WO2021086080A1 - Procédé de production d'oligosaccharides contenant du maltotétraose - Google Patents

Procédé de production d'oligosaccharides contenant du maltotétraose Download PDF

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WO2021086080A1
WO2021086080A1 PCT/KR2020/014954 KR2020014954W WO2021086080A1 WO 2021086080 A1 WO2021086080 A1 WO 2021086080A1 KR 2020014954 W KR2020014954 W KR 2020014954W WO 2021086080 A1 WO2021086080 A1 WO 2021086080A1
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maltotetraose
weight
enzyme
seq
strain
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Korean (ko)
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안신혜
박부수
김진하
이상희
박종진
최은수
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주식회사 삼양사
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas

Definitions

  • the present invention relates to a method for producing maltotetraose-containing saccharides, wherein the saccharides contain maltotetraose in a specific amount, and more specifically, maltotetraose-converting amylase enzyme or a microorganism producing the enzyme is used. It relates to a method for producing maltotetraose-containing saccharides containing terraose in a specific amount.
  • Maltooligosaccharide is a functional sugar used in the food technology field because it has properties such as providing a soft texture and masking function of food.
  • Maltooligosaccharides include maltose (G2, maltose), maltotriose (G3, maltotriose), maltotetraose (G4, maltotetraose) and maltopentaose (G5), maltohexaose (G6, maltohexaose), and maltoheptaose ( G7, maltoheptaose), maltooctaose (G8, maltooctaose), maltononaose (G9, maltononaose) and the like.
  • Maltotetraose (G4) is a substance composed of ⁇ -1,4 bonds of 4 glucose, and when added to a product, it plays a role in giving a soft body feeling, and is a sugar with less coloring and browning than general starch syrup. In addition, it improves the luster and moisturizing power of the product, has excellent inhibitory effect on starch and protein denaturation, and has prebiotic properties. Therefore, studies to prepare maltotetraose-containing saccharides containing oligosaccharides that improve hygroscopicity, quality and storage stability by controlling the maltotetraose content in maltooligosaccharides, have good flavor expression, and give a soft body feeling are continuously required. Has become.
  • a maltotetraose syrup having a maltotetraose content of 30% by weight or more may be prepared using starch, dextrin, etc., and a monosaccharide and disaccharide content of 10% or less.
  • An example of the present invention is to provide a method for producing maltotetraose-containing saccharides comprising 30 to 60% by weight of maltotetraose, based on 100% by weight of the solid content of the saccharide.
  • Another example of the present invention is to provide a composition for producing maltotetraose-containing saccharides containing 30 to 60% by weight of maltotetraose, based on 100% by weight of the solid content of the saccharide.
  • Another example of the present invention is to provide an enzyme for producing maltotetraose-containing saccharides containing 30 to 60% by weight of maltotetraose based on 100% by weight of the solid content of the saccharide using a polysaccharide as a substrate.
  • Pseudomonas stutzeri having the activity of converting maltotetraose from polysaccharides so that the maltotetraose content is 30 to 60% by weight based on 100% by weight of the solid content of the saccharide.
  • Pseudomonas stutzeri having the activity of converting maltotetraose from polysaccharides so that the maltotetraose content is 30 to 60% by weight based on 100% by weight of the solid content of the saccharide.
  • An example of the present invention is a maltotetraose-producing amylase enzyme, a microorganism that produces a maltotetraose-producing amylase enzyme, a microbial cell of the microbe, a microbial lysate of the microbe, a culture of the microbe, a culture supernatant of the microbe, and culture It relates to a method for producing maltotetraose-containing saccharides, comprising the step of producing maltotetraose-containing saccharides using at least one selected from the group consisting of a concentrate of a supernatant and powders thereof.
  • a further example of the present invention is a maltotetraose-producing amylase enzyme, a microorganism that produces a maltotetraose-producing amylase enzyme, a microbial cell of the microbe, a microbial lysate of the microbe, a culture of the microbe, a culture supernatant of the microbe, and It relates to a composition for producing maltotetraose-containing saccharides comprising at least one selected from the group consisting of a concentrate of a culture supernatant and a powder thereof.
