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WO2021086035A1 - Fructose 6-phosphate 4-épimérase et son utilisation - Google Patents

Fructose 6-phosphate 4-épimérase et son utilisation Download PDF

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Publication number
WO2021086035A1
WO2021086035A1 PCT/KR2020/014879 KR2020014879W WO2021086035A1 WO 2021086035 A1 WO2021086035 A1 WO 2021086035A1 KR 2020014879 W KR2020014879 W KR 2020014879W WO 2021086035 A1 WO2021086035 A1 WO 2021086035A1
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Prior art keywords
phosphate
enzyme
tagatose
fructose
microorganism
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English (en)
Korean (ko)
Inventor
한은진
정인석
최은수
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Samyang Corp
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Samyang Corp
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Priority claimed from KR1020200141721A external-priority patent/KR102513451B1/ko
Publication of WO2021086035A1 publication Critical patent/WO2021086035A1/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
    • 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

Definitions

  • the present invention relates to a fructose 6-phosphate 4-epimerization enzyme and a phosphorylated ketohexose production using the same, and a ketohexose can be prepared using a fructose 6-phosphate 4-epimerization enzyme.
  • Phosphorylated saccharide epimerase is an epimerase bonded to carbon in various phosphorylated saccharides, and ketohexose-6-phosphate epimerase can epimerize C3 or C4.
  • the ketohexose may be one or more ketohexose selected from the group consisting of fructose, allulose, sorbose, and tagatose.
  • Epimerase of fructose-6-phosphate includes 3-epimerase and 4-epimerase.
  • fructose-6-phosphate 4-epimerase is an enzyme capable of producing tagatose 6-phosphate by 4-epimerization (4-epimerization, 4 carbon epimerization) of fructose (D-fructose).
  • the fructose 6-phosphate 4-epimerization enzyme has a certain level of reaction equilibrium, so that the final conversion rate is only at the level of 20 to 35%. Therefore, in the case of preparing high-purity tagatose through a substrate transfer reaction using a fructose 6-phosphate 4-epimerase, an additional process of separating and removing fructose from the final reaction solution is required.
  • the enzyme used In the case of industrially using starch or maltodextrin as a substrate to produce ketohexose, such as fructose, the enzyme used must have high industrial production conditions, particularly heat stability, and must have the highest conversion rate, and must also contain sugar. Since it is used as a substrate, browning of sugar easily occurs under alkaline conditions, so it is necessary to satisfy conversion reaction conditions that prevent browning of sugars as much as possible.
  • an enzyme that satisfies at least one suitable substrate conversion rate, thermal stability of the enzyme, and enzyme reaction conditions, and produces ketohexose using fructose as a raw material, and a method for producing ketohexose using the same are urgently needed.
  • An example of the present invention is to provide a composition and method for preparing a fructose 6-phosphate 4-epimerase and tagatose 6-phosphate using the same.
  • a further example of the present invention is a nucleic acid molecule encoding a fructose 6-phosphate 4-epimerase.
  • a vector into which the nucleic acid molecule is introduced, and a transformed prokaryotic cell are provided.
  • a further example of the present invention is a fructose 6-phosphate 4-epimerization enzyme protein, a microorganism expressing the enzyme, a microorganism of the strain, a culture of the strain, a lysate of the lysate, a supernatant of the lysate, and the A method for producing tagatose-6-phosphate comprising converting fructose 6-phosphate to tagatose 6-phosphate using at least one selected from the group consisting of extracts of cells, cultures, lysates, and supernatant. to provide.
  • a further example of the present invention is a fructose 6-phosphate 4-epimerization enzyme protein, a microorganism expressing the enzyme, a microorganism of the strain, a culture of the strain, a lysate of the lysate, a supernatant of the lysate, and the It provides a composition for producing tagatose 6-phosphate comprising at least one selected from the group consisting of an extract of cells, cultures, lysates and supernatant.
  • a further example of the present invention is a fructose 6-phosphate 4-epimerization enzyme protein, a microorganism expressing the enzyme, a microorganism of the strain, a culture of the strain, a lysate of the lysate, a supernatant of the lysate, and the Converting fructose 6-phosphate to tagatose-6-phosphate using at least one selected from the group consisting of extracts of cells, cultures, lysates, and supernatant, and removing phosphate groups from the tagatose 6-phosphate It provides a method for producing tagatose comprising the step of.
  • a further example of the present invention is a fructose 6-phosphate 4-epimerase, a microorganism expressing the enzyme, a cell of the strain, a culture of the strain, a lysate of the lysate, a supernatant of the lysate, and the cell It provides a composition for producing tagatose, comprising at least one selected from the group consisting of an extract of a culture, a lysate and a supernatant, and a dephosphorylation enzyme of tagatose 6-phosphate.
