5-dihydroxy-2-oxopentanoate ((R)-4,5-dihydroxy-2-oxopentanoate = 2-keto-3-deoxy-arabonate (KDA) and / or (S)-4,5-dihydroxy-2-oxopentanoate = 2-keto-3-deoxy-xylonate (KDX)) to pyruvate and glycolaldehyde involves a dehydratase and an aldolase.

[0036] The dehydratases used in the present method are from the following group: EC 4.2.1.5 (arabonate dehydratase), 4.2.1.6 (galactonate dehydratase), 4.2.1.7 (altronate dehydratase), 4.2.1.8 (mannonate dehydratase), 4.2.1.9 (dihydroxy acid dehydratase), 4.2.1.25 (L-arabonate dehydratase), 4.2.1.39 (gluconate dehydratase), 4.2.1.40 (glucuronate dehydratase), 4.2.1.42 (galactarate dehydratase), 4.2.1.67 (D-fuconate dehydratase), 4.2.1.70 (galactonate dehydratase), 4.2.1.82 (galactonate dehydratase), 4.2.1.83 (galactonate dehydratase), 4.2.1.84 (galactonate dehydratase), 4.2.1.85 (galactonate dehydratase), 4.2.1.86 (galactonate dehydratase), 4.2.1.87 (galactonate dehydratase), 4.2.1.88 (galactonate dehydratase), 4.2.1.89 (galactonate dehydratase), 4.2.1.90 (galactonate dehydratase), 4.2.1.91 (dihydroxy acid dehydratase), 4.2.1.92 (dihydroxy acid dehydratase), 4.2.1.93 (L-arabonate dehydratase), 4.2.1.94 (gluconate dehydratase), 4.2.1.95 (galactonate dehydratase), 4.2.1.96 (galactonate dehydratase), 4.2.1. The enzyme may be from one of the group 4.2.1.68 (L-fuconate dehydratase), 4.2.1.82 (xylonate dehydratase), 4.2.1.140 (gluconate / galactonate dehydratase), 4.2.1.146 (L-galactonate dehydratase), 4.2.1.156 (L-talarate dehydratase), 4.2.1.158 (galactarate dehydratase, D-threo-forming), and 4.2.1.176 (L-lyxonate dehydratase), with group 4.2.1.25 (L-arabonate dehydratase) being particularly preferred.

[0037] The aldolases used in the present method are those in the following group: EC 4.1.2.18 (2-dehydro-3-deoxy-L-pentonate aldolase), 4.1.2.20 (2-dehydro-3-deoxyglucuronate aldolase), 4.1.2.21 (2-dehydro-3-deoxy-6-phosphogalactonate aldolase), 4.1.2.28 (2-dehydro-3-deoxy-D-pentonate aldolase), 4.1.2.29 (5-dehydro-2-deoxyphosphogluconate aldolase), 4.1.2.51 (2-dehydro-3-deoxy-D-gluconate aldolase), 4.1.2.52 (4-hydroxy-2-o The enzyme may be derived from one of 4.1.2.53 (2-keto-3-deoxy-L-rhamnonate aldolase), 4.1.2.54 (L-threo-3-deoxy-hexolose aldolase), 4.1.2.55 (2-dehydro-3-deoxy-phosphogluconate / 2-dehydro-3-deoxy-6-phosphogalactonate aldolase), and 4.1.3.39 (4-hydroxy-2-oxovalerate aldolase), with 4.1.2.55 (2-dehydro-3-deoxy-phosphogluconate / 2-dehydro-3-deoxy-6-phosphogalactonate aldolase) being particularly preferred.

[0038] The dehydrogenase used for the reduction of pyruvate to lactate is preferably from the group EC 1.1.1.27 (L-lactate dehydrogenase).

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

[0040] The enzymatic strategy presented herein minimizes the number of enzymes, thus enabling a highly efficient and cost-effective / price-competitive bioproduction process.

