EP3325638A1 - Procédé de production enzymatique de produits de réduction et d'oxydation de sucres mixtes - Google Patents

Procédé de production enzymatique de produits de réduction et d'oxydation de sucres mixtes

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Publication number
EP3325638A1
EP3325638A1 EP16741925.8A EP16741925A EP3325638A1 EP 3325638 A1 EP3325638 A1 EP 3325638A1 EP 16741925 A EP16741925 A EP 16741925A EP 3325638 A1 EP3325638 A1 EP 3325638A1
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EP
European Patent Office
Prior art keywords
sugar
mixture
xylose
stage
acid
Prior art date
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Pending
Application number
EP16741925.8A
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German (de)
English (en)
Inventor
Ortwin Ertl
Bernd Mayer
Alexander DYBOV
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Annikki GmbH
Original Assignee
Annikki GmbH
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Filing date
Publication date
Application filed by Annikki GmbH filed Critical Annikki GmbH
Publication of EP3325638A1 publication Critical patent/EP3325638A1/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/58—Aldonic, ketoaldonic or saccharic acids
    • C—CHEMISTRY; METALLURGY
    • C13—SUGAR INDUSTRY
    • C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
    • C13K1/00—Glucose; Glucose-containing syrups
    • C13K1/02—Glucose; Glucose-containing syrups obtained by saccharification of cellulosic materials
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C3/00—Pulping cellulose-containing materials
    • D21C3/20—Pulping cellulose-containing materials with organic solvents or in solvent environment
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    • C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01046—L-Arabinose 1-dehydrogenase (1.1.1.46)
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    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01047—Glucose 1-dehydrogenase (1.1.1.47)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01116—D-Arabinose 1-dehydrogenase (1.1.1.116)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01117—D-Arabinose 1-dehydrogenase (NAD(P)+)(1.1.1.117)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01175—D-Xylose 1-dehydrogenase (1.1.1.175)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01179—D-Xylose 1-dehydrogenase (NADP+) (1.1.1.179)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03004—Glucose oxidase (1.1.3.4)

Definitions

  • the present invention relates to a process for recovering n + a oxidation and reduction products from a mixture of n sugars selected from the group consisting of C5 and C6 sugars.
  • US 2004/0173533 A1 has described a process for the chromatographic separation of a mixture consisting of sugars, preferably of xylose and glucose.
  • the separation results in separate currents: one of them is enriched with xylose, the other with glucose.
  • the sugar mixture is preferably produced by hydrolysis of biomass.
  • a separation of sugars, sugar alcohols, carbohydrates and mixtures thereof is described in EP 1 490 521 Bl, wherein in at least one step a weakly basic anion exchange resin (crosslinked polyacrylic acid polymer or epichlorohydrin-triethylenetetramine resin) is used in the chromatographic separation ,
  • a weakly basic anion exchange resin crosslinked polyacrylic acid polymer or epichlorohydrin-triethylenetetramine resin
  • Another approach to sugar fractionation is an (optional) selective conversion of sugar into components that can be easily separated due to different physical or chemical properties (polarity, solubility, etc.).
  • Fractionation of xylose and arabinose takes place from a mixture of sugars.
  • the Fractionation occurs by conversion of the sugars to the mixture of xylose monoacetal and arabinose diacetal. Subsequently, xylose monoacetal is separated from arabinose diacetal by liquid-liquid extraction.
  • WO 2011/133536 A1 describes a process in which C5 and / or C6 aldose sugar in sugar hydrolyzate is brought into contact with a catalyst in order to convert the sugars into ketose isomers. Furthermore, the isomerized C5 and / or C6 ketoses were brought into contact with a complexing agent (CA), with the ketoses binding to the CA and producing a ketose-CA conjugate. Ketose-CA conjugate could be selectively separated from the sugar mixture.
  • CA complexing agent
  • a portion of the sugars in the sugar mixture are selectively converted to sugar acids.
  • glucose solution to an enzymatic system having catalase and glucose oxidase activity, wherein hydrogen peroxide is added in stoichiometric amount to oxidize all of the glucose.
  • Electrodialysis is performed to separate gluconic acid from the medium and to recover glucose oxidase.
