WO2013117251A1 - Procédé de régénération enzymatique de cofacteurs redox - Google Patents

Procédé de régénération enzymatique de cofacteurs redox Download PDF

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Publication number
WO2013117251A1
WO2013117251A1 PCT/EP2012/067781 EP2012067781W WO2013117251A1 WO 2013117251 A1 WO2013117251 A1 WO 2013117251A1 EP 2012067781 W EP2012067781 W EP 2012067781W WO 2013117251 A1 WO2013117251 A1 WO 2013117251A1
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Prior art keywords
reaction
group
oxidized
regeneration
cofactor
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PCT/EP2012/067781
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German (de)
English (en)
Inventor
Ortwin Ertl
Nicole STAUNIG
Marta SUT
Bernd Mayer
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Annikki Gmbh
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Application filed by Annikki Gmbh filed Critical Annikki Gmbh
Priority to TW102103119A priority Critical patent/TW201343623A/zh
Priority to ARP130100266A priority patent/AR089841A1/es
Priority to SA113340270A priority patent/SA113340270B1/ar
Priority to JP2014556034A priority patent/JP6329086B2/ja
Priority to LTEP13703568.9T priority patent/LT2812439T/lt
Priority to CA2862384A priority patent/CA2862384C/fr
Priority to PCT/EP2013/052313 priority patent/WO2013117584A1/fr
Priority to BR112014019287-1A priority patent/BR112014019287B1/pt
Priority to JP2014556035A priority patent/JP6189334B2/ja
Priority to UAA201409679A priority patent/UA117453C2/uk
Priority to US14/376,512 priority patent/US9644227B2/en
Priority to HUE13703568 priority patent/HUE044690T2/hu
Priority to PL13703568T priority patent/PL2812439T3/pl
Priority to IN7015DEN2014 priority patent/IN2014DN07015A/en
Priority to RSP20191245 priority patent/RS59314B1/sr
Priority to SG11201404614XA priority patent/SG11201404614XA/en
Priority to PT137035697T priority patent/PT2812440T/pt
Priority to DK13703569.7T priority patent/DK2812440T3/da
Priority to ES13703569T priority patent/ES2746698T3/es
Priority to NZ627477A priority patent/NZ627477A/en
Priority to LTEP13703569.7T priority patent/LT2812440T/lt
Priority to CA2863137A priority patent/CA2863137C/fr
Priority to PCT/EP2013/052316 priority patent/WO2013117585A1/fr
Priority to EP13703568.9A priority patent/EP2812439B1/fr
Priority to MX2014009455A priority patent/MX358771B/es
Priority to SI201331576T priority patent/SI2812440T1/sl
Priority to AU2013218042A priority patent/AU2013218042B2/en
Priority to HUE13703569A priority patent/HUE046250T2/hu
Priority to EP13703569.7A priority patent/EP2812440B1/fr
Priority to PL13703569T priority patent/PL2812440T3/pl
Priority to ES13703568T priority patent/ES2742381T3/es
Priority to RS20191065A priority patent/RS59114B1/sr
Priority to RU2014136162A priority patent/RU2635087C2/ru
Priority to CN201380008572.8A priority patent/CN104136620A/zh
Priority to US14/376,527 priority patent/US9902981B2/en
Priority to SI201331563T priority patent/SI2812439T1/sl
Priority to KR1020147023136A priority patent/KR102022137B1/ko
Priority to PT13703568T priority patent/PT2812439T/pt
Priority to MYPI2014702060A priority patent/MY172493A/en
Priority to CN202110638003.8A priority patent/CN113337568A/zh
Publication of WO2013117251A1 publication Critical patent/WO2013117251A1/fr
Priority to PH12014501770A priority patent/PH12014501770A1/en
Priority to HK15104308.2A priority patent/HK1204010A1/xx
Priority to HK15104309.1A priority patent/HK1204011A1/xx
Priority to US15/490,416 priority patent/US10370691B2/en
Priority to HRP20191514 priority patent/HRP20191514T1/hr
Priority to HRP20191693 priority patent/HRP20191693T1/hr

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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
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/36—Dinucleotides, e.g. nicotineamide-adenine dinucleotide phosphate
    • 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
    • C12P33/00—Preparation of steroids
    • 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
    • C12P33/00—Preparation of steroids
    • C12P33/12—Acting on D ring
    • C12P33/16—Acting at 17 position
    • 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
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
    • C—CHEMISTRY; METALLURGY
    • C13—SUGAR INDUSTRY
    • C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
    • C13K11/00—Fructose

Definitions

  • Redoxkomineen in its reduced form is obtained and the other in its oxidized form.