[0041] The following examples more fully illustrate preferred embodiments of the invention. [Example]

[0042] material 2-Keto-3-deoxyxylose lithium salt, sodium pyruvate, sodium L-lactate, and glycolaldehyde dimer, IPTG (isopropyl β-D-thiogalactopyranoside), and HEPES (2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid) were obtained from Sigma-Aldrich, and magnesium chloride hexahydrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, NAD + NADH disodium salt and sodium dodecyl sulfate (SDS) were obtained from Carl Roth, lysozyme and methanol were obtained from PanReac AppliChem (ITW Reagents), triethanolamine was obtained from Chem-Lab NV, and sodium glycolate was obtained from Alfa Aesar. Potassium L-arabonate and potassium D-xylonate were prepared from the corresponding sugars according to literature methods (Moore & Link, 1940).

[0043] Enzyme production For recombinant enzyme production in Escherichia coli strains, genomic DNA or its synthetic equivalent, adapted to E. coli codon usage, was first used as a template to amplify the gene to be expressed by PCR with specific oligonucleotides containing additional restriction endonuclease recognition sequences, which were then isolated from the reaction mixture. After digestion of the nucleic acid with the restriction enzymes SphI and HindIII, the gene fragment encoding the target enzyme was ligated into the SphI and HindIII-cleaved backbone of the expression vector pQE70-Kan. The ligation product was transformed into chemically competent E. coli Top10F cells, and the resulting colonies were used for plasmid isolation and restriction analysis.

[0044] The results of the cloning process were verified by restriction enzyme digestion and DNA sequencing. The resulting construct carries the target gene under an IPTG-inducible T5 promoter.

[0045] For overexpression of the enzyme in E. coli, the resulting expression plasmid was transformed into competent expression cells RB791. After incubation at 37°C for 24 hours, the resulting colonies were inoculated into LB medium for expression testing.

[0046] The next day, grow the expression culture at an optical density (OD) of 0.02. 550 seeded at an OD of 0.3 550 The mixture was shaken at 37°C until an OD of 0.5 was reached. The temperature was then lowered to 25°C. 550 Once the ATP concentration reached 0.1 mM, the cultures were induced with 0.1 mM IPTG. After 22 hours, the cultures were harvested (using centrifugation to separate the cell pellet from the medium) and analyzed for expression of the recombinant enzyme using SDS gel electrophoresis and activity assays (utilizing either the usage test or the optical enzyme assay).

[0047] Preparation of cell suspension To prepare the cell suspension, the cell pellet produced according to the method described above was weighed into a suitable container and buffer was added while stirring in an ice bath (see Table 1 for the buffer system used). The biomass mass fraction typically reached 20%, with the remainder consisting of buffer.

[0048] Preparation of homogenates by sonication disruption Lysozyme was added to the cell suspension prepared above at a concentration of 0.5 mg / ml. A Branson Sonifier 450 was used for cell disruption. The suspension was transferred to a 5 ml Eppendorf vial. The metal tip of the device was then submerged in the suspension, after which the suspension was subjected to three cycles of 15 ultrasonic pulses each (device settings: timer = 15; duty cycle = 50; output control = 3-5). In this way, a homogenate was obtained as a mixture of disrupted cells and buffer.

[0049] Preparation of lysates by centrifugation The homogenate prepared above was centrifuged (Eppendorf Centrifuge 5417R) at 4°C and 16,000 rpm for 10 minutes to separate insoluble cell fragments and obtain a lysate.

[0050] [Table 1]

[0051] Analysis method High-Performance Anion-Exchange Chromatography HPAEC (High-Performance Anion Exchange Chromatography) was used to determine substrate conversion and product concentrations. For this purpose, a Dionex ICS6000 system equipped with an AS-AP autosampler was used. Measurement of organic acids or their anions (xylonate, arabonate, 2-keto-3-deoxy-xylonate, 2-keto-3-deoxy-arabonate, lactate, and pyruvate), respectively, was performed by conductivity detection (CD) coupled with electrolytic regeneration inhibitors on a Dionex AERS 500 in external water mode. A Dionex IonPac AS11-HC-4 μm column, along with an appropriate precolumn and NaOH gradient, was used for analyte separation. The mobile phase was further pretreated with a Dionex ATC Anion Trap Column.

[0052] High-performance liquid chromatography HPLC (High Performance Liquid Chromatography) was used to quantify glycolaldehyde and glycolic acid. Detection was performed using a refractive index detector. A Phenomenex Rezex ROA-Organic Acid H+ (8%) column was used with the corresponding precolumn and eluted isocratically with 1 mM sulfuric acid.

