Method for producing an aqueous solution containing alkaline salt of glycolic acid and lactic acid

Abstract

The invention relates to a method for producing an aqueous solution containing alkaline salt of glycolic acid and lactic acid in that alkaline salt of xylonic acid and / or arbonic acid is treated with an enzyme system in vitro in an aqueous solution, said enzyme system containing dehydratase, aldolase, glycolaldehyde dehydrogenase, lactate dehydrogenase, and NAD+ as cofactors. The enzyme system is then separated.

Description

[0001] Process for the preparation of an aqueous solution containing an alkali salt of glycolic acid and lactic acid

[0002] The invention relates to a process for producing an aqueous solution containing an alkali salt of glycolic acid and lactic acid. Such solutions are used in the cosmetics industry as a base for skin peeling products.

[0003] The invention is a carbon-efficient enzymatic process for producing chemicals from renewable carbon sources. Specifically, it involves the atom-efficient production of alkali glycolate and alkali lactate using an enzymatic process in which D-xylonate and L-arabonate, the C1 oxidation products of the pentoses D-xylose and L-arabinose, are converted to the C1 building block glycolic acid (glycolate) and the C1 building block lactic acid (lactate).

[0004] 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.

[0005] Background of the invention

[0006] 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 considered problematic, especially when used in cosmetic products (e.g., skin peels (Sharad, 2013)), there is a need for alternative synthesis methods.

[0007] Chemo-enzymatic methods for glycolic acid production include 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. Another method is the conversion of ethylene glycol to glycolic acid using a microbial whole-cell biocatalyst (Gao et al., 2014). In this second case, the starting material is also generally chemically produced in an energy-intensive manner. One chemical process for the production of glycolic acid that uses a renewable raw material as the starting material 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 obtained by pyrolysis of biomass – an energy-intensive process in which the original composition of the biomass is destroyed and the synthetic potential of the raw material remains unused.

[0008] To develop sustainable production processes for chemicals from biomass, less energy-intensive methods are needed that utilize the existing diversity of the biomaterial. In addition to efficient, gentle methods for the depolymerization of renewable raw materials into shorter-chain sugars, efficient technologies are needed to further convert these carbohydrate intermediates into chemical products. A particular challenge lies in efficiently and as completely as possible converting the mixtures of substances resulting from depolymerization into valuable material streams. Straws, wood, and other plant-based raw materials generally contain hemicellulose and cellulose, from which monomer sugars can be released in varying compositions, primarily the pentoses xylose and arabinose, the hexoses glucose, mannose, and galactose, as well as acids derived from the sugars (e.g., glucuronic acid).Methods for the efficient separation of C6 and C5 sugars allow these material stream types to be further treated separately (e.g., WO 2011 / 014894). For example, corn hulls contain 10% arabinose, 16% xylose, 64% glucose, 4% galactose, and 2% mannose, based on total sugars (Hromädkovä & Ebringerovä, 1995), whereas wheat straw contains 5% arabinose, 30% xylose, 56% glucose, 1% galactose, and 2% mannose (Collins et al., 2014). These percentages demonstrate that the ratios of arabinose to xylose differ significantly in the two biomasses (corn hulls: 1:1.6 and wheat straw: 1:6).

[0009] Currently, biotechnological efforts primarily focus on processes aimed at converting biomass into chemical products through fermentation, i.e., the conversion of substances during the growth of microbes in a reactor. Despite all technical advances in the field, such as metabolic engineering and heterologous pathway expression, these fermentative whole-cell processes are limited by the physiological limitations of cellular production (tolerance to solvents, temperature, mass transport, and high substrate and product concentrations, as well as by-products from other metabolic pathways in the cell) (Claassens et al., 2019). These intracellular processes, and in particular the energy requirements for the metabolic (often carbon-emitting) steps, lead to long process times and, on the other hand, to the achievable carbon yield being significantly below the theoretically possible.In addition, these processes generally use biosynthetic pathways that release CO2 from the substrate, so that even the theoretical carbon yield in the product is only a fraction of the carbon used: While 1-4 carbons are lost for Cg sugars, depending on the pathway, the figure is 1-3 for C5 sugars. Typical key data of such processes are summarized in the literature (Salusjärvi et al., 2019).

