WO2021080277A1 - Novel enzyme for producing d-threonine, and method for stereospecifically producing d-threonine by using same - Google Patents

Novel enzyme for producing d-threonine, and method for stereospecifically producing d-threonine by using same Download PDF

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WO2021080277A1
WO2021080277A1 PCT/KR2020/014292 KR2020014292W WO2021080277A1 WO 2021080277 A1 WO2021080277 A1 WO 2021080277A1 KR 2020014292 W KR2020014292 W KR 2020014292W WO 2021080277 A1 WO2021080277 A1 WO 2021080277A1
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threonine
seq
amino acid
enzyme
producing
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PCT/KR2020/014292
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French (fr)
Korean (ko)
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이승구
염수진
김하성
박성현
이대희
나유진
이혜원
권길광
안정웅
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한국생명공학연구원
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/02—Aldehyde-lyases (4.1.2)
    • C12Y401/02042—D-Threonine aldolase (4.1.2.42)

Definitions

  • the present invention relates to a novel D-threonine producing enzyme.
  • amino acids in nature have alpha carbons that exhibit optical activity, and are divided into L-amino acids and D-amino acids according to their stereospecificity.
  • Most of the proteins in nature are composed of L-amino acids, but exceptionally, the components of microbial peptidoglycans, peptide-based antibiotics, and physiologically active substances of higher plants are composed of D-amino acids. have.
  • D-threonine is an intermediate or precursor for synthesizing physiologically active substances such as neurotransmitters, vaccines, synthetic sweeteners, antibiotics, and hormones. It is widely used, and thus, methods for producing D-threonine have been developed.
  • Methods of producing D-threonine can be largely divided into chemical synthesis methods and biological synthesis methods using enzymes or microorganisms.
  • threonine production by chemical synthesis since the product is obtained as a racemic mixture of D-threonine and L-threonine, it is difficult to undergo another complicated optical purification process to obtain pure D-threonine. have.
  • the chemical synthesis method as described above also has a problem that serious environmental pollution is caused due to by-products generated in the process.
  • D-threonine can be produced stereospecifically without environmental pollution, while not only having high activity and production, but also dramatically reducing the ratio of impurities such as D-allothreonine to produce pure D-threonine.
  • Research and development of a novel D-threonine-producing enzyme is still in need.
  • the present invention is capable of stereospecific production of only D-threonine, not a mixture of L-threonine and D-threonine, while overcoming the conventional problem that D-allotreonine is produced together, thereby producing D-threonine with high purity.
  • An object of the present invention is to provide a novel enzyme with characteristics and a method for producing D-threonine using the same.
  • an aspect of the present invention is the 147th arginine, 179th glycine of D-threonine aldolase derived from Filomicrobium marinum. And it provides a D-threonine-producing enzyme in which at least one amino acid selected from the group consisting of 312th serine is substituted with alanine.
  • another aspect of the present invention provides a gene encoding the D-threonine producing enzyme.
  • another aspect of the present invention provides a recombinant expression vector containing the gene and a transformant in which the expression vector is introduced into a host cell.
  • another aspect of the present invention comprises the steps of culturing the transformant; And separating the D-threonine-producing enzyme from the culture of the transformant.
  • another aspect of the present invention is a composition for producing D-threonine comprising the D-threonine-producing enzyme as an active ingredient, and the D-threonine-producing enzyme reacted with glycine and acetaldehyde. It provides a method for producing D-threonine in an in vitro containing;
  • another aspect of the present invention provides a method for producing D-threonine in vivo, comprising culturing the transformant in the presence of glycine and acetaldehyde. do.
  • the D-threonine-producing enzyme of the present invention converts glycine and acetaldehyde into D-threonine as substrates, reducing the ratio and production amount of D-allothreonine that can be produced together, thereby producing D-threonine with high purity. There is an effect.
  • 1 is a molecular mass characteristic of a protein having an amino acid sequence of SEQ ID NO: 1, overexpressed in a transformant, purified and isolated, by SDS-PGAE (A) and gel-filtration chromatography (B). This is the result of analysis.
  • FIG. 2 is a graph showing the degree of conversion of D-threonine by a protein having the amino acid sequence of SEQ ID NO: 1.
  • FIG. 3 is a graph confirming the type (A) of metal cations and the concentration (B) of metal cations affecting the enzyme activity of the protein having the amino acid sequence of SEQ ID NO: 1.
  • Figure 5 is a graph confirming the effect of pH on the enzyme activity of the protein having the amino acid sequence of SEQ ID NO: 1, MOPS (pH 6.5 to 7.5), HEPES (pH 7.5 to 8.0), EPPS (pH 8.0 to 8.5) as a buffer solution. ) And CHES (pH 8.5 to 10) buffer solutions, respectively.
  • PBP pyridoxal-5-phosphate
  • FIG. 7 is a photograph showing the X-ray crystal structure (A) of the protein enzyme having the amino acid sequence of SEQ ID NO: 1 and the residue (B) predicted to be involved in the diastereomer ratio change at the active site.
  • WT 9 is a wild-type protein (WT) having the amino acid sequence of SEQ ID NO: 1, the relative specific activity and diastereomers (D-threonine and D-allotreonine) of each protein in which one amino acid residue of SEQ ID NO: 1 is replaced with another. This is a graph confirming the rate change.
  • Figure 10 is a wild-type protein having the amino acid sequence of SEQ ID NO: 1 (Wild-type), SEQ ID NOs: 3, 4, 5 substituted with alanine, and each protein having a sequence substituted with an amino acid other than alanine was confirmed by SDS-PAGE. It is the result.
  • Figure 11 is a wild-type protein having the amino acid sequence of SEQ ID NO: 1 (Wild-type), SEQ ID NOs: 3, 4, 5 substituted with alanine, and relative specific activity and portions of each protein having a sequence substituted with an amino acid other than alanine. This is a graph confirming the change in the ratio of stereoisomers (D-threonine and D-allotreonine).
  • FIG. 12 is a result of confirming by SDS-PAGE a wild-type protein having the amino acid sequence of SEQ ID NO: 1 (Wild-type) and each protein having the amino acid sequence of SEQ ID NOs: 9 to 11 into which multiple alanine substitutions have been introduced.
  • A is SEQ ID NO: 1 (WT)
  • B is SEQ ID NO: 4 (G179A)
  • C is SEQ ID NO: 10 (G179A + S312A)
  • A is SEQ ID NO: 1 (WT)
  • B is SEQ ID NO: 4 (G179A)
  • C is the ratio of acetaldehyde as a substrate to the proteins having the amino acid sequence of SEQ ID NO: 10 (G179A + S312A) is D-threonine This is a graph confirming the effect on production.
  • FIG. 16A is SEQ ID NO: 1 (WT)
  • B is SEQ ID NO: 4 (G179A)
  • C is SEQ ID NO: 10 (G179A + S312A) by reacting proteins having the amino acid sequence of the amino acid sequence under optimal substrate conditions, D-threonine and D- This is a graph comparing the production of allotreonine.
  • the "D-threonine-producing enzyme” of the present invention means an enzyme having the activity of producing D-threonine from glycine and acetaldehyde as shown in the following scheme.
  • One aspect of the present invention is the 147th arginine, 179th glycine and 312th serine of D-threonine aldolase derived from Filomicrobium marinum. It provides a D-threonine-producing enzyme in which at least one amino acid selected from the group consisting of alanine is substituted.
  • the D-threonine-producing enzyme is that the 147th arginine of the D-threonine aldolase is substituted with alanine, the 179th glycine is substituted with alanine, the 312th serine is substituted with alanine, or The 179th glycine and the 312th serine may be substituted with alanine, respectively.
  • another aspect of the present invention provides a gene of a D-threonine-producing enzyme encoding the D-threonine-producing enzyme.
  • the D-threonine-producing enzyme may catalyze a reaction for producing D-threonine using glycine and acetaldehyde as substrates.
  • the D-threonine aldolase derived from Filomicrobium marinum may include the amino acid sequence of SEQ ID NO: 1, and the D-threonine aldolase is within a range that does not affect the function of the protein.
  • Amino acid residue deletion, insertion, substitution, or a combination thereof may be a variant of an amino acid having a different sequence, or fragments.
  • Amino acid exchange at the protein and peptide level that does not totally alter the activity of the D-threonine aldolase is known in the art. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, and farnesylation.
  • the D-threonine aldolase includes a protein having an amino acid sequence substantially identical to that of a protein comprising the amino acid sequence of SEQ ID NO: 1, and a variant thereof or an active fragment thereof.
  • the amino acid sequence of SEQ ID NO: 1 may be encoded by the nucleotide sequence of SEQ ID NO: 2.
  • the D-threonine producing enzyme of the present invention may include any one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11,
  • the D-threonine-producing enzyme may be mutants or fragments of amino acids having different sequences by deletion, insertion, substitution, or a combination of amino acid residues within a range that does not affect the function of the protein. Amino acid exchange at the protein and peptide level that does not totally alter the activity of the D-threonine producing enzyme of the present invention is known in the art.
  • the present invention has an amino acid sequence that is substantially identical to a protein comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. Proteins and variants or active fragments thereof.
  • the term'X#Y' is clearly recognized in the art, and'#' represents a substituted position with respect to the amino acid number of the protein, and'X' is a wild-type protein. Represents an amino acid found at that position in the amino acid sequence of, and'Y' represents a substituted amino acid at that position.