  • An example of the present invention relates to a fructose 6-phosphate 4-epimerase and its use, and in more detail, a fructose 6-phosphate 4-epimerization enzyme derived from a microorganism, a microorganism expressing the enzyme protein , A nucleic acid molecule encoding the enzyme protein, a recombinant vector and a transformed microorganism comprising the nucleic acid molecule, and to the production of phosphorylated glucose using the same.
  • the tagatose 6-phosphate or tagatose production method according to the present invention is environmentally friendly because it uses microorganisms or enzymes obtained therefrom, and converts the production of tagatose from fructose into a method that has not been previously available through a simple enzymatic reaction, and production costs It can greatly reduce the production effect while maximizing the production effect.
  • An example of the present invention relates to a composition for producing tagatose 6-phosphate or a composition for producing tagatose using a microorganism-derived fructose 6-phosphate 4-epimerase or a microorganism expressing the enzyme protein.
  • An example of the present invention relates to a method for producing tagatose 6-phosphate or a method for producing tagatose using a microorganism-derived fructose 6-phosphate 4-epimerase or a microorganism expressing the enzyme protein.
  • the active ingredient of the composition is a fructose 6-phosphate 4-epimerase, a microorganism expressing the enzyme protein, a transformed microorganism expressing the enzyme protein, the microbial cells of the microbe, the microbial lysate of the microbe, It may be one or more selected from the group consisting of a culture product, a culture supernatant of the microorganism, a concentrate of the culture supernatant of the microorganism, and a powder thereof.
  • the culture contains an enzyme produced from a microorganism that produces a fructose 6-phosphate 4-epimerase, and may be in a cell-free form that includes the microorganism cells or does not contain the cells.
  • the lysate refers to a lysate obtained by crushing the microbial cells of a fructose 6-phosphate 4-epimerase-producing enzyme or a supernatant obtained by centrifuging the lysate, and the fructose 6-phosphate 4-epimerase It includes enzymes produced from microorganisms that produce them.
  • an example of the present invention is 50% or more, 60% or more, 70% or more, 80% or more, 90% or more of the amino acid sequence of SEQ ID NO: 1, 4, or 7 , To a fructose 6-phosphate 4-epimerase comprising an amino acid sequence of at least 95%, at least 97% or at least 99%.
  • an amino acid sequence showing the homology of the numerical value it may be included without limitation as long as it is a protein constituting a fructose 6-phosphate 4-epimerase that is substantially identical to or corresponding to the enzyme.
  • sequence having such homology is an amino acid sequence that substantially exhibits a fructose 6-phosphate 4-epimerase function
  • protein variants in which some sequences are deleted, modified, substituted or added are also included within the scope of the present invention.
  • the enzyme protein may be encoded by the nucleotide sequence of SEQ ID NO: 3, 6 or 9 or the nucleotide sequence of SEQ ID NO: 2, 5, or 8, or the nucleotide sequence of SEQ ID NO: 3, 6 or 9 or SEQ ID NO: 2, It is encoded by a nucleotide sequence having at least 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more sequence identity with the nucleotide sequence of 5 or 8 I can.
  • homology means the degree to which a given amino acid sequence or base sequence is matched, and may be expressed as a percentage.
  • a homologous sequence thereof having the same or similar activity as a given amino acid sequence or base sequence is expressed as "% homology", “% identity” or “% identity”.
  • a specific example of the present invention is a fructose 6-phosphate 4-epimerase protein is a fructose 6-phosphate 4-epimerase protein (CE_FP4E) derived from Caldisericum exile comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: Fructose 6-phosphate 4-epimerase protein (KO_FP4E) derived from Kosmotoga olearia comprising the amino acid sequence of 4, or fructose 6-phosphate 4-epimerase derived from Limnochorda pilosa comprising the amino acid sequence of SEQ ID NO: 7 It may be (LP_FP4E).
  • the fructose 6-phosphate 4-epimerase protein according to the present invention catalyzes the reaction of converting the fourth carbon into tagatose 6-phosphate by epimerizing the fourth carbon in the fructose 6-phosphate substrate.
  • the conversion rate of fructose 6-phosphate 4-epimerase according to an example from fructose 6-phosphate to tagatose 6-phosphate is 20% or more, 25% or more, 30% or more, or 35% or more
  • the upper limit may be 60% or less, 50% or less, or 40% or less, and for example, may have a range according to a combination of the lower limit and the upper limit of the conversion rate.
  • the fructose 6-phosphate 4-epimerase may increase its activity in the presence of a specific metal ion.
  • concentration of the metal ion is preferably 0.1 mM to 20 mM, 0.2 to 20 mM, 0.5 to 20 mM, 1 to 20 mM, 0.1 mM to 15 mM, 0.2 to 15 mM, 0.5 to 15 mM, 1 to 15 mM , 0.1 mM to 10 mM, 0.2 to 10 mM, 0.5 to 10 mM, 1 to 10 mM, 0.1 mM to 5 mM, 0.2 to 5 mM, 0.5 to 5 mM, or 1 to 5 mM.
