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WO2016072800A1 - Procédé de préparation du psicose - Google Patents

Procédé de préparation du psicose Download PDF

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Publication number
WO2016072800A1
WO2016072800A1 PCT/KR2015/011954 KR2015011954W WO2016072800A1 WO 2016072800 A1 WO2016072800 A1 WO 2016072800A1 KR 2015011954 W KR2015011954 W KR 2015011954W WO 2016072800 A1 WO2016072800 A1 WO 2016072800A1
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Prior art keywords
mannitol
fructose
dehydrogenase
epimerase
psychos
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English (en)
Korean (ko)
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김선원
최민진
정성희
이대윤
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Gyeongsang National University GNU
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Gyeongsang National University GNU
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Priority to CN201580060676.2A priority Critical patent/CN107109443B/zh
Priority to US15/524,362 priority patent/US10266862B2/en
Priority claimed from KR1020150156115A external-priority patent/KR101754060B1/ko
Publication of WO2016072800A1 publication Critical patent/WO2016072800A1/fr
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    • 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
    • C12P19/02—Monosaccharides

Definitions

  • the present invention relates to a process for producing a psychos capable of separating the psychos with high purity.
  • D-psicose is an epimer of carbon number 3 of fructose (D-fructose), which is sweet like ordinary sugars, but is not metabolized in the human body. It is a functional sugar that can be used as a sugar substitute functional sweetener. In addition, it has the function of reducing the abdominal obesity by inhibiting the enzyme activity involved in lipid synthesis in the liver, and is a sugar currently being studied as a therapeutic agent for diabetes and atherosclerosis.
  • Ken Izumori et al Used galacitol, D-tagatose or D-talitol by utilizing microbial cell reactions. It has been shown that it is possible to produce psychocos from. However, these substrates also have the disadvantage that they are relatively rare in nature and have a high cost.
  • D-tagatose-3-epimerase of isolated microorganism Pseudomonas cichorii ST-24 is produced and purified from recombinant E. coli.
  • fructose has properties similar to that of psychos, even though it produces a psychose, it is not easy to separate it from fructose.
  • An object of the present invention is to provide a method for producing a psychose capable of separating the psychoses in high purity.
  • An object of the present invention is to provide a method for producing a psychos with improved production capacity of the psychos.
  • One aspect includes the steps of converting the fructose to mannitol by adding mannitol dehydrogenase to a mixture of fructose and cyclose; And separating the mannitol from the mixture.
  • Representative methods for the production of psychos include a chemical method of converting fructose to psychos using a catalyst, and a gene encoding an enzyme that catalyzes the conversion of fructose to psychos, is introduced into a microorganism to convert fructose into psychos. And biological methods.
  • fructose is used as a substrate, and fructose and psychocos have similar properties, which makes it difficult to separate the psychocos from the reaction mixture.
  • a mannitol dehydrogenase is added to a mixture of fructose and psycose to convert the fructose to mannitol.
  • the mixture of fructose and psychos is a mixture of converted psychos and unconverted fructose after the conversion of fructose to psychos in a chemical or biological manner.
  • the present invention may further comprise the step of preparing a mixture of the fructose and the psychic by reacting the substrate fructose and its epimerase.
  • the epimerase may be cycos-3-epimerase, specifically, derived from Agrobacterium tumefaciens (Agrobacterium tumefaciens) derived from the cocos-3-epimerase or the wife Rostines caccae (Anaerostipes caccae) It may be a psychocos-3-epimerase.
  • the gene encoding the epimerase is a gene encoding a cosmos-3-epimerase derived from Agrobacterium tumefaciens of SEQ ID NO: 1, or the wife rostrophes kaka of SEQ ID NO: 2 caccae) may be a gene encoding a cosmos-3-epimerase.
  • Agrobacterium tumefaciens-derived psychos-3-epimerase may have an amino acid sequence of SEQ ID NO: 3, and the wife rostipes carcae-derived psychos-3-epimerase may have the amino acid sequence of SEQ ID NO: 4 Can have
  • the gene encoding the epimerase may be a gene encoding the amino acid sequence of the cyclic cos-3-epimerase of SEQ ID NO: 5 in that the epimerase has better high temperature stability.
  • the amino acid sequence of SEQ ID NO: 5 is an amino acid sequence of amino acid sequence of Agrobacterium tumefaciens-derived cosmos-3-epimerase from amino acid 33 to leucine, amino acid 213 to cysteine is excellent thermal stability .
  • the inventors of the present invention confirmed that the cyclos-3-epimerase derived from the genus Clostridium has excellent thermal stability.
  • the cyclos-3-epimer derived from the genus Clostridium was high in thermal stability. In the case of one, it was confirmed that one amino acid had a corresponding sequence or both.
  • amino acid sequence may be a sequence in which the 32nd amino acid is substituted with leucine in the amino acid sequence of SEQ ID NO: 6, or the 196th amino acid is substituted with cysteine.
  • SEQ ID NO: 6 lists the sequence indicated by the box in FIG. 7 and is used in the present invention Agrobacterium tumefaciens, wife Rostifescaca, Clostridium boltea, Clostridium hilemo derived from psychos-3- Epimerase amino acid sequences (SEQ ID NOs: 3, 4, 9, 10). Therefore, the microorganism may be substituted with a gene encoding the amino acid sequence, but is not limited thereto.
  • the epimerase may be a cosmos-3-epimerase derived from the genus Clostridium, and the gene encoding the same may be Clostridium boltheea of SEQ ID NO.
  • Clostridium bolteae may be a gene encoding a cosmos-3-epimerase, or a gene encoding a Clostridium hylemonae (Clostridium hylemonae) derived cosmos-3-epimerase.
  • the gene may be a gene encoding a cosmos-3-epimerase derived from Clostridium hilemo.
  • Clostridium bolteae-derived cosmos-3-epimerase may have an amino acid sequence of SEQ ID NO: 9
  • clostridium hilemo derived cycos-3-epimerase may have an amino acid sequence of SEQ ID NO: 10 have.
  • the cos-3-epyrase derived from wife rostitis caca the 32nd amino acid is substituted with leucine or the 196th amino acid is substituted with cysteine in the amino acid sequence of SEQ ID NO: 6
  • Cycos-3-epimerases having the sequence shown above, Cycos-3-epimerases from Clostridium bolteae and Cycos-3-epimerases from Clostridium hylemonae Has a pH lower than 7 indicating optimal activity.
  • the reaction of fructose and epimerase may be carried out at a temperature of 20 to 90 °C, preferably from 40 to 90 °C in terms of maximizing the production capacity of the psychos.
  • Specific examples include 40 to 50 ° C, 40 to 60 ° C, 40 to 70 ° C, 40 to 80 ° C, 40 to 90 ° C, 45 to 60 ° C, 45 to 70 ° C, 45 to 80 ° C, 45 to 90 ° C, 50 to 70 ° C, 50 to 80 ° C, 50 to 90 ° C, 55 to 60 ° C, 55 to 70 ° C, 55 to 80 ° C, 55 to 90 ° C, 60 to 70 ° C, 60 to 80 ° C, 60 to 90 ° C, 70 to 80 ° C, 70 to 90 ° C, 75 to 80 ° C, 75 to 90 ° C, 80 to 90 ° C and the like. If the reaction temperature is greater than 90 °C epimerase or microorganisms to be described later may be thermally damaged
  • the reaction is preferably performed in a microorganism. As the reaction temperature increases, it is more difficult to implement the pymerase due to the heat degeneration of the pycos, but when the reaction is carried out in the microorganism, the epimerase is protected by the microorganism, so the reaction is performed at high temperature. This is possible.