  • maltotetraose-containing saccharide is “malto-oligosaccharide-containing saccharide”, and may be a malto-oligosaccharide-containing saccharide containing a specific amount of maltotetraose.
  • the "maltooligosaccharide” may be included as a saccharide containing maltooligosaccharide, and the saccharide containing maltooligosaccharide has a sum of G3 to G7 of 40% by weight or more, 50% by weight or more, and 60% by weight or more, based on the saccharide solid content, It may mean a maltooligosaccharide containing saccharide of 70% by weight or more, 80% by weight, 90% by weight or more, 95% by weight or more, 99% by weight or more, or 100% by weight, but is not limited thereto.
  • the maltooligosaccharide-containing saccharide may be a syrup containing maltooligosaccharide.
  • the maltooligosaccharide-containing saccharide may include monosaccharides and disaccharides, saccharides of G3 to G7, and saccharides of G8 or more.
  • malto-oligosaccharides are maltotriose (G3, maltoriose), maltotetraose (G4, maltotetraose), maltopentaose (G5, maltopentaose), maltohexaose (G6, maltohexaose), maltoheptaose (G7, maltoheptaose).
  • % The sum of such as 40% by weight or more, 50% by weight or more, 60% by weight or more, 70% by weight or more, 80% by weight or more, 90% by weight or more, 95% by weight or more, 99% by weight or more, or 100% by weight compared to 1 g of syrup It is referred to as %.
  • the malto-oligosaccharide-containing saccharide may be a maltotetraose (G4)-containing saccharide, specifically a maltotetraose-containing saccharide containing maltotetraose (G4) in a specific content or more, for example, maltotetraose ( G4) may mean a saccharide containing 30 to 60% by weight, but is not limited thereto.
  • the maltotetraose-containing saccharide contains maltotetraose (G4) in 30 to 60% by weight, 30 to 55% by weight, 30 to 54% by weight, 30 based on 100% by weight of the solid content of the saccharide.
  • the maltotetraose-containing saccharide has an upper limit of 10% by weight or less, 9% by weight or less, 8% by weight or less, or 7% by weight, based on 100% by weight of the solid content of the maltotetraose-containing saccharide.
  • the lower limit of the monosaccharide and disaccharide content may be 1% by weight or more, 2% by weight or more, 3% by weight or more, 4% by weight or more, 5% by weight or more, 5.5% by weight or more, 6% by weight or more, or 6.5% by weight. It may be more than that, and the monosaccharide and disaccharide content of the maltotetraose-containing saccharide may be set by a combination of the upper limit value and the lower limit value.
  • the maltotetraose-containing saccharide contains maltotetraose in an amount of 30 to 60% by weight, based on 100% by weight of the solid content of the maltotetraose-containing saccharide, and contains 25 to 55% by weight of a saccharide having a DP of 10 or more, , The content of the remaining saccharides may be 15 to 45% by weight.
  • the maltotetraose-containing saccharide may be a maltotetraose-containing liquid or powder, and may be, for example, a syrup.
  • the method for producing maltotetraose-containing saccharides is 10 to 50% by weight, 10 to 40% by weight, 10 to 35% by weight, 20 to 50% by weight, 20 to 40% by weight, 20 to 35
  • a starch raw material starch solution
  • maltotetraose-producing amylase enzyme is added to 1 to 20 units per 1 g of starch raw material solid content, 1 to 15
  • it may be to react at a reaction temperature of 40 to 70°C, 50 to 70°C, 55 to 70°C, 55 to 65°C, or 55 to 60°C.
  • the substrate used in the step of producing maltotetraose-containing saccharides according to the present invention may be at least one selected from the group consisting of starch raw materials and hydrolyzates thereof.
  • the starch raw material may be one or more selected from the group consisting of potato starch, sweet potato starch, corn starch, wheat starch, tapioca starch, rice starch, amylose, and amylopectin.
  • the decomposition product of the starchy raw material may be at least one selected from the group consisting of soluble starch, dextrin, amylodextrin, erythrodextrin, acrodextrin, and maltodextrin.