  • glucose is converted to gluconic acid, whereby glucose in aqueous solution is oxidized with oxygen.
  • Glucose solution is passed through a glucose oxidase and catalase-containing catalyst fixedly attached to an appropriate support.
  • a selective oxidation of glucose in the presence of fructose is described, with the resulting gluconic acid then by means of
  • a method for producing gluconic acid and its salts is known from CA 2 194 859, wherein glucose at a concentration of 15% or more to gluconic acid at a temperature of 10 ° C or higher in the presence of glucose oxidase and catalase is implemented.
  • the reaction is carried out using an excess of catalase activity relative to the oxidase activity.
  • No. 7,923,226 B2 describes a process for the preparation of 1,2,4-butanetriol, wherein xylose is also oxidized to xylonolactone / xylonic acid.
  • this patent does not disclose a recycling system for the redox cofactor reduced in the reaction.
  • Fermentative processes are also used for the preparation of sugar acids (for example, Buchert et al., 1988, Toivari et al., 2012b).
  • xylitol Extraction of xylitol from xylose is described.
  • a genetically modified strain of Candida tropicalis is used.
  • the regeneration of the cofactor of xylose reductase is not specified and is taken from the total metabolism of the cells.
  • a disadvantage is that in addition glucose is added to the cultures.
  • a large part of the sugar used is converted into biomass and is not used for product formation. Above all, the stoichiometrically possible amount of xylitol is not obtained relative to the xylose used.
  • Enzymes are described in the literature which allow the conversion of L-arabonate via L-2-keto-3-deoxyarabonate and alpha-ketoglutarate semialdehyde to alpha-ketoglutarate (Watanabe et al., 2006). Furthermore, enzymes are described which the
  • WO 2014/076012 Al inter alia, a method is described in which the arabinose is oxidized enzymatically from a mixture of arabinose and xylose (in a molar ratio of about 10 to 90) to arabinolactone or arabic acid and the xylose is reduced enzymatically to xylitol in substantially equimolar ratio.
  • the described molar ratio of arabinose and xylose is typical of a mixture of sugars obtained by digestion of lignocellulosic biomass and
  • hemicellulose-containing material can be obtained.
  • the object of the present invention is to fractionate the very complex sugar mixtures, which are often produced from biomass, with high purity and high yield, irrespective of the number and structure of the sugars. Furthermore, the present invention has the object, a possibility for the further implementation of fractionated To provide sugar / sugar acids, in particular xylose, xylonic acid, arabinose, arabic acid, to other products, in particular to xylitol and ⁇ -ketoglutarate.
  • the object of the present invention is achieved by a method according to claim 1.
  • the present invention first provides a process for obtaining n + a
  • n is at least 2 and a is at least 1,
  • At least a portion of the unreacted in the first stage sugar is half enzymatically oxidized and the remaining half is enzymatically reduced.
  • substantially the entire amount of the sugar not reacted in the first stage is half oxidized enzymatically and the remaining half is enzymatically reduced.
  • the two sugars oxidized or reduced in the first stage are reacted in a substantially equimolar amount.
  • the inventive method is based on a coupling of enzymatic oxidation and reduction reactions of sugars such that from a sugar mixture separable sugar oxidation products (such as sugar acids) and sugar reduction products (such as sugar alcohols) are obtained.
  • a sugar mixture separable sugar oxidation products (such as sugar acids) and sugar reduction products (such as sugar alcohols) are obtained.
  • sugars contained therein are not present in an equimolar ratio. If - as in the
  • WO 2014/076012 A1 describes - in a first stage, an enzymatic oxidation of the one sugar and an enzymatic reduction of the other sugar takes place in substantially equimolar ratio, so remains a part of existing in the original mixture in a larger amount of sugar in unreacted form in the mixture.
  • this unreacted part of this sugar is now subjected to enzymatic oxidation and enzymatic reduction.
  • the first stage may comprise several substeps:
  • the sugar A in a first step of the first step the sugar A can be completely oxidized or reduced and the sugar C in equimolar ratio
  • the sugar B are completely oxidized or reduced and again a portion of the sugar C is correspondingly reduced or oxidized.