  • the amino acid sequence of SEQ ID NO: 3 is that the 312th serine of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine (S312A).
  • the amino acid sequence of SEQ ID NO: 4 is that glycine at the 179th of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine (G179A).
  • the amino acid sequence of SEQ ID NO: 5 is one in which arginine at the 147th of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine (R147A).
  • amino acid sequence of SEQ ID NO: 9 arginine at the 147th of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, and glycine at the 179th is substituted with alanine (R147A, G179A).
  • the amino acid sequence of SEQ ID NO: 10 is that glycine at the 179th of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine and serine at the 312th is substituted with alanine (G179A, S312A).
  • the amino acid sequence of SEQ ID NO: 11 is that the 147th arginine of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, the 179th glycine is substituted with alanine, and the 312th serine is substituted with alanine (R147A, G179A, S312A). .
  • the D-threonine producing enzyme of the present invention can produce D-threonine and D-allotreonine, which is a diastereoisomer thereof.
  • the D-threonine producing enzyme of the present invention can produce D-threonine so that the proportion of D-threonine in the reaction product is 85% or more, and specifically 87.5% or more, 90% or more, 92% or more, 95% or more, It can be produced to be 97% or more, 97.5% or more, 98% or more, 98.5% or more, or 99% or more.
  • the D-threonine-producing enzyme can produce more D-threonine than D-allothreonine so that the diastereomeric excess (de) calculated by the following formula is 75% or more, and specifically de Is 77% or more, 79% or more, 80% or more, 82% or more, 85% or more, 87% or more, 90% or more, 92% or more, or 93% or more.
  • the gene encoding the D-threonine producing enzyme of the present invention is composed of any one nucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14 Can be.
  • the gene encoding the D-threonine-producing enzyme of the present invention and its mutant or active fragment thereof is variously modified in the coding region within a range that does not change the amino acid sequence of the enzyme and its mutant or active fragment thereof expressed from the coding region. This can be achieved, and various mutations can be made within a range that does not affect the expression of the gene even in portions other than the coding region, and such mutant genes are also included in the scope of the present invention.
  • the present invention consists of a nucleotide sequence substantially identical to a gene consisting of any one nucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14 It includes a gene and a fragment of the gene.
  • Genes consisting of the substantially identical nucleotide sequence means those having sequence homology of 80% or more, preferably 90% or more, and most preferably 95% or more, but are not limited thereto, and sequence homology of 80% or more. And, if the encoded protein has the same enzymatic activity, it is included in the present invention.
  • nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof. It is also included in the scope of the present invention.
  • nucleotide sequences may be single-stranded or double-stranded, and may be DNA molecules or RNA molecules.
  • Another aspect of the present invention provides a recombinant expression vector containing the gene of the D-threonine producing enzyme of the present invention and a transformant into which the recombinant expression vector has been introduced.
  • the recombinant expression vector of the present invention includes a gene encoding the D-threonine-producing enzyme, and thus can be usefully used as a vector capable of producing the D-threonine-producing enzyme.
  • the description of the D-threonine-producing enzyme is the same as described in the'new D-threonine-producing enzyme and its genes'.
  • the expression vector includes, but is not limited to, a plasmid vector, a cozmid vector, a bacteriophage vector, and a viral vector.
  • the recombinant expression vector contains an expression control sequence such as a promoter, a terminator, or an enhancer, or a sequence for secretion, depending on the type of host cell for which the D-threonine producing enzyme of the present invention is to be produced. It can be combined according to the purpose as appropriate.
  • the expression vector may further include a selection marker for selecting a host cell into which the vector has been introduced, and may include an origin of replication in the case of a replicable expression vector.
  • the recombinant expression vector may include a sequence for facilitating purification of the expressed protein, and specifically, the gene encoding the tag for separation and purification so as to be operable to the gene encoding the D-threonine producing enzyme of the present invention is Can be connected.
  • the tags for separation and purification may be used alone, such as GST, poly-Arg, FLAG, histidine-tag, c-myc, or two or more of them may be sequentially connected.
  • the gene encoding the D-threonine-producing enzyme can be cloned through the restriction enzyme cleavage site, and when the gene encoding the protein-cleaving enzyme recognition site is used in the vector, the gene and the frame of the D-threonine-producing enzyme are different. It is connected to fit (in frame), when the enzyme is obtained and then digested with a protein cleavage enzyme, the original form of D-threonine-producing enzyme can be prepared.
  • the recombinant expression vector may be prepared by inserting a gene encoding a D-threonine-producing enzyme into a plasmid vector pET28a(+), and in addition to pET28a(+) used in the preparation of the cloning vector, various vectors for prokaryotic cells or Since vectors for eukaryotic cells (pPIC and pPICZ, etc.) are known, various expression vectors other than the above vectors can be used depending on the purpose of expression.
  • the recombinant expression vector containing the gene encoding the D-threonine producing enzyme of the present invention is introduced.
  • the description of the D-threonine producing enzyme and its gene is the same as described in the'new D-threonine producing enzyme and its gene'.
  • a transformant can be prepared by transforming the recombinant expression vector according to the present invention into any suitable host cell selected from the group consisting of bacteria, yeast, E. coli, fungi, plant cells, and animal cells depending on the purpose of expression.
  • the host cell may be E. coli ( E. coli BL21(DE3), DH5 ⁇ , etc.) or yeast cells ( Saccharomyces genus, Pichia genus, etc.).
  • E. coli E. coli BL21(DE3), DH5 ⁇ , etc.
  • yeast cells Saccharomyces genus, Pichia genus, etc.
  • a known technique that is, a heat shock method, an electric shock method, or the like can be used.
  • the D-threonine-producing enzyme Since the protein expressed from the transformant is the D-threonine-producing enzyme of the present invention, the D-threonine-producing enzyme can be easily mass-produced by mass-culturing the transformant to express the gene.
  • Another aspect of the present invention provides a method for preparing a D-threonine-producing enzyme using a transformant into which a recombinant expression vector containing a gene encoding a D-threonine-producing enzyme of the present invention has been introduced.
  • the method for producing a D-threonine-producing enzyme of the present invention comprises the steps of culturing a transformant into which a recombinant expression vector containing a gene encoding the D-threonine-producing enzyme of the present invention has been introduced; And separating the D-threonine-producing enzyme from the culture of the transformant.
  • the method for preparing the D-threonine-producing enzyme may further include inducing expression of a gene encoding the D-threonine-producing enzyme in the cultured transformant.
  • a gene encoding a tag for separation and purification may be additionally linked to the N-terminus of the gene encoding the D-threonine-producing enzyme, thereby making it possible to obtain a D-threonine-producing enzyme.
  • a protein cleavage enzyme cleavage site may be additionally linked to the N-terminus, and thus, purification of a recombinant D-threonine-producing enzyme may be possible.
  • the cultivation of the transformant may be performed according to a known method, and conditions such as cultivation temperature, cultivation time, and pH of the medium may be appropriately adjusted.
  • the culture method may include batch culture, continuous culture, and fed-batch culture. The culture medium used must adequately meet the requirements of the specific strain.
  • Separation of the D-threonine-producing enzyme from the culture of the transformant may be performed through a method commonly performed in the art, such as centrifugation and filtration.
  • the D-threonine-producing enzyme isolated by the above method can be purified in a conventional manner, such as salting out (eg, ammonium sulfate precipitation, sodium phosphate precipitation), solvent precipitation (protein fraction precipitation using acetone, ethanol, etc.) , Dialysis, gel filtration, ion exchange, chromatography such as reverse phase column chromatography, ultrafiltration, and the like may be used alone or in combination to purify the enzyme of the present invention.
  • Another aspect of the present invention provides a composition for producing D-threonine for producing D-threonine with high purity.
  • the composition for producing D-threonine includes the enzyme for producing D-threonine of the present invention as an active ingredient.
  • the description of the D-threonine-producing enzyme is the same as described in the'new D-threonine-producing enzyme and its gene', and the D-threonine-producing enzyme has an activity capable of producing D-threonine with high purity, A composition containing the enzyme can be used for efficient production of D-threonine.
  • the composition for producing D-threonine may further include glycine and acetaldehyde, which are substrates for D-threonine producing enzymes.
  • the glycine and acetaldehyde may be included in a sufficient amount according to the amount of desired D-threonine, and the glycine and acetaldehyde are in a ratio of 1:1 to 10:1, such as a ratio of 2:1 to 8:1, 3 It may be included in a ratio of :1 to 5:1, or in a ratio of 4:1 to 4.5:1.
  • the composition may further contain a metal cation and/or pyridoxal-5-phosphate together with the D-threonine-producing enzyme.
  • the metal cation may be a divalent metal cation, and the divalent metal cation may be a manganese cation or a magnesium cation.
  • Another aspect of the present invention provides a method for producing D-threonine in vitro.
  • the method for producing D-threonine in vitro includes the step of reacting the D-threonine producing enzyme of the present invention with glycine and acetaldehyde.
  • the description of the D-threonine-producing enzyme is the same as described in the'new D-threonine-producing enzyme and its genes'.
  • the glycine and acetaldehyde may be injected from the outside, and the glycine and acetaldehyde must be injected in a sufficient amount according to the amount of the desired D-threonine.
