EP4695410A2 - Procédé de production d'allitol - Google Patents

Procédé de production d'allitol

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Publication number
EP4695410A2
EP4695410A2 EP24716415.5A EP24716415A EP4695410A2 EP 4695410 A2 EP4695410 A2 EP 4695410A2 EP 24716415 A EP24716415 A EP 24716415A EP 4695410 A2 EP4695410 A2 EP 4695410A2
Authority
EP
European Patent Office
Prior art keywords
seq
acid sequence
nucleic acid
amino acid
allitol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24716415.5A
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German (de)
English (en)
Inventor
Nicole STAUNIG
Maria Dupont
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Annikki GmbH
Original Assignee
Annikki GmbH
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Filing date
Publication date
Priority claimed from EP23167347.6A external-priority patent/EP4446421A1/fr
Priority claimed from EP23219719.4A external-priority patent/EP4574983A1/fr
Application filed by Annikki GmbH filed Critical Annikki GmbH
Publication of EP4695410A2 publication Critical patent/EP4695410A2/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
    • 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
    • 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
    • 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
    • C12N9/90—Isomerases (5.)
    • 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
    • C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
    • 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
    • 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)
    • C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)
    • 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)
    • C12Y101/01047—Glucose 1-dehydrogenase (1.1.1.47)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y501/00—Racemaces and epimerases (5.1)
    • C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)

Definitions

  • the present invention relates to an enzymatic process for the production of the rare sugar alcohol allitol from D-fructose.
  • Allitol is a hexavalent sugar alcohol that is rarely found in nature and has been found, for example, in the leaves of rosemary willows (Itea sp.) (Hough & Stacey, 1963). It is achiral and therefore forms an interface between the D- and L-hexoses in the so-called Izumoring strategy (Izumori, 2006; Hassanin et al., 2017). Allitol can therefore serve as a precursor for the production of D-psicose (Gullapalli et al., 2007; Poonperm et al., 2007) or L-psicose (Takeshita et al., 1996).
  • allitol can also be used as a sweetener due to its sweet taste (Hassanin et al., 2017).
  • WO 2020195106 Al describes a possible use of allitol as an anti-obesity agent (slimming agent).
  • Allitol can be obtained from D-fructose in two chemical conversion steps: 1) epimerization of D-fructose to D-psicose (C3 epimer) and 2) reduction of D-psicose to allitol.
  • Ketose-3-epimerases can be divided into three groups depending on their substrate specificity: 1) D-tagatose-3-epimerase (DTE), 2) D-psicose-3-epimerase (DPE) or D-allulose-3-epimerase (DAE) and 3) L-ribulose-3-epimerase (LRE).
  • DTE D-tagatose-3-epimerase
  • DPE D-psicose-3-epimerase
  • DAE D-allulose-3-epimerase
  • LRE L-ribulose-3-epimerase
  • an equilibrium ratio is formed between the two epimers. Depending on the reaction conditions (temperature between 40 and 70 °C, pH between 6 and 11), this is between 80:20 and 62.5:37.5 (D-fructose: D-psicose).
  • Many of the epimerases also require a divalent metal ion such as Mn 2+ or Co 2+ (toxic
  • D-psicose to allitol can be carried out microbially with Enterobacter agglomerans strain 221e (Muniruzzaman et al., 1995) or with Klebsiella oxytoca G4A4 (Han et al., 2014).
  • the NAD-dependent ribitol dehydrogenase (RDH; EC 1.1.1.56), which is responsible for the conversion of the pentavalent sugar alcohol ribitol to the ketopentose D-ribulose, also catalyzes the reduction of D-psicose to allitol.
  • D-sorbitol the reduction product of D-fructose
  • GDH glucose dehydrogenase
  • NAD(P) + NAD(P)H
  • the lactone hydrolyzes in an aqueous environment to D-gluconic acid/D-gluconate.