  • the entire unreacted portion of the sugar C would then preferably be reduced by half and oxidized.
  • oxidation products are sugar acids or
  • a mixture of substances containing xylose and at least one further sugar preferably selected from the group consisting of C5 sugars, such as e.g. Arabinose, lyxose, ribose and C6 sugars, e.g. Allose, altrose, glucose, mannose, idose, galactose and talose.
  • C5 sugars such as e.g. Arabinose, lyxose, ribose and C6 sugars, e.g. Allose, altrose, glucose, mannose, idose, galactose and talose.
  • the mixture of sugars contains xylose and arabinose, wherein xylose is in excess.
  • Such mixtures are obtained in particular by degradation of a hemicellulose-containing material which has been obtained by digesting a lignocellulosic material, in particular if the lignocellulosic material is a material selected from the group consisting of straw, in particular wheat straw, bagasse, energy grasses, in particular elephant grass, switch grass, and / or Husks, especially lemmas, is.
  • the molar ratio of xylose and arabinose in mixtures thus obtained may be about 9: 1.
  • Oxidation equivalents can be provided by the simultaneous oxidative production of the arabic acid.
  • the entire unreacted xylose is preferably reduced by half enzymatically to xylonic acid and half to xylitol, (unless the arabic acid has already been separated off) a mixture of 1 part of arabic acid, 4 parts of xylonic acid and 5 parts results xylitol.
  • the original sugar mixture elegantly produces a mixture of easily separable oxidation and reduction products which are either themselves valuable substances (such as xylitol) or can be further processed into value products.
  • resulting arabic acid and / or resulting xylonic acid is further processed to ⁇ -ketoglutaric acid.
  • This further processing can preferably be carried out enzymatically:
  • xylonic acid this can by means of a xylonic acid dehydratase first to D-2-keto-3-deoxyxylonat, then by means of a D-2-keto-3-deoxyxylonat dehydratase to alpha- Ketoglutarklaldehyd (alpha-KGSA) and by means of an alpha -KGSA dehydrogenase can be further converted to alpha-ketoglutarate.
  • Suitable representatives of the enzyme classes L-arabic acid dehydratase and L-2-keto-3-deoxyarabonate dehydratase are available, for example, from Azospirillum brasiliense.
  • Suitable representatives of the enzyme classes xylonic acid dehydratase and D-2-keto-3-deoxyxylonate dehydratase are obtainable, for example, from Caulobacter crescentus.
  • Suitable alpha-ketoglutaric semialdehyde dehydrogenases are available, for example, from Azospirillum brasiliense or from Caulobacter crescentus.
  • a further preferred embodiment of the method according to the invention is characterized in that the mixture containing xylose and arabinose additionally contains glucose.
  • the glucose contained in the mixture is oxidized to gluconic acid.
  • arabinose preferably after separation of the gluconic acid, enzymatically oxidized to arabic acid.
  • the entire unreacted xylose is half oxidized enzymatically to xylonic acid and half reduced to xylitol, (unless the arabic acid and / or the gluconic acid have already been separated off) a mixture of 1 part of arabic acid results, 0.4 part of gluconic acid, 3.8 parts of xylonic acid and 5.2 parts of xylitol.
  • the sugar mixture contains glucose in excess of the other existing sugar (s) and is at least partially recovered from the glucose sorbitol. It's supposed to be from an exemplary mix of
  • a mixture of this order of magnitude can be obtained, for example, in the digestion of wood and subsequent enzymatic degradation (Berrocal et al., 2004)
  • energy grasses can be obtained a sugar mixture with high glucose content. This results in e.g. following procedure:
  • the steps of the first stage may be consecutive or (partly) simultaneous.
  • a separation of sugar acids can take place after individual steps. If, in the second stage, preferably half of the total unreacted glucose is enzymatically oxidized to gluconic acid and half reduced to sorbitol, (unless the sugar acids have already been separated off) a mixture of 1.4 parts of mannonic acid, 0.7 part of galactonic acid, 0 is obtained , 7 parts of xylonic acid, 0.4 parts of arabic acid, 1.9 parts of gluconic acid and 5.1 parts of sorbitol.