  • the glycine and acetaldehyde may be reacted with the enzyme in a ratio of 1:1 to 10:1, for example, in a ratio of 2:1 to 8:1, a ratio of 3:1 to 5:1, or 4:1 to It can be reacted in a ratio of 4.5:1.
  • the injection of glycine and acetaldehyde may be performed continuously.
  • D-threonine may be continuously produced by the D-threonine producing enzyme.
  • the step of reacting the D-threonine-producing enzyme with glycine and acetaldehyde includes metal cation and/or pyridoxal-5-phosphate with the D-threonine-producing enzyme to enhance the activity of the D-threonine-producing enzyme. It may be to react by including more.
  • the metal cation may be a divalent metal cation, and the divalent metal cation may be a manganese cation or a magnesium cation.
  • the reacting step may be performed at a temperature of 20° C. to 55° C., for example, 22° C. to 45° C., or 25° C. to 40° C., but is not limited thereto.
  • the reaction may be carried out at a pH of 7 to 10, such as a pH of 8 to 9.5, or a pH of 8.5 to 9, but is not limited thereto.
  • the method for producing D-threonine in vitro may further include recovering D-threonine from the reaction product of the D-threonine producing enzyme, glycine and acetaldehyde.
  • Another aspect of the present invention provides a method for producing D-threonine in vivo.
  • the method for producing D-threonine in vivo includes culturing the transformant of the present invention in the presence of glycine and acetaldehyde.
  • the description of the transformant is the same as described in the'expression vector of the D-threonine-producing enzyme, the transformant, and a method for producing a D-threonine-producing enzyme using the same.'
  • the glycine and acetaldehyde In the step of culturing the transformant in the presence of glycine and acetaldehyde, the glycine and acetaldehyde must be present in a sufficient amount according to the amount of the desired D-threonine, and exist in a ratio of 1:1 to 10:1. And, for example, in a ratio of 2:1 to 8:1, in a ratio of 3:1 to 5:1, or in a ratio of 4:1 to 4.5:1.
  • the glycine and acetaldehyde may be artificially supplied from the outside.
  • glycine and acetaldehyde may be added and supplied together in a medium used for culturing the transformant, or an environment in which glycine and acetaldehyde are produced may be provided.
  • the environment in which glycine and acetaldehyde are produced may be provided by transforming genes encoding at least one enzyme involved in the production of glycine and acetalde
  • the cultivation of the transformant as described above may be performed according to an appropriate medium and culture conditions known in the art. Those of ordinary skill in the art can easily adjust and use the medium and culture conditions according to the type of host cell of the selected transformant.
  • the culture method may include a batch type, continuous type, fed-batch type, or a combination culture thereof.
  • the medium may contain various carbon sources, nitrogen sources, and trace element components.
  • the carbon source is, for example, glucose, sucrose, lactose, fructose, maltose, starch, carbohydrates such as cellulose, soybean oil, sunflower oil, castor oil, fats such as coconut oil, palmitic acid, stearic acid, fatty acids such as linoleic acid, Alcohols such as glycerol and ethanol, organic acids such as acetic acid, or combinations thereof.
  • the cultivation may be performed using glucose as a carbon source.
  • the nitrogen source is an organic nitrogen source and urea such as peptone, yeast extract, broth, malt extract, corn steep liquor (CSL), and soybean meal, an inorganic nitrogen source such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, or Combinations of these may be included.
  • the medium may include, for example, potassium dihydrogen phosphate and dipotassium hydrogen phosphate as a source of phosphorus, and a corresponding metal salt such as sodium-containing salt, magnesium sulfate, and iron sulfate.
  • amino acids, vitamins, and suitable precursors may be included in the medium.
  • the medium or individual components may be added to the culture medium in a batch or continuous manner.
  • the generation of air bubbles can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester during culture.
  • a metal cation and/or pyridoxal-5-phosphate may be further included in the medium to enhance the activity of the D-threonine-producing enzyme of the present invention prepared by culturing by the transformant.
  • the metal cation may be a divalent metal cation, and the divalent metal cation may be a manganese cation or a magnesium cation.
  • the cultivation of the transformant as described above may be carried out at 20 °C to 50 °C, for example, it may be carried out at 25 °C to 45 °C, or 30 °C to 40 °C.
  • the cultivation of the transformant is performed in a temperature range of less than 20° C. or more than 50° C., a sufficient amount of an intermediate product is not produced, and as a result, the production amount of D-threonine, which is a final product, may not be sufficient.
  • cultivation of the transformant as described above may be performed at pH 5 to pH 10, for example, pH 6 to pH 9, or pH 6.5 to pH 8, but is not limited thereto.
  • the pH conditions for culturing the transformants as described above can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the culture medium of the transformant.
  • the culture pH condition of the transformant is out of the above range, the growth of the transformant is inhibited, so that the expression of the D-threonine-producing enzyme decreases, and thus the production of D-threonine may be reduced.
  • the method for producing D-threonine in vivo may further include inducing expression of a gene encoding the D-threonine-producing enzyme in the cultured transformant.
  • the method for producing D-threonine in vivo may further include recovering D-threonine from the culture of the transformant.
  • the gene of the nucleotide sequence of SEQ ID NO: 2 was synthesized by requesting Bioneer, and a primer was designed for amplifying the D-threonine sequence using the synthesized gene as a template.
  • the nucleotide sequence of SEQ ID NO: 2 was amplified by performing PCR using a pair of forward primers (5'-tggtgccgcgcggcagccatatgcgcgcaccagcacggct-3') and reverse primers (5'-ggtggtggtggtggtgctcgagctagaacacacacaacctcgcgc-3').
  • the PCR product containing the nucleotide sequence of SEQ ID NO: 2 amplified as described above using Gibson assembly (New England Biolabs, USA) was used for vector amplification forward sequence (5'-gcgcgaggttgtgtgttctagctcgagcaccaccaccaccacc-3') and reverse sequence (5' -agccgtgctggtgcgcgcatatggctgccgcgcggcacca-3') was inserted into the multiple cloning site of the amplified plasmid vector pET28a(+) (Novagen, USA).
  • Example [1-1] The transformants stored frozen in Example [1-1] were pre-inoculated in 3 ml of LK solid medium (LB medium + 2 ⁇ g/ml kanamycin) and cultured at 37° C. for 10 hours or longer.
  • LK solid medium LB medium + 2 ⁇ g/ml kanamycin
  • One colony appearing in the solid medium was inoculated into a test tube containing 3 ml of LK solid medium through colony separation, and seed culture was performed for 12 hours in a shaking incubator at 37°C.
  • 2 ml of the seed cultured culture solution was added to a 1,000 ml flask containing 200 ml of LK medium to perform main culture, and the final concentration when the absorbance at 600 nm was 0.6 was 0.1 mM.
  • the culture medium of the transformant in which the overexpression of the protein having the amino acid sequence of SEQ ID NO: 1 is induced was dispensed into 50 ml of a conical tube (SPL Life Sciences Co., Ltd., Korea), and To the pellet from which the supernatant was separated by centrifugation at 3,000 rpm for 20 minutes at 4°C, 10 ml of Profinia 1X Lysis buffer (Bio-Rad Laboratories, USA) was added, and sonication (sonication) By disrupting the cells by the method, a cell lysate of the transformant was obtained.
  • the cell lysate obtained as described above was centrifuged again at 14,000 rpm for 20 minutes at 4° C. to obtain a supernatant, and high-speed protein liquid chromatography equipped with an IMAC Kit ® His tag adsorption column (Bio-Rad Laboratories, USA)
  • a protein having an overexpressed amino acid sequence of SEQ ID NO: 1 was isolated from the supernatant obtained as described above using Fast Protein Liquid Chromatography (Bio-Rad Laboratories, USA).
  • the thus-separated protein was mixed with a 4X protein dye mixed with 100 ⁇ l of 2-mercaptoethanol and 900 ⁇ l of Laemmli Sample Buffer (#1610747, Bio-Rad Laboratories, USA) at a ratio of 3:1. , Put in boiling water and heated for 10 minutes.
  • the protein is denatured and has a linear amino acid sequence, so it can be separated according to its molecular weight through SDS-PAGE. It was confirmed that the protein having the amino acid sequence of SEQ ID NO: 1 of 40.88 kDa to which His-Tag is attached was overexpressed by the above-described series of processes, and purified and separated from the cell lysate of the transformant (Fig. 1A). .
  • the protein having the amino acid sequence of SEQ ID NO: 1 purified and isolated through Example 1 has an activity as a'D-threonine producing enzyme' as shown in the following scheme, and the activity was confirmed by reacting glycine and acetaldehyde. .
  • Example [1-2] 0.01 mg/ml of the protein having the amino acid sequence of SEQ ID NO: 1 purified and isolated in Example [1-2] was mixed with 100 mM glycine and 100 mM acetaldehyde, and 100 ⁇ M pyridoxal-5. -Added to 50 mM CHES buffer (pH 9.0) containing phosphoric acid (pyridoxal-5-phosphate, PLP) and 1 mM MnCl 2 , reacted at 37° C. for 10 minutes, and then at 100° C. for 10 minutes. After the reaction was terminated by boiling water, the concentration of D-threonine was measured in the buffer solution at which the reaction was completed.