  • starch-degrading enzymes such as cyclodaxtrin glucanotransferase (EC 2.4.1.19), ⁇ -amylase (EC 3.2.1.1), ⁇ -amylase (EC 3.2.1.2), glucose Maltotetraose as a product is produced by using in combination with amylase (EC 3.2.1.3), ⁇ -glucosidase (EC 3.2.1.20), pullulanase (EC 3.2.1.41), or isoamylase (EC 3.2.1.68), and the like. You can change the composition of the artifact or increase the maltotetraose content.
  • the content of maltotetraose high content is reduced to 60 to 80 w/w%. Can be increased.
  • a starch deberanching enzyme such as pullulanase or isoamylase
  • the maltotetraose-containing saccharide according to an embodiment of the present invention may be prepared using a microorganism that produces an amylase enzyme having maltotetraose conversion activity or an enzyme having maltotetraose conversion activity.
  • the microorganism producing the maltotetraose-producing amylase enzyme according to an embodiment of the present invention may be a mutant strain obtained by improving the wild-type strain.
  • the microorganism producing the maltotetraose-producing amylase enzyme may be a mutant strain obtained by improving the wild-type Pseudomonas stutzeri strain.
  • the strain producing the maltotetraose-producing amylase enzyme may be a Pseudomonas stutzeri SYC-G4 strain having accession number KCCM12578P.
  • the mutant strain improved from the wild-type Pseudomonas stutzeri strain is an enzyme having the same or different sequence as the wild-type maltotetraose-producing amylase enzyme (SEQ ID NO: 3) derived from the wild-type Pseudomonas stutzeri strain.
  • amino acid sequence of SEQ ID NO: 3 the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 9, the amino acid sequence of SEQ ID NO: 11, and the amino acid sequence of SEQ ID NO: 13 It is possible to produce an enzyme comprising one or more amino acid sequences selected from the group consisting of, or consisting of one or more amino acid sequences selected from the group.
  • the mutant strain increases the expression level of the enzyme when culturing the strain due to mutagenesis, so that the cells of the mutant strain, the cell lysate of the strain, Enzyme titer per unit volume (ml) of the culture of the strain, the culture supernatant of the strain, the concentrate of the culture supernatant of the strain, etc. , It may be an increase in enzyme titer per unit volume (ml) of the culture supernatant of the wild-type strain and the concentrate of the culture supernatant of the wild-type strain.
  • Maltotetraose-producing amylase enzyme may have an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13.
  • the maltotetraose-producing amylase enzyme activity, maltotetraose-producing amylase enzyme activity, or maltotetraose conversion activity is to produce maltooligosaccharide using starch or a hydrolyzate thereof as a substrate, and the maltotetraose contained in the maltotetraose
  • the content may be active having a value of 30 to 60% by weight.
  • Maltotetraose-producing amylase enzyme is 2 times or more, 3 times or more, 5 times or more, 10 times or more, 15 compared to maltotetraose-producing amylase enzyme derived from wild-type Pseudomonas stutzeri strain. It may have a maltotetraose production activity of at least 20 times, at least 25 times, at least 30 times, at least 31 times, or at least 32 times.