  • Enzyme-catalyzed redox reactions are used in industrial processes, for example in the preparation of chiral alcohols, ⁇ -amino acids and ⁇ -hydroxy acids.
  • the majority of enzymes used in industrial redox reactions use cofactors such as NADH or NADPH.
  • cofactors such as NADH or NADPH.
  • those of particular interest are those in which the redox cofactors are restored by in situ cofactor regeneration systems. The reason for this is that the use of only catalytic amounts of expensive cofactors (NAD (P) + / NAD (P) H) is possible.
  • NAD (P) + / NAD (P) H The accessibility of suitable dehydrogenases and other enzymes has led to the development of various cofactor regeneration systems.
  • enzyme-coupled, substrate-coupled, in vivo natural cofactor regeneration systems in living organisms
  • photochemically, chemically or electro-enzymatically The method described here relates to an enzyme-linked regeneration system.
  • Advantages of enzyme-coupled systems are the high selectivity, the applicability for the production of different products and the high reuse rate of the total turnover number (TTN).
  • HSDH Hydroxysteroid dehydrogenase
  • 7ß-HSDH 7ß-HSDH
  • 12a-HSDH 12a-HSDH
  • the cofactor regeneration system used was a lactate dehydrogenase (NAD + -dependent) as well as a glucose dehydrogenase (NADP + -dependent).
  • Cosubstrates used were pyruvate and glucose.
  • glucose dehydrogenase NADP + partially reversed NAD + , hindering oxidation.
  • 12.5 mM (-0.5%) of the substrate cholic acid was used, making the process economically uninteresting.
  • Alcohol dehydrogenases S- and R-specific with different cofactor specificity achieved.
  • NADP was regenerated by NADPH oxidase (hydrogen peroxide producing) and NADH by formate dehydrogenase.
  • the co-substrates used were formate and oxygen. In the system were 4 enzymes without
  • a disadvantage of the method is the very low concentration of the substrate used of 0.2-0.5%, which is not suitable for industrial purposes.
  • Alcohol dehydrogenases with opposite stereoselectivities and different cofactor specificities were used.
  • the cofactors were regenerated by means of a so-called “hydride transfer system” using only one additional enzyme to regenerate the cofactors, various enzymes, e.g.
  • Known enzymatic cofactor regeneration systems for individual redox reactions include, for example
  • Formate dehydrogenase for NADH formate as cosubstrate
  • hydrogenase for NADH and NADPH H 2 as cosubstrate
  • NADH oxidase for NADH (0 2 as cosubstrate)
  • phosphite dehydrogenase for NADH phosphite as cosubstrate
  • NAD (P) H or NAD (P) by various enzymes such as
  • Lactate dehydrogenase (pyruvate as cosubstrate), alcohol dehydrogenase from T. brockii (isopropanol as cosubstrate), alcohol dehydrogenase from L. brevis, L. minor, Leuconostoc carnosum, T. ethanolicus, Clostridium beijerinckii regenerated.
  • these known methods relate only to the isolated individual reactions for the oxidation of
  • a cofactor regeneration system for NADH using malate dehydrogenase (“malate enzyme") has been previously described (Can, J. Chem., Eng, 1992, Volume 70, p 306-312), and has been published in the publication for the reductive amination of pyruvate Used alanine dehydrogenase. The pyruvate formed during cofactor regeneration was subsequently used in the product-forming reaction.
  • WO 2004/022764 also describes regenerating NADH by malate dehydrogenase. Unlike in the previously described publication, the pyruvate formed in the oxidative decarboxylation of malate was discontinued.
  • Phosphite dehydrogenase from Pseudomonas sp. used. Again, this is an individual reaction to product formation.
  • the object of the present invention was to provide a process for regenerating the redox cofactors NAD + / NADH and NADP + / NADPH to thereby have two or more perform enzymatically catalyzed redox reactions in a reaction mixture economically.