  • phosphoric acid pyridoxal-5-phosphate
  • the generated D-threonine was analyzed through HPLC after OPA/NAC pretreatment (OPA/NAC derivatization).
  • OPA o-phthalaldelhyde
  • NAC N-acetylcysteine
  • Example 2 since the protein having the amino acid sequence of SEQ ID NO: 1 acts as a D-threonine-producing enzyme, conditions affecting its activity were identified, and the protein having the amino acid sequence of SEQ ID NO: 1 Optimal activity conditions were derived.
  • Example [1-2] Mg/ml, 100 mM glycine, 100 mM acetaldehyde, and 100 ⁇ M pyridoxal-5-phosphate in 50 mM CHES buffer (pH 9.0) containing 1 mM EDTA or metal cations (Mn 2+ , Mg 2+ ). , Co 2+ , Zn 2+ ) and reacted at 35° C. for 10 minutes, followed by bathing at 100° C. for 10 minutes to terminate the reaction. And in the same manner as in Example 2, the concentrations of D-threonine and D-allothreonine were measured in the buffer solution in which the reaction was terminated as described above, and their relative values were compared.
  • the activity of the D-threonine-producing enzyme was improved when manganese cation and magnesium cation were added among various types of metal cations, and among them, when manganese cation was used. It was confirmed that the activity of the D-threonine-producing enzyme was the most excellent (Fig. 3A).
  • Example [1-2] In order to confirm the effect of the concentration of metal cation on the enzyme activity of the protein having the amino acid sequence of SEQ ID NO: 1, 0.01 mg of the protein having the amino acid sequence of SEQ ID NO: 1 purified and isolated in Example [1-2] /Ml, 100 mM glycine, 100 mM acetaldehyde, and 100 ⁇ M pyridoxal-5-phosphate in 50 mM CHES buffer solution (pH 9.0) with manganese cations 0 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1 Mm, 2.5 mM, and 5 mM were added respectively and reacted at 35° C. for 10 minutes, followed by bathing at 100° C. for 10 minutes to terminate the reaction. And in the same manner as in Example 2, the concentrations of D-threonine and D-allothreonine were measured in the buffer solution in which the reaction was terminated as described above, and their relative values were compared.
  • the protein having the amino acid sequence of SEQ ID NO: 1 exhibits the activity of the D-threonine-producing enzyme only in an environment in which a manganese cation is present (FIG. 3B).
  • Example [1-2] 100 mM of glycine and 100 mM of acetaldehyde were added to 50 mM CHES buffer (pH 9.0) containing 100 ⁇ M of pyridoxal-5-phosphoric acid and 1 mM of Mn 2+, and then at 25°C to 55°C. The reaction was performed at a temperature for 10 minutes, and a bath was performed at 100° C. for 10 minutes to terminate the reaction. And, in the same manner as in Example 2, the concentrations of D-threonine and D-allotreonine were measured in the buffer solution in which the reaction was terminated as described above, and their relative values were compared.
  • the enzymatic reaction was performed at a temperature of 35° C. for 10 minutes, and then bathed at 100° C. for 10 minutes to terminate the reaction. And in the same manner as in Example 2, the concentrations of D-threonine and D-allothreonine were measured in the buffer solution in which the reaction was terminated as described above, and their relative values were compared.
  • Example [1-2] 0 ⁇ M, 0.1 ⁇ M of pyridoxal-5-phosphate in 50 mM CHES buffer solution (pH 9.0) containing 0.01 mg/ml of protein having the sequence, 100 mM glycine, 100 mM acetaldehyde, and 1 mM Mn 2+, 0.5 ⁇ M, 2.5 ⁇ M, 5 ⁇ M, 25 ⁇ M, 50 ⁇ M, 100 ⁇ M, and 200 ⁇ M were added respectively to react at 35° C.
  • Example 3 As confirmed in Example 3, as it was confirmed that the protein having the amino acid sequence of SEQ ID NO: 1 has high activity as a D-threonine-producing enzyme, the enzyme was improved for the purpose of specifically synthesizing D-threonine. I did.
  • residues predicted to be involved in diastereomer changes in the active site in the crystal structure of the protein enzyme having the amino acid sequence of SEQ ID NO: 1 were selected, and the ratio of the activity to the diastereomer by substituting the residues with alanine Changes were analyzed.
  • residues that improve stereospecificity for D-threonine were derived, and it was confirmed that the stereospecificity for D-threonine was improved to 99% or more.
  • the protein structure having the amino acid sequence of SEQ ID NO: 1 was obtained based on the previously reported D-threonine aldolase X-ray crystal structure derived from Alcaligenes xylosoxidans (FIG. 7), and was determined. At the active site of the structure, amino acid residues that interact with substrates (glycine and acetaldehyde), pyridoxal-5-phosphate and manganese ions at a distance of 4 angstroms were selected.
  • each primer was designed based on the nucleotide sequence of SEQ ID NO: 2 and synthesized by requesting Macrogen.
  • the DNA containing the nucleotide sequence of SEQ ID NO: 2 is used as a template, and each PCR product amplified by performing PCR using each of the primer pairs is ligated using Phusion, Q5, and KOD polymerases.
  • Each of the ends of the nucleotide sequence was ligated by the kination method, and through sequencing (Macrogen, Inc.), it was confirmed that the corresponding residues were accurately substituted with nucleotide sequences encoding amino acids such as alanine.
  • the corresponding nucleotide sequences were transformed into E. coli C2566 strain (Novagen, USA), respectively, and stored frozen by adding a 20% glycerin solution.
  • Example [4-1] the transformants frozen in Example [4-1] were pre-inoculated in 3 ml of LK solid medium (LB medium + kanamycin 25 ⁇ g/ml) and at 37°C. Incubated for more than 10 hours.
  • One colony appearing in the solid medium was inoculated into a test tube containing 3 ml of LK solid medium in the process of separating a single colony, and seed culture was performed for 12 hours in a shaking incubator at 37°C.
  • 2 ml of the seed cultured culture solution was added to a 1,000 ml flask containing 200 ml of LK medium to perform main culture, and the final concentration when the absorbance at 600 nm was 0.6 was 0.1 mM.
  • IPTG isopropyl-1-thio- ⁇ -D-galactopyranoside
  • the stirring speed was adjusted to maintain 200 rpm and the culture temperature at 37°C, and after the addition of IPTG, the stirring speed was adjusted to 150 rpm and the culture temperature to 18°C, followed by incubation for 20 hours.
  • the culture medium of the transformant in which the overexpression of each of the single substituted proteins was induced was dispensed into a 50 ml conical tube (SPL Life Sciences Co., Ltd., Korea), and then at 4° C. at 3,000 rpm. By centrifuging for 20 minutes to separate the supernatant, 10 ml of Profinia 1X Lysis buffer (Bio-Rad Laboratories, USA) was added, and the cells were disrupted by sonication. , To obtain a cell lysate of the transformant (cell lysate).
  • the cell lysate obtained as described above was centrifuged again at 14,000 rpm for 20 minutes at 4° C. to obtain a supernatant, and high-speed protein liquid chromatography equipped with an IMAC Kit ® His tag adsorption column (Bio-Rad Laboratories, USA) The single substituted proteins overexpressed were isolated from the supernatant using Fast Protein Liquid Chromatography (Bio-Rad Laboratories, USA).
  • Example [4-1] The activity of each of the single substituted proteins obtained in Example [4-1] and the diastereomer ratio change of the reaction product catalyzed by them were confirmed.
  • 0.01 mg/ml of the proteins obtained in Example [4-1] were mixed with 100 mM glycine and 100 mM acetaldehyde, and 100 ⁇ M of pyridoxal-5-phosphate (pyridoxal-5).
  • -phosphate, PLP) and 1 mM MnCl 2 were added to 50 mM CHES buffer (pH 9.0), and reacted at 37° C. for 10 minutes. After the reaction was terminated by bathing at 100° C. for 10 minutes, the concentration of D-threonine was measured in the buffer solution at which the reaction was completed.
  • the amount of D-threonine produced was analyzed through HPLC after OPA/NAC pretreatment (OPA/NAC derivatization).
  • OPA o-phthalaldelhyde
  • NAC N-acetylcysteine
  • the mobile phase A and the mobile phase B were maintained at a ratio of 3:7 for 0 to 3 minutes, and the mobile phase A and mobile phase B at a ratio of 7:3 for 3 to 10 minutes.
  • commercially available D-threonine Sigma-aldrich, USA
  • D-allotreonine TOKYO CHEMICAL INDUSTRY CO., LTD., Japan
  • R147A, G179A, and S312A having the amino acid sequence of SEQ ID NO: 5, 4, and 3 have lower specific activity compared to the wild-type having the amino acid sequence of SEQ ID NO: 1, but the diastereomer excess (de) is wild-type 55.4 Compared to %, it was confirmed that it was significantly improved to 79.5%, 93.2%, and 79.5%, respectively. Therefore, it was confirmed that R147, G179, and S312 are residues involved in the diastereomer ratio change.
  • the DNA containing the nucleotide sequence of SEQ ID NO: 2 is used as a template, and each PCR product amplified by performing PCR using each of the primer pairs is ligated using Phusion, Q5, and KOD polymerases. Each end of the nucleotide sequence was ligated by the kination method, and through sequencing (Macrogen, Inc.), it was confirmed that the corresponding residues were correctly substituted with nucleotide sequences encoding amino acids of lysine, glutamine, valine, leucine, and threonine. . The corresponding nucleotide sequences were transformed into E. coli C2566 strain (Novagen, USA), respectively, and stored frozen by adding a 20% glycerin solution.