  • Zhao et al. (2022) used a multienzyme self-assembly system of DPE, RDH and glucose dehydrogenase (GDH; for cofactor regeneration) in combination with a glucose isomerase in a whole-cell system to produce allitol (15 g/L) directly from D-glucose (25 g/L).
  • GDH glucose dehydrogenase
  • Feng et al. (2023) describe an in v/vo process using an E. coli whole-cell catalyst with expressed DPE from Clostridium bolteae (requires the addition of 1 mM Co 2+ ), GDH from B. subtilis, and RDH from Providencia alcalifaciens for the simultaneous production of allitol and D-gluconic acid from D-fructose and D-glucose. Even with an E.
  • Hassanin et al. (2016) used a ribitol dehydrogenase (RDH) from Providentia alcalifaciens RIMD 1656011 and an FDH from Ogataea parapolymorpha DL-1 in the form of cell lysates to convert D-psicose (10 g/L) to allitol (94% in 6 h; addition of 2 mM NAD + ).
  • RDH ribitol dehydrogenase
  • the object is achieved according to the invention by forming D-psicose from D-fructose, which is present in an aqueous solution, by treating it with an epimerase in vitro, which is then reduced to allitol by treating it with an NAD(P)H-dependent oxidoreductase in vitro, whereby the NAD(P) + formed during the reduction is enzymatically converted back to NAD(P)H with a hydrogen donor. is reduced and is characterized by the use of a secondary alcohol as a hydrogen donor.
  • Decisive features of the present invention are that the process is carried out in vitro, i.e. not fermentatively, and that a secondary alcohol is used as hydrogen donor.
  • a secondary alcohol is an organic compound having a secondary alcohol group.
  • D-psicose is formed from D-fructose by treatment with an epimerase in vitro, which is then reduced to allitol in vitro using an NAD(P)H-dependent oxidoreductase. Allitol can then be separated from the solution by crystallization.
  • the process according to the invention is shown schematically in the attached Figure 1.
  • a preferred variant of the process according to the invention is that the secondary alcohol is 2-propanol (isopropanol).
  • 2-Propanol is a very cheap hydrogen donor for the regeneration of NAD(P)H and the oxidation product acetone is easily separated due to its volatility (Xu et al., 2021).
  • Acetone obtained from the exhaust gas stream can be heterogeneously catalytically hydrogenated back to 2-propanol (Al-Rabiah et al., 2022), either in the gas phase, in solution or in isopropanol/acetone/water mixtures, whereby in the future there could be an increasing focus on hydrogen from sustainable sources ("green hydrogen”).
  • the reduction of acetone to 2-propanol can also be accomplished enzymatically using alcohol dehydrogenase (in combination with enzymatic oxidation).
  • a further preferred variant of the process according to the invention consists in that the oxidized cofactor NAD(P) + formed by the reduction of D-psicose to allitol is reduced by means of a glucose dehydrogenase (GDH) and D-glucose to form D-gluconolactone, which, depending on the pH value in the aqueous medium, hydrolyzes to D-gluconic acid or D-gluconate.
  • GDH glucose dehydrogenase
  • D-gluconic acid is an important industrial chemical due to its wide range of possible applications, such as as a metal pickling agent (D-gluconic acid), acidifying agent (D-gluconic acid and D-gluconolactone), derusting agent (sodium gluconate) or dietary supplement (calcium gluconate, magnesium gluconate, iron gluconate) (Kornecki et al., 2020; EP 0132557 Bl).
  • a further preferred variant of the process according to the invention is characterized in that it is carried out as a one-pot reaction without isolation of the D-psicose.
  • the particularly preferred concentration of D-fructose is 100 - 250 g/l.
  • the most preferred concentration of D-glucose is 100 - 250 g/l.
  • the particularly preferred temperature range for the process according to the invention is between 25 and 45 °C.
  • the process according to the invention is preferably carried out in the pH range between 6.0 and 9.0, in particular between 7.0 and 8.5.
  • the enzymes are present in a suspension and/or in the homogenate and/or in the lysate of the corresponding cells that produce them, with lysates being particularly preferred.