[0010] For example, the fermentative production of glycolic acid from glucose via the glyoxylate shunt using metabolic engineering in recombinant E. coli strains is known (WO 2007 / 141316A2, WO 2007 / 140816A1, WO 2010 / 108909A1, WO 2011 / 036213A2). In one case, a 93-hour fermentation process resulted in the fermentative production of 31.3 g / l of glycolic acid in the fermentation supernatant, with yields of 0.22 g of glycolic acid per gram of glucose (WO 2007 / 141316A2). WO 2011 / 036213A2 then describes a further optimized fermentation process of the system, which achieves 53.9 g / l glycolic acid with a yield of 0.36 g glycolic acid per gram of glucose within 40 h through adapted process control.

[0011] Fermentative glycolic acid production from pentoses, e.g., via the combination of the ribulose-l-phosphate pathway and glyoxylate shunt, has also been described in the literature (Pereira et al., 2016). In this process, metabolic engineering of an E. coli strain results in a yield of 0.62 g of glycolic acid per gram of xylose. The final glycolic acid concentration after 85 h is 41 g / L in the fermentation broth, resulting in a yield of 61% of the theoretical yield (1.22 mol / mol). In general, the upper limit of fermentative approaches for glycolic acid production is currently 65.5 g / L final glycolic acid concentration and 90% of the theoretical yield (where 100% theoretical yield means the loss of one carbon atom per sugar molecule) when pure glucose is used as a substrate (Deng et al., 2018; Salusjärvi et al., 2019). Similar values ​​are achieved for pure xylose (44 g / L final glycolic acid concentration, 87% of the theoretical yield (Pereira et al., 2019)., 2016; Salusjärvi et al., 2019). However, with sugar mixtures (xylose + glucose), the achievable glycolic acid concentrations are an order of magnitude lower (Alkim et al., 2016; Salusjärvi et al., 2019). This is also the case in analogous systems that realize the fermentative conversion of sugars to ethylene glycol (Pereira et al., 2016; Salusjärvi et al., 2017; Uranukul et al., 2018; Salusjärvi et al., 2019).

[0012] In summary, existing fermentative systems generally accept the loss of at least one carbon per sugar monomer and only achieve low final product concentrations in sugar mixtures.

[0013] Cell-free conversion of metabolites was also demonstrated as early as 1897 by Eduard Buchner, who converted glucose into ethanol using a cell lysate of Saccharomyces cerevisiae (Buchner, 1897). In 1985, Welch and Scopes presented a cell-free system for ethanol production (Welch & Scopes, 1985), which, however, was not technologically viable due to a lack of specificity.

[0014] Since then, a number of other processes have been described to produce chemicals from purified enzymes (enzyme isolates). For example, alcohol dehydrogenases are used to produce high-quality chiral alcohols, where the cofactor NAD is regenerated, for example, by adding glucose and glucose dehydrogenase (Goldberg et al., 2007). Generally, glucose or formate dehydrogenases (GDHs or FDHs) and alcohol dehydrogenases (ADHs) are used for cofactor recycling (Schrittwieser et al., 2018).

[0015] In recent years, interest has shifted to processes that utilize highly selective conversions via enzyme cascade reactions to obtain the target chemical in a single step (one-pot). EP2700714A1, US8859247B2, and EP2204453B1 describe a portfolio of cascade reactions in which glucose is converted into two molecules of pyruvate in a cascade of five enzymatic transformations. Starting from glucose, the cascade follows the non-phosphorylating Entner-Doudoroff pathway to D-glyceraldehyde and pyruvate (sugar oxidation, dehydration, and aldol cleavage). D-glyceraldehyde is then further oxidized to D-glycerate and subsequently dehydrated to pyruvate. This then serves as a platform for further transformations to a range of amino acids and alcohols.The enzymes in the cascade include a glucose dehydrogenase, a promiscuous dihydroxy acid dehydratase (catalyzes dehydration of gluconate and D-glycerate), a 2-keto-3-deoxygluconate aldolase, and an aldehyde dehydrogenase. Using a promiscuous dehydrogenase that accepts both glucose and D-glyceraldehyde as substrates, the system can be reduced to three enzymes.

