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WO2020067786A1 - Nouvelle fructose-4-épimérase et procédé de production de tagatose l'utilisant - Google Patents

Nouvelle fructose-4-épimérase et procédé de production de tagatose l'utilisant Download PDF

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Publication number
WO2020067786A1
WO2020067786A1 PCT/KR2019/012618 KR2019012618W WO2020067786A1 WO 2020067786 A1 WO2020067786 A1 WO 2020067786A1 KR 2019012618 W KR2019012618 W KR 2019012618W WO 2020067786 A1 WO2020067786 A1 WO 2020067786A1
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Prior art keywords
amino acid
fructose
epimerase
tagatose
substituted
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Korean (ko)
Inventor
이영미
박을수
박일향
신선미
양성재
윤란영
최은정
김성보
박승원
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CJ CheilJedang Corp
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CJ CheilJedang Corp
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Priority claimed from KR1020190099827A external-priority patent/KR102308173B1/ko
Application filed by CJ CheilJedang Corp filed Critical CJ CheilJedang Corp
Priority to US17/252,705 priority Critical patent/US11753646B2/en
Priority to EP19864869.3A priority patent/EP3798312A4/fr
Publication of WO2020067786A1 publication Critical patent/WO2020067786A1/fr
Anticipated expiration legal-status Critical
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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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N9/88—Lyases (4.)
    • 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 application relates to a fructose-4-epimerase enzyme variant having improved conversion activity or stability, and a method for preparing tagatose using the same.
  • Tagatose has a natural sweet taste that is almost indistinguishable from sugar, and its physical properties are similar to sugar.
  • Tagatose is a natural sweetener that is present in small amounts in foods such as milk, cheese, cacao, sweet fruits such as apples and tangerines, and has a calorie of 1.5 kcal / g, 1/3 of sugar and GI (Glycemic index, blood sugar). The index is 3, which is 5% of sugar, but it has a sweetness similar to the taste of sugar and has various health functionalities, so it can be used as an alternative sweetener that can satisfy both health and taste at the same time.
  • the present inventors have discovered a novel variant protein comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1, wherein the variant protein has conversion activity like the wild type of SEQ ID NO: 1, or conversion activity or stability compared to wild type This application was completed by confirming this improvement and increasing the production capacity of tagatose.
  • One object of the present application is to provide a fructose-4-epimerase enzyme variant in which one or more amino acid residues are substituted in a fructose-4-epimerase comprising the amino acid sequence of SEQ ID NO: 1.
  • Another object of the present application is to provide a polynucleotide encoding the fructose-4-epimerase enzyme variant.
  • Another object of the present application is to provide a vector comprising the polynucleotide.
  • Another object of the present application is to provide a microorganism comprising the above variant.
  • Another object of the present application is fructose-4-epimerase or fructose-4-epimerase enzyme variant; Microorganisms including it; Or it is to provide a composition for producing tagatose containing one or more cultures of the microorganism.
  • Another object of the present application is fructose-4-epimerase or fructose-4-epimerase enzyme variant; Microorganisms expressing this; It is to provide a method for producing tagatose, comprising reacting fructose in the presence of a culture of the microorganism or fructose-4-epimerase derived therefrom.
  • the fructose-4-epimerase enzyme variant of the present application is industrially capable of producing tagatose having excellent properties, and has a high economical effect by converting the fructose, a common sugar, to tagatose.
  • CJ_KO_F4E tagatose-diphosphate aldolase enzyme
  • 2 and 3 is a graph showing the residual activity according to the change in time at a temperature condition of 60 °C relative to evaluate the thermal stability of the variants.
  • fructose-4-epimerase enzyme variant comprising one or more amino acid substitutions in the amino acid sequence of the fructose-4-epimerase.
  • fructose-4-epimerase enzyme variant comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1.
  • fructose-4-epimerization enzyme in the present application is an enzyme having fructose-4-epimerization activity that epimerizes the 4th carbon position of fructose to convert fructose to tagatose.
  • tagatose can be produced using fructose as a substrate, it can be included without limitation, and can be used in combination with 'D-fructose C4-epimerase.
  • EC 4.1.2.40 tagatose diphosphate aldolase or tagatose-diphosphate aldolase class II accessory protein can be included as fructose-4-epimerase if it has the activity of converting fructose to tagatose as a substrate.
  • the tagatose-diphosphoric acid aldolase was previously prepared with D-tagatose 1,6-bisphosphate as a substrate as shown in [Scheme 1] and glycerone phosphate. It is known as an enzyme that produces D-glyceraldehyde 3-phosphate.
  • tagatose-6-phosphate kinase (EC 2.7.1.144) has the activity of converting fructose to tagatose as a substrate, it may be included as a fructose-4-epimerase.
  • the tagatose-6-phosphate kinase was previously ADP and D-tagatose 6-phosphate (D-tagatose 6-phosphate) as a substrate, as shown in [Scheme 2], ADP and D-tagatose 1,6- It is known as an enzyme that produces D-tagatose 1,6-bisphosphate.
  • the fructose-4-epimerase, tagatose-diphosphate aldolase, tagatose-6-phosphate kinase of the present application may be a heat-resistant microorganism-derived enzyme or a variant thereof, for example, Kosmotoga olearia ), Thermanaerothrix daxensis , Rhodothermus profundi , Rhodothermus marinus , Limnochorda pilosa , Caldithrix abyssi , Caldilinea aerophila , Thermoanaerobacter thermohydrosulfuricus , Acidobacteriales bacterium , Caldicellulosiruptor kronotskyensis Thermo aerobacterium thermosaccharolyticum , or Pseudoalteromonas sp.
  • H103 a derived enzyme or a variant thereof, but is not limited thereto.