[0053] Determination of enzyme activity (optical-enzyme assay) Enzyme activity in the homogenate or lysate, respectively, was determined using a Shimadzu UV-1900 spectrophotometer. NADH formation or consumption, respectively, was monitored by tracking the change in absorbance at a wavelength of 340 nm. 0.2 mM cofactor (NADH) was added. +Measurements were carried out using NADH (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 adjusted to the desired pH using 100 mM TEA-HCl buffer (870 μl). After temperature equilibration of the cuvette, 10 μl of homogenate or lysate (diluted or undiluted) and 100 μl of substrate solution were added, and the measurement was started immediately. Measurements were routinely carried out at 25°C. The enzyme activity of the lysate was determined using the extinction coefficient of NADH at 340 nm (ε = 6220 Lmol -1 cm -1 ) can be used to determine U / ml (based on the volume of lysate) or U / g (based on the biomass used for production). 1 U corresponds to 1 μmol of substrate conversion per minute (1 U = 1 μmol / min = 1.67 x 10 -8 kat).

[0054] General information on handling reaction solutions The aqueous solution containing glycolate and lactate prepared according to the present invention can be obtained by removing cellular components by denaturation (e.g., by heat treatment), followed by filtration or centrifugation after the reaction is complete. Membrane filtration can also be performed to remove smaller cellular components. The filtrate thus obtained can then be concentrated, for example, using a rotary evaporator.

[0055] The following examples explain in more detail preferred variants of the method according to the invention: The suspensions, homogenates and lysates used in these examples were prepared according to the methods described above.

[0056] Example 1 Treatment of xylonate / arabonate mixtures of different compositions (9:1 and 2:1 m / m) by enzyme systems. The following components were prepared in two 2 ml Eppendorf vials (vial 1 and vial 2): ​​100 μl of 2000 mM TEA-HCl buffer (pH 8.5), 200 μl of deionized water, 40 μl of dehydratase lysate, 40 μl of aldolase lysate, 2 U of glycolaldehyde dehydrogenase lysate, and 2 U of lactate dehydrogenase lysate. The reaction was initiated by adding 100 μl of potassium xylonate and potassium arabonate solution to each Eppendorf vial. The solution for vial 1 contained 9 parts potassium xylonate (225.7 g / L) and 1 part potassium arabonate (25.0 g / L), and the solution for vial 2 contained 2 parts potassium xylonate (166.3 g / L) and 1 part potassium arabonate (84.0 g / L). The batches were incubated with continuous shaking (1200 rpm, Eppendorf Thermomixer) for a total of 48 hours at 30° C. The total volume of each batch was 500 μl.

[0057] For processing, a 40 μl batch was combined with 160 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 maximum g-force for 10 min. The clear supernatant was diluted 1:250 and analyzed using HPAEC (conductivity detection). For HPLC, 150 μl of the clear supernatant was transferred to an HPLC vial for analysis (RI detection). The results are shown in the table below.

[0058] [Table 2]

[0059] [Table 3]

[0060] The table above shows that the enzyme system can process different mixtures of xylonate and arabonate with similar efficiency. This is important because the corresponding pentoses (xylose and arabinose) can be released from biomass and occur in various ratios. Therefore, they can be oxidized without prior separation, and the resulting mixture of sugar acids can be directly converted to glycolate and lactate by the enzyme system.

[0061] Example 2 Effect of different enzyme preparations (suspension, homogenate or lysate) on the processing of xylonate / arabonate (9:1 w / w) mixtures by enzyme systems The following components were prepared in a 2 ml Eppendorf vial: 100 μl of 2000 mM TEA-HCl buffer (pH 8.5), 200 μl of deionized water, 40 μl of dehydratase preparation (see table below for details), 40 μl of aldolase preparation (see table below for details), 2 U of glycolaldehyde dehydrogenase preparation, and 2 U of lactate dehydrogenase lysate. The reaction was initiated by adding 100 μl of a solution containing 9 parts potassium xylonate (225.7 g / L) and 1 part potassium arabonate (25.0 g / L). The batch was incubated at 30° C. with continuous shaking (1200 rpm, Eppendorf Thermomixer) for a total of 48 hours. The total volume was 500 μl.