  • This object is achieved according to the present invention in a method of the type mentioned in that a process for the enzymatic regeneration of the redox cofactors NAD + / NADH and NADP + / NADPH in a one-pot reaction, wherein as a result of at least two further in the same reaction mixture expired enzymatically catalyzed redox reactions (product formation reactions) of one of the two
  • Ri is a straight-chain or branched-chain (Ci-C 4 ) alkyl group or for (Ci-C 4 ) -Carboxyalkyl distr, is reduced, and
  • R 2 and R 3 are independently selected from the group consisting of H, (QC ⁇ -alkyl, wherein alkyl is straight-chain or branched, (Ci-Cö ⁇ alkenyl, wherein alkenyl is straight-chain or branched and contains one to three double bonds, aryl, especially C ⁇ -C 12 aryl, carboxyl, or (Ci-C4) carboxyalkyl, in particular, cycloalkyl, including C3 -CG cycloalkyl,
  • a method provided in accordance with the present invention is also referred to herein as a "method according to (the present invention)".
  • the present invention provides a process according to the present invention for the enzymatic regeneration of the redox cofactors NAD + / NADH and NADP + / NADPH in a one-pot reaction, resulting in at least two further enzymatically catalyzed redox reactions occurring in the same reaction mixture
  • Ri is a substituted or unsubstituted C 1 -C 4 alkyl group, and b) a compound of the general one in the regeneration of the reduced cofactor
  • R 2 and R 3 are independently selected from the group consisting of
  • alkenyl is straight or branched chain
  • R in a method according to the present invention 2 and R 3 are independently selected from the group consisting of H, (C 1 -C ö) -alkyl, wherein alkyl is straight chain or branched (C 1 -C ö) Alkenyl, wherein alkenyl is straight-chain or branched and contains one to three double bonds, aryl, in particular C 6 -C 12 aryl, carboxyl, or (C 1 -C 4 ) -carboxyalkyl.
  • a process according to the present invention represents a substantial improvement in processes in which compounds are both enzymatically oxidized and reduced, as this makes it possible to obtain the necessary oxidation and reduction reactions as well as the associated reactions for cofactor regeneration to run a reaction and at the same time much higher
  • NAD + denotes the oxidized form and NADH denotes the reduced form of nicotinamide adenine dinucleotide while NADP + denotes the oxidized form and NADPH denotes the reduced form of nicotinamide adenine dinucleotide phosphate.
  • oxidation reaction (s) and “reduction reaction (s)” herein are meant those enzyme-catalyzed redox reactions which are not part of the cofactor regeneration and are involved in the formation of the product in a process of the present invention. and "reduction reaction (s)” are grouped together under the term “product formation reactions.”
  • the product formation reactions in a process of the present invention each include at least one
  • NAD + is used as a cofactor for the oxidation reaction (s)
  • NADPH is the cofactor for the reduction reaction (s).
  • NADP + is the cofactor for the
  • Reduction reaction oxidation reaction (s) and reduction reaction (s) can be carried out either simultaneously in time or sequentially, preferably in parallel, in the same reaction batch.
  • Substrates here are those compounds which are used for the purpose of product formation.
  • Kosubstrate here are those compounds that are implemented in the cofactor regeneration.
  • both a substrate and a plurality of substrates can be used. This reduction and / or
  • a process according to the present invention is suitable for a large number of reactions, for example for configuration inversion of stereoisomeric hydroxy compounds by oxidation to the corresponding ketone and subsequent reduction to the opposite stereospecific hydroxy compound.
  • one-pot reaction herein is meant a process wherein two or more enzymatic redox reactions involved in product formation and two enzymatic systems for cofactor regeneration occur in a reaction batch without isolating an intermediate.
  • Bile acids here all esters derived therefrom. Furthermore, here (partially) compounds provided with protective groups are included in the naming of the underlying substances.
  • a process according to the present invention is characterized in that the oxidation reaction and
  • a process according to the present invention is characterized in that both oxidation reaction and reduction reaction take place on the same molecular backbone.