  • Kosmotoga olearia Kosmotoga olearia
  • Thermosaka loliticum Thermoanaerobacterium thermosaccharolyticum
  • Pseudoalteromonas sp. H103 SEQ ID NO: 5
  • Thermanaerothrix daxensis SEQ ID NO: 7
  • Acidobacteriales bacterium SEQ ID NO: 9
  • Rhodothermus profundi SEQ ID NO: 9
  • Rhodothermus marinus SEQ ID NO: 13
  • Limnochorda pilosa SEQ ID NO: 15
  • Caldithrix abyssi SEQ ID NO: 17
  • Caldicellulos Derived from Syrupter Chronoskiensis SEQ ID NO: 19
  • Caldilinea aerophila SEQ ID NO: 21
  • Thermoanaerobacter thermohydrosulfuricus SEQ ID NO: 23
  • the fructose-4-epimerase, tagatose-diphosphate aldolase, or tagatose-6-phosphate kinase has SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 , 21, 23 amino acid sequence or an amino acid sequence having 70% or more homology or identity therewith, but is not limited thereto. More specifically, the fructose-4-epimerase of the present application is at least 60%, 70% with the amino acid sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 , 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity.
  • SEQ ID NO: 1 refers to an amino acid sequence having fructose-4-epimerase activity.
  • the SEQ ID NO: 1 can be obtained from the known database of NCBI GenBank or KEGG (Kyoto Encyclopedia of Genes and Genomes).
  • Cosmoto may be derived from Kosmotoga olearia , and more specifically, may be a polypeptide / protein including the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.
  • a sequence having the same activity as the amino acid sequence may be included without limitation.
  • it may include the amino acid sequence of SEQ ID NO: 1 or more than 70% homology (homology) or identity (identity) with it, but is not limited thereto.
  • the amino acid sequence has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with SEQ ID NO: 1 and SEQ ID NO: 1 Amino acid sequence.
  • amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the protein it is obvious that a protein having an amino acid sequence in which some sequences are deleted, modified, substituted or added is also included within the scope of the present application.
  • proteins having a substituted, conservative or added amino acid sequence can also be used in the present application.
  • a sequence that does not change the function of the protein before and after the amino acid sequence is added, a naturally occurring mutation, a potential mutation thereof, or a conservative substitution It is not excluded, and it is obvious that even within such a sequence addition or mutation, it is within the scope of the present application.
  • tagatose is a kind of ketohexose among monosaccharides and is used interchangeably with "D-tagatose”.
  • fructose-4-epimerase enzyme variant refers to a fructose-4-epimerase enzyme variant comprising one or more amino acid substitutions in the amino acid sequence of a polypeptide having fructose-4-epimerase activity.
  • the amino acid substitution is 8, 20, 23, 25, 26, 29, 45, 51, 53, 63, 86, 91, 97, 110 from the N-terminus.
  • the amino acid at one or more positions selected from the group consisting of the second may include those substituted with other amino acids, but is not limited thereto.
  • the 'N position' of the present application may include an N position and an amino acid position corresponding to the N position (Correspoding). Specifically, an amino acid position corresponding to any amino acid residue in a mature polypeptide disclosed in a specific amino acid sequence may be included.
  • the specific amino acid sequence may be any one of the amino acid sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23.
  • the amino acid position corresponding to the N-position or the amino acid position corresponding to any amino acid residue in the mature polypeptide disclosed in the specific amino acid sequence is Needle Program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et. al., 2000, Trends Genet. 16: 276-277), Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), specifically version 5.0.0 or later.
  • the parameters used may be a gap open penalty of 10, a gap extension penalty of 0.5 and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • sequence-based comparisons are unable to detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615)
  • Other pairwise sequence comparison algorithms can be used. Greater susceptibility in sequence-based searches can be achieved using a search program that uses probabilistic representation of a polypeptide family (profile) to search the database.
  • the PSI-BLAST program can produce profiles and detect distant homologs through an iterative database search process (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402).
  • the 'other amino acid' is not limited as long as it is other than the amino acid corresponding to each position.
  • 'Amino acids' are classified into four types according to the nature of the side chain: acidic, basic, polar (hydrophilic) and non-polar (hydrophobic).
  • the amino acid at each position is a non-polar amino acid, glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), phenylalanine ( F), tryptophan (W), and proline (P); Polar amino acids serine (S), threonine (T), cysteine (C), tyrosine (Y), asphaltic acid (D), and glutamine (Q); Acidic amino acids asparagine (N), and glutamic acid (E); It may be a protein substituted with one or more amino acids selected from the group consisting of basic amino acids lysine (K), arginine (R), and histidine (H), but is not limited thereto.
  • the amino acid at the 8th position may be substituted with a non-polar amino acid, a polar amino acid, an acidic amino acid, or a basic amino acid, and more specifically, alanine (A), glutamic acid (E), histidine (H), leucine (L) ), Proline (P), glutamine (Q) or valine (V).
  • the amino acid at position 20 may be substituted with a basic amino acid, and more specifically, may be substituted with arginine (R).
  • the amino acid at position 23 may be substituted with a polar amino acid, and more specifically, may be substituted with cysteine (C).
  • the amino acid at position 25 may be substituted with a non-polar amino acid, a polar amino acid, an acidic amino acid, or a basic amino acid, and more specifically, alanine (A), valine (V), serine (S), asphaltic acid (D), Histidine (H), phenylalanine (F), leucine (L), glycine (G), asparagine (N), methionine (M), glutamic acid (E), glutamine (Q), proline (P), lysine (K), It may be substituted with tyrosine (Y), arginine (R), tryptophan (W), isoleucine (I), or threonine (T).
  • the amino acid at the 26th position may be substituted with a non-polar amino acid or a polar amino acid, and more specifically, alanine (A), threonine (T), or valine (V) may be substituted.
  • the amino acid at position 29 may be substituted with a non-polar amino acid, a polar amino acid, an acidic amino acid, or a basic amino acid, and more specifically, tryptophan (W), cysteine (C), lysine (K), alanine (A), glutamic acid (E), leucine (L), proline (P), glutamine (Q), serine (S) or valine (V) may be substituted.
  • the amino acid at position 45 may be substituted with a non-polar amino acid, a polar amino acid, an acidic amino acid, or a basic amino acid, and more specifically, alanine (A), glutamine (Q), valine (V), lysine (K), glutamic acid (E), or may be substituted with methionine (M).