[0016] A similar cascade exists for the pentose xylose, known as the Dahms pathway (Dahms, 1974). Xylose is first oxidized to 1,4-xylonolactone, which is opened to xylonate under the action of a lactonase. A dehydratase converts xylonate to 2-keto-3-deoxyxylonate, which is cleaved by an aldolase into pyruvate and glycolaldehyde. Arabinose can also be converted via this pathway in organisms such as Sulfolobus solfataricus thanks to promiscuous enzymes in the pathway (Kopp et al., 2020).

[0017] Pyruvate is a key intermediate in cellular metabolism and serves, for example, as a precursor to the amino acid alanine or acetyl-CoA, which is involved in the citric acid cycle. Reduction of pyruvate produces lactic acid or lactate, which has a wide range of applications. Lactic acid is used, for example, as an acidifier in the food industry, as a descaling agent, pH regulator, or cleaning agent in the chemical industry, as an additive in anti-acne creams or moisturizers in the cosmetics industry, and as a precursor for acrylic acid or ethyl lactate (Wee et al., 2006). Furthermore, lactic acid (like glycolic acid) can also be used for skin exfoliation (chemical peeling) (Smith, 1996).The bifunctional lactic acid can also be polymerized to produce polylactide (PLA), a biodegradable and biocompatible plastic that has applications in the packaging industry, textile industry, electronics and medicine ( Bal la et al., 2021).

[0018] Boer et al. presented an in vitro variant of the Dahms pathway using purified enzyme isolates, in which xylose or xylonolactone (1 mM substrate concentration) is converted to ethylene glycol (reduction of glycolaldehyde), glycolic acid (oxidation of glycolaldehyde), or lactate (reduction of pyruvate). The main focus of the study was the application of a spectrophotometric assay to evaluate the efficiency of the cascade, which is based on the formation or consumption of NADH. For this purpose, 2 mM NAD was added as an external cofactor. Furthermore, the study investigated the role of lactonase (from Caulobacter crescentus) in the Dahms pathway. The data suggest that the spontaneous ring opening of xylonolactone is rate-limiting, especially at pH 7. Only the addition of lactonase noticeably accelerates the overall kinetics of the cascade, which can also be achieved by increasing the pH value (Boer et al., 2019).

[0019] WO 2014 / 162063A1 discloses the fermentation of glycolic acid and lactic acid from the pentose xylose in a metabolically engineered eukaryotic system (S. cerevisiae). However, the product concentrations achieved are far below the theoretical yield. For example, in the cofermentation of glucose (10 g / l) and xylose (20 g / l) over a fermentation time of 2 days, only 0.927 g / l of lactic acid and 0.696 g / l of glycolic acid are obtained; the concentrations achieved from fermentations of pure sugar are even lower.

[0020] This is where the present invention comes in, which aims to provide a process for producing an aqueous solution containing both an alkali salt of glycolic acid and lactic acid, which has high yields and can be carried out at higher concentrations.

[0021] Detailed description of the invention

[0022] This goal is achieved by a process in which an alkali salt of xylonic acid and / or arabonic acid in an aqueous solution is treated in vitro with an enzyme system comprising a dehydratase, an aldolase, a glycolaldehyde dehydrogenase, a lactate dehydrogenase and NAD + as a cofactor, after which the enzyme system is separated. Surprisingly, it has been shown that the objectives of the invention can be achieved if the reaction is not carried out fermentatively, but rather if the enzymes are contained as such in the aqueous solution, i.e., if the reaction is carried out in vitro.