  • microorganism may be a cell that can be cultured in a liquid medium.
  • the microorganism may be to express the epimerase intrinsically or by transformation.
  • epimerase may be produced in the microorganism so that the production of the psychos by the reaction in the microorganism of the fructose and the epimerase may be continuously performed.
  • the gene encoding the epimerase is a cycose-derived from Agrobacterium tumefaciens of SEQ ID NO: 1 It may be a gene encoding 3-epimerase, or a gene encoding a cyclic cos-3-epimerase derived from Anaerostipes caccae of SEQ ID NO: 2.
  • the gene encoding the epimerase may be a gene encoding the amino acid sequence of the cyclic cos-3-epimerase of SEQ ID NO: 5 in that the epimerase has better high temperature stability.
  • the gene encoding the epimerase in terms of excellent high temperature stability and the ability to produce cosmos may be a gene encoding the cosmos-3-epimerase derived from Clostridium bolteae of SEQ ID NO: 7, Or a gene encoding a cosmos-3-epimerase derived from Clostridium hylemonae of SEQ ID NO: 8.
  • the microorganism may be cultured in a liquid medium as prokaryotic or eukaryotic cells, and may be cultured at the aforementioned high temperature.
  • the microorganism may be, for example, a bacterium, fungus, or a combination thereof.
  • the bacterium may be a gram positive bacterium, a gram negative bacterium, or a combination thereof, and may be preferably a gram positive bacterium in terms of increasing psychocosal productivity.
  • Gram-negative bacteria can be of the genus Escherichia.
  • Gram-positive bacteria can be of the genus Bacillus, Corynebacterium, Genus Actinomyces, Lactobacillus or combinations thereof.
  • the fungus may be yeast, genus Cleveromyces, or a combination thereof.
  • thermophiles having high thermal stability are preferable.
  • it may be of the genus Corynebacterium, Actinomyces, more preferably coding for the above-mentioned epimerase in Corynebacterium glutamicum, most preferably Corynebacterium glutamicum ATCC 13032.
  • the gene may be introduced.
  • the microorganism of the genus Escherichia may be Escherichia coli, and specifically, genes encoding epimerase may be introduced into DH5 ⁇ , MG1655, BL21 (DE), S17-1, XL1-Blue, BW25113, or a combination thereof.
  • E. coli may be inactivated one region consisting of the gene encoding the endogenous 6-phosphoplactokinase and allose metabolic operon.
  • 6-phosphoplactokinase may be, for example, one having a nucleotide sequence of SEQ ID NO: 11, and 6-phosphoplactokinase may be one having an amino acid sequence of SEQ ID NO: 12.
  • Genes constituting the allose metabolic operon are rpiB, alsR, alsB, alsA, alsC, alsE and alsK, one or more of these genes may be inactivated.
  • the rpiB, alsR, alsB, alsA, alsC, alsE, and alsK genes may be, for example, each having a nucleotide sequence of SEQ ID NO: 13, 14, 15, 16, 17, 18, and 19.
  • the rpiB, alsR, alsB, alsA, alsC, alsE, and alsK genes may be encoding the amino acid sequences of SEQ ID NOs: 20, 21, 22, 23, 24, 25, and 26, respectively.
  • activation means that the expression of the gene is reduced or not expressed.
  • the “inactivation” can be made by methods known in the art. For example, it may be inactivated by homologuous recombination. The homologous recombination may be mediated by, for example, transposon mutagenesis or P1 transduction.
  • the microorganism of the genus Corynebacterium may be Corynebacterium glutamicum, specifically, a gene encoding epimerase may be introduced into Corynebacterium glutamicum ATCC 13032.
  • the microorganisms of the genus Corynebacterium are the ptsF (EII Fru , fruA, NCgl1861, GI: 19553141, EC 2.7.1.69) genes, which are PTS transport systems that transfer endogenous di-fractose into di-fractose 1-phosphate and transport them into cells. May be missing or inactivated.
  • ptsF EII Fru , fruA, NCgl1861, GI: 19553141, EC 2.7.1.69
  • the ptsF gene may have a nucleotide sequence of SEQ ID NO: 27 and may encode an amino acid sequence of SEQ ID NO: 28.
  • Psychoses are generated from D-fructose, so depletion or inactivation of the gene can inhibit the phosphorylation of fructose, which can significantly improve the production efficiency of psychos.
  • microorganism of the genus Corynebacterium may be a deletion or inactivation of the mtlD (NCgl0108, GI: 19551360, EC 1.1.1.67) gene encoding mannitol 2-dehydrogenase.
  • the mtlD gene may have a nucleotide sequence of SEQ ID NO: 29, or may encode an amino acid sequence of SEQ ID NO: 30.
  • the microorganism When the reaction of the fructose and its epimerase is carried out in a microorganism, the microorganism may be cultured in a medium containing fructose.
  • the medium may be a nutrient medium containing yeast extract and nitrogen sources, such as 2YT medium, LB medium, TB medium.
  • the fructose concentration contained in the medium is not particularly limited, and may be included, for example, at a concentration of 1% (w / v) to 80% (w / v), and, for example, 1% (w / v) within the above range.
  • 1% (w / v) 1% (w / v) within the above range.
  • 10% (w / v) to 80% (w / v) 20% (w / v) to 80% (w / v), 30% (w / v) to 80 % (w / v), 40% (w / v) to 80% (w / v) and the like.
  • carbon sources including glucose, glycerol and the like
  • Nitrogen sources including ammonia, urea, and the like
  • Essential metal ions such as sodium, potassium, calcium, magnesium, manganese and cobalt
  • It may be a life medium (defined medium) commonly used in the art including vitamins and the like.
  • the culture can be continuous, semi-continuous, or batch type culture.
  • the microorganism has a turbidity of cells (measured at 600 nm absorbance, OD 600 ) in a medium containing fructose in a range of 0.01 to 300, for example, 1 to 300, 10 to 300, 20 to 300, 5 to 300, or 40 to 40. It may be inoculated at a concentration of 300.
  • a cell containing a high concentration of the enzyme it is possible to efficiently convert fructose to a psychos in a medium containing fructose at a high concentration in the medium.
  • the culturing may be performed by further adding a substance for inducing the expression of a gene encoding epimerase.
  • the material for inducing the expression of the gene is not particularly limited and may be a material commonly used in the art.
  • the production of the psychos may be performed in a medium containing only inorganic salts for the supply of fructose and cofactors as substrates.
  • the reaction may be carried out in a medium containing only inorganic salts for the supply of fructose and cofactors as substrates.
  • the inorganic salt may be, for example, manganese salt or cobalt salt. Cobalt salt is preferable in view of showing a more improved production rate of psychos, and manganese salt is preferable in terms of safely utilizing the produced psychos as a food.