  • A alanine
  • Q glutamine
  • V valine
  • K lysine
  • E glutamic acid
  • M methionine
  • the amino acid at position 51 may be substituted with a non-polar amino acid, a polar amino acid, an acidic amino acid, or a basic amino acid, and more specifically, glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), phenylalanine (F), tryptophan (W), proline (P), serine (S), cysteine (C), tyrosine (Y), asphaltic acid (D), glutamine (Q), It may be substituted with asparagine (N), glutamic acid (E), lysine (K), arginine (R), or histidine (H).
  • the amino acid at position 53 may be substituted with a non-polar amino acid, a polar amino acid, or a basic amino acid, and more specifically, tryptophan (W), phenylalanine (F), cysteine (C), lysine (K), arginine (R) , Glycine (G), serine (S), leucine (L), threonine (T), or proline (P).
  • the amino acid at position 63 may be substituted with a non-polar amino acid, a polar amino acid, or an acidic amino acid.
  • proline (P), alanine (A), methionine (M), valine (V), glutamic acid (E) , Or leucine (L), and the amino acid at position 86 may be substituted with a non-polar amino acid, a polar amino acid, or a basic amino acid, and more specifically, arginine (R), valine (V), methionine It may be substituted with (M), alanine (A), leucine (L), or glycine (G).
  • the amino acid at position 91 may be substituted with a non-polar amino acid or a polar amino acid, and more specifically, may be substituted with phenylalanine (F), tryptophan (W), or tyrosine (Y).
  • the amino acid at position 97 may be substituted with a non-polar amino acid, a polar amino acid, an acidic amino acid, or a basic amino acid, and more specifically, leucine (L), proline (P), tyrosine (Y), glutamic acid (E), lysine. It may be substituted with (K).
  • the amino acid at position 110 may be substituted with a polar amino acid, and more specifically, may be substituted with tyrosine (Y).
  • the amino acid at position 133 may be substituted with a non-polar amino acid, a polar amino acid, an acidic amino acid, or a basic amino acid, and more specifically, valine (V), leucine (L), proline (P), glutamine (Q), asparagine (N) or glutamic acid (E).
  • the amino acid at position 144 may be substituted with a non-polar amino acid or a polar amino acid, and more specifically, alanine (A), valine (V), isoleucine (I), phenylalanine (F), or serine (S). It can be.
  • the amino acid at position 146 may be substituted with a non-polar amino acid, a polar amino acid, or a basic amino acid, and more specifically, leucine (L), isoleucine (I), proline (P), glutamine (Q), or histidine (H) ).
  • the amino acid at position 151 may be substituted with a non-polar amino acid, and more specifically, may be substituted with glycine (G).
  • the amino acid at position 155 may be substituted with a non-polar amino acid, and more specifically, may be substituted with glycine (G).
  • the amino acid at position 167 may be substituted with a non-polar amino acid, a polar amino acid, or a basic amino acid, and more specifically, valine (V), glycine (G), alanine (A), arginine (R), leucine (L) , Threonine (T), may be substituted with asphaltic acid (D).
  • the amino acid at position 172 may be substituted with a non-polar amino acid, and more specifically, may be substituted with alanine (A) or threonine (T).
  • the amino acid at position 173 may be substituted with a non-polar amino acid, a polar amino acid, or an acidic amino acid.
  • alanine (A), valine (V), threonine (T), glutamic acid (E) or asphaltic acid It may be substituted with (D).
  • the amino acid at position 174 may be substituted with a non-polar amino acid, a polar amino acid, or a basic amino acid, and more specifically, glycine (G), valine (V), leucine (L), methionine (M), phenylalanine (F) , Tryptophan (W), serine (S), tyrosine (Y), asphaltic acid (D), lysine (K), or arginine (R).
  • the amino acid at position 181 may be substituted with a non-polar amino acid or a basic amino acid, and more specifically, glycine (G), alanine (A), leucine (L), isoleucine (I), proline (P), lysine ( K), or arginine (R).
  • the amino acid at position 191 may be substituted with a non-polar amino acid, a polar amino acid, or a basic amino acid, and more specifically, glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I) , Serine (S), threonine (T) or arginine (R).
  • the amino acid at position 239 may be substituted with a non-polar amino acid, an acidic amino acid, or a basic amino acid, and more specifically, glycine (G), alanine (A), valine (V), leucine (L), tryptophan (W) , Proline (P), glutamic acid (E), or lysine (K).
  • the amino acid at position 263 may be substituted with a non-polar amino acid, a polar amino acid, an acidic amino acid, or a basic amino acid, and more specifically, alanine (A), leucine (L), glutamine (Q), glutamic acid (E), or It may be substituted with lysine (K).
  • the amino acid at position 266 may be substituted with a non-polar amino acid, a polar amino acid, or a basic amino acid, and more specifically, glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I) , Tryptophan (W), proline (P), cysteine (C), tyrosine (Y), asphaltic acid (D), or arginine (R).
  • the amino acid at position 285 may be substituted with a non-polar amino acid, a polar amino acid, or an acidic amino acid.
  • the amino acid at position 294 may be substituted with a non-polar amino acid, and more specifically, may be substituted with glycine (G).
  • the amino acid at position 298 may be substituted with a non-polar amino acid, and more specifically, may be substituted with glycine (G).
  • the amino acid at position 308 may be substituted with a non-polar amino acid or a basic amino acid, and more specifically, alanine (A), valine (V), leucine (L), isoleucine (I), tryptophan (W), arginine ( R), or histidine (H).
  • the amino acid at position 315 may be substituted with a non-polar amino acid, a polar amino acid, or a basic amino acid, and more specifically, alanine (A), valine (V), leucine (L), proline (P), asphaltic acid (D) ), Or histidine (H).
  • the amino acid at position 316 may be substituted with a non-polar amino acid, a polar amino acid, an acidic amino acid, or a basic amino acid, and more specifically, valine (V), leucine (L), methionine (M), proline (P), bit It may be substituted with leonine (T), asparagine (N), lysine (K), or arginine (R).
  • the amino acid at position 317 may be substituted with a non-polar amino acid, a polar amino acid, or a basic amino acid, and more specifically, valine (V), isoleucine (I), serine (S), asphaltic acid (D), arginine (R) ), Or histidine (H).