  • CE_FP4E, KO_FP4E, and LP_FP4E are commonly increased in activity by Co, Mg, and Ni ions, the greatest decrease in activity by Fe ions, and exhibited a decrease in activity by Zn ions.
  • CE_FP4E exhibits a property of increasing activity by Ca, Co, Mg, Mn, and Ni ions, and decreasing activity by Fe and Zn ions, compared to the control to which no metal ions were added.
  • KO_FP4E exhibits a property of increasing activity by Co, Mg, Mn, and Ni ions, and decreasing activity by Ca, Fe, and Zn ions, compared to the control to which no metal ions were added.
  • LP_FP4E exhibits a property of increasing activity by Co, Mg, and Ni ions, and decreasing activity by Ca, Fe, Mn, and Zn ions, compared to the control to which no metal ions were added.
  • the fructose 6-phosphate 4-epimerase may have enzyme activity at a reaction temperature of 40 to 75°C, preferably 45 to 75°C or 50 to 75°C, and/or a reaction pH of 6.5 to 8.0. .
  • CE_FP4E is stable at 40 to 75°C and may have an activity of 75% or more of the maximum activity of the enzyme in a temperature range of 55 to 75°C, and has a maximum activity at 70°C.
  • KO_FP4E is stable at 40 to 75°C, and the activity increases with increasing temperature, and may have an activity of 75% or more of the maximum enzyme activity in a temperature range of 55 to 75°C.
  • LP_FP4E is stable at 40 to 75°C and has an activity of 50% or more compared to the maximum activity in the range of 55-80°C, or has an activity of 75% or more compared to the maximum activity in the range of 65-75°C Have.
  • the fructose 6-phosphate 4-epimerase exhibits maximum activity at pH 7.0 for CE_FP4E and at pH 7.5 for KOF6PE and LPF6PE, and 75% or more of the maximum activity in the range of pH 6.5-8.0 suitable for commercialization processes. Have activity.
  • a nucleic acid molecule encoding the fructose 6-phosphate 4-epimerase is provided.
  • a specific example of a nucleic acid encoding a fructose 6-phosphate 4-epimerase according to the present invention includes the nucleotide sequence of SEQ ID NO: 3, 6 or 9 or the nucleotide sequence of SEQ ID NO: 2, 5, or 8, or , Or at least 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more with the nucleotide sequence of SEQ ID NO: 3, 6 or 9 or the nucleotide sequence of SEQ ID NO: 2, 5, or 8 It may comprise a nucleotide sequence having at least 99% sequence identity.
  • the present invention provides a vector or transformant comprising a nucleic acid molecule encoding the fructose 6-phosphate 4-epimerase of the present application.
  • the term "transformation” means introducing a vector containing a nucleic acid encoding a target protein into a host cell so that the protein encoded by the nucleic acid can be expressed in the host cell.
  • the transformed nucleic acid can include all of them, whether inserted into the chromosome of the host cell or located outside the chromosome, as long as it can be expressed in the host cell.
  • the nucleic acid includes DNA and RNA encoding the target protein.
  • the nucleic acid may be introduced in any form as long as it can be introduced into a host cell and expressed.
  • the nucleic acid may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all elements necessary for self-expression.
  • the expression cassette may generally include a promoter operably linked to the nucleic acid, a transcription termination signal, a ribosome binding site, and a translation termination signal.
  • the expression cassette may be in the form of an expression vector capable of self-replicating.
  • the nucleic acid may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.
  • operably linked in the above means that a promoter sequence that initiates and mediates transcription of a nucleic acid encoding a protein of interest of the present application and the gene sequence are functionally linked.
  • the method of transforming the vector of the present application includes any method of introducing a nucleic acid into a cell, and may be performed by selecting an appropriate standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and Lithium acetate-DMSO method, and the like, but is not limited thereto.
  • electroporation calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and Lithium acetate-DMSO method, and the like, but is not limited thereto.
  • the host cell it is good to use a host having high DNA introduction efficiency and high expression efficiency of the introduced DNA, but may be, for example, E. coli, but is not limited thereto.
  • a further example of the present invention is a fructose 6-phosphate 4-epimerization enzyme protein, a microorganism expressing the enzyme, a microorganism of the strain, a culture of the strain, a lysate of the lysate, a supernatant of the lysate, and the It provides a composition for producing tagatose 6-phosphate comprising at least one selected from the group consisting of an extract of cells, cultures, lysates and supernatant.