  • the medium containing only fructose and inorganic salt may be a liquid medium in which fructose and inorganic salt are dissolved in a solvent.
  • the solvent may be water, for example.
  • metabolites such as organic acids of microorganisms, etc.
  • the medium in addition to psychose, so that the medium may be gradually acidified.
  • the medium containing only fructose and inorganic salts according to the present invention does not contain a buffer solution, in such a case, it is more preferable to use a cycos-3-epimerase having a low optimum active pH (for example, below pH 7). Do.
  • the method of producing the psychos of the present invention may further include inducing the microorganisms to have dormant cells by culturing the microorganisms in a medium not containing the fructose before the reaction between the fructose and the epimerase.
  • Induction to have the dormant cells can be carried out by culturing the microorganism to a stationary phase in a medium that does not contain fructose.
  • a resting cell refers to a cultured cell which is no longer proliferating.
  • the stationary phase stops the division and proliferation of cells after the exponential phase when the cells are cultured, and does not show an increase in cell population, and the synthesis and decomposition of cellular components are balanced. Means status.
  • the dormant cells according to the present invention refers to a cell in which growth is completed and the expression of epimerase in the cell is sufficiently performed.
  • the expression amount of epimerase is maximum. It is possible to maximize the production of psychocos.
  • the medium not containing fructose may be the same medium as the above-mentioned medium containing fructose, except that no fructose is included.
  • the present invention may further include the step of recovering the microorganism after the reaction of the fructose and its epimerase and reusing it for conversion of the other substrate to the psychos.
  • the reaction between fructose and its epimerase is carried out in a microorganism, and even when exposed to high temperature, the epimerase is protected by the microorganism and still exhibits enzymatic activity.
  • the microorganisms can be recovered and reused for the conversion of another substrate to psychos.
  • the number of reuse is not limited, and hundreds of times or thousands of times can be reused.
  • thermophiles having high thermal stability in terms of high enzymatic activity upon reuse.
  • it may be of the genus Corynebacterium, Actinomyces, more preferably Corynebacterium glutamicum, and most preferably Corynebacterium glutamicum ATCC 13032
  • the gene encoding epimerase may be introduced.
  • the present invention reacts the mixture of fructose and psycose with mannitol dehydrogenase to convert fructose not reacted with epimerase to mannitol.
  • the reaction mixture may be a supernatant obtained by separating a mixture of fructose and psychos from a microorganism.
  • the mannitol dehydrogenase may be mannitol-2-dehydrogenase.
  • Leukonostoke Pseudomecenteroides ATCC 12291 Leuconostoc pseudomesenteroides ATCC 12291) -derived mannitol-2-dehydrogenase (GenBank: CAD31644.1, GI: 28865823), Leuconostoc mesenteroides ) mannitol-2-dehydrogenase (GenBank: ACT22631.1, GI: 253317413), mannitol-2-dehydrogenase (GenBank: AAC45771.1, GI: 2338764) from Rhodobacter sphaeroides or Pseudomonas flu Oresense DSM 50106 ( Pseudomonas mannitol-2-dehydrogenase (GenBank: AAC04472.1, GI: 2293418) derived from fluorescens
  • the mannitol-2-dehydrogenase derived from Leuconostock Pseudomethenteroides ATCC 12291 may have the amino acid sequence of SEQ ID NO: 43, and the mannitol-2-dehydrogenase derived from leuconosstock mesenteroides has the amino acid sequence of SEQ ID NO: 44
  • the Rhodobacter sphaeroides-derived mannitol-2-dehydrogenase may have an amino acid sequence of SEQ ID NO: 45
  • the Pseudomonas fluorescens DSM50106-derived mannitol-2-dehydrogenase may have an amino acid sequence of SEQ ID NO: 46.
  • the reaction of the mixture of fructose and psychose with mannitol dehydrogenase can be carried out in the presence of a NADH (reduced form of Nicotinamide Adenine Dinucleotide (NAD)) source.
  • NADH reduced form of Nicotinamide Adenine Dinucleotide
  • NADH is a coenzyme of mannitol dehydratase and can significantly increase the conversion of fructose to mannitol when NADH is supplied to mannitol dehydratase. As a result, fructose in the reaction solution is reduced, so that the psychos can be more easily separated.
  • NADH sources can be used without limitation, known in the art, for example can be formic acid and formic acid dehydrogenase.
  • Formic acid dehydrogenase can be used without limitation formic acid dehydrogenase known in the art, for example, formic acid dehydrogenase (FDH, GenBank: AB072394.1, derived from Mycobacterium vaccae N10) GI: 15982576).
  • FDH formic acid dehydrogenase
  • the formic acid dehydrogenase derived from Mycobacterium vaccae N10 may have an amino acid sequence of SEQ ID NO: 49.
  • reaction of the reaction mixture with mannitol dehydrogenase can be carried out in a microorganism.
  • the microorganism may be cultured in a liquid medium as prokaryotic or eukaryotic cells, and may be cultured at the aforementioned high temperature.
  • the microorganism may be, for example, a bacterium, fungus, or a combination thereof.
  • the bacterium may be a gram positive bacterium, a gram negative bacterium, or a combination thereof, and may be preferably a gram positive bacterium in terms of increasing psychocosal productivity.
  • Gram-negative bacteria can be of the genus Escherichia.
  • Gram-positive bacteria can be of the genus Bacillus, Corynebacterium, Genus Actinomyces, Lactobacillus or combinations thereof.
  • the fungus may be yeast, genus Cleveromyces, or a combination thereof.
  • Cycos is used in food as a sugar substitute functional sweetener
  • the microorganism is preferably a strain listed as GRAS (Generally Recognized As Safe).
  • GRAS Generally Recognized As Safe
  • it may be of the genus Corynebacterium, more specifically Corynebacterium glutamicum, most preferably Corynebacterium glutamicum ATCC 13032.
  • the microorganism may be to express the mannitol dehydrogenase intrinsically or by transformation.
  • mannitol dehydrogenase is produced in the microorganism so that the production of mannitol by the reaction in the microorganism of the reaction mixture and mannitol dehydrogenase can be carried out continuously.
  • the leuconosstock pseudomethenoteroides KCTC 3652 ( Leuconostoc pseudomesenteroides Mannitol-2-dehydrogenase from KCTC 3652) (GenBank: CAD31644.1, GI: 28865823), mannitol-2-dehydrogenase from Leuconostoc mesenteroides (GenBank: ACT22631.1, GI: 253317413) , Rhodobacter mannitol-2-dehydrogenase derived from sphaeroides (GenBank: AAC45771.1, GI: 2338764) or mannitol-2-dehydrogenase derived from Pseudomonas fluorescens DSM 50106 (GenBank: AAC04472.1, GI: 2293418) ) Can
  • the microorganism may be to express the formic acid dehydrogenase intrinsically or by transformation.
  • formic acid dehydrogenase can be produced in the microorganism to produce NADH from formic acid.
  • NADH acts as a coenzyme of mannitol dehydrogenase, thereby significantly increasing the conversion rate of fructose to mannitol.