  • the amino acid at position 323 may be substituted with a non-polar amino acid, a polar amino acid, or a basic amino acid, and more specifically, glycine (G), valine (V), leucine (L), methionine (M), asphaltic acid (D) ), Arginine (R), or histidine (H).
  • the amino acid at position 336 may be substituted with a non-polar amino acid or a basic amino acid, and more specifically, may be substituted with glycine (G), alanine (A), or arginine (R).
  • the amino acid at position 347 may be substituted with a non-polar amino acid, a polar amino acid, or an acidic amino acid, and more specifically, glycine (G), proline (P), serine (S), tyrosine (Y), asphaltic acid (D) ), Asparagine (N), or phenylalanine (F).
  • the amino acid at position 359 may be substituted with a non-polar amino acid, a polar amino acid, an acidic amino acid, or a basic amino acid, and more specifically, glycine (G), alanine (A), valine (V), asphaltic acid (D), It may be substituted with asparagine (N) or arginine (R).
  • the amino acid at position 367 may be substituted with a non-polar amino acid or a basic amino acid, and more specifically, glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), or arginine It may be substituted with (R).
  • the amino acid at position 385 may be substituted with a non-polar amino acid or a basic amino acid, and more specifically, may be substituted with alanine (A) or arginine (R).
  • the amino acid at position 386 may be substituted with a non-polar amino acid, a polar amino acid, or a basic amino acid, and more specifically, alanine (A), valine (V), leucine (L), isoleucine (I), serine (S) , Threonine (T), asphaltic acid (D), arginine (R), or histidine (H).
  • the amino acid at position 388 may be substituted with a non-polar amino acid, a polar amino acid, or a basic amino acid, and more specifically, glycine (G), valine (V), isoleucine (I), serine (S), threonine ( T), asphaltic acid (D), or arginine (R).
  • the amino acid at position 389 may be substituted with a non-polar amino acid, a polar amino acid, an acidic amino acid, or a basic amino acid, and more specifically, glycine (G), valine (V), methionine (M), serine (S), asphalt It may be substituted with acid (D), glutamic acid (E), lysine (K), or arginine (R).
  • the amino acid at position 410 may be substituted with a non-polar amino acid, and more specifically, may be substituted with alanine (A), valine (V), leucine (L), or threonine (T).
  • the amino acid at position 414 may be substituted with a non-polar amino acid, a polar amino acid, or an acidic amino acid, and more specifically, may be substituted with proline (P), glutamine (Q), or glutamic acid (E).
  • the amino acid at position 417 may be substituted with a non-polar amino acid, a polar amino acid, or a basic amino acid, and more specifically, glycine (G), alanine (A), valine (V), leucine (L), methionine (M) , Proline (P), serine (S), asphaltic acid (D), or arginine (R) may be substituted, but is not limited thereto.
  • the fructose-4-epimerase variant has the recited sequence listed above for conservative substitution and / or modification of one or more amino acids other than the amino acid at a specific position being replaced by another amino acid. sequence), but may include a polypeptide in which the functions or properties of the protein are maintained.
  • conservative substitution in this application means to replace one amino acid with another amino acid having similar structural and / or chemical properties.
  • the variant may retain one or more biological activities, but may have one or more conservative substitutions, for example. Conservative substitutions have little or no effect on the activity of the resulting polypeptide.
  • a variant type in which one or more amino acids other than the amino acid at the specific position described above may be modified may include deletion or addition of amino acids having minimal influence on the properties and secondary structure of the polypeptide.
  • the polypeptide can be conjugated with a signal (or leader) sequence of the protein N-terminal that is involved in the translation of a protein co-translationally or post-translationally.
  • the polypeptide may be conjugated with other sequences or linkers to identify, purify, or synthesize the polypeptide.
  • the variant may be at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 70%, 80%, 85%, 90%, other than the mutation of SEQ ID NO.
  • Amino acids having at least 99% homology or identity.
  • the variation of SEQ ID NO: 1 is as described above, and the homology or identity thereof may have homology or identity at positions other than the aforementioned variation.
  • the fructose-4-epimerase enzyme variant is characterized by improved conversion activity or stability compared to wild type.
  • conversion activity refers to epimerization of position 4 of D-fructose (fructose) to tagatose
  • stability means an enzyme having high heat resistance and thermal stability.
  • the fructose-4-epimerase enzyme variant of the present application may be an enzyme having high heat resistance. Specifically, the fructose-4-epimerase enzyme variant of the present application exhibits 50% to 100%, 60% to 100%, 70% to 100%, or 75% to 100% of the maximum activity at 50 ° C to 70 ° C. Can be represented. More specifically, the fructose-4-epimerase enzyme variant of the present application is 80% to 100% or 85% to maximal activity at 55 ° C to 60 ° C, 60 ° C to 70 ° C, 55 ° C, 60 ° C, or 70 ° C 100% of activity.
  • variant positions of the variants and the mutated amino acids are as described in Tables 1 to 6, for example, but are not limited thereto.
  • Another aspect of the present application is to provide a polynucleotide encoding the fructose-4-epimerase enzyme variant, or a vector comprising the polynucleotide.
  • polynucleotide is a polymer of nucleotides in which a nucleotide monomer (monomer) is long chained by a covalent bond, a DNA or RNA strand of a certain length or more, and more specifically, the mutant protein. Means a polynucleotide fragment that encodes.
  • the polynucleotide encoding the fructose-4-epimerase enzyme variant of the present application may be included without limitation as long as it is a polynucleotide sequence encoding the fructose-4-epimerase enzyme variant of the present application.
  • the polynucleotide encoding the fructose-4-epimerase enzyme variant of the present application may be a polynucleotide sequence encoding the amino acid sequence, but is not limited thereto.
  • the polynucleotide may be variously modified in the coding region within a range that does not change the amino acid sequence of the protein due to the degeneracy of the codon or in consideration of the codon preferred in the organism to express the protein. . Therefore, it is obvious that a polynucleotide that can be translated into a polypeptide composed of the amino acid sequence or a polypeptide having homology or identity thereto by codon degeneracy may also be included.
  • a probe that can be prepared from a known gene sequence for example, a sequence that hybridizes under strict conditions with complementary sequences for all or part of the nucleotide sequence, is limited if it is a sequence encoding the fructose-4-epimerase enzyme variant. Can be included without.