  • a process according to the present invention is characterized in that the compound of the formula I (2-oxo acid) pyruvate (cosubstrate) is used, which is reduced by means of a lactate dehydrogenase to lactate, that is, in the regeneration reaction , which converts the reduced cofactor back into its original oxidized form by means of a
  • Lactate dehydrogenase pyruvate is reduced to lactate.
  • a process according to the present invention is characterized in that as the compound of the formula II
  • 2-propanol isopropyl alcohol, IPA
  • cosubstrate is used, which is oxidized by means of an alcohol dehydrogenase to acetone, that is, in the regeneration reaction, which converts the oxidized cofactor back into its original reduced form by means of an alcohol dehydrogenase.
  • 2-propanol is oxidized to acetone.
  • a method according to the invention is characterized in that oxygen is used, which is reduced by means of an NADH oxidase.
  • a process according to the present invention is characterized in that secondary alcohol is malate
  • Malatdehydro genese (“malate enzyme") is oxidized to pyruvate and C0 2 , for example, that in the regeneration reaction, the oxidized cofactor back to its original converted into reduced form by means of a malate dehydrogenase malate to pyruvate and C0 2 is oxidized.
  • the resulting pyruvate is reacted in this embodiment in a further redox reaction, which does not serve for product formation, but represents the second cofactor regeneration reaction.
  • a process according to the present invention is characterized in that it is used to carry out in each case at least one oxidation reaction and at least one reduction reaction in the same reaction mixture of compounds of the general formula
  • R 4 is hydrogen, a methyl group, a hydroxy group or an oxo group
  • R5 is hydrogen, a hydroxy group, an oxo group or a methyl group
  • R 6 is hydrogen or a hydroxy group
  • R 7 is hydrogen, -COR 13 , wherein R 13 is an unsubstituted or substituted with a hydroxy group C 1 -C 4 alkyl group, or a substituted, in particular with a
  • R 6 and R 7 together represent an oxo group
  • R 8 is hydrogen, a methyl group, a hydroxy group or an oxo group
  • R 9 is hydrogen, a methyl group, a hydroxy group or an oxo group
  • Rn is hydrogen, a methyl group, a hydroxy group, an oxo group or halogen
  • R 12 is hydrogen, a hydroxy group, an oxo group or a methyl group, where the structural element
  • the substrate (s) for the reduction reaction (s) involved in the product formation is / are present in a concentration of ⁇ 5 (w / v) in the reaction mixture.
  • a method according to the invention is characterized in that an enzymatic conversion of dehydroepiandrosterone (DHEA) to testosterone takes place.
  • DHEA dehydroepiandrosterone
  • a process according to the invention is characterized in that an enzymatic epimerization of hydroxysteroid compound 3a, 7a-dihydroxy-5 ⁇ -cholanic acid (chenodeoxycholic acid, CDC) by oxidation to ketolithocholic acid (KLC) and reduction to 3a, 7 ⁇ - Dihydroxy-5 ⁇ -cholanic acid (ursodeoxycholic acid, UDC) is carried out by means of two oppositely stereospecific hydroxysteroid dehydrogenases.
  • a method according to the present invention is characterized in that a C 5 - or C 6 sugars used as the substrate, for example, that the process for isomerization of C 5 - or C ö sugars is used.
  • a process according to the present invention is characterized in that an isomerization of glucose takes place by reduction to sorbitol and oxidation to fructose, for example that the process is used for the isomerization of glucose by reduction to sorbitol and subsequent oxidation to fructose ,
  • a process according to the invention is preferably carried out in an aqueous system, wherein it is possible that the substrate for oxidation and
  • Reduction reaction is partly unresolved in the form of a suspension and / or as a second liquid phase.
  • a process according to the present invention is characterized in that the substrate (s) for the oxidation reaction (s) involved in the product formation are present in a concentration of at least 5% (w / v) and more, preferably 7%. (w / v) and more, more preferably 9% (w / v) and more in the
  • Reaction mixture is present / present.
  • a process according to the present invention is characterized in that a total of> 70, in particular> 90, conversion is achieved in the product formation reactions.
  • a buffer may be added to the aqueous system.
  • Suitable buffers include, for example, potassium phosphate, tris-HCl, and glycine, having a pH of from 5 to 10, preferably from 6 to 9.