  • Maltotetraose-producing amylase enzyme is 20 to 600 U/ml, 30 to 600 U/ml, 35 to 600 U/ml, 40 to 600 U/ml, 50 to 600 U/ml, 100 to 600 U/ml, 150 to 600 U/ml, 200 to 600 U/ml, 300 to 600 U/ml, 350 to 600 U/ml, 380 to 600 U/ml, 390 to 600 U/ml, 400 to 600 U/ml, 430 to 600 U/ml, 450 to 600 U/ml, 20 to 550 U/ml, 30 to 550 U/ml, 35 to 550 U/ml, 40 to 550 U/ml, 50 to 550 U /ml, 100-550 U/ml, 150-550 U/ml, 200-550 U/ml, 300-550 U/ml, 350-550 U/ml, 380-550 U/ml, 390-550 U/ ml, 400 to 550
  • the maltotetraose-producing amylase enzyme may have a stable maltotetraose conversion activity of the enzyme in a specific pH range, and for example, the maltotetraose conversion activity may be stable in a pH range of 5 to 10, 5 To 9.5, 5 to 9, 5 to 8.5, 5 to 8, 5 to 7.9, 5 to 7.8, 5 to 7.7, 5 to 7.6, 5 to 7.5, 5.5 to 10, 5.5 to 9.5, 5.5 to 9, 5.5 to 8.5 , 5.5 to 8, 5.5 to 7.9, 5.5 to 7.8, 5.5 to 7.7, 5.5 to 7.6, 5.5 to 7.5, 6 to 10, 6 to 9.5, 6 to 9, 6 to 8.5, 6 to 8, 6 to 7.9, 6 To 7.8, 6 to 7.7, 6 to 7.6, 6 to 7.5, 6.5 to 10, 6.5 to 9.5, 6.5 to 9, 6.5 to 8.5, 6.5 to 8, 6.5 to 7.9, 6.5 to 7.8, 6.5 to 7.7, 6.5 to 7.6
  • the maltotetraose-producing amylase enzyme has a maltotetraose (G4) conversion rate at an enzyme reaction pH of 7.5, greater than 1 to 1.5 times, and more than 1 to 1.4 a conversion rate of maltotetraose (G4) at an enzyme reaction pH of 5.8. Times, more than 1 times to 1.3 times, more than 1 times to 1.2 times, more than 1 times to 1.1 times, 1.01 to 1.5 times, 1.01 to 1.4 times, 1.01 to 1.3 times, 1.01 to 1.2 times, 1.01 to 1.1 times, 1.05 to It may be 1.5 times, 1.05 to 1.4 times, 1.05 to 1.3 times, 1.05 to 1.2 times, or 1.05 to 1.1 times (FIG. 5).
  • the maltotetraose-producing amylase enzyme may have a stable maltotetraose conversion activity of the enzyme in a specific temperature range, for example, the maltotetraose conversion activity is stable in a temperature range of 40 to 70°C, 40 to 65°C, 40 to 64°C, 40 to 63°C, 40 to 62°C, 40 to 61°C, 40 to 60°C, 45 to 70°C, 45 to 65°C, 45 to 64°C, 45 to 63°C, 45 to 62°C, 45 to 61°C, 45 to 60°C, 50 to 70°C, 50 to 65°C, 50 to 64°C, 50 to 63°C, 50 to 62°C, 50 to 61°C, 50 to 60°C, 55 to 70°C, 55 to 65°C, 55 to 64°C, 55 to 63°C, 55 to 62°C, 55 to 61°C, 55 to 60°C, 56 to 70°C
  • the maltotetraose-producing amylase enzyme has a maltotetraose (G4) conversion rate at an enzyme reaction temperature of 60° C., greater than 1 to 1.5 times, or more than 1 times the conversion rate of maltotetraose (G4) at an enzyme reaction temperature of 50° C.
  • G4 maltotetraose
  • the maltotetraose-producing amylase enzyme may be a cell culture solution, a supernatant, an isolated and purified enzyme, or a powdered enzyme.
  • the maltotetraose-producing amylase enzyme according to an embodiment of the present invention may be a powdered enzyme.
  • the maltotetraose-producing amylase enzyme according to an embodiment of the present invention may be a powdered enzyme, and the enzyme may have increased activity by powdering.
  • the maltotetraose-producing amylase enzyme is more than 1 times, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, It may be increased by 9 times or more, or 10 times or more, and in this case, the upper limit of the increase rate of the activity by powdering may be 20 times or less, 15 times or less, 12 times or less, or 11 times or less.
  • Maltotetraose-producing amylase enzyme according to an embodiment of the present invention may be characterized in that the activity after storage at a specific temperature range for a certain period of time is similar to the initial activity, or is increased than the initial activity, so that long-term storage is possible.
  • the maltotetraose-producing amylase enzyme according to an embodiment of the present invention has a relative activity after storage for a certain time in a specific temperature range, 60% or more, 65% or more, 70% or more, 75% or more of the initial activity.
  • the maltotetraose-producing amylase enzyme may have an activity higher than that of the initial enzyme after being stored for a certain time in a specific temperature range.
  • the specific temperature range is 0 to 50°C, 0 to 45°C, 0 to 40°C, 0 to 35°C, 5 to 50°C, 5 to 45°C, 5 to 40°C, 5 to 35°C, 8 to 50°C, It may be in a temperature range of 8 to 45°C, 8 to 40°C, or 8 to 35°C.