  • stringent condition refers to a condition that enables specific hybridization between polynucleotides. These conditions are specifically described in the literature (eg, J. Sambrook et al., Homology). For example, genes with high homology or identity, 70% or more, 80% or more, 85% or more, specifically 90% or more, more specifically 95% or more, more specifically In the case of hybridization between genes having at least 97%, particularly specifically at least 99% homology or identity, and not hybridization between genes with less homology or identity, or washing of conventional southern hybridization At salt concentrations and temperatures corresponding to the conditions 60 ° C, 1 X SSC, 0.1% SDS, specifically 60 ° C, 0.1 X SSC, 0.1% SDS, more specifically 68 ° C, 0.1 X SSC, 0.1% SDS, Conditions for washing once, specifically 2 to 3 times can be enumerated.
  • Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization.
  • the term “complementary” is used to describe the relationship between nucleotide bases that are hybridizable to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine.
  • the present application can also include isolated nucleic acid fragments complementary to the entire sequence, as well as substantially similar nucleic acid sequences.
  • polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55 ° C. and using the above-described conditions.
  • the Tm value may be 60 ° C, 63 ° C or 65 ° C, but is not limited thereto, and may be appropriately adjusted by a person skilled in the art according to the purpose.
  • the appropriate stringency to hybridize a polynucleotide depends on the length and degree of complementarity of the polynucleotide, and variables are well known in the art (see Sambrook et al., Supra, 9.50-9.51, 11.7-11.8).
  • the term 'homology' or 'identity' refers to the degree of correlation with two given amino acid sequences or nucleotide sequences and may be expressed as a percentage.
  • sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, and default gap penalties established by the program used can be used together.
  • Substantially, homologous or identical sequences are generally at least about 50%, 60%, 70%, 80% of the entire or full-length sequence in medium or high stringent conditions. Or it can be hybridized to 90% or more. Hybridization also contemplates polynucleotides containing degenerate codons instead of codons in the polynucleotide.
  • the homology, similarity or identity of a polynucleotide or polypeptide is, for example, Smith and Waterman, Adv. Appl. As known in Math (1981) 2: 482, for example, Needleman et al. (1970), J Mol Biol. 48: 443 can be determined by comparing sequence information using a GAP computer program.
  • the GAP program defines the total number of symbols in the shorter of the two sequences, divided by the number of similar aligned symbols (ie, nucleotides or amino acids).
  • the default parameters for the GAP program are (1) Binary Comparison Matrix (contains values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: Weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or gap open penalty 10, gap extension penalty 0.5); And (3) no penalty for the end gap.
  • the term “homology” or “identity” refers to relevance between sequences.
  • the term "vector” is a DNA preparation containing a nucleotide sequence of a polynucleotide encoding the target variant protein operably linked to a suitable regulatory sequence so that the target variant protein can be expressed in a suitable host.
  • the regulatory sequence may include a promoter capable of initiating transcription, any operator sequence to regulate such transcription, a suitable mRNA ribosome binding site sequence, and a sequence that regulates termination of transcription and translation. After transformation into a suitable host cell, the vector can replicate or function independently of the host genome and can be integrated into the genome itself.
  • the vector used in the present application is not particularly limited as long as it can be replicated in the host cell, and any vector known in the art can be used.
  • Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophage.
  • pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pBR-based, pUC-based, and pBluescriptII-based plasmid vectors.
  • pGEM system pTZ system, pCL system and pET system.
  • pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like can be used.
  • a polynucleotide encoding a target mutant protein in a chromosome may be replaced with a mutated polynucleotide through a vector for intracellular chromosomal insertion. Insertion of the polynucleotide into the chromosome can be made by any method known in the art, for example, homologous recombination, but is not limited thereto.
  • a selection marker for checking whether the chromosome is inserted may be further included. Selection markers are used to select cells transformed with a vector, that is, to confirm whether a target nucleic acid molecule is inserted, and selectable phenotypes such as drug resistance, nutritional demand, resistance to cytotoxic agents, or expression of surface variant proteins. Markers to give can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit different expression traits, so that the transformed cells can be selected.
  • the present application is to provide a microorganism that produces tagatose, including the mutated protein or a polynucleotide encoding the mutated protein.
  • the microorganism containing the mutant protein and / or the polynucleotide encoding the mutant protein may be a microorganism prepared by transformation with a vector containing the polynucleotide encoding the mutant protein, but is not limited thereto. .
  • transformation in the present application means that a vector containing a polynucleotide encoding a target protein is introduced into a host cell so that the protein encoded by the polynucleotide in the host cell can be expressed.
  • the transformed polynucleotide may include all of them, whether they can be inserted into the host cell chromosome or located outside the chromosome, as long as it can be expressed in the host cell.
  • the polynucleotide includes DNA and RNA encoding a target protein. The polynucleotide may be introduced into a host cell and expressed as long as it can be expressed in any form.
  • the polynucleotide 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 include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, which are operably linked to the polynucleotide.
  • the expression cassette may be in the form of an expression vector capable of self-replicating.
  • the polynucleotide may be introduced into a host cell in its own form and may be operably linked to a sequence required for expression in the host cell, but is not limited thereto.
  • operably linked in the above means that the promoter sequence and the gene sequence to initiate and mediate the transcription of the polynucleotide encoding the target variant protein of the present application are functionally linked.
  • a polynucleotide encoding the fructose-4-epimerase enzyme variant or a vector comprising the polynucleotide.
  • the microorganism may be a microorganism that produces the fructose-4-epimerase variant or tagatose.
  • microorganism including fructose-4-epimerase enzyme variant used in the present application may mean a microorganism that has been recombined so that the fructose-4-epimerase enzyme variant of the present application is expressed.
  • a host capable of expressing the variant by transforming it with a vector comprising a polynucleotide encoding a fructose-4-epimerase variant, or a vector comprising a polynucleotide encoding a fructose-4-epimerase variant Cell or microorganism.
  • the microorganism is a microorganism expressing a fructose-4-epimerase enzyme variant comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1, wherein the amino acid substitutions are at one or more positions from the N-terminus. It may be a microorganism that expresses a variant protein having one or more amino acids substituted and having fructose-4-epimerase activity, but is not limited thereto.