  • the formic acid dehydrogenase can be, for example, a formate dehydrogenase (FDH, GenBank: AB072394.1, GI: 15982576) from Mycobacterium vaccae N10.
  • FDH formate dehydrogenase
  • the microorganism may be to express the glucose transport protein (Glucose Transport Protein, GLF) intrinsically or by transformation.
  • GLF glucose transport Protein
  • the inflow of fructose as a substrate into the microorganism may be increased, thereby increasing the amount of mannitol production.
  • fructose in the reaction solution is reduced, so that the psychos can be more easily separated.
  • Glucose transport proteins can be used without limitation, glucose transport proteins known in the art, for example, glucose transport proteins from Zymomonas mobilis subsp.mobilis ZM4, ATCC 31821 or KCTC 1534 (GenBank: AAG29864. 1; GI: 11095424).
  • the glucose transport protein from Zymomonas mobilis subsp.mobilis ZM4, ATCC 31821 or KCTC 1534 may have an amino acid sequence of SEQ ID NO: 50.
  • the microorganism When the reaction of the fructose and its epimerase is carried out in a microorganism, the microorganism may be cultured in the above-mentioned medium.
  • the medium further includes formic acid.
  • the medium may be a condition of pH 6 to 7.5, more preferably pH 6.5 to 7.0.
  • mannitol productivity is excellent, it is possible to more easily separation of the psychos.
  • the pH in the above range can be obtained by using sodium acetate, PIPES, sodium phosphate buffer solution, or the like as a solvent of the liquid medium, or by using water.
  • the step of converting fructose to mannitol may be performed in an open reaction system.
  • the CO 2 produced by the reaction between formic acid and formic acid dehydrogenase can be released to keep the formic acid dehydrogenase active.
  • the medium since the pH of the medium may be increased by the consumption of formic acid, the medium preferably includes a buffer solution as a liquid solvent, and more preferably, a PIPES buffer solution may be used.
  • the method of producing the psychos of the present invention may further include inducing the microorganisms to have dormant cells by culturing the microorganism in a medium not containing the fructose before the reaction of the reaction mixture and mannitol dehydrogenase. .
  • the present invention may further comprise the step of recovering the microorganism after the reaction of the reaction mixture and mannitol dehydrogenase, and reusing the conversion of the other substrate to mannitol.
  • Fructose and psycose have similar physical properties and are difficult to separate, but mannitol has different physical properties from psychic, and can easily separate psycose from mannitol.
  • the separation method is not particularly limited, and examples thereof include centrifugation, filtration, crystallization, ion exchange chromatography, and the like, and since mannitol and psychose have a large difference in solubility in solvents, preferably mannitol and It can be separated by crystallization according to the difference in solubility in the solvent of the psychos.
  • the second solvent may be crystallized by adding a second solvent different from the first solvent to the reaction solution containing the first solvent, or crystallizing the mannitol by heating and concentrating the solvent in the reaction solution.
  • the said solvent is not specifically limited, For example, water, aqueous salt solution, ethanol, hexane, acetone, etc. are mentioned. These can be used individually or in mixture of 2 or more types.
  • mannitol and psychos can also be separated by liquid chromatography.
  • Psychos and fructose have similar physical properties and have similar retention times in the column of liquid chromatography, so that the area of the peak phase overlaps, but the psychos and mannitol can be easily separated because they have different physical properties and are separated into the peak phase.
  • an aspect of the present invention provides a method for producing a psychose comprising the step of converting the fructose to mannitol by adding a mannitol dehydrogenase to the mixture of fructose and psychose.
  • the step of converting the fructose to mannitol by adding mannitol dehydrogenase to the mixture of fructose and psychocos is carried out by converting fructose into psychos in a chemical or biological manner, as described above, and then converting the psychos and unconverted fructose. It can be carried out by adding mannitol dehydrogenase to the mixture of to convert fructose to mannitol.
  • reaction of the reaction mixture with mannitol dehydrogenase may be carried out in a microorganism.
  • the specific method is as described above.
  • Phenose is an epimer of Fructose No. 3 carbon, which is sweet like ordinary sugars but is not metabolized in the human body, and almost calorie is almost zero sugar and can be used as a sugar replacement functional sweetener for diabetic and obese patients.
  • fructose is high in calories and can be a major cause of diabetes by overlying insulin sensitivity.
  • mannitol unlike fructose, is low in calories, poorly absorbed by the body, and requires long-term metabolism, and is a component used as a sweetener in diabetic patients.
  • the mannitol dehydrogenase when added to the mixture of fructose and psychose to convert the fructose to mannitol, even when used as a sweetener in a mixture of psychose and mannitol, without separating psychose into mannitol, diabetes, obesity, etc. It can be used as a sugar substitute functional sweetener which reduces the possibility of causing metabolic syndrome.
  • the present invention may further comprise the step of preparing a mixture of the fructose and the psychic by reacting the fructose as a substrate and its epimerase.
  • the step of reacting the substrate fructose with its epimerase to prepare a mixture of the fructose and the psychos may be carried out according to the above-described method.
  • the present invention may further comprise the step of recovering the microorganism after the reaction of the fructose and its epimerase and reusing it for the conversion of other substrates to the psychos, which is likewise carried out according to the method described above. Can be.
  • the method of the present invention it is possible to separate the psychos with high purity, and the yield of the production of the psychos is remarkably improved.
  • the yield and speed of the production of the psychos are improved.
  • Figure 1 shows the measurement of the amount of psychos produced from the fructose as a substrate according to the reaction temperature of the dormant cell transformation reaction from the Corynebacterium glutamicum transformants introduced with the cosmos-3-epimerase.
  • Figure 2 shows the measurement of the production of psychos from the fructose as a substrate according to the reaction temperature of the dormant cell transformation reaction from E. coli MG1655 transformant introduced with cyclose-3-epimerase.
  • FIG. 3 shows the recovery of the cells after the Pseudomonas-producing dormant cell transformation reaction at 60 ° C. for 3 hours using the Corynebacterium glutamicum and Escherichia coli MG1655 transformants incorporating cyclose-3-epimerase. It is shown by measuring the yield of the psychos obtained by the reaction under the same reaction conditions.
  • Figure 4 shows the production of psychos from fructose according to the composition change of the psychos production reaction medium used for the dormant cell transformation reaction of the Corynebacterium glutamicum transformants introduced with the Psyco-3- epimerase will be.
  • Figure 5 shows the composition derived from the Pseudomonas cycos-3-epimerase and the composition of the Pseudomonas cyclic medium during the dormant cell transformation reaction of the Corynebacterium glutamicum transformants introduced with the Psycho-3-Epimerase It shows the output of the psychos according.
  • FIG. 6 is a diagram showing the derivation of Pseudo-3-Epimerase-derived strain and Pseudomonas spp. During the dormant cell transformation of the Corynebacterium glutamicum transformants incorporating Psycho-3-Epimerase. The output is shown.
  • Figure 7 compares the amino acid sequence of cyclose-3-epimerase from various strains.
  • Figure 8 shows the production of psychos according to the number of reuse of Corynebacterium glutamicum incorporating cycos-3-epimerase from the genus Clostridium.