  • the specified time period is 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 5 weeks or more, 6 weeks or more, 7 weeks or more, 8 weeks or more, 9 weeks or more, 10 weeks or more, 11 weeks or more, 12 weeks It can be more than, 13 weeks or more, 14 weeks or more, 15 weeks or more, 16 weeks or more, 17 weeks or more, 18 weeks or more, 19 weeks or more, or 20 weeks or more.
  • the upper limit of the storage period is 100 weeks or less, 80 weeks It may be 60 weeks or less, 52 weeks or less, 50 weeks or less, 40 weeks or less, 30 weeks or less, 25 weeks or less, or 20 weeks or less.
  • Maltotetraose-producing amylase enzyme is the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 9, the amino acid sequence of SEQ ID NO: 11, and It may contain or have one or more selected from the group consisting of the amino acid sequence of SEQ ID NO: 13.
  • the maltotetraose-producing amylase enzyme is the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 9, the amino acid sequence of SEQ ID NO: 11, or SEQ ID NO: 13 It may be a polypeptide consisting of the amino acid sequence of.
  • the maltotetraose-producing amylase enzyme may be encoded by the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12.
  • Another example of the present invention provides a Pseudomonas stutzeri strain having an activity of converting maltotetraose from a polysaccharide, wherein the strain is an enzyme reactant (maltotetraose-containing saccharide) with a solid content of 100 weight.
  • the maltotetraose content is 30 to 60% by weight, 30 to 55% by weight, 30 to 54% by weight, 30 to 53% by weight, 30 to 52% by weight, 30 to 51% by weight, 35 to 60% by weight %, 35 to 55% by weight, 35 to 54% by weight, 35 to 53% by weight, 35 to 52% by weight, 35 to 51% by weight, 40 to 60% by weight, 40 to 55% by weight, 40 to 54% by weight, 40 to 53% by weight, 40 to 52% by weight, 40 to 51% by weight, 45 to 60% by weight, 45 to 55% by weight, 45 to 54% by weight, 45 to 53% by weight, 45 to 52% by weight, 45 to 51% by weight, 48 to 60% by weight, 48 to 55% by weight, 48 to 54% by weight, 48 to 53% by weight, 48 to 52% by weight, 48 to 51% by weight, 48.5 to 60% by weight, 48.5 to 55% by weight %, 48.5 to 54% by weight, 48.5 to 53% by weight, 48.5 to
  • the Pseudomonas stutzeri strain has an upper limit of the content of monosaccharides and disaccharides of 10% by weight or less, 9% by weight or less, 8% by weight or less, or 7% by weight or less, based on 100% by weight of the solid content of saccharides.
  • the activity of converting maltotetraose from polysaccharides may have the activity of converting maltotetraose from polysaccharides, and the lower limit of the content of monosaccharides and disaccharides is 1% by weight or less, 2% by weight or less, 3% by weight or more, 4% by weight or more, 5% by weight or more, 5.5% by weight It may have an activity of converting maltotetraose from polysaccharides to be above, 6% by weight or more, or 6.5% by weight, and the Pseudomonas stutzeri strain is within a range set by a combination of the upper limit and the lower limit. It may have an activity of converting maltotetraose from polysaccharides so as to have monosaccharide and disaccharide content.
  • the Pseudomonas stutzeri strain according to an embodiment of the present invention may be a mutant strain improved from a wild-type Pseudomonas stutzeri strain.
  • the Pseudomonas stutzeri strain may be a Pseudomonas stutzeri SYC-G4 strain having accession number KCCM12578P.
  • Pseudomonas stutzeri strain according to an embodiment of the present invention, 40 to 70 °C, 40 to 65 °C, 40 to 64 °C, 40 to 63 °C, 40 to 62 °C, 40 to 61 °C, 40 to 60 °C , 45 to 70°C, 45 to 65°C, 45 to 64°C, 45 to 63°C, 45 to 62°C, 45 to 61°C, 45 to 60°C, 50 to 70°C, 50 to 65°C, 50 to 64°C , 50 to 63°C, 50 to 62°C, 50 to 61°C, 50 to 60°C, 55 to 70°C, 55 to 65°C, 55 to 64°C, 55 to 63°C, 55 to 62°C, 55 to 61°C , 55 to 60°C, 56 to 70°C, 56 to 65°C, 56 to 64°C, 56 to 63°C, 56 to 62°C, 56 to 61°C, 56 to 60
  • Pseudomonas stutzeri having the activity of converting maltotetraose from polysaccharides so that the maltotetraose content is 30 to 60% by weight based on 100% by weight of the solid content of the saccharide. It relates to a composition for producing maltotetraose-containing saccharides, comprising at least one selected from the group consisting of bacterial cells, enzymes produced by the strain, cultures of the strain, and lysate of the strain.