  • the fructose-4-epimerase enzyme variant of the present application transforms the DNA expressing the enzyme or variant thereof of the present application into a strain such as E. coli , and then cultivates it to obtain a culture, and the culture is obtained. It may be obtained by crushing and purifying through a column or the like.
  • the transformation strains include, but are not limited to, Escherichia coli , Corynebacterum glutamicum , Aspergillus oryzae , or Bacillus subtilis . Does not.
  • the microorganism of the present application is a microorganism capable of producing the fructose-4-epimerase of the present application, including the nucleic acid of the present application or the recombinant vector of the present application
  • both prokaryotic and eukaryotic microorganisms may be included.
  • Escherichia genus, Erwinia genus, Serratia genus, Providencia genus, Corynebacterium genus and Brevibacterium genus Belonging to the microorganism strain may be included, but is not limited thereto.
  • the microorganism of the present application may include all microorganisms capable of expressing the fructose-4-epimerase of the present application by various known methods in addition to the introduction of the nucleic acid or vector.
  • the culture of the microorganism of the present application may be prepared by culturing a microorganism expressing the fructose-4-epimerase of the present application in a medium.
  • culturing means growing the microorganism under appropriately controlled environmental conditions.
  • the process of culturing the microorganism is not particularly limited, but may be performed by a known batch culture method, a continuous culture method, a fed-batch culture method, or the like.
  • the culture conditions are not particularly limited, but using a basic compound (e.g. sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (e.g. phosphoric acid or sulfuric acid) to a proper pH (e.g. pH 5 to 9, specifically Can adjust pH 6 to 8, most specifically pH 6.8), and maintain aerobic conditions by introducing oxygen or an oxygen-containing gas mixture into the culture.
  • the culture temperature may be maintained at 20 to 45 ° C, specifically 25 to 40 ° C, and cultured for about 10 to 160 hours, but is not limited thereto.
  • the culture medium used is sugar and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g. soybean oil, sunflower seeds) Oil, peanut oil and coconut oil), fatty acids (e.g. palmitic acid, stearic acid and linoleic acid), alcohols (e.g. glycerol and ethanol) and organic acids (e.g. acetic acid) can be used individually or in combination. , But is not limited to this.
  • Nitrogen sources include nitrogen-containing organic compounds (e.g.
  • peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea), or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and Ammonium nitrate) may be used individually or in combination, but is not limited thereto.
  • the phosphorus source potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the corresponding sodium-containing salt may be used individually or in combination, but are not limited thereto.
  • the medium may contain other metal salts (eg, magnesium sulfate or iron sulfate), essential growth-promoting substances such as amino acids and vitamins.
  • a fructose-4-epimerase comprising the amino acid sequence of SEQ ID NO: 1 or the fructose-4-epimerase enzyme variant; Microorganisms including it; Or it provides a composition for producing tagatose comprising a culture of the microorganism.
  • composition for producing tagatose of the present application may further include fructose.
  • composition for tagatose production of the present application may further include any suitable excipients commonly used in the composition for tagatose production.
  • excipients may be, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizers or isotonic agents, but are not limited thereto.
  • the composition for producing tagatose of the present application may further include a metal ion or a metal salt.
  • the metal of the metal ion or metal salt may be a metal containing a divalent cation.
  • the metal of the present application may be nickel (Ni), iron (Fe), cobalt (Co), magnesium (Mg), or manganese (Mn). More specifically, the metal salt MgSO 4 , FeSO 4 , NiSO 4 , NiCl 2 , CoSO 4 , MgCl 2 , MnCl 2 or MnSO 4 .
  • a fructose-4-epimerase comprising the amino acid sequence of SEQ ID NO: 1 or a microorganism comprising the fructose-4-epimerase enzyme variant; Or it provides a method for producing tagatose comprising the step of converting fructose into tagatose by contacting the culture thereof with fructose.
  • the contact of the present application may be performed at pH 5.0 to pH 9.0 conditions, at 30 ° C to 80 ° C temperature conditions, and / or for 0.5 to 48 hours.
  • the contacting of the present application may be performed under pH 6.0 to pH 9.0 conditions or pH 7.0 to pH 9.0 conditions.
  • the contact of the present application is 35 °C to 80 °C, 40 °C to 80 °C, 45 °C to 80 °C, 50 °C to 80 °C, 55 °C to 80 °C, 60 °C to 80 °C, 30 °C to 70 °C, 35 °C to 70 °C, 40 °C to 70 °C, 45 °C to 70 °C, 50 °C to 70 °C, 55 °C to 70 °C, 60 °C to 70 °C, 30 °C to 65 °C, 35 °C to 65 °C, 40 °C to 65 °C, 45 °C ⁇ 65 °C, 50 °C ⁇ 65 °C, 55 °C ⁇ 65 °C, 30 °C -60 °C, 35 °C -60 °C, 35 °
  • the contact of the present application is from 0.5 hour to 36 hours, from 0.5 hour to 24 hours, from 0.5 hour to 12 hours, from 0.5 hour to 6 hours, from 1 hour to 48 hours, from 1 hour to 36 hours, 1 For hours to 24 hours, for 1 hour to 12 hours, for 1 hour to 6 hours, for 3 hours to 48 hours, for 3 hours to 36 hours, for 3 hours to 24 hours, for 3 hours to 12 hours, for 3 hours
  • the contact of the present application can be performed in the presence of a metal ion or metal salt.
  • the metal ions or metal salts that can be used are as in the above-described embodiments.
  • the manufacturing method of the present application may further include separating and / or purifying the prepared tagatose.
  • the separation and / or purification may use methods conventionally used in the technical field of the present application. Non-limiting examples include dialysis, precipitation, adsorption, electrophoresis, ion exchange chromatography and fractional crystallization.
  • the purification may be performed by only one method, or two or more methods may be performed together.
  • the manufacturing method of the present application may further include a step of performing decolorization and / or desalination before or after the separation and / or purification step.
  • a step of performing decolorization and / or desalination By performing the decolorization and / or desalination, tagatose with better quality can be obtained.