[0023] The term "enzyme system" refers to the totality of the four enzymes, i.e. dehydratase, aldolase, glycolaldehyde dehydrogenase, lactate dehydrogenase and NAD +as a cofactor. These four enzymes, along with the cofactor, can be added as such (enzyme isolates) to the aqueous solution. However, it is also possible to suspend cells expressing the specified enzymes in the aqueous solution. In this case, the term "enzyme system" refers to a cell suspension. Furthermore, the term "enzyme system" refers to a homogenate that can be obtained from the cell suspension, for example, by subjecting the cells to ultrasound to cause them to burst. Furthermore, the term "enzyme system" also refers to lysates that are obtained from the homogenates by separating the solid cell components. Finally, the term "enzyme system" also refers to enzymes when they are immobilized in or on a matrix.

[0024] A major advantage of the process according to the invention is that the conversion can also be carried out under non-physiological conditions, such as high substrate concentrations, i.e. at concentrations under which a fermentative process cannot operate.

[0025] Another great advantage of the process according to the invention is that it can be used at low NAD + concentrations because it is intrinsically regenerated in the process. The intrinsic regeneration is possible because the formation of glycolate from glycolaldehyde is an oxidation, which NAD + to form NADH, while the formation of lactate from pyruvate is a reduction which consumes NADH, whereby NAD + This intrinsic regeneration is due to the high price of NAD +a decisive advantage and allows the process to be carried out on a large scale.

[0026] The reaction scheme is shown in the attached figure, which also shows the intrinsic regeneration. Oxidation and reduction can be achieved by NAD + but equally also by NADH, so that for the purposes of the present claims and description NAD + also stands for the corresponding NADH.

[0027] In a further preferred variant of the method according to the invention, the enzymes forming the enzyme system are present in a suspension, in the homogenate and / or in the lysate of the corresponding cells forming them. It has been shown that when using suspensions, lysates and / or homogenates, the cofactor of the dehydrogenases does not need to be added separately, because the small amounts of cofactor present in the suspension, lysate and / or homogenate are sufficient due to the intrinsic regeneration of NAD. + are already sufficient to allow the final step of oxidation and reduction to take place.

[0028] Due to the intrinsic cofactor regeneration, the system thus does not require the addition of an organic cosubstrate, which would impair energy and carbon efficiency and complicate downstream processing. The process according to the invention thus extends the core reactions of the Dahms pathway, namely dehydration and aldol cleavage, to a self-contained redox system, which uses the same cofactor pair (NAD + / NADH) glycolaldehyde is oxidized to glycolic acid and pyruvate is reduced to lactate.

[0029] In this context, suspension means a suspension of resting cells. These are harvested after cultivation (separated from the nutrient medium) and suspended in a suitable buffer system. In contrast to fermentative processes, which also work with whole cells, the resting cells can no longer grow due to the removal of carbon sources and nutrients; instead, they only serve to convert substrates (Lin & Tao, 2017). In this context, homogenate refers to a physically and / or chemically treated 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 Production Enzymes for details).

[0030] According to a preferred embodiment of the process according to the invention, a potassium salt is used as the alkali salt.

[0031] In further preferred embodiments of the process according to the invention, the concentration of the alkali salt of xylonic acid and / or arabonic acid in the aqueous solution is 50 - 300 g / l, 100 - 200 g / l and finally 150 - 250 g / l.

[0032] In addition, it is preferred that the process according to the invention is carried out at a temperature between 20 and 50 °C, between 25 and 40 °C and even more preferably between 30 and 40 °C.

[0033] The most preferred pH range of the reaction is between 7.5 and 8.5.

[0034] According to a further preferred aspect of the invention, all reaction steps of the process, as well as the regeneration of the cofactors, take place in a single reaction vessel (one-pot reaction), thus avoiding the costly isolation of intermediates. In this embodiment, all enzymes are used at the beginning of the reaction.