  • the Pseudomonas stutzeri strain is as described above.
  • the malto-oligosaccharide-containing saccharide and the maltotetraose-containing saccharide are as described above.
  • the culture contains an enzyme produced from the Pseudomonas stutzeri strain, and may be in a cell-free form that includes the strain or does not include the strain.
  • the lysate refers to a lysate obtained by crushing the Pseudomonas stutzeri strain or a supernatant obtained by centrifuging the lysate, and contains an enzyme produced from the Pseudomonas stutzeri strain.
  • the present invention provides a mutant strain having a significantly increased maltotetraose production activity and an enzyme produced by the strain, thereby producing an equivalent level of maltotetraose-containing saccharides despite the significant decrease in the amount of enzyme added compared to the conventional maltotetraose production method. It is possible to provide a way to do it.
  • FIG. 3 is a diagram showing a graph showing the activity (Activity, U/ml) of maltotetraose synthase of a mutant strain according to an embodiment of the present invention.
  • FIG. 4 is a view showing the result of performing SDS-PAGE analysis of the culture supernatant of a mutant strain according to an example of the present invention.
  • FIG. 5 is a diagram showing the amount of maltotetraose produced by reaction pH of a maltotetraose-producing enzyme of a mutant strain according to an embodiment of the present invention.
  • FIG. 6 is a diagram showing the amount of maltotetraose produced by reaction temperature of a maltotetraose-producing enzyme of a mutant strain according to an embodiment of the present invention.
  • FIG. 7 is a diagram showing the enzyme activity of maltotetraose synthase of a mutant strain according to an embodiment of the present invention for each storage period.
  • FIG. 8 is a diagram showing the results of HPLC analysis of maltotetraose-containing saccharides produced using a mutant strain of accession number KCCM12578P.
  • Example 1 Isolation of wild strains having maltotetraose conversion activity
  • Each of the microorganisms cultured in the above (1) was centrifuged to obtain a supernatant, and the supernatant was used as a crude enzyme. Using 1% soluble starch as a substrate, it was reacted with Crude enzyme at 40°C for 20 minutes, and the amylase activity of the enzyme was confirmed through the DNS method and HPLC analysis.
  • the Pseudomonas stutzeri wild strain was cultured and centrifuged to recover the culture supernatant.
  • potato starch was used as a dextrin as a substrate, and dissolved by heating at a concentration of 1% in 100 mM sodium phosphate buffer pH 7.0 and McIlvaine buffer pH 5.5, respectively.
  • 500 ⁇ l of the P. stutzeri culture supernatant was added so that the amount of enzyme added to 500 ⁇ l of the substrate was 8 units per g of the substrate, followed by reaction at 50°C.
  • the reaction result was confirmed through HPLC analysis, and 42A (Bio-rad) was used as the column.
  • a mutant strain having increased activity of the G4 amylase enzyme, which specifically produces a large amount of maltotetraose, was prepared from the wild P. stutzeri strain (parent strain) isolated in Example 1.
  • the glycerol stock of P. stutzeri wild strain was inoculated in M9 minimal salt liquid medium containing soluble starch, and then first cultured at 30° C. for 24 hours. 50ul of the primary culture solution was taken, inoculated into 3ml of a new medium, and cultured at 30°C for 12 hours. The concentration of the cells was confirmed by measuring the absorbance at 600 nm of the culture medium, and the cells were recovered to select excellent mutant strains with increased enzyme activity when reaching the logarithmic growth phase.