  • the manufacturing method of the present application may further include the step of converting to tagatose of the present application, separating and / or purifying, or crystallizing tagatose after decoloring and / or desalting.
  • the crystallization can be performed using a conventional crystallization method.
  • crystallization may be performed using a cooling crystallization method.
  • the manufacturing method of the present application may further include the step of concentrating tagatose prior to the crystallization step.
  • the concentration can increase the crystallization efficiency.
  • the manufacturing method of the present application comprises contacting unreacted fructose with an enzyme of the present application, a microorganism expressing the enzyme, or a culture of the microorganism after the separation and / or purification of the present application, After the crystallization step, the step of reusing the mother liquor from which the crystals are separated may be further included in the separation and / or purification step, or a combination thereof.
  • tagatose can be obtained in a higher yield and the amount of fructose discarded can be reduced, which is an economical advantage.
  • fructose-4-epimerase To prepare fructose-4-epimerase, Cosmoto obtained amino acid sequence (SEQ ID NO: 1) and gene information derived from Kosmotoga olearia to obtain E. coli expression vectors and transforming microorganisms (transformants). It was prepared. It was confirmed that the sequence can be used as a fructose-4-epimerase that converts fructose to tagatose (FIG. 1).
  • E.coli BL21 (DE3) a strain for E. coli expression, and named E.coli BL21 (DE3) / CJ_KO_F4E.
  • E.coli BL21 (DE3) / CJ_KO_F4E was deposited on March 24, 2017 under the accession number KCCM11999P.
  • the E.coli BL21 (DE3) / CJ_KO_F4E was inoculated into a culture tube containing 5 ml of LB liquid medium containing ampicillin, and seed culture was performed in a shake incubator at 37 ° C. until absorbance was 2.0 at 600 nm.
  • the culture was carried out by inoculating the cultured culture of the seed culture into a culture flask containing a liquid medium containing LB and a protein expression regulator, lactose.
  • the stirring speed during the culture process was 180 rpm, and the culture temperature was maintained at 37 ° C.
  • the culture medium was centrifuged at 4 ° C for 20 minutes at 8,000 rpm, and the cells were recovered.
  • the recovered cells were washed twice with 50 mM Tris-HCl (pH8.0) buffer solution, and resuspended in 50 mM NaH2PO4 (pH 8.0) buffer solution containing 10 mM imidazole and 300 mM NaCl.
  • the suspended cells were crushed using a sonicator, and the cell lysate was centrifuged at 4 ° C for 20 minutes at 13,000 rpm and only the supernatant was taken.
  • the supernatant was purified using His-taq affinity chromatography, and a 50 mM NaH2PO4 (pH 8.0) buffer solution containing 20 mM imidazole and 300 mM NaCl was flowed at a 10-fold amount of the filler to remove non-specific binding capable proteins.
  • the 50mM NaH2PO4 (pH8.0) buffer solution containing the final 250mM imidazole and 300mM NaCl was passed through and purified by elution, and the enzyme for enzyme characterization was obtained after dialysis with 50mM Tris-HCl (pH 8.0) buffer solution.
  • Example 1-1 In order to measure the activity of the enzymes obtained in Example 1-1, 30% by weight fructose was used, 50mM Tris-HCl (pH 8.0), 1mM CoSO 4 , 20mg / ml tablets isolated from Example 2 The enzyme was added to react for 2 hours at a temperature of 60 ° C. The concentration of tagatose converted by CJ_KO_F4E and the conversion rate from fructose to tagatose were 16.0%.
  • Conversion rate tagatose weight / initial fructose weight X 100
  • fructose-4-epimerization was performed through an error-prone PCR using fructose-4-epimerase gene derived from Kosmotoga olearia as a template.
  • a variant library of enzymes was constructed. Specifically, using the Diversify random mutagenesis kit (ClonTech), a random mutation was induced to cause 2 to 3 mutations per 1000 base pairs of fructose-4-epimerase gene, and PCR reaction conditions are shown in Table 1 and It is shown in 2.
  • a gene library encoding a variant of fructose-4-epimerase was constructed, and then inserted into E.coli BL21 (DE3).
  • Reaction solution composition Addition amount ( ⁇ l) PCR Grade Water 36 10X TITANIUM Taq Buffer 5 MnSO4 (8 mM) 4 dGTP (2 mM) One 50X Diversify dNTP Mix One Primer mix One Template DNA One TITANIUM Taq Polym.
  • E.coli BL21 (DE3) with pBT7-C-His plasmid containing the variant gene of the fructose-4-epimerase, containing 0.2 mL of LB liquid medium containing ampicillin antibiotic Inoculated into a deep well rack, and cultured for more than 16 hours in a 37 ° C shake incubator.
  • the culture solution obtained as a result of the seed culture was inoculated in a culture deep well rack containing a liquid medium containing LB and a protein expression regulator, lactose, to perform the main culture.
  • the seed culture and main culture were carried out at a stirring speed of 180 rpm and 37 ° C. Next, the culture medium was centrifuged at 4 ° C for 20 minutes at 4,000 rpm, and then the cells were recovered.
  • a colorimetric method capable of specifically quantifying D-fructose was used. Specifically, after mixing the 70% polylinic acid solution (folin-ciocalteu reagent, SIGMA-ALDRICH) and the substrate reaction complete solution at a ratio of 15: 1, react at 80 ° C for 5 minutes and measure at 900nm for comparative analysis with OD value Did.
  • the color development method was evaluated as being effective, and was used for screening the improved activity of the prepared library.
  • the library was used for screening variants with improved activity, and specifically, variants having activity (conversion of D-fructose to D-tagatose) were selected when comparing relative activity with wild-type enzyme (SEQ ID NO: 1). These genes were analyzed for amino acid mutations after sequencing.
  • Recombinant expression vector pBT7-C-His-KO (expressing a recombinase with 6xHis-tag bound to wild-type C-terminus) prepared for expression of E. coli BL21 (DE3) of the wild-type enzyme gene was selected in Example 2 It was used as a template for the saturation mutation method for the production of 47 additional mutant strain libraries. Inverse PCR-based saturation mutagenesis was used in consideration of variation in diversity and yield of variants (2014. Anal. Biochem.