  • a further example of the present invention is a fructose 6-phosphate 4-epimerase, a microorganism expressing the enzyme, a cell of the strain, a culture of the strain, a lysate of the lysate, a supernatant of the lysate, and the cell It provides a composition for producing tagatose, comprising at least one selected from the group consisting of an extract of a culture, a lysate and a supernatant, and a dephosphorylation enzyme of tagatose 6-phosphate. Tagatose produced in this way can be usefully used by being added to functional foods and pharmaceuticals.
  • the culture of the strain includes an enzyme produced from a strain that produces the fructose-6-phosphate epimerase, and may be in a cell-free form with or without the strain.
  • the lysate refers to a lysate obtained by crushing the strain producing the fructose-6-phosphate epimerase or a supernatant obtained by centrifuging the lysate, and the strain producing the fructose-6-phosphate epimerase It contains enzymes produced from.
  • the strain producing the fructose-6-phosphate epimerase used is a cell of the strain, a culture of the strain, a lysate of the lysate, a supernatant of the lysate, And it is used to mean one or more selected from the group consisting of extracts thereof.
  • reaction temperature and reaction pH conditions using the enzyme or the microorganism producing the enzyme are described above in the reaction temperature and reaction pH conditions of the enzyme. As shown.
  • the fructose-6-phosphate is preferably obtained by hexokinase treatment of fructose or a fructose-containing substance, but is also included in the case where it is provided by other chemical synthesis methods.
  • the composition for producing tagatose 6-phosphate or the composition for producing tagatose is a hexokinase enzyme that converts fructose into fructose-6-phosphate, a transforming microorganism expressing the enzyme protein, the microorganism cells, the It may include at least one selected from the group consisting of a lysate of microorganisms, a culture of the microorganisms, a culture supernatant of the microorganisms, a concentrate of the culture supernatant of the microorganisms, and powders thereof.
  • the fructose-6-phosphate may be prepared from glucose-6-phosphate, and the composition for producing ketohexose is a glucose-6-phosphate isomerase that converts glucose-6-phosphate into fructose-6-phosphate by isomerizing glucose-6-phosphate. It may further include.
  • the glucose-6-phosphate may be prepared by directly phosphorylating glucose or may be converted from glucose-1-phosphate.
  • Glucose may be glucose obtained by treating a glucose-producing amylase such as starch or a starch hydrolyzate, for example, dextrin, or the like, and glucose-1-phosphate may be obtained by treating the glucose with a phosphorylating enzyme.
  • the composition for producing ketohexose may further include an enzyme system for producing glucose-6-phosphate.
  • the enzyme contained in the composition for producing tagatose of the present invention and the substrate used for producing tagatose are not limited.
  • the composition for producing tagatose of the present invention includes (a) (i) starch, maltodextrin, sucrose or a combination thereof, glucose, glucose-1-phosphate, glucose-6-phosphate, or fructose-6-phosphate; (ii) phosphate; (iii) tagatose-6-phosphate dephosphorylation enzyme; (iv) glucose-6-phosphate-isomerase; (v) phosphoglucomutase or glucose phosphorylation enzyme; And/or (vi) ⁇ -glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase, sucrose phosphorylase, ⁇ -amylase, pullulanase, isoamylase, glucoamylase or sucrase. ; Or (b) may further include a microorganism expressing the enzyme of item (a) or
  • starch / maltodextrin phosphorylase (starch / maltodextrin phosphorylase, EC 2.4.1.1) and ⁇ -glucan phosphorylase of the present invention are glucose from starch or maltodextrin by transferring phosphate to glucose. Any protein may be included as long as it has an activity of producing -1-phosphate.
  • the sucrose phosphorylase (EC 2.4.1.7) of the present invention may include any protein as long as it has the activity of producing glucose-1-phosphate from sucrose by transferring phosphate to glucose.
  • Starch liquor enzymes of the present invention ⁇ -amylase ( ⁇ -amylase, EC 3.2.1.1), pullulanse (EC 3.2.1.41), glucoamylase (EC 3.2.1.3) and isoamylase (isoamylase) May include any protein as long as it has the activity of converting starch or maltodextrin into glucose.
  • Sucrase (EC 3.2.1.26) of the present invention may include any protein as long as it has an activity of converting sucrose into glucose.
  • the phosphoglucomutase (EC 5.4.2.2) of the present invention may include any protein as long as it has an activity of converting glucose-1-phosphate into glucose-6-phosphate.
  • Glucokinase may include any protein as long as it has the activity of converting phosphate to glucose and converting it into glucose-6-phosphate.
  • the glucose kinase may be a polyphosphate-dependent glucose kinase.
  • Glucose-6-phosphate isomerase may include any protein as long as it has an activity to convert glucose-6-phosphate into fructose-6-phosphate.
  • composition for producing tagatose 6-phosphate or composition for producing tagatose is phytase performing a dephosphorylation reaction from tagatose-6-phosphate, or tagatose-6-phosphate dephosphorylation enzyme (tagatose- 6-phosphate phosphatase) may be additionally included, but is not limited thereto.