  • LpMDH is a mannitol dehydrogenase derived from leuconosstock pseudomethenoterides
  • LmMDH is a mannitol dehydrogenase derived from leuconosstock mecetheroides
  • RsMDH is a mannitol dehydrogenase derived from Rhodobacter spheroides
  • PfMDH is a mannitol dehydrogenase derived from Pseudomonas fluorescens It is shown.
  • FIG. 10 is a diagram showing the amount of cyclose (A) remaining without reacting by mannitol dehydrogenase (A) and mannitol production amount (B) converted from fructose when a mixed sugar composed of sicose and fructose is provided as a substrate.
  • FIG. 11 is a chart showing mannitol production amount (A) and pH change (B) when using a conversion medium containing a buffer solution having a different pH and using a conversion medium containing water without a buffer solution.
  • W is water without buffer
  • SA sodium aceteate buffer at pH 5
  • P is PIPES buffer at pH 6
  • SP sodium phosphate buffer at pH 6.5.
  • FIG. 12 is a chart comparing mannitol production amount (A) and pH change (B) according to the use of a conical tube as a closed reaction container and a test tube as an open reaction container.
  • FIG. 13 is a graph showing mannitol production when water or pH 6 PIPES buffer solution is used as a conversion medium in a test tube that is an open reaction container.
  • GLF 14 is a chart comparing mannitol production according to the introduction of glucose transport protein (GLF).
  • 15 is a chart showing the concentrations of fructose, mannitol, and psycos after separating mannitol crystallized from the mannitol conversion reaction mixed sugar solution according to the ethanol addition amount.
  • 16 is a chart showing the concentrations of fructose, mannitol, and psychos after separating mannitol crystallized as the volume of the solution of the mixed sugar per mannitol conversion reaction through evaporation is reduced.
  • FIG. 17 is a diagram comparing the resolution patterns on high performance liquid chromatography of mixed sugars in the reaction solution with and without additional mannitol conversion reaction after the Pycos production process.
  • PCES208 J. Microbiol. Biotechnol., 18: 639-647, 2008
  • E. coli-Corynebacterium shuttle vector was modified and used to construct a pSGT208 shuttle vector in which a terminator and a lac promoter were inserted.
  • Psycho-3-epimerase is a dpe gene of Agrobacterium tumefaciens str. C58; taxid: 176299; GenBank NID: NC_003062, ATCC33970 for the production of psychoses in Corynebacterium glutamicum. (AGR_L_260, GI: 15890243, SEQ ID NO: 1) was introduced into the pSGT208 shuttle vector thus prepared and used.
  • amplification of the dpe gene from the Agrobacterium tumefaciens genome using primers 1 of SEQ ID NO: 31 and primer 2 of SEQ ID NO: 32 was carried out by restriction enzymes KpnI and BamHI and inserted into the same site of pSGT208 shuttle vector.
  • PS208-dpe recombinant shuttle vector containing the cyclic cosine epimerase was prepared.
  • lac promoter was replaced with the trc promoter derived from pTrc99a in pS208-dpe in order to increase the expression level of Pycos-3-epimerase in Corynebacterium glutamicum. It was named pS208cT-dpe. .
  • the recombinant vectors pS208-dpe, pS208cT-dpe and pSGT208 vector, which are negative controls, were introduced into the wild type Corynebacterium glutamicum ATCC 13032 and transformed. It was used for the production of psychocos from fructose. Transformation followed the method specified in the Handbook of Corynebacterium glutamicum (Lothar Eggeling et al., ISBN 0-8493-1821-1, 2005 by CRC press).
  • Corynebacterium glutamicum transformants prepared above were inoculated in 5 ml of LB medium (Difco) containing 20 ⁇ g / ml kanamycin to ensure high concentration of cells and cultured at 30 ° C. and 250 rpm.
  • the main culture was incubated in a 500 mL Erlenmeyer flask with 100 mL volume for 12 hours at 30 ° C. and 180 rpm to induce sufficient cell mass and sufficient expression of protein.
  • the obtained culture solution was centrifuged to remove the supernatant, and the cells were recovered, and the cell concentration was resuspended to 40 OD 600 in the same medium containing 40% (w / v) fructose as a substrate. , 37, 50, 60 or 70 °C, the dormant cell conversion reaction at 180rpm conditions.
  • the concentrations of fructose and psycose were measured using high performance liquid chromatography (HPLC).
  • HPLC was used SCL-10A (Shimadzu, Japan) equipped with a Kromasil 5 NH 2 column (4.6 mm x 250 mm), and the mobile phase was separated at 40 ° C. while flowing at 1.5 mL / min using 75% acetonitrile and then RI. Analysis was performed using a Reflective Index detector. Under the above conditions, the retention time of fructose was 5.5 minutes, and the psychocos was 4.6 minutes.
  • FIG. 1 The measurement result is shown in FIG. Referring to Figure 1, the Corynebacterium glutamicum ATCC13032 strain with the pSGT208cT-dpe shuttle vector was transformed in a medium containing 40% fructose, and as a result, the production rate of the psychos was remarkably increased. It seems to be faster and production increases. Particularly, in the experimental group reacted at 50, 60 and 70 ° C., the reaction equilibrium of the cosmos-3-epimerase enzyme was reached within approximately 3 hours to produce about 120 g / L of psychose, which is the psyche-3-epi. It can be seen that the conversion rate and yield of cycos from fructose of merase are temperature dependent.
  • the yield increased sharply from 50 ° C, which is significantly higher than the temperature required for the normal enzymatic reaction, and it is believed that the reaction between the enzyme and the substrate changed at that temperature.
  • E. coli MG1655 which lacks the pfkA (SEQ ID NO: 11) and als2 (SEQ ID NOs: 14, 15, 16, 17, 18, and 19) genes, was used to block the Pseudolysis pathway.
  • E. coli MG1655 transformant prepared above was inoculated in 5 ml of LB medium (Difco) containing 100 ⁇ g / ml of empicillin to incubate at 37 ° C. and 250 rpm to obtain high concentration of cells.
  • the culture was inoculated in 2YT medium containing / L glucose and 100 ⁇ g / ml of empicillin.
  • the main culture was incubated in a 500 mL Erlenmeyer flask with 100 mL volume for 12 hours at 37 ° C. and 180 rpm to induce sufficient cell mass and sufficient expression of protein.
  • the obtained supernatant was centrifuged to remove the supernatant and the cells were recovered, and E. coli minimal medium M9 medium containing 11.3 g M9 minimal salts (Difco), 0.1 mL 1 M, containing 40% (w / v) fructose as a substrate.
  • the cell concentration was resuspended in CaCl 2 , 2mL 1M MgSO 4 , 1mL 100mM MnSO 4 5H 2 O) at 40 OD 600 , and the dormant cell conversion reaction was performed at 37, 60 or 70 ° C. and 180 rpm, respectively.
  • the concentrations of fructose and psycose were analyzed according to the method described in Example 1 above. The measurement results are shown in FIG. 2.
  • the E. coli MG1655 ( ⁇ pfkA, als2) strain which introduced the pTPE vector, was also subjected to a conversion reaction in a medium containing 40% fructose. Seems to do. Particularly, in the experimental group reacted at 60 and 70 ° C., the reaction equilibrium of psychocos-3-epimerase was reached in about 2 hours as in the experiment with Corynebacterium, and produced about 120 g / L of psychos.