  • 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, but only serve to convert substrates (Lin & Tao, 2017).
  • Homogenate in this context means a physically and/or chemically treated suspension (e.g. treated by pressure, lysozyme or ultrasound), whereby the cell components are released from the cells.
  • a lysate is obtained when the insoluble cell components of the homogenate are removed, for example by filtration or centrifugation (see Production of enzymes & Production of lysates for details).
  • the enzymes can also be modified at the N-terminus with a water-soluble polymer such as polyethylene glycol, immobilized in or on a solid matrix, or be part of a fusion protein.
  • the enzymes can be in powder form, in lyophilized or spray-dried form.
  • only enzymes from the enzyme groups epimerases and oxidoreductases are used for the conversion of the starting material, with one or more of these enzymes being selected from each of these groups.
  • the epimerase used in the process can originate from one of the groups EC 5.1.3.30 (D-psicose-3-epimerase) or EC 5.1.3.31 (D-tagatose-3-epimerase/L-ribulose-3-epimerase), the former being particularly preferred.
  • the enzyme used to reduce D-psicose comes from the group of oxidoreductases, with a short-chain dehydrogenase/reductase being particularly preferred.
  • the NAD(P)H-dependent oxidoreductase for reducing D-psicose to allitol preferably comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 2, SEQ ID No. 8 or SEQ ID No. 10 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 1, SEQ ID No. 7 or SEQ ID No. 9 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 7 or SEQ ID No. 9.
  • an oxidoreductase whose amino acid sequence is at least 80% identical to SEQ ID No. 2, SEQ ID No. 8 or SEQ ID No. 10 or which is encoded by a nucleic acid which has an identity to SEQ ID No. 1, SEQ ID No. 7 or SEQ ID No. 9 of at least 80% or which binds under stringent conditions to a nucleic acid molecule with the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 7 or SEQ ID No. 9.
  • the oxidoreductases listed here for the reduction of D-psicose to allitol preferably comprise or consist of an amino acid sequence which has an identity to SEQ ID No. 2, SEQ ID No. 8 or SEQ ID No. 10 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%.
  • the oxidoreductase according to the invention for the reduction of D-psicose to allitol particularly preferably comprises or consists of the amino acid sequence SEQ ID No. 2, SEQ ID No. 8 or SEQ ID No. 10.
  • the oxidoreductases for reducing D-psicose to allitol preferably comprise or consist of an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 1, SEQ ID No. 7 or SEQ ID No. 9 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%.
  • the nucleic acid which encodes the oxidoreductase according to the invention for reducing D-psicose to allitol comprises or consists of the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 7 or SEQ ID No. 9.
  • identity refers to the percentage of identical nucleotide or amino acid matches between at least two sequences, using a standardized algorithm for aligning nucleotide or amino acid sequences ("alignment"). Such an algorithm can, in a standardized and reproducible manner, insert gaps (“gap”) in the compared sequences in order to optimize the alignment between two sequences and thus achieve a more meaningful comparison of the two sequences.
  • Percent identity between sequences can be determined using one or more computer algorithms or programs known in the art or described herein.
  • the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) provided by the National Center for Biotechnology Information (NCBI) is used to determine identity.
  • the BLAST suite of software includes several programs, including a tool called "BLAST 2 Sequences” which is used for direct pairwise comparison of two nucleotide or amino acid sequences. "BLAST 2 Sequences” can also be accessed and used interactively on the Internet via the NCBI World Wide Web site.
  • W word length
  • E expectation
  • M amino acid sequence
  • B BLOSUM62 scoring matrix
  • the oxidoreductases for reducing D-psicose to allitol preferably comprise an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ. ID NO:1, SEQ. ID NO:7, or SEQ ID NO:9.
  • stringent conditions refer to conditions under which so-called specific hybrids are formed, but not nonspecific hybrids.