  • the tagatose-6-phosphate dephosphorylation enzyme usable in the present invention may include any protein as long as it has an activity of converting tagatose-6-phosphate to tagatose. More specifically, the tagatose-6-phosphate dephosphorylation enzyme may be a protein having an activity of irreversibly converting tagatose-6-phosphate into tagatose.
  • the composition for producing tagatose 6-phosphate or the composition for producing tagatose expresses tagatose-6-phosphate phosphatase, and the tagatose-6-phosphate dephosphorylation enzyme. It may further include a culture of microorganisms.
  • a further example of the present invention is a fructose 6-phosphate 4-epimerization enzyme protein, a microorganism expressing the enzyme, a microorganism of the strain, a culture of the strain, a lysate of the lysate, a supernatant of the lysate, and the A method for producing tagatose-6-phosphate comprising converting fructose 6-phosphate to tagatose 6-phosphate using at least one selected from the group consisting of extracts of cells, cultures, lysates, and supernatant. to provide.
  • a further example of the present invention is a fructose-6-phosphate 4-epimerase protein, a microorganism expressing the enzyme, a microorganism of the strain, a culture of the strain, a lysate of the lysate, a supernatant of the lysate, and Converting fructose-6-phosphate to tagatose-6-phosphate using at least one selected from the group consisting of extracts of the cells, cultures, lysates, and supernatant, and in the tagatose-6-phosphate It provides a method for producing tagatose, comprising the step of removing a phosphate group.
  • the method may further comprise the step of preparing fructose-6-phosphate from fructose or a fructose-containing material using hexokinase, and/or expressing the enzyme. It may further include the step of preparing fructose-6-phosphate by contacting the microorganism or the culture of the microorganism.
  • the manufacturing method of the present invention is before the step of converting the fructose-6-phosphate to tagatose-6-phosphate, glucose-6-phosphate to glucose-6-phosphate-isomerase, the glucose-6-phosphate-isomerization It may further include the step of converting the glucose-6-phosphate into fructose-6-phosphate by contacting a culture of the enzyme-expressing microorganism or the glucose-6-phosphate-isomerase-expressing microorganism.
  • the manufacturing method of the present invention prior to the step of converting the glucose-6-phosphate to fructose-6-phosphate, glucose-1-phosphate (Glucose-1-phosphate) phosphoglucomutase, the phosphoglucomuta It may further include the step of converting the glucose-1-phosphate into glucose-6-phosphate by contacting the culture of the enzyme-expressing microorganism or the phosphoglucomutase-expressing microorganism.
  • the manufacturing method of the present invention comprises a glucose phosphorylating enzyme in glucose, a microorganism expressing the glucose phosphorylating enzyme, or a microorganism expressing the glucose phosphorylating enzyme before the step of converting the glucose-6-phosphate into fructose-6-phosphate. It may further include the step of converting the glucose into glucose-6-phosphate by contacting the culture of the phosphate and phosphate.
  • the production method of the present invention is prior to the step of converting glucose-1-phosphate of the present invention to glucose-6-phosphate, starch, maltodextrin, sucrose, or a combination thereof, ⁇ -glucan phosphorylase, starch phosphorylase , Maltodextrin phosphorylase or sucrose phosphorylase; Microorganisms expressing the phosphorylase; Or it may further include the step of converting the starch, maltodextrin, sucrose, or a combination thereof to glucose-1-phosphate by contacting the culture of the microorganism expressing the phosphorylase and phosphate.
  • the production method of the present invention is prior to the step of converting glucose of the present invention into glucose-6-phosphate, starch, maltodextrin, sucrose, or a combination thereof with ⁇ -amylase, pullulanase, glucoamylase, sucrase or isoamylase. ; Microorganisms expressing the amylase, fluranase, or sucrase; Or it may further include the step of converting the starch, maltodextrin, sucrose, or a combination thereof into glucose by contacting the culture of the microorganism with the amylase, fluranase or sucrase.
  • the production method of the present invention is a culture of a microorganism expressing 4- ⁇ -glucanotransferase, the 4- ⁇ -glucanotransferase, or a microorganism expressing the 4- ⁇ -glucanotransferase in glucose
  • the step of converting the glucose into starch, maltodextrin or sucrose may additionally be included.
  • the step of removing the phosphate from the tagatose-6-phosphate may include a tagatose-6-phosphate phosphatase, a microorganism expressing the tagatose-6-phosphate dephosphorylation enzyme, or the tagatose.
  • Tagatose can be prepared by carrying out a culture of microorganisms expressing tos-6-phosphate dephosphorylation enzyme.
  • a step of converting the tagatose-6-phosphate to tagatose is performed by contacting the tagatose-6-phosphate with a phytase, a microorganism expressing it, or a culture of the microorganism.
  • tagatose can be produced.