[0062] For processing, a 40 μl batch was combined with 160 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 maximum g-force for 10 min. The clear supernatant was diluted 1:250 and analyzed using HPAEC (conductivity detection). For HPLC, 150 μl of the clear supernatant was transferred to an HPLC vial for analysis (RI detection). The results are shown in the table below.

[0063] [Table 4]

[0064] [Table 5]

[0065] [Table 6]

[0066] The table above shows that various enzyme formulations (suspension, homogenate, and lysate) can be used in the enzyme system. The highest conversion is achieved when all enzymes are added in lysate form (see Vial 1 in Example 1).

[0067] Example 3 NAD on the processing of xylonate / arabonate (9:1 w / w) mixture by enzyme systems. + Effects of Addition In a 2 ml Eppendorf vial (vial 1), the following components were prepared: 100 μl of 2000 mM TEA-HCl buffer (pH 8.5), 200 μl of deionized water, 40 μl of dehydratase lysate, 40 μl of aldolase lysate, 2 U of glycolaldehyde dehydrogenase lysate, and 2 U of lactate dehydrogenase lysate. In a second 2 ml Eppendorf vial (vial 2), a similar mixture was prepared, but with 10 μl of 10 mM NAD + solution, and 190 μl of deionized water instead of 200 μl was added. The reaction was initiated by adding 100 μl of a solution containing 9 parts potassium xylonate (225.7 g / L) and 1 part potassium arabonate (25.0 g / L) to both Eppendorf vials. The batches were incubated at 30° C. with continuous shaking (1200 rpm, Eppendorf Thermomixer) for a total of 48 hours. The total volume of each batch was 500 μl.

[0068] For processing, a 40 μl batch was combined with 160 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 maximum g-force for 10 min. The clear supernatant was diluted 1:250 and analyzed using HPAEC (conductivity detection). For HPLC, 150 μl of the clear supernatant was transferred to an HPLC vial for analysis (RI detection). The results are shown in the table below.

[0069] [Table 7]

[0070] [Table 8]

[0071] The above table shows the amount of cofactor (0.2 mM NAD + ) does not provide any advantage in terms of conversion under these conditions.

[0072] Example 4 Treatment of xylonate / arabonate (9:1 w / w) mixture (substrate concentration: 100 g / l) by enzyme system The following components were prepared in a 2 ml Eppendorf vial: 150 μl of 2000 mM TEA-HCl buffer (pH 8.5), 90 μl of deionized water, 60 μl of dehydratase lysate, 60 μl of aldolase lysate, 4 U of glycolaldehyde dehydrogenase lysate, and 4 U of lactate dehydrogenase lysate. The reaction was initiated by adding 100 μl of a solution containing 9 parts potassium xylonate (450.1 g / L) and 1 part potassium arabonate (51.7 g / L). The batch was incubated at 30° C. with continuous shaking (1200 rpm, Eppendorf Thermomixer) for a total of 48 hours. The total volume of the batch was 500 μl.

[0073] For processing, a 20 μl batch was combined with 180 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 maximum g-force for 10 min. The clear supernatant was diluted 1:250 and analyzed using HPAEC (conductivity detection). For HPLC, 150 μl of the clear supernatant was transferred to an HPLC vial for analysis (RI detection). The results are shown in the table below.

[0074] [Table 9]

[0075] The table above shows that this method is also suitable for converting more concentrated substrate solutions.

[0076] Example 5 Glycolaldehyde dehydrogenase and / or lactate dehydrogenase, and NAD + Treatment of a mixture of glycolaldehyde and pyruvate with In a 2 ml Eppendorf vial, the following components were prepared: 100 μl of 500 mM TEA-HCl buffer (pH 8.5; final concentration 100 mM), 0.1 U of lactate dehydrogenase (as lysate) and / or 0.1 U of glycolaldehyde dehydrogenase (as lysate), and cofactor solution (NADH, NAD + ). The final enzyme units and cofactor concentrations in the batches are listed in the table below. All batches were filled with the appropriate amount of deionized water to a total volume of 400 μl.

[0077] The reaction was initiated by adding 100 μl of substrate solution (50 mM sodium pyruvate and 50 mM glycolaldehyde in deionized water). The batches were incubated for 1 hour at 30° C. and 1200 rpm in an Eppendorf Thermomixer. The total volume of each batch was 500 μl.