  • PCR products amplified through PCR as described above were linked to the ends of the nucleotide sequence by ligation/kination method using Phusion, Q5, and KOD polymerases, respectively, and through nucleotide sequencing (Macrogen Co., Ltd.), the corresponding It was confirmed that the residues were correctly substituted with nucleotide sequences encoding multiple alanines.
  • the corresponding nucleotide sequences were transformed into E. coli C2566 strain (Novagen, USA), respectively, and stored frozen by adding a 20% glycerin solution.
  • the protein was prepared in the same manner as in Examples [4-2] and [4-3]. Expression and purification were confirmed (FIG. 12), and enzyme reactions and analysis were performed and compared with specific activity. As a result, it was confirmed that when both amino acid residues G179 and S312 were substituted with alanine, the diastereomer excess rate (d.e.) increased to 99.4%, thereby improving the diastereomer specificity (FIG. 13). Therefore, when both G179 and S312 amino acids were substituted with alanine, it was confirmed that D-threonine-specific production activity was improved.
  • Example [4-3] 0.01 mg/ml of wild-type, R147A, G179A, and S312A enzymes each having the purified amino acid sequence of SEQ ID NOs: 1, 5, 4, 3 were added to 50 mM CHES (pH 9.0. ), 0.1 mM pyridoxal-5-phosphate, 1 mM manganese ion, and reacted under the conditions of 35° C., and 0, 1, 2.5, 5, 10, 25, 50, and 100 mM of D-threonine or D-allo When reacted with threonine for 10 minutes, the degree of decomposition thereof was measured by HPLC.
  • Example [4-2] 50 mM CHES buffer solution (pH 9.0) containing 100 ⁇ M of -5-phosphate and 1 mM Mn 2+ was reacted at 35° C. for 15, 30, 60, 90, 120, 150, and 180 minutes. And in order to terminate the reaction, the reaction solution was bathed at 100° C. for 10 minutes, and through HPLC analysis via OPA/NAC derivatization, which is the same method as in Example [4-3], D-threonine and D- The concentration of allothreonine was measured and its relative values were compared.
  • D-threonine was specifically produced in the enzyme G179A and the enzyme G179A+S312A having the amino acid sequences of SEQ ID NOs: 4 and 10 compared to the wild type having the amino acid sequence of SEQ ID NO: 1. It was confirmed that the enzyme G179A + S312A having the amino acid sequence of produced 46 mM D-threonine at a ratio of 95% diastereomeric excess in 60 minutes (FIG. 16).
  • the enzymes having the amino acid sequences of SEQ ID NOs: 4 and 10 of the present invention each produced 16.4 mM and 18.7 mM D-threonine in 10 minutes, respectively, 95% and 98% diastereomers. It can be specifically synthesized with an excess of isomers.
  • the enzyme having the amino acid sequence of SEQ ID NO: 10 (G179A+S312A) is capable of synthesizing D-threonine with the same level of purity as commercially available D-threonine. -It has the best specificity for threonine.

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Abstract

The present invention relates to a novel enzyme for producing D-threonine. When reactions are carried out by using the enzyme of the present invention, the ratio of diastereomer D-allo-threonine, which can be produced together with D-threonine, is lowered and higher purity D-threonine can be obtained.

Description

신규 D-트레오닌 생산 효소 및 이를 이용한 D-트레오닌의 입체특이적 생산 방법New D-threonine-producing enzyme and method for stereospecific production of D-threonine using the same
본 발명은 신규한 D-트레오닌 생산 효소에 관한 것이다.The present invention relates to a novel D-threonine producing enzyme.
자연계에 존재하는 아미노산들은 대부분 광학활성을 나타내는 알파탄소를 가지고 있으며 이의 입체특이성에 따라 L-아미노산과 D-아미노산으로 나뉜다. 자연계에 존재하는 대부분의 단백질은 L-아미노산으로 구성되어 있으나, 예외적으로 미생물의 펩티도글리칸이나 펩티드계 항생물질의 구성성분 및 고등식물의 생리활성물질의 구성성분 등은 D-아미노산으로 구성되어 있다.Most of the amino acids in nature have alpha carbons that exhibit optical activity, and are divided into L-amino acids and D-amino acids according to their stereospecificity. Most of the proteins in nature are composed of L-amino acids, but exceptionally, the components of microbial peptidoglycans, peptide-based antibiotics, and physiologically active substances of higher plants are composed of D-amino acids. have.
한편, 현재까지의 연구에 의하면, 상기와 같은 D-아미노산 중에서도 D-트레오닌은 신경전달물질, 백신, 합성감미료, 항생제, 호르몬 등의 생리활성물질을 합성하는 중간물질 또는 전구체로서 식품과 의약분야에서 널리 이용되고 있으며, 이에 D-트레오닌을 생산하기 위한 방법들이 개발되어 왔다.Meanwhile, according to studies to date, among the D-amino acids described above, D-threonine is an intermediate or precursor for synthesizing physiologically active substances such as neurotransmitters, vaccines, synthetic sweeteners, antibiotics, and hormones. It is widely used, and thus, methods for producing D-threonine have been developed.
D-트레오닌을 생산하는 방법은 크게 화학적 합성법, 그리고 효소나 미생물을 이용하는 생물학적 합성법으로 나눌 수 있다. 화학적 합성법에 의한 트레오닌 생산의 경우, D-트레오닌 및 L-트레오닌이 혼합된 상태(racemic mixture)로 생성물이 얻어지기 때문에, 순수한 D-트레오닌을 얻기 위해서는 또 다른 복잡한 광학적 순수화 과정을 거쳐야 하는 어려움이 있다. 뿐만 아니라, 상기와 같은 화학적 합성 방법은 그 과정에서 발생하는 부산물들로 인해 심각한 환경오염이 유발되는 문제도 있다. Methods of producing D-threonine can be largely divided into chemical synthesis methods and biological synthesis methods using enzymes or microorganisms. In the case of threonine production by chemical synthesis, since the product is obtained as a racemic mixture of D-threonine and L-threonine, it is difficult to undergo another complicated optical purification process to obtain pure D-threonine. have. In addition, the chemical synthesis method as described above also has a problem that serious environmental pollution is caused due to by-products generated in the process.
생물학적인 합성법에 의한 트레오닌 생산의 경우, 상기와 같은 다단계의 합성단계의 필요성 문제나 환경오염의 문제는 없으나, D-트레오닌을 특이적으로 생산할 수 있는 종래 알려진 D-트레오닌 생산 효소들은 그 활성이 적고 D-트레오닌의 생산성이 낮아 산업적으로 이용하기에 불충분한 문제점이 있다. 또한, D-트레오닌과 함께 D-알로트레오닌(D-allothreonine)이 거의 1:1의 비율로 혼합된 상태의 생성물을 생산하는 문제점이 있어, 순수한 D-트레오닌만의 생산이 어려운 단점이 있다.In the case of threonine production by biological synthesis, there is no problem of necessity or environmental pollution of the multi-step synthesis step as described above, but the previously known D-threonine-producing enzymes capable of specifically producing D-threonine have little activity. Due to the low productivity of D-threonine, there is a problem that is insufficient for industrial use. In addition, there is a problem in producing a product in a state in which D-allothreonine and D-allothreonine are mixed in a ratio of almost 1:1, and thus it is difficult to produce only pure D-threonine.
이에, 화학적 합성법을 대체하여 입체특이적으로 D-트레오닌을 환경오염 없이 생산할 수 있으면서도, 활성 및 생산량이 높을 뿐만 아니라 D-알로트레오닌과 같은 불순물의 비율을 획기적으로 감소시켜 순수한 D-트레오닌을 생산할 수 있는 신규한 D-트레오닌 생산 효소의 연구와 개발이 더욱 필요한 실정이다.Therefore, by replacing the chemical synthesis method, D-threonine can be produced stereospecifically without environmental pollution, while not only having high activity and production, but also dramatically reducing the ratio of impurities such as D-allothreonine to produce pure D-threonine. Research and development of a novel D-threonine-producing enzyme is still in need.
본 발명은 L-트레오닌과 D-트레오닌의 혼합물이 아닌 D-트레오닌만을 입체특이적으로 생산할 수 있으면서도, D-알로트레오닌이 함께 생산되는 종래의 문제점을 극복하여 높은 순도로 D-트레오닌을 생산할 수 있는 특징이 있는 신규한 효소와 이를 이용한 D-트레오닌의 생산 방법을 제공하는 것을 목적으로 한다.The present invention is capable of stereospecific production of only D-threonine, not a mixture of L-threonine and D-threonine, while overcoming the conventional problem that D-allotreonine is produced together, thereby producing D-threonine with high purity. An object of the present invention is to provide a novel enzyme with characteristics and a method for producing D-threonine using the same.