  • stringent conditions comprise hybridization in 6xSSC (sodium chloride/sodium citrate) at 45°C and then washing with 0.2 to 1xSSC, 0.1% SDS at 50 to 65°C; or such conditions may comprise hybridization in 1xSSC at 65 to 70°C and then washing with 0.3xSSC at 65 to 70°C.
  • Hybridization can be performed by conventionally known methods such as those described by J. Sambrook et al. in Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).
  • One aspect of the present invention relates to the use of an oxidoreductase for reducing D-psicose to allitol, wherein the oxidoreductase comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having at least 80% identity to SEQ ID No. 2, SEQ ID No. 8 or SEQ ID No. 10, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID No. 1, SEQ ID No. 7 or SEQ ID No. 9, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 7 or SEQ ID No. 9.
  • the alcohol dehydrogenase (ADH) used for cofactor regeneration can come from one of the groups EC 1.1.1.1 (NAD-dependent ADH) and EC 1.1.1.2 (NADP-dependent ADH).
  • the NAD(P)-dependent alcohol dehydrogenase for cofactor regeneration preferably comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 4 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 3 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 3.
  • Particularly suitable for cofactor regeneration in general is an alcohol dehydrogenase whose amino acid sequence is at least 80% identical to SEQ ID No. 4 or which is encoded by a nucleic acid which has an identity of at least 80% to SEQ ID No. 3 or which binds under stringent conditions to a nucleic acid molecule with the nucleic acid sequence SEQ ID No. 3.
  • the alcohol dehydrogenase for cofactor regeneration mentioned here preferably comprises an amino acid sequence which has an identity to SEQ ID No. 4 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%.
  • the alcohol dehydrogenase according to the invention for cofactor regeneration particularly preferably comprises the amino acid sequence SEQ ID No. 4 or consists of this.
  • the alcohol dehydrogenase for cofactor regeneration preferably comprises or consists of an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 3 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%.
  • the nucleic acid encoding the alcohol dehydrogenase for cofactor regeneration according to the invention comprises or consists of the nucleic acid sequence SEQ ID No. 3.
  • One aspect of the present invention relates to the use of an alcohol dehydrogenase for cofactor regeneration, wherein the alcohol dehydrogenase comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 4 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 3, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 3.
  • the glucose dehydrogenase (GDH) used for cofactor regeneration can come from one of the groups EC 1.1.1.47 (glucose-l-dehydrogenase), EC 1.1.1.118 (glucose-l-dehydrogenase (NAD + )), EC 1.1.1.119 (glucose-l-dehydrogenase (NADP + )) or EC 1.1.1.360 (glucose/galactose-l-dehydrogenase).
  • the NAD(P)-dependent glucose dehydrogenase for cofactor regeneration preferably comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 6 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 5 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 5.
  • Particularly suitable for cofactor regeneration in general is a glucose dehydrogenase whose amino acid sequence is at least 80% identical to SEQ ID No. 6 or which is encoded by a nucleic acid which has an identity of at least 80% to SEQ ID No. 5 or which binds under stringent conditions to a nucleic acid molecule with the nucleic acid sequence SEQ ID No. 5.
  • the glucose dehydrogenase for cofactor regeneration mentioned here preferably comprises an amino acid sequence which has an identity to SEQ ID No. 6 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%.
  • the glucose dehydrogenase according to the invention for cofactor regeneration particularly preferably comprises the amino acid sequence SEQ ID No. 6 or consists of this.
  • the glucose dehydrogenase for cofactor regeneration preferably comprises an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 5 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%.
  • the nucleic acid which encodes the glucose dehydrogenase for cofactor regeneration according to the invention comprises or consists of the nucleic acid sequence SEQ ID No. 5.
  • glucose dehydrogenase for cofactor regeneration, wherein the glucose dehydrogenase comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 6 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 5 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 5.
  • the cofactor NAD(P) + which is formed during the reduction of D-psicose to allitol, is reduced to NAD(P)H by means of formate and a formate dehydrogenase with the formation of CO2 (cofactor regeneration).