[0035] The present invention is also based on the surprising finding that the yield of glycolate, for example, is higher when lactate is formed simultaneously by using lactate dehydrogenase, compared to the sole production of glycolate (and vice versa, see Example 5 below).

[0036] 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 aldolase.

[0037] The dehydratase used in the process can be selected from one of the groups 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 (glucarate dehydratase), 4.2.1.42 (galactarate dehydratase), 4.2.1.67 (μ-fuconate dehydratase), 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.

[0038] The aldolase used in the process can be selected from one of the groups EC 4.1.2.18 (2-dehydro-3-deoxy-L-pentonate aldolase), 4.1.2.20 (2-dehydro-3-deoxyglucarate 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-oxoheptandioate aldolase), 4.1.2.53 (2-keto-3-deoxy-L-rhamnonate aldolase), 4.1.2.54 (L-threo-3-deoxy-hexylosonate 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 the group 4.1.2.55 (2-dehydro-3-deoxy-phosphogluconate / 2-dehydro-3-deoxy-6-phosphogalactonate aldolase) being particularly preferred.

[0039] The dehydrogenase used to reduce pyruvate to lactate preferably comes from groups EC 1.1.1.27 (L-lactate dehydrogenase).

[0040] The dehydrogenase used for the oxidation of glycolaldehyde to glycolic acid preferably comes from group EC 1.2.1.21 (glycolaldehyde dehydrogenase).

[0041] The enzymatic strategy presented here minimizes the number of enzymes and thus allows a highly efficient and cost-effective / price-competitive bioproduction process.

[0042] The following examples describe preferred embodiments of the invention in more detail.

[0043] Materials

[0044] 2-Keto-3-deoxyxylonic acid lithium salt, sodium pyruvate, sodium L-lactate and glycolaldehyde dimer, IPTG (isopropyl-ß-D-thiogalactopyranoside) and HEPES (2-(4-(2-hydroxyethyl)-l-piperazinyl)-ethanesulfonic acid) were purchased from Sigma-Aldrich, magnesium chloride hexahydrate, potassium dihydrogen phosphate, di-potassium hydrogen phosphate, NAD +NADH disodium salt and sodium dodecyl sulfate (SDS) were purchased from Carl Roth. Lysozyme and methanol were purchased from PanReac AppliChem (ITW Reagents). Triethanolamine was purchased from Chem-Lab NV. Sodium glycolate was purchased from Alfa Aesar. Potassium L-arabonate and potassium D-xylonate were prepared from the corresponding sugars according to the literature procedure (Moore & Link, 1940).

[0045] Production of enzymes

[0046] 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, synthetically adapted to the codon usage of E. coli, as a template, together with specific oligonucleotides additionally carrying recognition sequences for restriction endonucleases. The gene fragment encoding the target enzyme was isolated from the reaction mixture. After nucleic acid digestion 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 cells (ToplOF), and the resulting colonies were used for plasmid isolation and restriction analysis. The result of the cloning step was verified by restriction enzyme digestion and DNA sequencing.The resulting construct carries the target gene under the IPTG-inducible T5 promoter.

[0047] 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.

[0048] 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).

[0049] Production of cell suspensions

[0050] To prepare cell suspensions, the cell pellets prepared according to the above procedure were weighed into a suitable container, mixed with buffer (see Table 1 for buffer systems used), and dissolved in an ice bath with stirring. The mass fraction of biomass was typically 20%, with the remainder being buffer.

[0051] Preparation of homogenates using sonifier digestion

[0052] 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 immersed in the suspension, after which the suspension was treated three times with 15 ultrasonic pulses each (device settings: Timer = 15; Duty Cycle = 50; Output Control = 3-5). This yielded a homogenate as a mixture of disrupted cells and buffer.

[0053] Preparation of lysates by centrifugation

[0054] The homogenate prepared above was centrifuged for 10 min at 4 °C and 16000 rpm (Eppendorf Centrifuge 5417R) to separate the insoluble cell fragments and obtain the lysate.