  • the step of converting the tagatose 6-phosphate may be performed at a reaction temperature of 40 to 75°C and a reaction pH of 6.5 to 8.0, and the reaction temperature is 40 to 75°C, 45 To 75°C, or 50 to 75°C.
  • the step of converting to tagatose 6-phosphate may be performed in the presence of one or more metal ions selected from the group consisting of Co, Mg, and Ni ions.
  • the method of removing the phosphate further comprises converting tagatose into tagatose by reacting phytase or a dephosphorylating enzyme of tagatose-6-phosphate on tagatose 6-phosphate.
  • the tagatose 6-phosphate may be removed by other enzymes or chemical methods.
  • the fructose 6-phosphate 4-epimerase of the present invention has stability at pH and temperature in an industrially useful range, and can produce tagatose in high yield using economical raw materials such as starch or maltodextrin, It can be widely used in sugar-related health food and pharmaceutical industries.
  • 1 is an HPLC analysis result confirming the generation of tagatose for the three enzymes CE_FP4E, KO_FP4E, and LP_FP4E according to an example of the present invention.
  • FIG. 2 is a graph showing the relative activity of tagatose production of CE_FP4E enzyme according to a reaction temperature change according to an example of the present invention.
  • FIG. 3 is a graph showing the relative activity of tagatose production of KO_FP4E enzyme according to a reaction temperature change according to an example of the present invention.
  • FIG. 4 is a graph showing the relative activity of tagatose production of LP_FP4E enzyme according to a reaction temperature change according to an example of the present invention.
  • FIG. 5 is a graph showing the relative activity of tagatose production of CE_FP4E enzyme according to a change in reaction pH conditions according to an example of the present invention.
  • FIG. 6 is a graph showing the relative activity of tagatose production of KO_FP4E enzyme according to a change in reaction pH conditions according to an example of the present invention.
  • FIG. 7 is a graph showing the relative activity of tagatose production of LP_FP4E enzyme according to a change in reaction pH conditions according to an example of the present invention.
  • FIG. 8 is a graph showing the relative activity of the CE_FP4E enzyme to produce tagatose according to the presence conditions of various metal ions according to an example of the present invention.
  • FIG. 9 is a graph showing the relative activity of the KO_FP4E enzyme to produce tagatose according to the presence conditions of various metal ions according to an example of the present invention.
  • FIG 10 is a graph showing the relative activity of the LP_FP4E enzyme to produce tagatose according to conditions in which various metal ions exist according to an example of the present invention.
  • 11 is an HPLC for confirming whether tagatose is produced for two enzymes including an enzyme of D-tagatose bisphosphate aldolase derived from Rubrobacter indicoceani (RI) and Fructose 1,6-bisphosphate aldolase derived from Lachnospiraceae bacterium (LB). This is the result of the analysis.
  • the synthesized polynucleotide was inserted into the pET-28a vector using restriction enzymes NheI and HindIII (NEB), and transformed into an E. coli ER2566 strain to produce a recombinant expression vector.
  • NheI and HindIII NEB
  • the recovered cells were turbid in lysis buffer (300 mM NaCl, 10 mM imidazole, 50 mM Tris-HCl, pH 8.0), and then 20 at 4°C using a sonic vibrator (Ultrasonic prosessor, ColeParmer). Crushed for minutes. The supernatant was collected by centrifugation at 13,000 rpm for 20 minutes, passed through a Ni-NTA column (Ni-NTA Superflow, Qiagen) equilibrated with a lysis buffer, and then 50 mM Tris-containing 300 mM NaCl and 20 mM immidazole. The target protein was eluted by sequentially flowing 50 mM Tris-HCl (pH 8.0) buffer solution containing HCl (pH 8.0), 300 mM NaCl, and 200 mM immidazole.
  • lysis buffer 300 mM NaCl, 10 mM imidazole, 50 mM Tris-HCl, pH 8.0
  • Example 1 In order to analyze the conversion activity of fructose 6-phosphate to tagatose 6-phosphate, the enzyme purified in Example 1 was used as 20 mM fructose 6-phosphate and 50 mM sodium phosphate (pH 7.0) After addition to the buffer solution, the reaction was carried out at 60°C for 1 hour, and the enzyme activity was stopped by heating at 100°C for 5 minutes.
  • Tagatose was prepared by treating the enzyme reaction solution with phytase to remove a phosphate group from the resulting tagatose 6-phosphate.
  • the reaction product solution was measured as the amount of tagatose produced using High-Performance Liquid Chromatography (HPLC).
  • HPLC analysis conditions are water, temperature 80 °C, flow rate 0.6 mL/min using RID (Refractive Index Detector, Agilent 1260 RID) of HPLC (Agilent, USA) equipped with SUGAR SP0810 column (Shodex). Performed.