  • the liquid culture was centrifuged (12,000 rpm, 10 minutes) to recover only the cells, and washed twice with TM buffer (pH 7.2) containing 7.9 g/L of 50mM Tris-HCl, and 2.0 g/L of 8mM Magnesium sulfate. The ingredients were removed.
  • the washed cells were diluted with sterile distilled water, and 100ul was dispensed on M9 agar plate (Table 4), smeared, and incubated at 30°C. After irradiating UV with a wavelength of 254 nm for a certain period of time (10 to 30 seconds) with a 15cm interval from the plate medium coated with the cells, it was confirmed whether colonies were grown by incubating at 30°C.
  • the strains selected from the plate medium were inoculated into a 14 mL round bottom tube in which 3 mL of the production medium (Table 5) was dispensed, and cultured at 30° C. for 3 days. A part of the culture medium was taken, centrifuged, the supernatant was recovered, and the enzyme activity was finally confirmed using the DNS method.
  • a strain having a higher titer than the parent strain was plated on a starch agar plate to select several single colonies for strain stabilization. The selected colonies were stored at -70°C in glycerol stock.
  • the glycerol stock of the mutant strain selected in Example 2 stored at -70°C was inoculated into the seed medium according to Table 6 and seeded at 30°C for 24 hours.
  • Production medium #3 (Table 7) All 3 ml of seed culture solution were inoculated into a flask containing 40 ml, followed by main culture at 30°C for 4 days. The concentration of cells in the culture medium was measured, and after 1 ml of sample was centrifuged (12,000 rpm, 10 minutes), the activity of the G4 synthase in the supernatant was analyzed by DNS method. The results are shown in Table 8 and FIG. 3.
  • the activity of the mutant strain (Activity, U/ml) was significantly higher than that of the wild-type strain, and in particular, it was found that the Mutant E mutant strain has more than 30 times the activity of the wild-type strain.
  • the samples were diluted with distilled water to uniformly adjust the protein concentration and then subjected to SDS-PAGE analysis.
  • a 10 ⁇ 16% gradient gel plate was prepared and 10ul of the sample was loaded. After developing at 135V for 30 minutes, the gel was recovered, stained with staining buffer for 1 hour, and then stored in de-staining buffer for more than 24 hours. The results are shown in FIG. 4.
  • the protein molecular weight of G4 synthase was about 45 to 50 kDa. Based on the enzyme activity, protein concentration analysis and SDS-PAGE results, it was confirmed that the G4 amylase protein expression increased as the improvement progressed (Mutant A ⁇ E). In wild strains, as the G4 amylase activity of the mutant strain Mutant C increased, the enzyme expression level also increased, whereas the 8th mutant (Lane 5) Mutant D (lane 4) showed a significant increase in G4 amylase expression compared to the strain Mutant C. It was not done, but it was confirmed that the expression of the impurity protein was reduced.
  • strain Mutant E with the highest activity of G4 synthase was selected, and this strain was named Pseudomonas stutzeri SYC-G4 strain and deposited with the Korea Microbial Conservation Center (KCCM) to receive the deposit number KCCM12578P.
  • KCCM Korea Microbial Conservation Center
  • Example 1 The sequence of the 16s rRNA of the wild line isolated in Example 1 is shown in SEQ ID NO: 1. As a result, the wild strain isolated in Example 1 was identified as Pseudomonas stutzeri.
  • genomic DNA was extracted using the Genomic DNA Extraction Kit (Bioneer), and then the nucleotide sequence of the G4-amylase enzyme was analyzed and shown in Table 10.
  • the Pseudomonas stutzeri SYC-G4 strain prepared through mutation was cultured to analyze the properties of G4 synthase.
  • Pseudomonas stutzeri SYC-G4 was cultured to obtain a supernatant of active 890 U/ml and concentrated to 2,107 U/ml.
  • the concentrate of the cell culture supernatant was subjected to enzyme powdering using a spray dryer (BUCHI Mini spray dryer B-191), and enzyme activity before and after powdering and stability by storage temperature were checked.
  • the experiment was conducted by storing the powdered Pseudomonas stutzeri SYC-G4-derived G4 enzyme at a temperature of 8, 16, 25, or 35, respectively.
  • the activity of the enzyme was measured over time.