  • dormant cells for 3 hours in the presence of fructose in order to determine how long the activity of the fructose to convert the fructose to the psychos remain 3 hours after reaching the reaction equilibrium that maximizes the production of psychos.
  • the cells used for the production of the psychos were recovered and reused for the dorsal cell conversion reaction produced by the psychos.
  • the cell reuse dormant cell conversion reaction was repeated three times at 60 °C temperature.
  • the first dormant cell conversion reaction is R0
  • the first time dormant cell conversion reaction from which cells are recovered from the previous reaction solution is R1
  • the second reused dormant cell conversion reaction is R2
  • the third reused dormant cell conversion reaction is R3. Marked as. Culture conditions and analysis methods were performed in the same manner as in Example 1. The results are shown in FIG.
  • Example 1 in the conversion reaction of producing fructose from fructose in Corynebacterium glutamicum, the minimum medium containing 40% Fructose (1 g K 2 HPO 4 , 10 g (NH 4 ) 2 SO 4 , 0.4 g MgSO 4 7H 2 O, 20 mg FeSO 4 7H 2 O, 20 mg MnSO 4 5H 2 O, 50 mg NaCl, 2 g urea, 0.1 mg biotin, 0.1 mg thiamine). The components of the medium used for this conversion reaction were minimized to prepare a more economical and convenient medium, and the comparison of the cyclic productivity with the medium used in Example 1 was performed.
  • the components of the medium used for the Pseudomonas-producing dormant cell transformation reaction need only MnSO 4 , which is a cofactor of fructose and Pseudo- 3- Epimerase.
  • the whole gene of Anaerostipes caccae DSM 14662; taxid: 411490 was purchased from the poisonous DSMZ company.
  • Primers of SEQ ID NOs: 33 and 34 containing the entire sequence of the purchased genome as a template to include the cosmos-3-epamerase presumed gene (AP endonuclease; Sequence ID: gb
  • the first PCR was performed using the pair as a primer.
  • a second PCR was performed using primer pairs of SEQ ID NOs: 35 and 36, which specifically bind to the Pycos-3-epimerase gene, using the amplified PCR product as a template.
  • the obtained PCR product was inserted into the same enzyme site of pS208cT-dpe (vector described in Example 1 of Korean Patent Application No. 10-2013-0060703) using restriction enzymes BamHI and XbaI to prepare a recombinant vector pS208cT-AcDPE.
  • the prepared pS208cT-AcDPE vector was transformed by introducing into wild-type Corynebacterium glutamicum ATCC 13032, which was used for the production of cycos from fructose. Transformation followed the method specified in the Handbook of Corynebacterium glutamicum (Lothar Eggeling et al., ISBN 0-8493-1821-1, 2005 by CRC press).
  • the obtained recombinant Corynebacterium glutamicum strain was stored at -80 ° C and used for culturing.
  • Plasmid was obtained from Yakult Korea. PCR was carried out using primer pairs of SEQ ID NOs: 37 and 38 that specifically bind to the Pseudo-3-Epimerase gene.
  • the obtained PCR product was inserted into the same enzyme site of pS208cT-dpe (vector described in Example 1 of Korean Patent Application No. 10-2013-0060703) using restriction enzymes KpnI and XbaI to prepare a recombinant vector pS208cT-CbDPE.
  • the recombinant vector pS208cT-CbDPE vector thus produced was introduced and transformed into wild-type Corynebacterium glutamicum ATCC 13032 in the same manner as above, and was used for the production of cycos from fructose.
  • the obtained recombinant Corynebacterium glutamicum strain was stored at -80 ° C and used for culturing.
  • the entire gene of Clostridium hylemonae DSM 15053; taxid: 553973 was purchased from DSMZ, Germany.
  • SEQ ID NOs: 39 and 40 using the entire genome as a template, to include a polico-3-epimerase putative gene (dolichol monophosphate mannose synthase; Sequence ID: ref
  • the first PCR was performed using primer pairs of as primers.
  • the second PCR was performed using primer pairs of SEQ ID NOs: 41 and 42 which specifically bind to the cosmos-3-epimerase gene using the amplified PCR product as a template.
  • the obtained PCR product was inserted into the same enzyme site of pS208cT-dpe (vector described in Example 1 of Korean Patent Application No. 10-2013-0060703) using restriction enzymes BamHI and XbaI to prepare a recombinant vector pS208cT-ChDPE.
  • the prepared pS208cT-ChDPE vector was introduced into the wild-type Corynebacterium glutamicum ATCC 13032 and transformed in the same manner as above, and was used for the production of psychocos from fructose.
  • the obtained recombinant Corynebacterium glutamicum strain was stored at -80 ° C and used for culturing.
  • the transformants were inoculated in 2YT medium containing 20 ⁇ g / ml of kanamycin and cultured at 30 ° C and 250rpm, and then the transformants were inoculated in 2YT medium containing 20 ⁇ g / ml of kanamycin. .
  • the main culture was incubated in a slotted 300 ml Erlenmeyer flask at 60 ° C. for 7 hours at 30 ° C. and 180 rpm to induce sufficient cell mass and expression of proteins.
  • the cells obtained by the method of Example 1 were suspended in 2YT and continuously heated at 60 ° C. for 0, 3, 6, 9, 12, and 24 hours using a shake incubator. After each hour of heating, the cells were recovered, suspended in a simple conversion reaction medium containing only 20 ⁇ g / ml kanamycin, 0.1 mM manganese, and 40% (w / v) fructose. The reaction proceeded. Concentrations of fructose and psycose were measured in the same manner as described in Example 1. The results are shown in FIG.
  • a recombinant corynebacterium glutamicum incorporating previously used cyclose-3-epimerase derived from agrobacterium and cyclose-3-epimerase derived from wife rostipes is After 3 hours of heat at 60 ° C, it seems that they rarely produce Psychos.
  • recombinant Corynebacterium glutamicum which introduced cyclose-3-epimerase from the genus Clostridium, appears to maintain psychose production even after 24 hours of heat, producing psychose in high temperature processes. It is believed to be advantageous over Cycos-3-epimerase from Agrobacterium.
  • FIG. 1 A comparison between the amino acid sequence of Agrobacterium tumefaciens-derived psychos-3-epimerase and the amino acid sequence of cyclos-3-epimerase from Clostridium genus is shown in FIG. In the case of the high thermal stability of cyclos-3-epimerase from the genus Clostridium, one amino acid had a corresponding sequence or both.
  • Example 7 it was confirmed that the cyclose-3-epimerase derived from the genus Clostridium has high stability against high temperature, and among the recombinant strains incorporating the cosmos-3-epimerases derived from the two Clostridium spp.
  • Recombinant Corynebacterium glutamicum cells incorporating cyclose-3-epimerase in Clostridium hilemo typically showed the effect of fungal reuse after dormant cell conversion at high temperature.
  • Example 3 After the cells obtained in the same manner as described in Example 3 were subjected to dormant cell conversion for 3 hours at 60 ° C., the cells were recovered and subjected to the same dormant cell conversion reaction and the cells were reused three times in total ( Experiment on the same conditions as in Example 3).