  • RID Refractive Index Detector, Agilent 1260 RID
  • HPLC HPLC
  • FIG. 1 tagatose was generated for three enzymes: an enzyme derived from Caldisericum exile (hereinafter, CE_FP4E), an enzyme derived from Kosmotoga olearia (hereinafter, KO_FP4E), and an enzyme derived from Limnochorda pilosa (hereinafter, LP_FP4E).
  • CE_FP4E Caldisericum exile
  • KO_FP4E Kosmotoga olearia
  • LP_FP4E an enzyme derived from Limnochorda pilosa
  • the three enzymes each have an amino acid sequence (SEQ ID NO: 1, 4, 7) derived from Caldisericum exile, Kosmotoga olearia and Limnochorda pilosa and a polynucleotide encoding the protein CDS (SEQ ID NO: 2, 5, 8),
  • SEQ ID NO: 1, 4, 7 derived from Caldisericum exile, Kosmotoga olearia and Limnochorda pilosa
  • a polynucleotide encoding the protein CDS SEQ ID NO: 2, 5, 8
  • the codon usage of E. coli strain K-12 was requested by gene synthesis, and the polynucleotides used have SEQ ID NOs: 3, 6, and 9, respectively.
  • Example 1 To confirm the change in enzyme activity according to the reaction temperature change, the enzyme purified in Example 1 was added to a 50 mM sodium phosphate (pH 7.0) buffer solution containing 20 mM fructose 6-phosphate and 5 mM MgCl 2 Then, the enzyme reaction was carried out for 1 hour at 50-80°C, and the enzyme activity was stopped by heating at 100°C for 5 minutes.
  • a 50 mM sodium phosphate (pH 7.0) buffer solution containing 20 mM fructose 6-phosphate and 5 mM MgCl 2
  • the enzyme reaction was carried out for 1 hour at 50-80°C, and the enzyme activity was stopped by heating at 100°C for 5 minutes.
  • Tagatose was prepared by treating the enzyme reaction solution with phytase to remove a phosphate group from the resulting tagatose 6-phosphate.
  • the reaction product solution was measured as the amount of tagatose produced using High-Performance Liquid Chromatography (HPLC).
  • HPLC High-Performance Liquid Chromatography
  • CE_FP4E showed maximum activity at 70° C.
  • the activity of KO-FP4E increased with increasing temperature.
  • Both CE_FP4E and KO-FP4E showed more than 75% activity compared to the maximum activity in a wide range of 55-75°C.
  • LP-FP4E showed maximum activity at 65°C, showing 50% or more of the maximum activity in the range of 55-80°C, and 75% or more compared to the maximum activity in the range of 65-75°C. Showed activity.
  • Example 2 In order to confirm the change in enzyme activity according to the reaction pH change, the enzyme purified in Example 2 was used in a 50 mM buffer solution (pH 6.0, Sodium citrate buffer; pH 6.5-8.5, Tris-HCl buffer; pH 8.5-9.0, at various pHs). Glycine-NaOH buffer) was added with 20 mM fructose 6-phosphate and 5 mM MgCl 2 , followed by reaction at 60° C. for 1 hour, and heating at 100° C. for 5 minutes to stop the enzyme activity. Thereafter, tagatose was quantitatively analyzed as in Example 3. For the three enzymes CE_FP4E, KO_FP4E, and LP_FP4E, the relative activities of tagatose production according to the reaction temperature are shown in Figs.
  • CE_FP4E showed maximum activity at pH 7.0, and KOF6PE and LPF6PE at pH 7.5, and all three types were 75% or more compared to the maximum activity in the range of pH 6.5-8.0 suitable for commercialization processes. Showed activity.
  • Example 5 Analysis of enzyme activity according to metal ions
  • Example 2 In order to confirm the change in enzyme activity according to the type of metal ion, the enzyme purified in Example 2 was treated with 1 mM of CaCl 2 , CoCl 2 , FeSO 4 , MgCl 2 , MnCl 2 , NiSO 4 , and ZnSO 4 respectively, and 20 mM After the addition of a buffer solution of 6-phosphate and 50 mM sodium phosphate buffer (pH 7.0), the reaction was performed for 1 hour at 60°C. It was heated at 100° C. for 5 minutes to stop the enzyme reaction. Thereafter, tagatose was quantitatively analyzed as in Example 3. For the three enzymes CE_FP4E, KO_FP4E, and LP_FP4E, the relative activities of tagatose production according to the type of metal ion are shown in Figs. 8, 9, and 10, respectively.
  • the three enzymes CE_FP4E, KO_FP4E, and LP_FP4E increase their activity by Co, Mg and Ni ions in common, and the decrease in activity by Fe ions is the greatest, It shows the property of reducing activity by Zn ions.
  • CE_FP4E compared to the control group to which no metal ions were added, the activity of CE_FP4E increased by Ca, Co, Mg, Mn and Ni ions, and decreased by Fe and Zn ions. It shows the characteristic that it does.