  • the system may contain ions for stabilizing the enzymes, such as Mg 2+ or other additives , such as glycerol are added.
  • concentration of added cofactors NAD (P) + and NAD (P) H in a method of the present invention is usually between 0.001 mM and 10 mM, preferably between 0.01 mM and 1 mM.
  • Invention at a temperature of 10 ° C to 70 ° C, preferably from 20 ° C to 45 ° C are performed.
  • Hydroxysteroid dehydrogenases are understood as meaning those enzymes which catalyze the oxidation of hydroxy groups to the corresponding keto groups or, conversely, the reduction of keto groups to the corresponding hydroxy groups on the steroid skeleton.
  • Suitable hydroxysteroid dehydrogenases which can be used for redox reactions on hydroxysteroids are, for example, 3a-HSDH, 3 ⁇ -HSDH, 7a-HSDH, 7 ⁇ -HSDH or 17 ⁇ -HSDH.
  • Suitable enzymes having 7a-HSDH activity are obtainable, for example, from clostridia (Clostridium absonum, Clostridium sordelii), Escherichia coli or Bacteroides fragilis.
  • Suitable enzymes having 7 ⁇ -HSDH activity are available, for example, from Ruminococcus sp. or Clostridium absonum.
  • Suitable lactate dehydrogenases are available, for example, from Oryctolagus cuniculus.
  • Suitable alcohol dehydrogenases are obtainable, for example, from Lactobacillus kefir.
  • xylose reductase is available from Candida tropicalis.
  • Suitable sorbitol dehydrogenases are for example obtainable from sheep liver, Bacillus subtilis or Malus domestica.
  • NADH oxidases are available, for example, from Leuconostoc mesenteroides, Streptococcus mutans, Clostridium aminovalericum.
  • enzymes are preferably used as proteins overexpressed recombinantly in E. coli, furthermore preferably the
  • the enzyme unit 1 U corresponds to that amount of enzyme that is needed to implement 1 ⁇ substrate per min.
  • Fig. 1 shows the reaction scheme of the epimerization of chenodeoxycholic acid
  • Fig. 2 shows the reaction scheme of the epimerization of chenodeoxycholic acid
  • Fig. 3 shows the reaction scheme of the epimerization of chenodeoxycholic acid
  • Ursodeoxycholic acid via the intermediate 3a-hydroxy-7oxo-5 ⁇ -cholanic acid with cofactor regeneration using 2-propanol and oxygen.
  • Figure 4 shows the reaction scheme of isomerization of glucose to fructose with cofactor regeneration using 2-propanol and pyruvate.
  • Figure 5 shows the reaction scheme of isomerization of glucose to fructose with cofactor regeneration using 2-propanol and oxygen.
  • a 0.5 ml mixture contains 50 mg chenodeoxycholic acid, 12 U of recombinant 7 ⁇ -hydroxysteroid dehydrogenase from Escherichia coli, 6 U of recombinant 7 ⁇ -hydroxysteroid dehydrogenase from Ruminococcus torques, and 0.5 mM NAD + and 0.3 mM NADPH.
  • NAD + 6 U recombinant lactate dehydrogenase and 350 mM sodium pyruvate are used.
  • NADPH 6 U of the recombinant alcohol dehydrogenase from Lactobacillus kefir and initially 2.4% IPA (w / v) are used.
  • An open system is also used to allow the evaporation of acetone and to shift the reaction towards ursodeoxycholic acid.
  • 20 ⁇ 4-methyl-2-pentanol are added after 24 h.
  • 200 ⁇ M 2-pentanol and 1.6% (w / v) IPA are added.
  • the proportion of ursodeoxycholic acid in all bile acids in the reaction mixture is> 97%.
  • a 0.5 ml mixture contains 50 mg chenodeoxycholic acid, 20 U of the recombinant 7 ⁇ -hydroxysteroid dehydrogenase from Escherichia coli, 20 U of the recombinant 7 ⁇ -hydroxysteroid dehydrogenase from Ruminococcus torques and 1 mM NAD + and 1 mM NADPH.
  • An open system is also used to allow escape of the resulting C0 2 .
  • 20 U 7a-HSDH and 10 U lactate dehydrogenase were dosed in after 16 h and 40 h.