[0078] For processing, one 200 μl batch was combined with 200 μl MeOH and incubated at 60° C. for 20 minutes (1200 rpm, Eppendorf Thermomixer). After denaturation, the sample was centrifuged at maximum g-force for 10 minutes. The clear supernatant was diluted 1:100 and analyzed using HPAEC (conductivity detection) to determine pyruvate and lactate. For HPLC (RI detection), 150 μl of the clear supernatant was transferred to an HPLC vial for analysis (glycolate and glycolaldehyde determination*). The results are shown in the table below.

[0079] The concentrations of glycolaldehyde and pyruvate were 10 mM in all batches.

[0080] [Table 10]

[0081] This table shows that when both lactate dehydrogenase and glycolaldehyde dehydrogenase were used simultaneously, the yields of glycolate and lactate (batch 1) were increased compared to batches 3 (glycolaldehyde dehydrogenase only) and 4 (lactate dehydrogenase only) with the same NAD + The results show that the NADH concentration (10 mM) significantly increased the NADH concentration (lactate: 5.9 mM to 7.9 mM; glycolate: 4.7 mM to 9.9 mM).

[0082] literature 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

[0083] Panova, A., Mersinger, LJ, Liu, Q., Foo, T., Roe, DC, Spillan, WL, Sigmund, AE, Ben-Bassat, A., Wagner, LW, O'Keefe, D., Wu, S., Petrillo, K., Payne, MS, Breske, ST, Gallagher, FG & DiCosimo, R. (2007). Chemoenzymatic synthesis of glycolic acid. Synthesis of Glycolic Acid).Advanced Synthesis&Catalysis,349(8-9),1462-1474.https: / / doi.org / 10.1002 / adsc.200700061

[0084] 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

[0085] 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 / s12010-014-1114-9

[0086] Dapsens, PY, Mondelli, C., Kusema, BT, Verel, R. & Perez-Ramirez, J. (2014). A continuous process for glyoxal valorization using tailored Lewis-acid zeolite catalysts. Green Chemistry, 16(3), 1176-1186. https: / / doi.org / 10.1039 / C3GC42353K

[0087] Hromadkova, Z. & Ebringerova, A. (1995). Isolation and Characterization of Hemicelluloses of Corn Hulls. Chemical Papers 49(2), 97-101. https: / / chempap.org / ?id=7&paper=3556

[0088] Collins, SRA, Wellner, N., Martinez Bordonado, I., Harper, AL, Miller, CN, Bancroft, I. & Waldron, KW (2014). Variation in the chemical composition of wheat straw: the role of tissue ratio and composition. Biotechnology for Biofuels, 7, 121. https: / / doi.org / 10.1186 / s13068-014-0121-y

[0089] Claassens, NJ, Burgener, S., Vogeli, B., Erb, TJ & Bar-Even, A. (2019). A critical comparison of cellular and cell-free bioproduction systems. Current Opinion in Biotechnology, 60, 221-229. https: / / doi.org / 10.1016 / j.copbio.2019.05.003

[0090] Salusjarvi, L., Havukainen, S., Koivistoinen, O. & Toivari, M. (2019). Biotechnological production of glycolic acid and ethylene glycol: current state and perspectives. Applied Microbiology and Biotechnology, 103, 2525-2535. https: / / doi.org / 10.1007 / s00253-019-09640-2

[0091] Pereira, B., Li, Z.-J., De Mey, M., Lim, C.G., Zhang, H., Hoeltgen, C. & Stephanopoulos G. (2016). Efficient utilization of pentoses for bioproduction of the renewable two-carbon compounds ethylene glycol and glycolate. Metabolic Engineering, 34, 80-87. https: / / doi.org / 10.1016 / j.ymben.2015.12.004

[0092] Deng, Y., Ma, N., Zhu, K., Mao, Y., Wei, X. & Zhao, Y. (2018). Balancing the carbon flux distributions between the TCA cycle and glyoxylate shunt to produce glycolate at high yield and titer in Escherichia coli. Metabolic Engineering, 46, 28-36. https: / / doi.org / 10.1016 / j.ymben.2018.02.008

[0093] Alkim, C., Trichez, D., Cam, Y., Spina, L., Francois, JM & Walther, T. (2016). The synthetic xylulose-1 phosphate pathway increases production of glycolic acid from xylose-rich sugar mixtures. Biotechnology for Fuels, 9, 201. https: / / doi.org / 10.1186 / s13068-016-0610-2