  • a formate dehydrogenase which comprises or consists of the amino acid sequence SEQ ID No. 12, or a functional fragment of this formate dehydrogenase.
  • the formate dehydrogenase preferably used is preferably encoded by the nucleic acid sequence SEQ ID No. 11.
  • a "functional fragment" of the formate dehydrogenase comprises an N-terminally and/or C-terminally truncated variant of the formate dehydrogenase with the amino acid sequence SEQ ID No.
  • SEQ ID No. 12 MAKILCVLYDDPVDGYPKTYARDDLPKIDHYPGGQTLPTPKAIDFTPGALLGSVSGELGLRKYLEANGHTFV VTSDKDGPDSVFERELVDADVVISQPFWPAYLTPERIAKNLKLALTAGIGSDHVDLQSAIDRGITVAEVT YCNSISVAEHVVMM ILGLVRNYIPSHDWARKGGWNIADCVEHSYDLEGMTVGSVAAGRIGLAVLRRLAP FDVKLHYTDRHRLPEAVEKELGLVWHDTREDMYPHCDVVTLNVPLHPETEHM INDETLKLFKRGAYIVNT ARGKLADRDAIVRAIESGQLAGYAGDVWFPQPAPKDHPWRTMKWEGMTPHISGTSLSAQARYAAGTR EILECFFEGRPIRDEYLIVQGGALAGTGAHSYSKGNATGGSEEAAKFKKAG
  • the formate dehydrogenase used for cofactor regeneration comprises an amino acid sequence which is selected from the group consisting of: i) an amino acid sequence which has an identity to SEQ ID No. 12 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 11 of at least 80%, and iii) an amino acid sequence which is encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 11, or a functional fragment thereof.
  • the formate dehydrogenase preferably comprises an amino acid sequence which has an identity to SEQ ID No. 12 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%.
  • the formate dehydrogenase preferably comprises an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 11 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%.
  • a further aspect of the present invention relates to the use of a formate dehydrogenase for cofactor regeneration or a functional fragment thereof, wherein the formate dehydrogenase comprises or consists of an amino acid sequence which is selected from the group consisting of: i) an amino acid sequence which has an identity to SEQ ID No. 12 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 11, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 11.
  • D-Psicose was purchased from TCI and Hunan Garden Naturals Inc. (China), allitol was purchased from TCI, D-fructose, lysozyme and methanol were purchased from PanReac AppliChem (ITW Reagents), D-glucose, sodium gluconate, IPTG (isopropyl-ß-D-thiogalactopyranoside) were purchased from Sigma-Aldrich, potassium dihydrogen phosphate, di-potassium hydrogen phosphate, NAD + , NADH disodium salt and sodium dodecyl sulfate (SDS) were purchased from Carl Roth and triethanolamine (TEA) was purchased from Chem-Lab NV.
  • the gene to be expressed was first amplified in a PCR using the genomic DNA or its synthetic equivalent adapted to the codon usage of E. coli as a template together with specific oligonucleotides that additionally carry recognition sequences for restriction endonucleases and isolated from the reaction mixture. After nucleic acid digestion with the restriction enzymes Sphl and Hindlll, the gene fragment coding for the target enzyme was ligated into the backbone of the expression vector pQE70-Kan cut with Sphl and Hindlll. 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.
  • 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 tests.
  • the cell pellet prepared according to the above procedure was weighed into a suitable container and mixed with buffer and lysozyme (final concentration 0.5 mg/ml) (e.g. triethanolamine (TEA) - HCl) and dissolved with stirring.
  • buffer and lysozyme final concentration 0.5 mg/ml
  • the mass fraction of biomass is usually 20%, the rest is made up of the buffer.
  • the resulting homogenate was centrifuged for 10 min at 4 °C and 16000 rpm (Eppendorf Centrifuge 5417R) to separate the insoluble cell fragments and obtain the lysate.