상기의 목적을 달성하기 위하여, 본 발명의 일 측면은 필로마이크로비움 마리넘(Filomicrobium marinum) 유래 D-트레오닌 알돌라아제(D-threonine aldolase)의 147 번째 아르기닌(arginine), 179 번째 글리신(glycine) 및 312 번째 세린(serine)으로 이루어진 군으로부터 선택되는 하나 이상의 아미노산이 알라닌(alanine)으로 치환된, D-트레오닌 생산 효소를 제공한다.In order to achieve the above object, an aspect of the present invention is the 147th arginine, 179th glycine of D-threonine aldolase derived from Filomicrobium marinum. And it provides a D-threonine-producing enzyme in which at least one amino acid selected from the group consisting of 312th serine is substituted with alanine.
또한, 상기의 목적을 달성하기 위하여, 본 발명의 다른 측면은 상기 D-트레오닌 생산 효소를 암호화하는 유전자를 제공한다.In addition, in order to achieve the above object, another aspect of the present invention provides a gene encoding the D-threonine producing enzyme.
또한, 상기 목적을 달성하기 위하여, 본 발명의 또 다른 측면은 상기 유전자를 포함하는 재조합 발현벡터 및 상기 발현벡터가 숙주세포에 도입된 형질전환체를 제공한다.In addition, in order to achieve the above object, another aspect of the present invention provides a recombinant expression vector containing the gene and a transformant in which the expression vector is introduced into a host cell.
또한, 상기 목적을 달성하기 위하여, 본 발명의 또 다른 측면은 상기 형질전환체를 배양하는 단계; 및 상기 형질전환체의 배양물로부터 D-트레오닌 생산 효소를 분리하는 단계;를 포함하는 D-트레오닌 생산 효소의 제조 방법을 제공한다.In addition, in order to achieve the above object, another aspect of the present invention comprises the steps of culturing the transformant; And separating the D-threonine-producing enzyme from the culture of the transformant.
또한, 상기 목적을 달성하기 위하여, 본 발명의 또 다른 측면은 상기 D-트레오닌 생산 효소를 유효성분으로 포함하는 D-트레오닌 생산용 조성물, 및 상기 D-트레오닌 생산 효소를 글리신 및 아세트알데히드와 함께 반응시키는 단계;를 포함하는, 인 비트로(in vitro)에서의 D-트레오닌 생산 방법을 제공한다.In addition, in order to achieve the above object, another aspect of the present invention is a composition for producing D-threonine comprising the D-threonine-producing enzyme as an active ingredient, and the D-threonine-producing enzyme reacted with glycine and acetaldehyde. It provides a method for producing D-threonine in an in vitro containing;
아울러, 상기 목적을 달성하기 위하여, 본 발명의 또 다른 측면은 상기 형질전환체를 글리신 및 아세트알데히드의 존재 하에서 배양하는 단계를 포함하는, 인 비보(in vivo)에서의 D-트레오닌 생산 방법을 제공한다.In addition, in order to achieve the above object, another aspect of the present invention provides a method for producing D-threonine in vivo, comprising culturing the transformant in the presence of glycine and acetaldehyde. do.
본 발명의 D-트레오닌 생산 효소는 글리신과 아세트알데히드를 기질로 D-트레오닌으로 전환함에 있어, 함께 생산될 수 있는 D-알로트레오닌의 비율과 생산량을 감소시켜, D-트레오닌을 높은 순도로 생산할 수 있는 효과가 있다.The D-threonine-producing enzyme of the present invention converts glycine and acetaldehyde into D-threonine as substrates, reducing the ratio and production amount of D-allothreonine that can be produced together, thereby producing D-threonine with high purity. There is an effect.
도 1은 형질전환체에서 과발현되어 정제 및 분리된, 서열번호 1의 아미노산 서열을 갖는 단백질의 분자 질량 특성을 SDS-PGAE(A) 및 젤-여과 크로마토그래피(Gel-filtration chromatography)(B)로 분석한 결과이다.1 is a molecular mass characteristic of a protein having an amino acid sequence of SEQ ID NO: 1, overexpressed in a transformant, purified and isolated, by SDS-PGAE (A) and gel-filtration chromatography (B). This is the result of analysis.
도 2는 서열번호 1의 아미노산 서열을 갖는 단백질에 의한 D-트레오닌 전환 정도를 나타낸 그래프이다.2 is a graph showing the degree of conversion of D-threonine by a protein having the amino acid sequence of SEQ ID NO: 1.
도 3은 서열번호 1의 아미노산 서열을 갖는 단백질의 효소 활성에 영향을 미치는 금속 양이온의 종류(A)와 금속 양이온의 농도(B)를 확인한 그래프이다.3 is a graph confirming the type (A) of metal cations and the concentration (B) of metal cations affecting the enzyme activity of the protein having the amino acid sequence of SEQ ID NO: 1. FIG.
도 4는 온도가 서열번호 1의 아미노산 서열을 갖는 단백질의 효소 활성에 미치는 영향을 확인한 그래프이다. 4 is a graph confirming the effect of temperature on the enzyme activity of the protein having the amino acid sequence of SEQ ID NO: 1.
도 5는 pH가 서열번호 1의 아미노산 서열을 갖는 단백질의 효소 활성에 미치는 영향을 확인한 그래프로, 완충 용액으로 MOPS(pH 6.5 내지 7.5), HEPES(pH 7.5 내지 8.0), EPPS(pH 8.0 내지 8.5) 및 CHES(pH 8.5 내지 10) 완충 용액을 각각 이용하여 확인한 것이다.Figure 5 is a graph confirming the effect of pH on the enzyme activity of the protein having the amino acid sequence of SEQ ID NO: 1, MOPS (pH 6.5 to 7.5), HEPES (pH 7.5 to 8.0), EPPS (pH 8.0 to 8.5) as a buffer solution. ) And CHES (pH 8.5 to 10) buffer solutions, respectively.
도 6은 피리독살-5-인산(PLP)이 서열번호 1의 아미노산 서열을 갖는 단백질의 효소 활성에 미치는 영향을 확인한 그래프이다.6 is a graph confirming the effect of pyridoxal-5-phosphate (PLP) on the enzyme activity of the protein having the amino acid sequence of SEQ ID NO: 1.
도 7은 서열번호 1의 아미노산 서열을 갖는 단백질 효소의 X-ray 결정구조(A)와 활성부위에서 부분입체 이성질체 비율 변화에 관여할 것으로 예측되는 잔기(B)를 나타내는 사진이다.7 is a photograph showing the X-ray crystal structure (A) of the protein enzyme having the amino acid sequence of SEQ ID NO: 1 and the residue (B) predicted to be involved in the diastereomer ratio change at the active site.
도 8은 서열번호 1의 아미노산 서열을 갖는 야생형 단백질(Wild-type)과 서열번호 49 내지 69의 아미노산 서열을 갖는 각 단백질을 SDS-PAGE로 분석한 결과이다. (각각의 치환 단백질의 명칭을 'X#Y'라고 할 때 '#'는 단백질의 아미노산 번호와 관련하여 치환된 위치를, 'X'는 야생형 단백질의 아미노산 서열의 그 위치에서 발견되는 아미노산을, 'Y'는 그 위치에서의 치환된 아미노산을 의미하며, 이하 동일하다.)8 is a result of SDS-PAGE analysis of the wild-type protein having the amino acid sequence of SEQ ID NO: 1 (Wild-type) and each protein having the amino acid sequence of SEQ ID NOs: 49 to 69. (When the name of each substituted protein is'X#Y','#' is the substituted position in relation to the amino acid number of the protein,'X' is the amino acid found at that position in the amino acid sequence of the wild-type protein, 'Y' means a substituted amino acid at that position, and is the same hereinafter.)
도 9는 서열번호 1의 아미노산 서열을 갖는 야생형 단백질(WT), 상기 서열번호 1의 아미노산 잔기 하나를 다른 것으로 치환한 각 단백질의 상대 특이 활성도와 부분입체 이성질체(D-트레오닌 및 D-알로트레오닌) 비율 변화를 확인한 그래프이다. 9 is a wild-type protein (WT) having the amino acid sequence of SEQ ID NO: 1, the relative specific activity and diastereomers (D-threonine and D-allotreonine) of each protein in which one amino acid residue of SEQ ID NO: 1 is replaced with another. This is a graph confirming the rate change.
도 10은 서열번호 1의 아미노산 서열을 갖는 야생형 단백질(Wild-type), 알라닌으로 치환된 서열번호 3, 4, 5, 및 알라닌 외 다른 아미노산으로 치환된 서열을 갖는 각 단백질을 SDS-PAGE로 확인한 결과이다.Figure 10 is a wild-type protein having the amino acid sequence of SEQ ID NO: 1 (Wild-type), SEQ ID NOs: 3, 4, 5 substituted with alanine, and each protein having a sequence substituted with an amino acid other than alanine was confirmed by SDS-PAGE. It is the result.
도 11은 서열번호 1의 아미노산 서열을 갖는 야생형 단백질(Wild-type), 알라닌으로 치환된 서열번호 3, 4, 5, 및 알라닌 외 다른 아미노산으로 치환된 서열을 갖는 각 단백질의 상대 특이 활성도와 부분입체 이성질체(D-트레오닌 및 D-알로트레오닌) 비율 변화를 확인한 그래프이다.Figure 11 is a wild-type protein having the amino acid sequence of SEQ ID NO: 1 (Wild-type), SEQ ID NOs: 3, 4, 5 substituted with alanine, and relative specific activity and portions of each protein having a sequence substituted with an amino acid other than alanine This is a graph confirming the change in the ratio of stereoisomers (D-threonine and D-allotreonine).