  • KO_FP4E has a property of increasing activity by Co, Mg, Mn, and Ni ions, and decreasing activity by Ca, Fe, and Zn ions, compared to the control without the addition of metal ions.
  • LP_FP4E increases the activity by Co, Mg, and Ni ions, and decreases the activity by Ca, Fe, Mn, and Zn ions, compared to the control without the addition of metal ions. Represents.
  • D-tagatose bisphosphate aldolase (Genbank accession no. WP_119069933) derived from Rubrobacter indicoceani (RI), Fructose 1,6-bisphosphate aldolase derived from Lachnospiraceae bacterium (LB), class II (Genbank accession no. HCT64551 enzyme)
  • RI Rubrobacter indicoceani
  • LB Lachnospiraceae bacterium
  • HCT64551 enzyme The synthesized polynucleotide sequences of the D-tagatose bisphosphate aldolase (RI), Fructose 1,6-bisphosphate aldolase (LB), and the synthesized polynucleotide sequences are shown in SEQ ID NO 10 or SEQ ID NO 11, respectively.
  • the polynucleotide sequence of the D-tagatose bisphosphate aldolase (RI) was compared with the KO_FP4E polynucleotide sequence of SEQ ID NO: 6 or the polynucleotide sequence of LP_FP4E of SEQ ID NO: 9, respectively. As a result, the polynucleotide sequence identity was 56.47% or 53.12. %.
  • the amino acid sequence of the D-tagatose bisphosphate aldolase (RI) was compared with the amino acid sequence of KO_FP4E of SEQ ID NO: 4 or LP_FP4E of SEQ ID NO: 7, respectively, and the amino acid sequence identity was 48.64 and 47.87%.
  • Example 1 Based on the obtained polynucleotide sequences of SEQ ID NOs: 10 and 11, a large amount of genes were obtained in substantially the same manner as in Example 1. In substantially the same manner as in Example 1, the obtained gene was introduced into E. coli and expressed, and then the target protein was eluted. The finally obtained protein was converted to 50mM sodium phosphate buffer (pH 7.0) and stored for later use.
  • Example 2 In order to analyze the conversion activity from fructose 6-phosphate to tagatose 6-phosphate, in the same manner as in Example 2, an enzymatic reaction using fructose-6-phosphate as a substrate was performed using the obtained enzyme. I did. Tagatose was prepared by treating the enzyme reaction solution with a phosphatase to remove a phosphate group from the resulting tagatose 6-phosphate. The reaction product solution was measured as the amount of tagatose produced using High-Performance Liquid Chromatography (HPLC). HPLC analysis conditions were performed in substantially the same manner as in Example 2. The HPLC analysis conditions are shown in FIG. 11.

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Abstract

La présente invention concerne une fructose 6-phosphate 4-épimérase ayant une activité à un pH et à des températures situés dans des plages industriellement utiles et capable de produire du cétohexose avec un rendement élevé à l'aide d'une matière première économique telle que de l'amidon ou de la maltodextrine, une composition pour la production de cétohexose, et un procédé de production de cétohexose l'utilisant.
PCT/KR2020/014879 2019-10-31 2020-10-29 Fructose 6-phosphate 4-épimérase et son utilisation Ceased WO2021086035A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101480422B1 (ko) * 2013-07-29 2015-01-13 건국대학교 산학협력단 효소조합 반응에 의한 과당으로부터 타가토스 생산 방법 및 그 조성물
KR101610911B1 (ko) * 2013-05-09 2016-04-08 주식회사 삼양사 L-리불로스 5-인산 4-에피머화 효소를 이용한 과당에서 타가토스 생산
KR20180111666A (ko) * 2017-03-31 2018-10-11 씨제이제일제당 (주) 타가토스 생산용 조성물 및 이를 이용한 타가토스 제조방법
US20190017083A1 (en) * 2015-10-02 2019-01-17 Bonumose Llc Enzymatic production of d-tagatose

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101610911B1 (ko) * 2013-05-09 2016-04-08 주식회사 삼양사 L-리불로스 5-인산 4-에피머화 효소를 이용한 과당에서 타가토스 생산
KR101480422B1 (ko) * 2013-07-29 2015-01-13 건국대학교 산학협력단 효소조합 반응에 의한 과당으로부터 타가토스 생산 방법 및 그 조성물
US20190017083A1 (en) * 2015-10-02 2019-01-17 Bonumose Llc Enzymatic production of d-tagatose
KR20180111666A (ko) * 2017-03-31 2018-10-11 씨제이제일제당 (주) 타가토스 생산용 조성물 및 이를 이용한 타가토스 제조방법

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Title
DATABASE Protein GenPept; ANONYMOUS: "tagatose-bisphosphate aldolase [Limnochorda pilosa]", XP055806713, retrieved from NCBI *

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