  • 10 7 ⁇ -HSDH were dosed in after 20 h, 24 h, 44 h and 48 h.
  • a 0.5 ml mixture contains 50 mg chenodeoxycholic acid, 12 U of the recombinant 7 ⁇ -hydroxysteroid dehydrogenase from Escherichia coli, 7.5 U of the recombinant 7 ⁇ -hydroxysteroid dehydrogenase from Ruminococcus torques and 1 mM NAD + and 1 mM NADPH.
  • NAD + 20 U of the recombinant NADH oxidase from Clostridium aminovalericum are used.
  • NADPH 5 U of the recombinant alcohol dehydrogenase from Lactobacillus kefir and initially 2% IPA (w / v) are used.
  • the reaction is carried out in an aqueous potassium phosphate buffer (100 mM, pH 6) at 25 ° C with continuous shaking (850 rpm).
  • An open system is also used to allow the evaporation of acetone and to shift the reaction towards ursodeoxycholic acid.
  • 2% IPA are dosed after 18 h, 22 h, 26 h and 41 h and 5% IPA after 41 h and 48 h.
  • 20 U of NADH oxidase and after 41 h of 7.5 U of 7ß-hydroxysteroid dehydrogenase and 5 U alcohol dehydrogenase are replenished.
  • the proportion of ursodeoxycholic acid in all bile acids in the reaction mixture is about 95-98%.
  • a 0.5 ml batch contains 50 mg / ml glucose and 6 U / ml of the recombinant xylose reductase from Candida tropicalis and 0.1 mM NADP + .
  • 7% IPA and the recombinant alcohol dehydrogenase from Lactobacillus kefir are added.
  • the enzymes are used in the form of cell lysate.
  • the open system leads to the removal of the acetone, which drives the reaction towards sorbitol formation.
  • water and IPA also evaporate, so that they are dosed after 6 h and 21 h.
  • Rabbit muscle and 300 mM pyruvate used. The batch is made up to 0.5 ml with water. The reaction takes place for 24 h at 40 ° C and pH 9 under continuous
  • a 0.5 ml batch contains 50 mg / ml glucose, 6 U / ml of the recombinant xylose reductase from Candida tropicalis and 0.1 mM NADP + .
  • 7% IPA and the recombinant alcohol dehydrogenase from Lactobacillus kefir are added. The enzymes are used in the form of cell lysate.
  • the open system leads to the removal of the acetone, which drives the reaction towards sorbitol formation.
  • water and IPA also evaporate, so that they are dosed after 6 h and 21 h.
  • the reaction vessel is incubated at 60 ° C under vacuum to deactivate the enzymes and to evaporate IPA, as well as resulting acetone. After cooling to room temperature, the recombinant
  • 10 U / ml (final concentration) of the oxidase from Leuconostoc mesenteroides are used.
  • the batch is made up to 0.5 ml with water.
  • the enzymes are used in the form of cell lysate.
  • the batch is incubated for 10 min at 65 ° C to deactivate the enzymes and then centrifuged.
  • the supernatant is then filtered through a 0.2 ⁇ PVDF filter and analyzed by Ligand Exchange HPLC (Agilent Technologies Inc.).
  • Be separated sugars and polyols here via a lead column by Showa Denko KK (Shodex Sugar ® SP0810) / with a flow of 0.5 ml min water (VWR International Ltd., HPLC grade) at 80 ° C.
  • the detection is performed by means of refraction of light detector (RID, Agilent 1260 Infinity ®, Agilent Technologies Inc.).

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Abstract

Procédé de régénération enzymatique des cofacteurs redox NAD+/NADH et NADP+/NADPH dans une réaction en monoréacteur, un des deux cofacteurs redox étant obtenu dans sa forme réduite et l'autre cofacteur étant obtenu dans sa forme oxydée, en résultat d'au moins deux autres réactions redox (réactions de formations de produits) à catalyse enzymatique se déroulant lors du même processus de réaction, caractérisé en ce que a) lors de la réaction de régénération qui reconvertit le cofacteur réduit dans sa forme oxydée originelle, de l'oxygène ou un composé de formule générale R1C(O)COOH sont réduits, et b) lors de la réaction de régénération qui reconvertit le cofacteur oxydé dans sa forme réduite originelle, un composé de formule générale R2CH(OH)R3 est oxydé, R1, R2 et R3 pouvant avoir diverses significations dans les composés mentionnés.