  • the resulting D-sorbitol may be enzymatically or non-enzymatically, preferably enzymatically, e.g. be oxidized with a D-sorbitol dehydrogenase or with an enzyme with D-sorbitol dehydrogenase activity to D-fructose.
  • Redox cofactors NAD (P) possibly reduced by the enzyme can be regenerated by at least one other redox enzyme. As a result, the redox cofactors can be used in a substoichiometric amount.
  • the process of the present invention may be e.g. be used to obtain very pure D-fructose from a biomass hydrolyzate with glucose content. An easier separation of the other sugars from the mixture is made possible by the conversion into sugar acids.
  • the process of the present invention may be e.g. be used to obtain very pure D-fructose from a biomass hydrolyzate with glucose content. An easier separation of the other sugars from the mixture is made possible by the conversion into sugar acids.
  • Oxidation to sugar acids ready for the reduction of glucose to sorbitol.
  • a relatively high proportion of the glucose in the mixture can be converted to sorbitol without adding external substances for redox cofactor recycling.
  • the sorbitol thus obtained is available for the preparation of the valuable product fructose.
  • the enzymatic oxidation of the sugars carried out in the process according to the invention can be carried out by different classes of enzymes.
  • oxidases using oxygen
  • dehydrogenases using the oxidized redox cofactors NAD (P) +
  • enzymes are used which are dependent on redox cofactors. Particularly preferred are exclusively from
  • a further preferred embodiment of the method according to the invention is accordingly characterized in that at least in one of the two stages, preferably at least in the second stage, particularly preferably both in the first as well as in the second stage, at least one redox cofactor and at least one enzyme dependent on this redox cofactor is present in the reaction mixture.
  • arabinose in particular in the first stage, is oxidized to arabic acid.
  • an L-arabinose dehydrogenase can be used for the oxidation of L-arabinose.
  • Suitable L-arabinose dehydrogenases are available, for example, from Azospirillum brasiliense or from
  • xylose in particular a part of the xylose remaining in the solution after the first stage, is oxidized to xylonic acid.
  • oxidation of D-xylose can be by means of a D-xylose dehydrogenase.
  • Suitable xylose dehydrogenases are available, for example, from Caulobacter crescentus.
  • glucose in particular in the first stage, is oxidized to gluconic acid.
  • a D-glucose-1-dehydrogenase can be used for the oxidation of glucose.
  • a suitable D-glucose-1 dehydrogenase is available from Bacillus subtilis.
  • a glucose oxidase can be used for the oxidation of glucose.
  • a suitable D-glucose oxidase is available from Aspergillus niger.
  • xylose in particular a part of the xylose remaining in the solution after the first step, is reduced to xylitol.
  • D-xylose reductase the reduction of D-xylose can be catalyzed by a D-xylose reductase.
  • Suitable xylose reductases are, for example, obtainable from Candida tropicalis, Candida parapsilosis or Saccharomyces cerevisiae.
  • the cofactors NADH, NADPH, NAD + and / or NADP + are used.
  • NAD + denotes the oxidized form
  • NADH denotes the reduced form of nicotinamide adenine dinucleotide
  • NADP + denotes the oxidized form
  • NADPH denotes the reduced form of nicotinamide adenine dinucleotide phosphate.
  • the cofactors can either be added separately to the reaction, or they are part of others Components of the reaction, eg the enzymes used or a combination of these two sources is used. When redox cofactors are used, they are present in a process according to the invention in substoichiometric amounts relative to the substrates.
  • the redox cofactors oxidized or reduced during reduction or oxidation reactions can be returned to their original redox state (redox cofactor recycling) by suitable enzymatic reactions and can thus undergo several reaction cycles.
  • Enzymatic cofactor regeneration systems are in particular selected from the group consisting of alcohol dehydrogenases, sugar dehydrogenases, NAD (P) H oxidases, hydrogenases or lactate dehydrogenases with consumption of co-substrates, in particular ketones, aldehydes, sugars, pyruvic acid and its salts and / or oxygen or with generation of hydrogen.
  • redox cofactor recycling can be accomplished by another redox enzyme, e.g. by an alcohol dehydrogenase, an NAD (P) H oxidase or a sugar reductase, e.g. a xylose reductase.