  • the first dormant cell conversion reaction is R0
  • the first time dormant cell conversion reaction from which cells are recovered from the previous reaction solution is R1
  • the second reused dormant cell conversion reaction is R2
  • the third reused dormant cell conversion reaction is R3. Marked as. The results are shown in FIG.
  • a pSGT208 vector modified to facilitate cloning of the pCES208 vector which is an E. coli-Corinebacterium shuttle vector, was used (vector described in Example 1 of Korean Patent Application No. 10-2013-0060703).
  • the promoter used a trc promoter which eliminated the lac operator to ensure sustained expression.
  • Mannitol-producing enzymes include Leukonostock Pseudomethenteroides ATCC 12291 (or KCTC 3652, Leuconostoc pseudomesenteroides ATCC 12291), Leuconostoc mesenteroides, Rhodobacter sphaeroides and Pseudomonas An enzyme derived from 50106 (Pseudomonas fluorescens DSM 50106) was used.
  • Leukonostock Pseudomethenteroides strains were purchased from KCTC and purified as a template, followed by mannitol 2-dehydrogenase (MDH; GenBank: AJ486977.1, GI: 28865822, SEQ ID NO: 43, LpMDH). PCR was performed using primer pairs of SEQ ID NOs: 47 and 48 as primers.
  • Mannitol dehydrogenase derived from Leukonostock mesenteroides (GenBank: ACT22631.1, GI: 253317413, SEQ ID NO: 44, LmMDH), Rhodobacter spheroides derived mannitol dehydrogenase (GenBank: AAC45771.1, GI: 2338764, SEQ ID NO: 45, RsMDH) and Pseudomonas fluorescens derived mannitol dehydrogenase (GenBank: AAC04472.1, GI: 2293418, SEQ ID NO: 46, PfMDH) were synthesized from GenScript.
  • the obtained PCR product and the synthetic gene were inserted into the same restriction enzyme site of the pSGT208 vector in which the trc promoter from which the lac operator was removed using restriction enzymes KpnI and BamHI was inserted into the recombinant vectors pS208cT-LpMDH, pS208cT-LmMDH, pS208cT-RsMDH, and pS208cT- PfMDH was prepared.
  • the enzyme was introduced to help the continuous regeneration of NADH used as a coenzyme of mannitol dehydrogenase.
  • Formic acid dehydrogenase which regenerates NADH by oxidation of formic acid to carbon dioxide, was used as an enzyme to assist regeneration of NADH.
  • Forco acid dehydrogenase derived from Mycobacterium vaccae N10 formate dehydrogenase, FDH, GenBank: AB072394.1, GI: 15982576, SEQ ID NO: 49 was used, and the gene was synthesized from GenScript.
  • the synthesized gene was inserted into the same restriction enzyme sites of pS208cT-LpMDH, pS208cT-LmMDH, pS208cT-RsMDH, and pS208cT-PfMDH vectors constructed previously using restriction enzymes BamHI and XbaI.
  • -FDH, pS208cT-RsMDH-FDH and pS208cT-PfMDH-FDH were prepared.
  • PS208cT-LpMDH-FDH, pS208cT-LmMDH-FDH, pS208cT-RsMDH-FDH and pS208cT-PfMDH-FDH vectors prepared were wild-type Corynebacterium glutamicum ATCC 13032 (Corynebacterium glutamicum ATCC 13032).
  • Corynebacterium glutamicum ATCC 13032 was followeded the method specified in the Handbook of Corynebacterium glutamicum (Lothar Eggeling et al., ISBN 0-8493-1821-1, 2005 by CRC press).
  • the transformed recombinant Corynebacterium strain was stored at -80 ° C and used.
  • Mannitol production was performed using a dormant cell conversion reaction using high concentration of cells.
  • the prepared Corynebacterium glutamicum recombinant strain was inoculated in 2YT medium containing 20 ⁇ g / ml of kanamycin and cultured at 30 ° C and 250 rpm, followed by 20 ⁇ g / ml This culture was inoculated with 5-10% (v / v) of seed culture in 2YT medium containing kanamycin, 0.5% (v / v) glucose to induce sufficient cell mass and protein expression at 30 ° C and 180 rpm.
  • mannitol dehydrogenase is an enzyme having a low specificity for the substrate, not only fructose but also psychos may be recognized and reduced as a substrate. Therefore, it was confirmed that a strategy to easily separate fructose by converting only fructose into mannitol in a mixed sugar of psychocos and fructose was applicable.
  • Example 9 Using the recombinant Corynebacterium strain thus constructed, only the substrate was changed into a mixed sugar of psychocos and fructose, and the same procedure as in Example 9 was performed.
  • Mixed sugars were the ratio of fructose 7 to psychos 3 (v / v) that can be obtained after the fructose conversion reaction, and the concentration of the mixed sugars was 10% (v / v) as in Example 9. The results are shown in Figures 10 (A) and 10 (B).
  • FIG. 10 (A) shows the remaining psychocos concentration without being used as a substrate.
  • the initial concentration of about 30 g / L was maintained as it was during the 48-hour conversion reaction.
  • FIG. 10 (B) shows the amount of mannitol produced from 70 g / L fructose constituting 100 g / L mixed sugar and produced about 20 g / L during a 48-hour conversion reaction at a conversion temperature of 30 ° C.
  • FIG. Accordingly, it was confirmed that the mannitol dehydrogenase selectively used fructose as a substrate in the mixed sugar substrate solution in which both cocos and fructose are present. Therefore, it was confirmed that the concept of the present patent, which converts fructose remaining after fructose conversion from fructose to mannitol, facilitates the separation of psychocos, can be successfully applied.
  • mannitol production was compared according to pH. Since the mannitol conversion reaction is known to occur in weak acidity, a buffer solution to be maintained at a weakly acidic pH was selected. In addition, it was confirmed whether mannitol production was efficient even when using a conversion medium consisting only of water without a buffer solution.
  • the mannitol conversion reaction was carried out in a closed environment using a conical tube for the conversion reaction.
  • the experiment was conducted using a test tube that allows free access of the gas in order to confirm the effect of the CO 2 produced during the dehydrogenation process of formic acid and melting into the conversion reaction medium.
  • the experimental results are shown in FIGS. 12 (A) and 12 (B).
  • Figure 12 (A) shows the mannitol production obtained in the conical tube of the closed vessel reaction conditions and the test tube of the open vessel reaction conditions.
  • the mannitol production was found to be about 9 times less than in the open environment using the test tube.
  • the pH after 24 hours of the mannitol conversion reaction in the open environment was 9.2, which was much higher than the pH 7.2 in the closed environment. It is presumed that this is because CO 2 is not exhausted in a sealed environment and dissolved in carbonic acid in the medium, which hinders the rise of pH and the action of formic acid dehydrogenase.
  • the action of formic acid dehydrogenase has the effect of raising the pH of the medium through formic acid consumption.
  • CO 2 is well discharged, the action of formic acid dehydrogenase is also enhanced, and the pH is greatly increased.