PCT/EP2012/067781 2012-02-07 2012-09-12 Procédé de régénération enzymatique de cofacteurs redox WO2013117251A1 (fr)

Priority Applications (46)

Application Number Priority Date Filing Date Title
TW102103119A TW201343623A (zh) 2012-02-07 2013-01-28 使氧化還原輔因子經酶催化再生之方法
ARP130100266A AR089841A1 (es) 2012-02-07 2013-01-29 Procedimiento para la regeneracion enzimatica de los cofactores redox
SA113340270A SA113340270B1 (ar) 2012-02-07 2013-02-05 عملية للتجديد الإنزيمي لعوامل مشتركة خاصة بالأكسدة / الاختزال
IN7015DEN2014 IN2014DN07015A (fr) 2012-02-07 2013-02-06
MX2014009455A MX358771B (es) 2012-02-07 2013-02-06 Proceso para la regeneracion enzimatica de cofactores redox.
CA2862384A CA2862384C (fr) 2012-02-07 2013-02-06 Procede de regeneration enzymatique de cofacteurs redox
LTEP13703568.9T LT2812439T (lt) 2012-02-07 2013-02-06 Redokso kofaktorių fermentinio regeneravimo būdas
BR112014019287-1A BR112014019287B1 (pt) 2012-02-07 2013-02-06 processo para regeneração enzimática de cofatores de redox
JP2014556035A JP6189334B2 (ja) 2012-02-07 2013-02-06 グルコースからのフラン誘導体の製造方法。
UAA201409679A UA117453C2 (uk) 2012-02-07 2013-02-06 Спосіб ферментної регенерації окисно-відновних кофакторів nad+/nadh і/або nadp+/nadph
US14/376,512 US9644227B2 (en) 2012-02-07 2013-02-06 Process for the enzymatic regeneration of redox cofactors
HUE13703568 HUE044690T2 (hu) 2012-02-07 2013-02-06 Eljárás redox kofaktorok enzimes regenerálására
PL13703568T PL2812439T3 (pl) 2012-02-07 2013-02-06 Sposób enzymatycznej regeneracji kofaktorów redox
EP13703568.9A EP2812439B1 (fr) 2012-02-07 2013-02-06 Procédé destiné à la régénération enzymatique de co-facteurs redox
RSP20191245 RS59314B1 (sr) 2012-02-07 2013-02-06 Postupak za proizvodnju derivata furana od glukoze
SI201331576T SI2812440T1 (sl) 2012-02-07 2013-02-06 Postopek za izdelavo derivatov furana iz glukoze
PT137035697T PT2812440T (pt) 2012-02-07 2013-02-06 Método de produção de derivados de furano a partir de glicose
DK13703569.7T DK2812440T3 (da) 2012-02-07 2013-02-06 Fremgangsmåde til fremstilling af furanderivater af glucose
ES13703569T ES2746698T3 (es) 2012-02-07 2013-02-06 Procedimiento para la preparación de derivados de furano a partir de glucosa
NZ627477A NZ627477A (en) 2012-02-07 2013-02-06 Method for enzymatic redox cofactor regeneration
LTEP13703569.7T LT2812440T (lt) 2012-02-07 2013-02-06 Furano darinių, gaunamų iš glikozės, gamybos būdas
CA2863137A CA2863137C (fr) 2012-02-07 2013-02-06 Procede de production de derives de furane a partir de glucose
PCT/EP2013/052316 WO2013117585A1 (fr) 2012-02-07 2013-02-06 Procédé de production de dérivés de furane à partir de glucose
JP2014556034A JP6329086B2 (ja) 2012-02-07 2013-02-06 酸化還元補因子の酵素的な再生のためのプロセス
PCT/EP2013/052313 WO2013117584A1 (fr) 2012-02-07 2013-02-06 Procédé de régénération enzymatique de cofacteurs redox
SG11201404614XA SG11201404614XA (en) 2012-02-07 2013-02-06 Process for the enzymatic regeneration of redox cofactors
AU2013218042A AU2013218042B2 (en) 2012-02-07 2013-02-06 Method for enzymatic redox cofactor regeneration