  • a Dionex ICS6000 system with AS-AP autosampler was used to quantify D-gluconic acid/D-gluconate using HPAEC (High Performance Anion Exchange Chromatography). The measurement was carried out using conductivity detection (CD) coupled to a Dionex AERS 500 electrolytically regenerated suppressor in external water mode.
  • CD conductivity detection
  • a Dionex lonPac ASll-HC-4pm column with a corresponding pre-column and a NaOH gradient was used to separate the analytes.
  • the mobile phase was also pretreated with a Dionex ATC Anion Trap Column.
  • Enzyme activities in the lysates were determined using a Shimadzu UV-1900 spectrophotometer.
  • the formation or consumption of NAD(P)H was monitored at a wavelength of 340 nm via the change in absorption.
  • the measurements were carried out with 0.2 mM cofactor (NAD(P) + or NAD(P)H).
  • NAD(P) + or NAD(P)H cofactor + or NAD(P)H
  • 20 pl of a 10 mM stock solution of the cofactor were placed in a cuvette (Greiner bio-one semi-micro cuvette made of polystyrene) and the desired pH value was adjusted with 100 M TEA-HCl buffer (870 pl).
  • 10 pl of lysate (diluted or undiluted) and 100 pl of substrate solution were added to the cuvette and the measurement started immediately afterwards.
  • the mixtures were incubated under continuous shaking (Eppendorf Thermomixer, 35 °C, 800 rpm) for a total of 24 h.
  • the reaction was carried out in a Multifors table bioreactor (Infors AG).
  • a glass reactor (volume 1 1) with a stirrer and pH electrode was used as the vessel.
  • the pH was controlled by adding 5M NaOH or 1M H2SO4.
  • the reactor contents were stirred for 1 h at 70 °C.
  • the hot reaction mixture was filtered through a glass frit (P4, 10-16 pm) by applying a vacuum.
  • the filtrate was mixed with acetone (final volume fraction 50%) and stored overnight in the refrigerator at 4 °C.
  • the precipitate was filtered off (glass frit P4, 10-16 pm) and washed with ice-cold acetone.
  • the product was dried for 24 h in a vacuum drying cabinet at 50 °C.
  • An HPLC and HPAEC analysis of the product showed that allitol could be obtained in high purity (> 97%) from the D-gluconate-containing solution.

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Abstract

Procédé de production d'allitol par formation de D-psicose à partir de D-fructose, présent sous forme dissoute dans une solution aqueuse, par traitement avec une épimérase in vitro, lequel D-psicose est réduit en allitol in vitro par traitement avec une oxydoréductase NAD(P)H dépendante, le NAD(P)+ résultant de la réduction étant à nouveau réduit enzymatiquement en NAD(P)H avec un donneur d'hydrogène, caractérisé en ce que l'on utilise comme donneur d'hydrogène un alcool secondaire.
EP24716415.5A 2023-04-11 2024-04-11 Procédé de production d'allitol Pending EP4695410A2 (fr)

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EP23167347.6A EP4446421A1 (fr) 2023-04-11 2023-04-11 Procédé de préparation d'allitol
EP23219719.4A EP4574983A1 (fr) 2023-12-22 2023-12-22 Procédé de préparation d'allitol
PCT/EP2024/059807 WO2024213621A2 (fr) 2023-04-11 2024-04-11 Procédé de production d'allitol

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EP4695410A2 true EP4695410A2 (fr) 2026-02-18

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EP4680759A2 (fr) * 2023-03-15 2026-01-21 Annikki GmbH Procédé de production d'une solution aqueuse contenant du l-psicose
EP4446422A3 (fr) * 2023-03-15 2025-12-10 Annikki GmbH Procédé de préparation d'une solution aqueuse contenant de la l-psicose

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DE3326546A1 (de) 1983-07-22 1985-02-07 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V., 8000 München Verfahren zur kontinuierlichen enzymatischen herstellung von gluconsaeure oder ihren derivaten und sorbit und/oder mannit
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