도 12는 서열번호 1의 아미노산 서열을 갖는 야생형 단백질(Wild-type)과 다중 알라닌 치환이 도입된 서열번호 9 내지 11의 아미노산 서열을 갖는 각 단백질을 SDS-PAGE로 확인한 결과이다.FIG. 12 is a result of confirming by SDS-PAGE a wild-type protein having the amino acid sequence of SEQ ID NO: 1 (Wild-type) and each protein having the amino acid sequence of SEQ ID NOs: 9 to 11 into which multiple alanine substitutions have been introduced.
도 13은 서열번호 1의 아미노산 서열을 갖는 야생형 단백질(Wild-type)과 다중 알라닌 치환이 도입된 서열번호 9 내지 11의 아미노산 서열을 갖는 각 단백질의 상대 특이 활성도와 부분입체 이성질체(D-트레오닌 및 D-알로트레오닌) 비율 변화를 확인한 그래프이다.13 shows the relative specific activity and diastereomers of each protein having the amino acid sequence of SEQ ID NOs: 9 to 11 into which a wild-type protein having the amino acid sequence of SEQ ID NO: 1 (Wild-type) and multiple alanine substitutions have been introduced (D-threonine and This is a graph confirming the change in the ratio of D-Allothreonine).
도 14의 A는 서열번호 1(WT), B는 서열번호 4(G179A), C는 서열번호 10(G179A+S312A)의 아미노산 서열을 갖는 단백질들에 대해 기질인 글리신의 비율이 D-트레오닌 생산량에 미치는 영향을 확인한 그래프이다.In Figure 14, A is SEQ ID NO: 1 (WT), B is SEQ ID NO: 4 (G179A), C is SEQ ID NO: 10 (G179A + S312A), the ratio of the substrate glycine to the proteins having the amino acid sequence of D-threonine production This is a graph confirming the effect on
도 15의 A는 서열번호 1(WT), B는 서열번호 4(G179A), C는 서열번호 10(G179A+S312A)의 아미노산 서열을 갖는 단백질들에 대해 기질인 아세트알데히드의 비율이 D-트레오닌 생산량에 미치는 영향을 확인한 그래프이다.In Figure 15, A is SEQ ID NO: 1 (WT), B is SEQ ID NO: 4 (G179A), C is the ratio of acetaldehyde as a substrate to the proteins having the amino acid sequence of SEQ ID NO: 10 (G179A + S312A) is D-threonine This is a graph confirming the effect on production.
도 16의 A는 서열번호 1(WT), B는 서열번호 4(G179A), C는 서열번호 10(G179A+S312A)의 아미노산 서열을 갖는 단백질들을 최적 기질조건에서 반응시켜 D-트레오닌 및 D-알로트레오닌의 생산량을 비교한 그래프이다.In FIG. 16A is SEQ ID NO: 1 (WT), B is SEQ ID NO: 4 (G179A), C is SEQ ID NO: 10 (G179A + S312A) by reacting proteins having the amino acid sequence of the amino acid sequence under optimal substrate conditions, D-threonine and D- This is a graph comparing the production of allotreonine.
먼저, 본 발명의 명세서에서 이용된 용어를 설명한다.First, terms used in the specification of the present invention will be described.
본 발명의 "D-트레오닌 생산 효소"는 하기 반응식과 같이 글리신과 아세트알데히드로부터 D-트레오닌을 생산하는 활성을 가진 효소를 의미한다.The "D-threonine-producing enzyme" of the present invention means an enzyme having the activity of producing D-threonine from glycine and acetaldehyde as shown in the following scheme.
[반응식][Reaction Scheme]
Figure PCTKR2020014292-appb-I000001
Figure PCTKR2020014292-appb-I000001
이하, 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
1. 신규 D-트레오닌 생산 효소 및 그 유전자1. New D-threonine-producing enzyme and its gene
본 발명의 일 측면은 필로마이크로비움 마리넘(Filomicrobium marinum) 유래 D-트레오닌 알돌라아제(D-threonine aldolase)의 147 번째 아르기닌(arginine), 179 번째 글리신(glycine) 및 312 번째 세린(serine)으로 이루어진 군으로부터 선택되는 하나 이상의 아미노산이 알라닌(alanine)으로 치환된, D-트레오닌 생산 효소를 제공한다.One aspect of the present invention is the 147th arginine, 179th glycine and 312th serine of D-threonine aldolase derived from Filomicrobium marinum. It provides a D-threonine-producing enzyme in which at least one amino acid selected from the group consisting of alanine is substituted.
예를 들어, 상기 D-트레오닌 생산 효소는 상기 D-트레오닌 알돌라아제의 147 번째 아르기닌이 알라닌으로 치환된 것, 179 번째 글리신이 알라닌으로 치환된 것, 312 번째 세린이 알라닌으로 치환된 것, 또는 179 번째 글리신 및 312 번째 세린이 각각 알라닌으로 치환된 것일 수 있다.For example, the D-threonine-producing enzyme is that the 147th arginine of the D-threonine aldolase is substituted with alanine, the 179th glycine is substituted with alanine, the 312th serine is substituted with alanine, or The 179th glycine and the 312th serine may be substituted with alanine, respectively.
또한, 본 발명의 다른 측면은 상기 D-트레오닌 생산 효소를 암호화하는 D-트레오닌 생산 효소의 유전자를 제공한다.In addition, another aspect of the present invention provides a gene of a D-threonine-producing enzyme encoding the D-threonine-producing enzyme.
상기 D-트레오닌 생산 효소는 글리신 및 아세트알데히드를 기질로 하여, D-트레오닌을 생산하는 반응을 촉매할 수 있다.The D-threonine-producing enzyme may catalyze a reaction for producing D-threonine using glycine and acetaldehyde as substrates.
상기 필로마이크로비움 마리넘(Filomicrobium marinum) 유래 D-트레오닌 알돌라아제는 서열번호 1의 아미노산 서열을 포함하는 것일 수 있으며, 상기 D-트레오닌 알돌라아제는 단백질의 기능에 영향을 미치지 않는 범위 내에서, 아미노산 잔기의 결실, 삽입, 치환 또는 이들의 조합에 의해서 상이한 서열을 가지는 아미노산의 변이체들, 또는 단편들일 수 있다. 상기 D-트레오닌 알돌라아제의 활성을 전체적으로 변경시키지 않는 단백질 및 펩티드 수준에서의 아미노산 교환은 당해 분야에 공지되어 있다. 경우에 따라서는 인산화(phosphorylation), 황화(sulfation), 아크릴화(acrylation), 당화(glycosylation), 메틸화(methylation), 파네실화(farnesylation) 등으로 변형될 수 있다. 따라서 상기 D-트레오닌 알돌라아제는 서열번호 1의 아미노산 서열을 포함하는 단백질과 실질적으로 동일한 아미노산 서열을 갖는 단백질 및 이의 변이체 또는 이의 활성 단편을 포함한다. 상기 서열번호 1의 아미노산 서열은 서열번호 2의 염기 서열에 의해 암호화된 것일 수 있다.The D-threonine aldolase derived from Filomicrobium marinum may include the amino acid sequence of SEQ ID NO: 1, and the D-threonine aldolase is within a range that does not affect the function of the protein. , Amino acid residue deletion, insertion, substitution, or a combination thereof may be a variant of an amino acid having a different sequence, or fragments. Amino acid exchange at the protein and peptide level that does not totally alter the activity of the D-threonine aldolase is known in the art. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, and farnesylation. Accordingly, the D-threonine aldolase includes a protein having an amino acid sequence substantially identical to that of a protein comprising the amino acid sequence of SEQ ID NO: 1, and a variant thereof or an active fragment thereof. The amino acid sequence of SEQ ID NO: 1 may be encoded by the nucleotide sequence of SEQ ID NO: 2.
본 발명의 D-트레오닌 생산 효소는 서열번호 3, 서열번호 4, 서열번호 5, 서열번호 9, 서열번호 10 및 서열번호 11로 이루어진 군으로부터 선택되는 어느 하나의 아미노산 서열을 포함하는 것일 수 있으며, 상기 D-트레오닌 생산 효소는 단백질의 기능에 영향을 미치지 않는 범위 내에서, 아미노산 잔기의 결실, 삽입, 치환 또는 이들의 조합에 의해서 상이한 서열을 가지는 아미노산의 변이체들, 또는 단편들일 수 있다. 본 발명의 D-트레오닌 생산 효소의 활성을 전체적으로 변경시키지 않는 단백질 및 펩티드 수준에서의 아미노산 교환은 당해 분야에 공지되어 있다. 경우에 따라서는 인산화(phosphorylation), 황화(sulfation), 아크릴화(acrylation), 당화(glycosylation), 메틸화(methylation), 파네실화(farnesylation) 등으로 변형될 수 있다. 따라서 본 발명은 서열번호 3, 서열번호 4, 서열번호 5, 서열번호 9, 서열번호 10 및 서열번호 11로 이루어진 군으로부터 선택되는 어느 하나의 아미노산 서열을 포함하는 단백질과 실질적으로 동일한 아미노산 서열을 갖는 단백질 및 이의 변이체 또는 이의 활성 단편을 포함한다.The D-threonine producing enzyme of the present invention may include any one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, The D-threonine-producing enzyme may be mutants or fragments of amino acids having different sequences by deletion, insertion, substitution, or a combination of amino acid residues within a range that does not affect the function of the protein. Amino acid exchange at the protein and peptide level that does not totally alter the activity of the D-threonine producing enzyme of the present invention is known in the art. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, and farnesylation. Accordingly, the present invention has an amino acid sequence that is substantially identical to a protein comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. Proteins and variants or active fragments thereof.