  • a sugar reductase is used.
  • redox cofactor recycling can also be accomplished by another redox enzyme, e.g. by an alcohol dehydrogenase or a sugar dehydrogenase, e.g. a glucose dehydrogenase or an arabinose dehydrogenase.
  • a sugar dehydrogenase is used.
  • NADH oxidases are available, for example, from Clostridium aminovalericum or Streptococcus mutans.
  • Suitable alcohol dehydrogenases are available, for example, from Lactobacillus kefir or Thermoanaerobium brockii.
  • glucose is converted with a glucose dehydrogenase to gluconic acid (lactone), the redox cofactor recycling being carried out by a xylose reductase.
  • the resulting gluconic acid is separated from the mixture of substances in a further particular embodiment of the process according to the present invention.
  • arabinose is converted to arabic acid (lactone) with an arabinose dehydrogenase, the redox cofactor recycling being effected by a xylose reductase.
  • the resulting arabic acid is used in a further particular embodiment of the
  • the xylose remaining after the first stage is reduced to xylitol both in the presence of resulting sugar acids and, optionally, after sugar acid separation, with an enzyme, preferably a xylose reductase, more preferably an NAD (P) - dependent xylose reductase.
  • an enzyme preferably a xylose reductase, more preferably an NAD (P) - dependent xylose reductase.
  • P NAD
  • the remaining xylose becomes NAD (P) -dependent
  • Xylose reductase reduced to xylitol wherein the redox cofactor recycling is done by a xylose dehydrogenase, so that the remaining xylose is oxidized to xylonic acid simultaneously.
  • both in the first and in the second stage at least one redox cofactor and at least one enzyme dependent on this redox cofactor are preferably present in the reaction mixture.
  • at least one redox cofactor and at least one enzyme dependent on this redox cofactor are preferably present in the reaction mixture.
  • enzymes in each of two stages e.g. a reductase on the one hand and a
  • all the enzymes used are particularly preferably dependent on a redox cofactor which is also present in the mixture.
  • the redox cofactor is either already contained in the enzyme preparations in sufficient quantity or it is additionally added redox cofactor to the reaction.
  • the redox cofactor used in the first and / or second stage is further preferred in each case by reduction and reduction processes taking place in parallel
  • the enzymes used in the process can be obtained by recombinant expression.
  • the expert person is known to various systems, such as E. coli, Saccharomyces cerevisiae or Pichia pastoris.
  • E. coli is used; a competent person is familiar with the usual protocols for this purpose.
  • the enzymes can be used in intact cells, in permeabilized cells or in the form of cell lysates.
  • the enzymes may either be used directly or further purified, for example by chromatographic methods for protein purification, which may be found in the literature and / or known to a person skilled in the art.
  • a simple purification step eg centrifugation or filtration
  • first and second stages can be carried out in a one-pot reaction.
  • the two stages can take place at least partially simultaneously. Based on the described enzymatic reactions a simultaneous reaction of the two stages is possible.
  • Another embodiment of the method according to the invention comprises the removal of accumulating sugar acids from the mixture.
  • the removal of accumulating sugar acids for example, arabic acid, gluconic acid or xylonic acid
  • the hemicellulose-containing material by means of digestion of a
  • Digestion methods for lignocellulose-containing material can also Brön et al. (2011).
  • a method for the digestion or delignification of lignocellulose-containing material can also be found, for example, WO 2010/124312 A2 (Ertl et al., 2010).
  • lignocellulosic material includes in particular lignocellulosic biomass, eg annual or perennial plants or parts of annuals or perennials such as wood such as softwood or hardwood or (dry) grasses or parts of grasses. preferably grasses, straw, such as wheat straw, rye straw or maize straw, energy grasses such as barnyardgrass, miscanthus / miscanthus, abaca, sisal, bagasse or atypical
  • Lignocellulosic substrates such as corn spindles, husks, e.g. Lemmas, such as wheat husks, rice husks, particularly preferably straw, in particular wheat straw, bagasse, energy grasses, in particular elephant grass, switch grass, and / or husks, in particular lemmas.