  • 0.05g of the powdered enzyme was taken and re-suspensioned with 0.5ml 50mM sodium phosphate buffer (pH7.0).
  • the powdered G4 synthase the results of maintaining stable enzyme activity for a storage period of 20 weeks or more at each temperature (8°C, 16°C, 25°C, or 35°C) were obtained (FIG. 7). Therefore, it was confirmed that it is preferable to apply powdered enzymes for stable storage and distribution of enzymes for a long period of time when powdered.
  • Maltotetraose syrup containing high maltotetraose (G4) was prepared using the UV mutant strain Pseudomonas stutzeri SYC-G4.
  • the amount of enzyme added was determined in consideration of the color value of the final product and the sugar composition such that the total content of monosaccharides and disaccharides was less than 7% by weight, and the sugar composition of the syrup was confirmed after the reaction.
  • Maltodextrin (DE8-10) was used as the substrate, and the powdered enzyme prepared in Example 6 was used as the enzyme.
  • the initial pH of the reaction was titrated with 0.5N NaOH to become 6.2.
  • the maltodextrin was a solution having a solid content of 35% by weight.
  • the enzyme reaction was carried out at 60° C. by adding 10 units of the amount of enzyme to the solid weight (g) of the substrate. It was confirmed that the reaction time is suitable when the amount of enzyme used within 16 to 20 hours is 7.5 to 10 U/g, and the color value increases as the amount of enzyme used increases.
  • the enzymatic reaction results were performed using HPLC, and when the G4 oligosaccharide content was 45 to 50% or more based on the total amount of saccharides produced, the content of monosaccharides and disaccharides was confirmed to be less than 6 to 10% (Fig. 8 and Table 14).
  • the titer of the mutant enzyme was about 450 U/ml, which was about 30 times higher than that of the wild strain enzyme at about 14 U/ml (see Table 8), and the amount of enzyme added was reduced by 30 times compared to the wild strain.

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Abstract

La présente invention concerne la production d'oligosaccharides contenant du maltotétraose ayant une quantité particulière de maltotétraose.
PCT/KR2020/014954 2019-10-29 2020-10-29 Procédé de production d'oligosaccharides contenant du maltotétraose WO2021086080A1 (fr)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
KR960011095A (ko) * 1994-09-15 1996-04-20 쯔지 요시후미 기관의 공연비 제어장치
US20120301927A1 (en) * 2009-04-10 2012-11-29 Danisco Us Inc. Production Of Maltotetraose Syrup Using A Pseudomonas Saccharophila Maltotetraohydrolase Variant
KR20190082659A (ko) * 2018-01-02 2019-07-10 인그리디언코리아 유한회사 이소말토올리고당 조성물의 제조 방법

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JPH0789916B2 (ja) * 1987-03-28 1995-10-04 株式会社林原生物化学研究所 マルトテトラオ−ス生成アミラ−ゼとその製造方法

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KR960011095A (ko) * 1994-09-15 1996-04-20 쯔지 요시후미 기관의 공연비 제어장치
US20120301927A1 (en) * 2009-04-10 2012-11-29 Danisco Us Inc. Production Of Maltotetraose Syrup Using A Pseudomonas Saccharophila Maltotetraohydrolase Variant
KR20190082659A (ko) * 2018-01-02 2019-07-10 인그리디언코리아 유한회사 이소말토올리고당 조성물의 제조 방법

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Title
DATABASE PROTEIN 5 March 2012 (2012-03-05), ANONYMOUS: "glucan 1,4-alpha-maltohexaosidase [Pseudomonas stutzeri ATCC 14405 = CCUG 16156]", XP055808253, retrieved from NCBI Database accession no. EHY78281 *
HANA MAALEJ, HANEN BEN AYED, OLFA GHORBEL-BELLAAJ, MONCEF NASRI, NOOMEN HMIDET: "Production and Biochemical Characterization of a High Maltotetraose (G4) Producing Amylase from Pseudomonas stutzeri AS22", BIOMED RESEARCH INTERNATIONAL, HINDAWI PUBLISHING CORPORATION, vol. 2014, 1 January 2014 (2014-01-01), pages 1 - 11, XP055700311, ISSN: 2314-6133, DOI: 10.1155/2014/156438 *

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