[0055] Table 1. Donor organisms and digestion conditions for the enzymes used in the examples.

[0056] Analytical methods

[0057] High Performance Anion Exchange Chromatography

[0058] Substrate conversions and product concentrations were determined by HPAEC (High Performance Anion Exchange Chromatography). A Dionex ICS6000 system with an AS-AP autosampler was used for this purpose. The organic acids and their anions (xylonate, arabonate, 2-keto-3-deoxyxylonate, 2-keto-3-deoxyarabonate, lactate, and pyruvate) were measured using conductivity detection (CD) coupled to a Dionex AERS 500 electrolytically regenerated suppressor in external water mode. A Dionex IonPac AS11-HC-4pm column with a corresponding precolumn and a NaOH gradient was used to separate the analytes. The mobile phase was additionally pretreated with a Dionex ATC Anion Trap Column.

[0059] High Performance Liquid Chromatography

[0060] 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 with a corresponding precolumn was used for the measurement and eluted isocratically with 1 mM sulfuric acid.

[0061] Determination of enzyme activities (optical-enzymatic assay)

[0062] Enzyme activities in the homogenates and lysates were determined using a Shimadzu UV-1900 spectrophotometer. NADH formation and consumption were monitored at a wavelength of 340 nm via the change in absorption. 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). After tempering 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. The measurements were carried out at 25 °C as standard. The extinction coefficient of NADH 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 conversion per minute (1 U = 1 pmol / min = 1.67-10' 8 cat).

[0063] General information on the processing of the reaction solution

[0064] The glycolate- and lactate-containing aqueous solution obtained according to the invention is prepared by removing the cell components after the reaction has ended by denaturation (e.g., by applying heat) followed by filtration or centrifugation. Membrane filtration can also be performed to remove smaller cell components. The resulting filtrate can then be concentrated, for example, using a rotary evaporator.

[0065] The following examples describe preferred variants of the method according to the invention in more detail. The suspensions, homogenates, and lysates used in these examples were prepared according to the methods described above.

[0066] Example 1

[0067] Treatment of xylonate / arabonate mixtures in different compositions (9 / 1 and 2 / 1 m / m) with the enzyme system

[0068] The following components were placed in two 2 ml Eppendorf vials (Vial 1 and Vial 2): ​​100 μl of a 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 reactions were initiated by adding 100 μl each of a solution of potassium xylonate and potassium arabonate to both Eppendorf vials. The solution for vial 1 contained 9 parts potassium xylonate (225.7 g / l) and 1 part potassium arabonate (25.0 g / l); the solution for vial 2 contained 2 parts potassium xylonate (166.3 g / l) and 1 part potassium arabonate (84.0 g / l). The samples were incubated for a total of 48 h at 30 °C with continuous shaking (1200 rpm, Eppendorf Thermomixer). The total volume of each sample was 500 μl.

[0069] For processing, 40 μl of a sample was mixed with 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 samples were centrifuged at max. 1 g for 10 min. The clear supernatant was diluted 1:250 and measured using HPAEC (conductivity detection). For HPLC, 150 μl of the clear supernatant was transferred to HPLC vials and measured (RI detection). The results are presented in the table below. * Due to peak overlap in HPAEC, substrate conversion can only be given as a sum parameter.

[0070] ** The concentrations and masses of the products glycolate and lactate as well as their conversions were calculated for the respective free acids.

[0071] The tables above show that the enzyme system can process different ratios of xylonate and arabonate with similar efficiency. This is relevant because the corresponding pentoses (xylose and arabinose) can be released from biomass, where they occur in different ratios. These can thus be oxidized without prior separation, and the resulting mixtures of sugar acids can be directly converted by the enzyme system to glycolate and lactate.