  • the lignocellulosic material can be obtained by digestion with an alcohol, in particular with a CI_ 4 -alcohol, water and an alkali.
  • an alcohol in particular with a CI_ 4 -alcohol, water and an alkali.
  • a corresponding method is known, for example, from WO 2010/124312 A2 (Ertl et al, 2010).
  • Sugar-acid lactones included and vice versa.
  • the ratio of these two products is highly dependent on the sugar used and on the reaction conditions, e.g. from the reaction time or v.a. from the pH value.
  • Example 1 Xylanase treatment to obtain a sugar mixture from biomass. It was used from straw-made delignified pulp. A description of the preparation of the pulp can be taken from WO 2010/124312 A2 (Example 1). 10 g (dry weight) of the pulp were washed with dist. Water was resuspended to 10% consistency and it was adjusted with H 2 S0 4 pH 4.9. 1000 ⁇ l of xylanase Ecopulp TX800A (Ecopulp Finland Oy) were added and incubated at 50 ° C. for 16 h. A 1.5% sugar solution (w / v) is obtained which contains mainly glucose, xylose and arabinose in a ratio of about 2: 10: 1.
  • Examples 2 to 5 serve to illustrate the possibilities of selective enzymatic oxidation or reduction of sugars from a sugar mixture.
  • Example 2 Glucose oxidation with a glucose dehydrogenase (NADH oxidase for cofactor recirculation).
  • a sugar mixture (about 500 ⁇ ), which contains glucose, xylose and arabinose (sugar concentration about 1%), 18.5 mg NaHC0 3 was added. Thereafter, 30 ⁇ glucose dehydrogenase (activity about 300 U / ml), 10 ⁇ NADH oxidase (activity about 1140 U / ml) and 2.5 ⁇ NADH (concentration 100 mM) were added. The mixture was incubated at 25 ° C for about 17 hours. 86% of the glucose was converted to gluconic acid. The resulting solution was passed through a strong ion exchanger (Amberlyst A-26 (OH), Alfa Aesar). As a result, the resulting gluconic acid was completely separated from the mixture.
  • a strong ion exchanger Amberlyst A-26 (OH), Alfa Aesar
  • Example 3 Arabinose oxidation with an arabinosedehydro genese (NADH oxidase for cofactor Recvcling).
  • Example 5 Arabinose oxidation with an arabmose dehydrogenase (xylose reductase for cofactor recycling), xylose reduction with a xylose reductase (alcohol dehydrogenase for cofactor recycling).
  • the solution was stirred in a 200 ml round bottom flask at 35 ° C. (water bath) with a magnetic stirrer (200 rpm) for 20 min. Arabinose is complete
  • the cofactor used was already sufficiently present in the enzyme lysates used and did not have to be added separately.
  • the first stage is the oxidation of arabinose and reduction of an equimolar part of xylose.
  • the second step which proceeds at least partially in parallel, involves the oxidation of half of the unreacted xylose by arabinose dehydrogenase activity and reduction of the other half of the remaining xylose.
  • the reaction mixture comprised the following components: 364 ⁇ M dH 2 O, 2.5 ⁇ M NADPH solution (100 mM), 10 ⁇ M D-glucose solution (50% w / v), 100 ⁇ M D-xylose solution (50% w / v). v), 5 ⁇ glucose dehydrogenase (300 U / ml, measured with glucose), 19 ⁇ xylose reductase (160 U / ml), and 5.6 mg CaC0 3 .
  • the glucose dehydrogenase used also has some xylose dehydrogenase activity.
  • the reaction was gently shaken at 35 ° C and sampled at various times. By GC / MS, the content of sugars and the reaction products was determined.
  • Gluconic acid 10 mg / ml xylonic acid, 20 mg / ml xylitol, 70 mg / ml xylose.
  • composition of the reaction after 6 h 10 mg / ml gluconic acid, 40 mg / ml xylonic acid, 50 mg / ml xylitol, 10 mg / ml xylose.
  • Gluconobacter oxydans for production of xylonic acid from hemicellulose hydrolysates. Applied and Environmental Microbiology 28, 367-372.