[0094] Salusjarvi, L., Toivari, M., Vehkomaki, M.-L., Koivistoinen, O., Mojzita, D., Niemela, K., Penttila, M. & Ruohonen, L. (2017). Production of ethylene glycol or glycolic acid from D-xylose in Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, 101, 8151-8163. https: / / doi.org / 10.1007 / s00253-017-8547-3

[0095] Uranukul, B., Woolston, BM, Fink, GR & Stephanopoulos, G. (2018). Biosynthesis of monoethylene glycol in Saccharomyces cerevisiae utilizing native glycolytic enzyme. Metabolic Engineering, 51, 20-31. https: / / doi.org / 10.1016 / j.ymben.2018.09.012

[0096] Buchner, E. 1897.

[0097] Welch, P. & Scopes, R.K. (1985). Studies on cell-free metabolism: Ethanol production by a yeast glycolytic system reconstituted from purified enzymes. Journal of Biotechnology, 2(5), 257-273. https: / / doi.org / 10.1016 / 0168-1656(85)90029-X

[0098] Goldberg, K., Schroer, K., Lutz, S., & Liese, A. (2007). Biocatalytic ketone reduction—a powerful tool for the production of chiral alcohols—part I: processes with isolated enzymes. Applied Microbiology and Biotechnology, 76, 237-248. https: / / doi.org / 10.1007 / s00253-007-1002-0

[0099] Schrittwieser, JH, Velikogne, S., Hall, M. & Kroutil, W. (2018). Artificial Biocatalytic Linear Cascades for Preparation of Organic Molecules. Chemical Reviews, 118(1), 270-348. https: / / doi.org / 10.1021 / acs.chemrev.7b00033

[0100] Dahms, AS(1974). 3-Deoxy-D-pentulosonic acid aldolase and its role in a new pathway of D-xylose degradation. Biochemical and Biophysical Research Communications, 60(4), 1433-1439. https: / / doi.org / 10.1016 / 0006-291X(74)90358-1

[0101] Kopp, D., Bergquist, P.L., & Sunna, A. (2020). Enzymology of Alternative Carbohydrate Catabolic Pathways. Catalysts, 10(11), 1231. https: / / doi.org / 10.3390 / catal10111231

[0102] Wee, Y.-J., Kim, J.-N., & Ryu, H.-W. (2006). Biotechnological Production of Lactic Acid and Its Recent Applications. Food Technology & Biotechnology, 44(2), 163-172. https: / / www.ftb.com.hr / 80-volume-44-issue-no-2 / 445-biotechnological-production-of-lactic-acid-and-its-recent-applications

[0103] Smith, WP (1996). Epidermal and dermal effects of topical lactic acid. Journal of the American Academy of Dermatology, 35(3), 388-391. https: / / doi.org / 10.1016 / s0190-9622(96)90602-7

[0104] Balla, E., Daniilidis, V., Karlioti, G., Kalamas, T., Stefanidou, M., Bikiaris, ND, Vlachopoulos, A., Koumentakou, I. & Bikiaris, DN(2021). Poly(lactic acid): A versatile biobased polymer for the future with multifunctional properties - From monomer synthesis, polymerization techniques and molecular weight increase to PLA applications. Polymers, 13(11), 1822. https: / / doi.org / 10.3390 / polym13111822

[0105] Boer, H., Andberg, M., Pylkkanen, R., Maaheimo, H. & Koivula, A. (2019). In vitro reconstitution and characterization of the oxidative D-xylose pathway for production of organic acids and alcohols. AMB Express, 9, 48. https: / / doi.org / 10.1186 / s13568-019-0768-7

[0106] Lin, B. & Tao, Y. (2017). Whole-cell biocatalysts by design. Microbial Cell Factories, 16, 106. https: / / doi.org / 10.1186 / s12934-017-0724-7

[0107] Moore, S. & Link, KP (1940). CARBOHYDRATE CHARACTERIZATION: I. THE OXIDATION OF ALDOSES BY HYPOIODITE IN METHANOL II. THE IDENTIFICATION OF SEVEN ALDO-MONOSACCHARIDES AS BENZIMIDAZOLE DERIVATIVES. Journal of Biological Chemistry, 133, 293-311. https: / / doi.org / 10.1016 / S0021-9258(18)73312-7