[0072] Example 2

[0073] Influence of different enzyme formulations (suspension, homogenate or lysate) on the treatment of a xylonate / arabonate (9 / 1 m / m) mixture with the enzyme system

[0074] The following components were placed in a 2 ml Eppendorf vial: 100 μl of a 2000 mM TEA-HCl buffer (pH 8.5), 200 μl of deionized water, 40 μl of a dehydratase formulation (see the following tables for details), 40 μl of an aldolase formulation (see the following tables for details), 2 U of a glycolaldehyde dehydrogenase formulation, and 2 U of lactate dehydrogenase lysate. The reaction was initiated by adding 100 μl of a solution of 9 parts potassium xylonate (225.7 g / l) and 1 part potassium arabonate (25.0 g / l). The mixture was incubated for a total of 48 h at 30 °C with continuous shaking (1200 rpm, Eppendorf Thermomixer). The total volume was 500 μl.

[0075] For processing, 40 μl of the mixture was mixed with 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 samples were centrifuged at max. 1 g for 10 min. The clear supernatant was diluted 1:250 and measured using HPAEC (conductivity detection). For HPLC, 150 μl of the clear supernatant was transferred to HPLC vials and measured (RI detection). The results are presented in the table below.

[0076] * Due to peak overlap in HPAEC, substrate conversion can only be given as a sum parameter.

[0077] ** The concentrations and masses of the products glycolate and lactate as well as their conversions were calculated for the respective free acids.

[0078] The tables above show that different enzyme formulations (suspensions, homogenates, and lysates) can be used for the enzyme system. The highest conversion is achieved when all enzymes are added in the form of lysates (see vial 1 of Example 1).

[0079] Example 3

[0080] Influence of NAD + -Addition to the treatment of a xylonate / arabonate (9 / 1 m / m) mixture with the enzyme system

[0081] The following components were placed in a 2 ml Eppendorf vial (Vial 1): 100 μl of a 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. A similar mixture was added to a second 2 ml Eppendorf vial (Vial 2), but with 10 μl of a 10 mM NAD +solution and 190 μl of deionized water instead of 200 μl. The reactions were initiated by adding 100 μl of a solution of 9 parts potassium xylonate (225.7 g / l) and 1 part potassium arabonate (25.0 g / l) to each of the Eppendorf vials. The mixtures were incubated for a total of 48 h at 30 °C with continuous shaking (1200 rpm, Eppendorf Thermomixer). The total volume of the mixtures was 500 μl each.

[0082] For processing, 40 μl of a sample was mixed with 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 samples were centrifuged at max. 1 g for 10 min. The clear supernatant was diluted 1:250 and measured using HPAEC (conductivity detection). For HPLC, 150 μl of the clear supernatant was transferred to HPLC vials and measured (RI detection). The results are presented in the table below.

[0083] * Due to peak overlap in HPAEC, substrate conversion can only be given as a sum parameter.

[0084] ** The concentrations and masses of the products glycolate and lactate as well as their conversions were calculated for the respective free acids.

[0085] From the tables above it can be seen that the addition of cofactor (0.2 mM NAD + ) does not bring any advantages in terms of sales under these conditions.

[0086] Example 4

[0087] Treating a xylonate / arabonate (9 / 1 m / m) mixture (substrate concentration: 100 g / l) with the enzyme system

[0088] The following components were placed in a 2 ml Eppendorf vial: 150 μl of a 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 of 9 parts potassium xylonate (450.1 g / l) and 1 part potassium arabonate (51.7 g / l). The mixture was incubated for a total of 48 h at 30 °C with continuous shaking (1200 rpm, Eppendorf Thermomixer). The total volume of the mixture was 500 μl.

[0089] For processing, 20 μl of the mixture was mixed with 180 μl of ultrapure water and 200 μl of MeOH and incubated at 60 °C for 20 min (1200 rpm, Eppendorf Thermomixer). After denaturation, the samples were centrifuged at max. 1 g for 10 min. The clear supernatant was diluted 1:250 and measured using HPAEC (conductivity detection). For HPLC, 150 μl of the clear supernatant was transferred to HPLC vials and measured (RI detection). The results are presented in the table below.