  • a primer was prepared by using a total length of 33 bp with 15 bp of the front base, 3 bp of the substitution base, and 15 bp of the rear base of each site. PCR conditions were denatured at 94 ° C. for 2 minutes, then 30 ° C. denaturation at 30 ° C., annealing at 60 ° C.
  • the saturation mutant library gene prepared in 3-1 above was E. After transforming into coli BL21 (DE3), each transformed microorganism was inoculated into a culture tube containing 5 mL of LB liquid medium containing ampicillin antibiotic, and the absorbance at 600 nm was 2.0. Seed culture was performed in a 37 ° C shaking incubator. This culture was performed by inoculating the culture solution obtained as a result of the seed culture in a culture flask containing a liquid medium containing LB and a protein expression regulator, lactose.
  • the seed culture and main culture were carried out at a stirring speed of 180 rpm and 37 ° C.
  • the culture medium was centrifuged at 4 ° C for 20 minutes at 8,000 rpm, and the cells were recovered.
  • the recovered cells were washed twice with 50 mM Tris-HCl (pH8.0) buffer solution, and resuspended in 50 mM NaH2PO4 (pH 8.0) buffer solution containing 10 mM imidazole and 300 mM NaCl. Did.
  • the resuspended cells were crushed using a sonicator, centrifuged at 4 ° C for 20 minutes at 13,000 rpm, and only the supernatant was taken.
  • the supernatant was purified using His-taq affinity chromatography, and non-specific binding was performed by flowing a 50 mM NaH 2 PO 4 (pH 8.0) buffer solution containing 20 mM imidazole and 300 mM NaCl in a 10-fold amount of the filler. Possible proteins were removed. Subsequently, 50 mM NaH 2 PO 4 (pH8.0) buffer solution containing 250 mM imidazole and 300 mM NaCl was further flown and purified by elution, followed by dialysis with 50 mM Tris-HCl (pH 8.0) buffer solution. Each enzyme was obtained for enzyme characterization.
  • mutant strain of the present application has increased fructose-4-epimerization activity than the wild type.
  • a primer was prepared by using a total length of 33 bp with 15 bp of the front base, 3 bp of the substitution base, and 15 bp of the rear base of each site. PCR conditions were denatured at 94 ° C. for 2 minutes, then 30 ° C. denaturation at 30 ° C., annealing at 60 ° C.
  • the saturation mutation library prepared in 2-1 above After transforming the gene into E. coli BL21 (DE3), each transformed microorganism was inoculated into a culture tube containing 5 mL of LB liquid medium containing ampicillin antibiotic, and the absorbance was 2.0 at 600 nm. Seeds were cultured in a 37 ° C shaking incubator until ready.
  • This culture was performed by inoculating the culture solution obtained as a result of the seed culture in a culture flask containing a liquid medium containing LB and a protein expression regulator, lactose.
  • the seed culture and main culture were carried out at a stirring speed of 180 rpm and 37 ° C.
  • the culture medium was centrifuged at 4 ° C for 20 minutes at 8,000 rpm, and the cells were recovered.
  • the recovered cells were washed twice with 50 mM Tris-HCl (pH8.0) buffer solution, and resuspended in 50 mM NaH2PO4 (pH 8.0) buffer solution containing 10 mM imidazole and 300 mM NaCl. Did.
  • the resuspended cells were crushed using a sonicator, centrifuged at 4 ° C for 20 minutes at 13,000 rpm, and only the supernatant was taken.
  • the supernatant was purified using His-taq affinity chromatography, and non-specific binding was performed by flowing a 50 mM NaH 2 PO 4 (pH 8.0) buffer solution containing 20 mM imidazole and 300 mM NaCl in a 10-fold amount of the filler. Possible proteins were removed.
  • Example 5-2 To measure the fructose-4-epimerization activity of the recombinant mutants obtained in Example 5-2, 50 mM Tris-HCl (pH 8.0), 3 mM MnSO4 and 5 mg / mL each were added to 30% by weight fructose. And reacted at 60 ° C for 2 hours.
  • each 5 mg / mL was placed at 60 ° C for a minimum of 19 hours to a maximum of 90 hours, then left on ice for 5 minutes, and then 30% by weight Fructose was reacted by adding 50 mM Tris-HCl (pH 8.0) and 3 mM MnSO4.
  • fructose-4-epimerization conversion activity and stability of all mutant strains of the present application were increased than that of the wild type.
  • the activity results are shown in Table 6 below, and the stability results are shown in FIGS. 2 and 3.
  • the present inventors transformed into E.coil BL21 (DE3) strains to transform E.coil BL21 (DE3) / CJ_KO_F4E_M1 (C25S), E.coil BL21 (DE3) / CJ_KO_F4E_M2 (T51V), E.coil BL21 (DE3) / CJ_KO_F4E_M5
  • Each transformant (transformed microorganism) named (T317Y) was prepared, and the transformant was deposited on September 19, 2018 at the Korea Microorganism Conservation Center (KCCM), an international depository organization under the Budapest Treaty, accession number KCCM12320P.
  • KCCM Microorganism Conservation Center
  • E.coil BL21 (DE3) / CJ_KO_F4E_M1 E.coil BL21 (DE3) / CJ_KO_F4E_M1
  • KCCM12321P E.coil BL21 (DE3) / CJ_KO_F4E_M2
  • KCCM12324P E.coil BL21 (DE3) / CJ_KO_F4E_M5

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Abstract

La présente invention concerne une variante de tagatose-bisphosphate aldolase ayant une activité de conversion de tagatose et un procédé de production de tagatose l'utilisant.
PCT/KR2019/012618 2018-09-28 2019-09-27 Nouvelle fructose-4-épimérase et procédé de production de tagatose l'utilisant Ceased WO2020067786A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3859002A4 (fr) * 2018-09-28 2022-09-28 CJ Cheiljedang Corporation Nouvelle fructose-4-épimérase et procédé de production de tagatose l'utilisant
CN119320766A (zh) * 2024-12-19 2025-01-17 浙江工业大学 一种塔格糖-4-差向异构酶突变体及其应用

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