  • Example 12 As confirmed in Example 12, it was confirmed that the supply of NADH was abundant due to the active action of formic acid dehydrogenase when using the test tube. Therefore, in addition to the open environment, the pH was adjusted to weak acid so as to be suitable for mannitol production. The pH was corrected by adding PIPES (pH 6) buffer to the conversion medium to a final concentration of 300 mM, and the conversion temperature was 45 ° C. The results are shown in FIG.
  • Glucose transport protein also known as fructose influx
  • Glucose transport protein was obtained from KCTC (KCTC 1534) using an enzyme from Zymomonas mobilis subsp.mobilis ZM4 or ATCC 31821.
  • PCR was performed using the primers of SEQ ID NOS: 51 and 52 with a glucose transport protein (GenBank: AAG29864.1; GI: 11095424; SEQ ID NO: 50) of Zimomonas mobilis.
  • the obtained PCR product was inserted into the same restriction enzyme site of pJC1-1-cT-Cmp vector using restriction enzymes BamHI and XbaI to construct recombinant vector pJC1-1-cT-GLF-Cmp.
  • the expression vector (pJC1-1-cT-Cmp) used in the construction was modified from the E. coli-Corynebacterium shuttle vector, pJC1, introducing the promoter variable region and the MCS region of the pSGT208 vector, and further terminating the Corynebacterium. A terminator region was introduced to construct the pJC1-1 vector.
  • the pJC1-1-cT vector was constructed by replacing the lac promoter region including the lac operator of the pJC1-1 vector with the trc promoter, and using the kanamycin antibiotic resistance gene for expression with the pSGT208 vector used in the previous example.
  • the pJC1-1-cT-Cmp vector was finally constructed by substitution with the gene.
  • the previously prepared pJC1-1-cT-GLF-Cmp recombination vector was introduced into Corynebacterium glutamicum ATCC 13032 transformed with pS208cT-LpMDH-FDH, and the recombinant Corynebacterium strain was Stored at -80 °C was used.
  • the mannitol production experiment was carried out using the PIPES buffer conversion medium under the same conditions as in Example 13 using the recombinant Corynebacterium strain thus constructed, and the concentration of chloramphenicol was 5 ⁇ g / ml. The results are shown in FIG.
  • the fructose which is not easily separated from the psychos, is converted into mannitol, thereby making it possible to easily separate the cosmos from the mannitol, and it can be used as an expensive functional sugar as the mixed psychocos and mannitol itself.
  • fructose and psychocos are difficult to separate and purify because their physical properties are similar.
  • mannitol has a large difference in solubility between fructose and psycose, it can be easily separated and purified.
  • Fructose a substrate in the process of producing sicose, is converted to psychose at a conversion rate of about 30%. Therefore, when 400 g / L fructose is used, the reaction liquid after completion
  • the reaction solution was subjected to the mannitol conversion reaction of the remaining fructose using the recombinant Corynebacterium strain (pS208cT-MDH-FDH) of Example 13 to fructose (140 g / L), mannitol (140 g / L), and cycos ( 120 g / L) was mixed to obtain a final reaction solution.
  • pS208cT-MDH-FDH recombinant Corynebacterium strain
  • the concentrations of fructose and psychos in the mannitol conversion reaction solution are maintained through mannitol crystallization, whereas only the concentration of mannitol is significantly decreased. Therefore, by separating only mannitol through the addition of ethanol, it is possible to easily obtain a solution with an increased content of psychos.
  • the concentrations of fructose and psycose in the initial mixed sugar solution are concentrated and increased, while the concentration of mannitol is lowered. Therefore, if fructose is converted to mannitol, simply increasing the temperature can increase the proportion of psychose in the mixed sugar.
  • Example 1 The resolution of the mixed sugars in the reaction solution before and after the mannitol conversion reaction was observed using the HPLC analysis conditions of Example 1.
  • the analysis was carried out with the Pseudomonas production reaction solution (Cycos 120g / L, fructose 280g / L; Fig. 17 (A)), 50% mannitol conversion reaction solution (Cycos 120g / L, Fructose 140g / L, mannitol 140g / L; 17 (B)) and 100% mannitol conversion reaction solution (Cycos 120g / L, mannitol 280g / L; FIG. 17 (C)).
  • FIG. 17 (A) it can be seen that the psychoses and fructose have similar retention times and thus have large areas of overlapping peaks.
  • FIG. 17B the fructose is converted to 50% of mannitol having a longer retention time, and the overlapping interval between the psychos and the fructose is reduced.
  • FIG. 17 (C) when both fructose was converted into mannitol, it was confirmed that the psycose and mannitol were completely separated by the HPLC peak.
  • the HPLC column used in the above experiments is a laboratory level column.
  • Industrial HPLC columns are larger than laboratory level columns and can readily separate high concentrations of solution without dilution, but at a lower level.
  • the overlapping area between the psychos and fructose will be more apparent. Therefore, it would be advantageous to convert residual fructose to mannitol in order to effectively separate the psychos from fructose when using an industrial HPLC column.

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Abstract

La présente invention concerne un procédé de préparation du psicose et, plus spécifiquement, un procédé de préparation de psicose, comprenant les étapes consistant à : appliquer une mannitol déshydrogénase à un mélange de fructose et de psicose ce qui permet de convertir le fructose en mannitol ; et séparer le mannitol du mélange, grâce auquel il est possible d'augmenter la capacité de production en psicose et de séparer le psicose avec une pureté élevée.
PCT/KR2015/011954 2014-11-06 2015-11-06 Procédé de préparation du psicose Ceased WO2016072800A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0302970A1 (fr) * 1985-12-20 1989-02-15 Uop Inc. Procédé pour séparer le psicose d'un autre cétose
KR100832339B1 (ko) * 2006-12-11 2008-05-26 솔젠트 (주) 과당을 사이코스로 전환하는 신규한 시노리조비움 속균주와 이를 이용한 사이코스 생산법
KR20110041910A (ko) * 2009-10-16 2011-04-22 경상대학교산학협력단 사이코스 3-에피머라제 효소를 코딩하는 폴리뉴클레오티드를 포함하는 대장균 및 그를 이용하여 사이코스를 생산하는 방법
KR20110108185A (ko) * 2010-03-26 2011-10-05 씨제이제일제당 (주) Ｄ-사이코스 결정을 제조하는 방법
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Publication number Priority date Publication date Assignee Title
EP0302970A1 (fr) * 1985-12-20 1989-02-15 Uop Inc. Procédé pour séparer le psicose d'un autre cétose
KR100832339B1 (ko) * 2006-12-11 2008-05-26 솔젠트 (주) 과당을 사이코스로 전환하는 신규한 시노리조비움 속균주와 이를 이용한 사이코스 생산법
KR20110041910A (ko) * 2009-10-16 2011-04-22 경상대학교산학협력단 사이코스 3-에피머라제 효소를 코딩하는 폴리뉴클레오티드를 포함하는 대장균 및 그를 이용하여 사이코스를 생산하는 방법
KR20110108185A (ko) * 2010-03-26 2011-10-05 씨제이제일제당 (주) Ｄ-사이코스 결정을 제조하는 방법
WO2014168302A1 (fr) * 2013-04-09 2014-10-16 주식회사 삼양제넥스 Épimérase de d-psicose et procédé de production de psicose l'utilisant

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