HUE13703569A HUE046250T2 (hu) 2012-02-07 2013-02-06 Eljárás furán-származékok elõállítására glükózból
EP13703569.7A EP2812440B1 (fr) 2012-02-07 2013-02-06 Procédé pour la production de dérivés de furane à partir du glucose
PL13703569T PL2812440T3 (pl) 2012-02-07 2013-02-06 Sposób wytwarzania pochodnych furanu z glukozy
ES13703568T ES2742381T3 (es) 2012-02-07 2013-02-06 Procedimiento para la regeneración enzimática de cofactores redox
RS20191065A RS59114B1 (sr) 2012-02-07 2013-02-06 Postupak za enzimsku regeneraciju redoks kofaktora
RU2014136162A RU2635087C2 (ru) 2012-02-07 2013-02-06 Способ ферментной регенерации окислительно-восстановительных кофакторов
CN201380008572.8A CN104136620A (zh) 2012-02-07 2013-02-06 用于氧化还原辅因子的酶再生的方法
US14/376,527 US9902981B2 (en) 2012-02-07 2013-02-06 Process for the production of furan derivatives from glucose
SI201331563T SI2812439T1 (sl) 2012-02-07 2013-02-06 Postopek encimske regeneracije redoks kofaktorjev
KR1020147023136A KR102022137B1 (ko) 2012-02-07 2013-02-06 효소적 산화환원 보조인자의 재생 방법
PT13703568T PT2812439T (pt) 2012-02-07 2013-02-06 Método para a regeneração enzimática dos co-factores redox
MYPI2014702060A MY172493A (en) 2012-02-07 2013-02-06 Process for the enzymatic regeneration of redox cofactors
CN202110638003.8A CN113337568A (zh) 2012-02-07 2013-02-06 用于氧化还原辅因子的酶再生的方法
PH12014501770A PH12014501770A1 (en) 2012-02-07 2014-08-06 Method for enzymatic redox cofactor regeneration
HK15104308.2A HK1204010A1 (en) 2012-02-07 2015-05-06 Method for enzymatic redox cofactor regeneration
HK15104309.1A HK1204011A1 (en) 2012-02-07 2015-05-06 Method for the production of furan derivatives from glucose d-
US15/490,416 US10370691B2 (en) 2012-02-07 2017-04-18 Process for the enzymatic regeneration of redox cofactors
HRP20191514 HRP20191514T1 (hr) 2012-02-07 2019-08-22 Postupak enzimske regeneracije redoks kofaktora
HRP20191693 HRP20191693T1 (hr) 2012-02-07 2019-09-19 Postupak za proizvodnju derivata furana iz glukoze

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US9644227B2 (en) 2012-02-07 2017-05-09 Annikki Gmbh Process for the enzymatic regeneration of redox cofactors
US9902981B2 (en) 2012-02-07 2018-02-27 Annikki Gmbh Process for the production of furan derivatives from glucose
US10370691B2 (en) 2012-02-07 2019-08-06 Annikki Gmbh Process for the enzymatic regeneration of redox cofactors
US11339415B2 (en) 2012-02-07 2022-05-24 Annikki Gmbh Process for the enzymatic regeneration of redox cofactors
AT513721A1 (de) * 2012-12-10 2014-06-15 Annikki Gmbh Verfahren zur enzymatischen Regenerierung von Redoxkofaktoren
AT513721B1 (de) * 2012-12-10 2014-09-15 Annikki Gmbh Verfahren zur enzymatischen Regenerierung von Redoxkofaktoren
EP3234162A4 (fr) * 2014-12-16 2018-07-11 Newpek S.A. De C.V. Procédés enzymatiques pour la production d'isobutanol
US10550410B2 (en) 2014-12-16 2020-02-04 Newpek S.A. De C.V. Enzymatic methods for isobutanol production
CN105695551A (zh) * 2016-03-04 2016-06-22 苏州引航生物科技有限公司 一种制备去氢表雄酮的生物方法
CN112280818A (zh) * 2020-11-16 2021-01-29 济南大学 一种循环酶催化制备熊去氧胆酸的方法

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