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Abstract

La présente invention concerne un procédé d'obtention de n+a produits d'oxydation et de réduction à partir d'un mélange de n sucres choisis dans le groupe constitué par des sucres en C5 et C6, n valant au moins 2 et a étant au moins 1. Au moins deux des sucres du mélange sont présents dans un rapport non équimoléculaire entre eux. Dans une première étape, au moins un sucre présent dans un rapport non équimoléculaire est oxydé par voie enzymatique et en même temps au moins un des autres sucres présents dans un rapport sucre non-équimolaire est réduit par voie enzymatique et, dans la première étape, une partie d'au moins un des sucres présents dans un rapport non-équimolaire n'est pas converti. Le procédé de l'invention est caractérisé en ce que, dans au moins une deuxième étape, au moins une partie du sucre non converti dans la première étape est oxydée pour moitié par voie enzymatique et est réduit pour la moitié restante par voie enzymatique.
EP16741925.8A 2015-07-24 2016-07-20 Procédé de production enzymatique de produits de réduction et d'oxydation de sucres mixtes Pending EP3325638A1 (fr)

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WO2017202686A1 (fr) * 2016-05-23 2017-11-30 Annikki Gmbh Procédé de transformation enzymatique du d-glucose en d-fructose via le d-sorbitol
WO2019008131A1 (fr) * 2017-07-06 2019-01-10 Consejo Superior De Investigaciones Científicas Pseudomonas putida recombinant destiné à la production de d-xylonate à partir de d-xylose

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US2351500A (en) 1941-02-06 1944-06-13 Claude R Wickard Process for gluconic acid production
US2651592A (en) 1950-08-15 1953-09-08 Ben L Sarett Enzymatic process for producing gluconic acid
US3619396A (en) 1969-01-29 1971-11-09 Cpc International Inc Enzymatic production of gluconic acid
DE2214442C3 (de) 1972-03-24 1981-09-10 Boehringer Mannheim Gmbh, 6800 Mannheim Verfahren zur Überführung von Glucose in Gluconsäure
ATE216724T1 (de) 1995-05-12 2002-05-15 Dsm Nv Enzymatische produktion von glukonsäure und ihrer salzen
CA2359337C (fr) 1999-01-14 2005-09-13 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Procede de separation de sucres
EP1444368A1 (fr) 2001-07-24 2004-08-11 Arkenol, Inc. Separation du xylose et du glucose
FI20020592A (fi) 2002-03-27 2003-09-28 Danisco Sweeteners Oy Menetelmä sokereiden, sokerialkoholien, hiilihydraattien ja niiden seosten erottamiseksi niitä sisältävistä liuoksista
WO2005068642A2 (fr) 2003-10-01 2005-07-28 Board Of Trustees Operating Michigan State University Synthese d'enantiomeres 1,2,4-butanetriol issue de glucides
US7498430B2 (en) 2004-11-09 2009-03-03 Board Of Trustees Of Michigan State University Process for the preparation and separation of arabinose and xylose from a mixture of saccharides
FI20095278A0 (fi) 2009-03-18 2009-03-18 Valtion Teknillinen Ksylonihapon valmistus
TW201114906A (en) 2009-04-30 2011-05-01 Annikki Gmbh Process for the production of carbohydrate cleavage products from a lignocellulosic material
US9242222B2 (en) 2010-04-19 2016-01-26 The University Of Toledo Aldose-ketose transformation for separation and/or chemical conversion of C6 and C5 sugars from biomass materials
GB201116971D0 (en) * 2011-10-03 2011-11-16 Isis Innovation Cofactor regeneration system
TW201343623A (zh) * 2012-02-07 2013-11-01 Annikki Gmbh 使氧化還原輔因子經酶催化再生之方法
AT513562A1 (de) 2012-11-14 2014-05-15 Annikki Gmbh Verfahren zur Gewinnung von Zuckerderivaten
US10253340B2 (en) * 2013-03-27 2019-04-09 Annikki Gmbh Method for the isomerisation of glucose
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JP6732017B2 (ja) * 2015-07-24 2020-07-29 アニッキ ゲーエムベーハーＡｎｎｉｋｋｉ Ｇｍｂｈ 糖混合物から酸化生成物および還元生成物を得るための方法

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