[0108] Setubal, JC, two Santos , P , Goldman , BS , Ertesvag , H , Espin , G , Rubio , LM , Valla , S , Almeida , NF , Balasubramanian , D , Cromes , L , Curatti , L , Du , Z , Godsy , E , G oodner ,B ,Hellner-Burris ,K ,Hernandez ,JA ,Houmiel ,K ,Imperial ,J ,Kennedy ,C ,Larson ,TJ ,Latreille ,P ,Ligon ,LS ,Lu ,J ,Maerk ,M ,Miller ,NM,Norton,S.,O'Carroll,IP,Paulsen,I.,Raulfs,EC,Roemer,R.,Rosser,J.,Segura,D.,Slater,S.,Stricklin,SL,Studholme,DJ,Sun,J.,Viana,C J , Wallin , E. , Wang , B. , Wheeler , C. , Zhu , H. , Dean , DR , Dixon , R. & Wood , D. (2). 009).Specifically, the thermostatically smooth surfaces of the snowflakes can be removed Recombinant sludge·protozoa(Azotobacter). vinelandii) (Genome sequence of Azotobacter vinelandii,an obligate aerobe specialized to support various anaerobic metabolic processes).Journal of Bacteriology, 191(14), 4534-4545

[0109] Protein [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Accession number ACO81022.1, L-arabonate dehydratase [Azotobacter vinelandii DJ]; [Accessed 04.10.2022]. Available at https: / / www.ncbi.nlm.nih.gov / protein / 226721851

[0110] Wolterink-van Loo, S., Siemerink, MAJ, Perrakis, G., Kaper, T., Kengen, SWM & van der Oost, J. (2009). Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase. Archaea, 2(4), 233-239. https: / / doi.org / 10.1155 / 2009 / 194186

[0111] Hidalgo, E., Chen, Y.-M., Lin, ECC & Aguilar, J. (1991). Molecular cloning and DNA sequencing of the Escherichia coli K-12 ald gene encoding aldehyde dehydrogenase. Journal of Bacteriology, 173(19), 6118-6123. https: / / doi.org / 10.1128 / jb.173.19.6118-6123.1991

[0112] Zheng, Y., Guo, S., Guo, Z. & Wang, X. (2004). Effects of N-terminal deletion mutations on rabbit muscle lactate dehydrogenase. Biochemistry (Moscow), 69(4), 401-406. https: / / doi.org / 10.1023 / b:biry.0000026195.31821.e1

Claims

1. A method for producing an aqueous solution containing the alkali salts of glycolic acid and lactic acid by treating, in vitro, the alkali salts of xylonic acid and / or arabinonic acid in an aqueous solution with an enzyme system comprising a dehydratase, an aldolase, glycolaldehyde dehydrogenase, lactate dehydrogenase and NAD+ as a cofactor, and then separating the enzyme system.

2. 2. The method according to claim 1, wherein the concentration of the alkali salt of xylonic acid and / or arabinonic acid in the aqueous solution is between 50 and 300 g / l.

3. 3. The method according to claim 2, wherein the concentration of the alkali salt of xylonic acid and / or arabinonic acid in the aqueous solution is from 100 to 200 g / l.

4. 4. The method according to claim 3, wherein the concentration of the alkali salt of xylonic acid and / or arabinonic acid in the aqueous solution is between 150 and 250 g / l.

5. 5. The process according to any one of claims 1 to 4, characterized in that it is carried out at a temperature of from 20 to 50°C.

6. 6. The process according to claim 5, characterized in that it is carried out at a temperature of 25 to 40°C.

7. 7. The process according to claim 6, characterized in that it is carried out at a temperature of 30 to 40°C.

8. 8. The method according to any one of claims 1 to 7, characterized in that the enzymes forming the enzyme system are present as a lysate of the corresponding cells from which they are formed.

9. 8. The method according to any one of claims 1 to 7, characterized in that the enzymes forming the enzyme system are present as a homogenate of the corresponding cells forming them.

10. 8. The method according to any one of claims 1 to 7, characterized in that the enzymes forming the enzyme system are present as a suspension of the corresponding cells forming them.

11. 12. The method according to claim 11, characterized in that the pH is between 7.5 and 8.5.

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