[0090] * Due to peak overlap in the HPAEC, the conversion of the substrates can only be given as a sum parameter. ** The concentrations and masses of the products glycolate and lactate, as well as their conversions, were calculated for the respective free acids.

[0091] The table above shows that the process is also suitable for the conversion of more concentrated substrate solutions.

[0092] Example 5

[0093] Treating a mixture of glycolaldehyde and pyruvate with glycolaldehyde dehydrogenase and / or lactate dehydrogenase and NAD +

[0094] The following components were placed in 2 ml Eppendorf vials: 100 μl of a 500 mM TEA-HCl buffer (pH 8.5; final concentration 100 mM), 0.1 U lactate dehydrogenase (as lysate) and / or 0.1 U glycolaldehyde dehydrogenase (as lysate) and cofactor solution (NADH, NAD + The final enzyme units and cofactor concentrations in the preparations are listed in the table below. All preparations were made up to 400 μl with an appropriate amount of deionized water.

[0095] The reactions were initiated by adding 100 μl of a substrate solution (50 mM sodium pyruvate and 50 mM glycolaldehyde in deionized water). The mixtures were incubated for 1 h at 30 °C and 1200 rpm in an Eppendorf Thermomixer. The total volume of each mixture was 500 μl.

[0096] For processing, 200 μl of a sample was mixed with 200 μl MeOH and incubated at 60 °C for 20 min (1200 rpm, Eppendorf Thermomixer). After denaturation, the samples were centrifuged at max. 1 g for 10 min. The clear supernatant was diluted 1:100 and analyzed using HPAEC (conductivity detection) (determination of pyruvate and lactate). For HPLC (RI detection), 150 μl of the clear supernatant was transferred to HPLC vials and analyzed (determination of glycolate and glycolaldehyde*). The results are presented in the following table.

[0097] The concentrations of glycolaldehyde and pyruvate were 10 mM in all preparations.

[0098] * The glycolaldehyde concentration cannot be clearly determined due to peak overlap of lactate and glycolaldehyde in HPLC.

[0099] From the table it can be seen that when lactate dehydrogenase and glycolaldehyde dehydrogenase are used simultaneously, the yields of glycolate and lactate (batch 1) at the same NAD + / NADH concentrations (10 mM) were significantly increased compared to approaches 3 (glycolaldehyde DH only) and 4 (lactate dehydrogenase only) (lactate: 5.9 mM to 7.9 mM; glycolate: 4.7 mM to 9.9 mM). Literature

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Claims

Patent claims 1. A process for the preparation of an aqueous solution containing an alkali salt of glycolic acid and lactic acid, by treating an alkali salt of xylonic acid and / or arabonic acid in an aqueous solution in vitro with an enzyme system which comprises a dehydratase, an aldolase, a glycolaldehyde dehydrogenase, a lactate dehydrogenase and NAD + as a cofactor, after which the enzyme system is separated.

2. Process according to claim 1, characterized in that the concentration of the alkali salt of xylonic acid and / or arabonic acid in the aqueous solution is 50 - 300 g / l.

3. Process according to claim 2, characterized in that the concentration of the alkali salt of xylonic acid and / or arabonic acid in the aqueous solution is 100 - 200 g / l.

4. Process according to claim 3, characterized in that the concentration of the alkali salt of xylonic acid and / or arabonic acid in the aqueous solution is 150 - 250 g / l.

5. Process according to one of claims 1 to 4, characterized in that it is carried out at a temperature between 20 and 50 °C.

6. Process according to claim 5, characterized in that it is carried out at a temperature between 25 and 40 °C.

7. Process according to claim 6, characterized in that it is carried out at a temperature between 30 and 40 °C.

8. Method according to one of claims 1 to 7, characterized in that the Enzymes forming enzyme systems are present as lysates of the corresponding cells that form them.

9. Method according to one of claims 1 to 7, characterized in that the Enzymes forming enzyme systems are present as homogenates of the corresponding cells that form them.

10. Method according to 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. Process according to claim 11, characterized in that the pH value is between 7.5 and 8.5.

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