아미노산 서열에서의 치환된 아미노산과 관련하여 용어 'X#Y'는 본 기술 분야에서 자명하게 인식되는 것으로, '#'는 단백질의 아미노산 번호와 관련하여 치환된 위치를 나타내고, 'X'는 야생형 단백질의 아미노산 서열의 그 위치에서 발견되는 아미노산을 나타내고, 'Y'는 그 위치에서의 치환된 아미노산을 나타낸다.With respect to the substituted amino acid in the amino acid sequence, the term'X#Y' is clearly recognized in the art, and'#' represents a substituted position with respect to the amino acid number of the protein, and'X' is a wild-type protein. Represents an amino acid found at that position in the amino acid sequence of, and'Y' represents a substituted amino acid at that position.
상기 서열번호 3의 아미노산 서열은 서열번호 1의 아미노산 서열의 312 번째 세린(serine)이 알라닌(alanine)으로 치환된 것(S312A)이다.The amino acid sequence of SEQ ID NO: 3 is that the 312th serine of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine (S312A).
상기 서열번호 4의 아미노산 서열은 서열번호 1의 아미노산 서열의 179 번째 글리신(glycine)이 알라닌으로 치환된 것(G179A)이다.The amino acid sequence of SEQ ID NO: 4 is that glycine at the 179th of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine (G179A).
상기 서열번호 5의 아미노산 서열은 서열번호 1의 아미노산 서열의 147 번째 아르기닌(arginine)이 알라닌으로 치환된 것(R147A)이다.The amino acid sequence of SEQ ID NO: 5 is one in which arginine at the 147th of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine (R147A).
상기 서열번호 9의 아미노산 서열은 서열번호 1의 아미노산 서열의 147 번째 아르기닌이 알라닌으로 치환되고, 179 번째 글리신이 알라닌으로 치환된 것(R147A, G179A)이다.In the amino acid sequence of SEQ ID NO: 9, arginine at the 147th of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, and glycine at the 179th is substituted with alanine (R147A, G179A).
상기 서열번호 10의 아미노산 서열은 서열번호 1의 아미노산 서열의 179 번째 글리신이 알라닌으로 치환되고, 312 번째 세린이 알라닌으로 치환된 것(G179A, S312A)이다.The amino acid sequence of SEQ ID NO: 10 is that glycine at the 179th of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine and serine at the 312th is substituted with alanine (G179A, S312A).
상기 서열번호 11의 아미노산 서열은 서열번호 1의 아미노산 서열의 147 번째 아르기닌이 알라닌으로 치환되고, 179 번째 글리신이 알라닌으로 치환되고, 312 번째 세린이 알라닌으로 치환된 것(R147A, G179A, S312A)이다.The amino acid sequence of SEQ ID NO: 11 is that the 147th arginine of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, the 179th glycine is substituted with alanine, and the 312th serine is substituted with alanine (R147A, G179A, S312A). .
본 발명의 D-트레오닌 생산 효소는 D-트레오닌 및 이의 부분입체 이성질체(diastereoisomer)인 D-알로트레오닌을 생산할 수 있다. 본 발명의 D-트레오닌 생산 효소는 상기 반응 생성물 중 D-트레오닌의 비율이 85% 이상이 되도록 D-트레오닌을 생산할 수 있으며, 구체적으로 87.5% 이상, 90% 이상, 92% 이상, 95% 이상, 97% 이상, 97.5% 이상, 98% 이상, 98.5% 이상, 또는 99% 이상이 되도록 생산할 수 있다.The D-threonine producing enzyme of the present invention can produce D-threonine and D-allotreonine, which is a diastereoisomer thereof. The D-threonine producing enzyme of the present invention can produce D-threonine so that the proportion of D-threonine in the reaction product is 85% or more, and specifically 87.5% or more, 90% or more, 92% or more, 95% or more, It can be produced to be 97% or more, 97.5% or more, 98% or more, 98.5% or more, or 99% or more.
또한, 상기 D-트레오닌 생산 효소는 하기 식에 의해 계산되는 부분입체 이성질체 과잉률(diastereomeric excess, d.e.)이 75% 이상이 되도록 D-트레오닌을 D-알로트레오닌보다 더 많이 생산할 수 있으며, 구체적으로 d.e.가 77% 이상, 79% 이상, 80% 이상, 82% 이상, 85% 이상, 87% 이상, 90% 이상, 92% 이상, 또는 93% 이상이 되도록 생산할 수 있다.In addition, the D-threonine-producing enzyme can produce more D-threonine than D-allothreonine so that the diastereomeric excess (de) calculated by the following formula is 75% or more, and specifically de Is 77% or more, 79% or more, 80% or more, 82% or more, 85% or more, 87% or more, 90% or more, 92% or more, or 93% or more.
[식][expression]
d.e. (%) = ('D-트레오닌의 농도' - 'D-알로트레오닌의 농도') × 100 / ('D-트레오닌의 농도' + 'D-알로트레오닌의 농도')d.e. (%) = ('Concentration of D-Threonine'-'Concentration of D-Allothreonine') × 100 / ('Concentration of D-Threonine' +'Concentration of D-Allothreonine')
본 발명의 상기 D-트레오닌 생산 효소를 암호화하는 유전자는 서열번호 6, 서열번호 7, 서열번호 8, 서열번호 12, 서열번호 13 및 서열번호 14로 이루어진 군으로부터 선택되는 어느 하나의 염기 서열로 이루어진 것일 수 있다. 그러나 본 발명의 D-트레오닌 생산 효소 및 이의 변이체 또는 이의 활성 단편을 암호화하는 유전자는 암호화 영역으로부터 발현되는 상기 효소 및 이의 변이체 또는 이의 활성 단편의 아미노산 서열을 변화시키지 않는 범위 내에서 암호화 영역에 다양한 변형이 이루어질 수 있고, 암호화 영역을 제외한 부분에서도 유전자의 발현에 영향을 미치지 않는 범위 내에서 다양한 변이가 이루어질 수 있으며, 이러한 변이 유전자 역시 본 발명의 범위에 포함된다. 따라서 본 발명은 상기 서열번호 6, 서열번호 7, 서열번호 8, 서열번호 12, 서열번호 13 및 서열번호 14로 이루어진 군으로부터 선택되는 어느 하나의 염기 서열로 이루어진 유전자와 실질적으로 동일한 염기 서열로 이루어진 유전자 및 상기 유전자의 단편을 포함한다. 상기 실질적으로 동일한 염기 서열로 이루어진 유전자란 80％ 이상, 바람직하게는 90％ 이상, 가장 바람직하게는 95％ 이상의 서열 상동성을 갖는 것들을 의미하나, 이에 한정되는 것은 아니며, 80% 이상의 서열 상동성을 가지며 암호화된 단백질이 동일한 효소 활성을 가진다면 본 발명에 포함된다. 상기와 같이, 본 발명의 D-트레오닌 생산 효소를 암호화하는 유전자는 이와 동등한 활성을 갖는 단백질을 암호화하는 한, 하나 이상의 핵산 염기가 치환, 결실, 삽입 또는 이들의 조합에 의해 변이될 수 있으며, 이들 또한 본 발명의 범위에 포함된다. 상기와 같은 염기 서열들은 단일 가닥 또는 이중 가닥일 수 있으며, DNA 분자 또는 RNA 분자일 수 있다.The gene encoding the D-threonine producing enzyme of the present invention is composed of any one nucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14 Can be. However, the gene encoding the D-threonine-producing enzyme of the present invention and its mutant or active fragment thereof is variously modified in the coding region within a range that does not change the amino acid sequence of the enzyme and its mutant or active fragment thereof expressed from the coding region. This can be achieved, and various mutations can be made within a range that does not affect the expression of the gene even in portions other than the coding region, and such mutant genes are also included in the scope of the present invention. Therefore, the present invention consists of a nucleotide sequence substantially identical to a gene consisting of any one nucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14 It includes a gene and a fragment of the gene. Genes consisting of the substantially identical nucleotide sequence means those having sequence homology of 80% or more, preferably 90% or more, and most preferably 95% or more, but are not limited thereto, and sequence homology of 80% or more. And, if the encoded protein has the same enzymatic activity, it is included in the present invention. As described above, as long as the gene encoding the D-threonine-producing enzyme of the present invention encodes a protein having equivalent activity, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof. It is also included in the scope of the present invention. Such nucleotide sequences may be single-stranded or double-stranded, and may be DNA molecules or RNA molecules.
2. D-트레오닌 생산 효소의 발현벡터, 형질전환체 및 이를 이용한 D-트레오닌 생산 효소의 제조 방법2. Expression vector of D-threonine-producing enzyme, transformant, and method for producing D-threonine-producing enzyme using the same
본 발명의 또 다른 측면은 본 발명의 D-트레오닌 생산 효소의 유전자가 포함된 재조합 발현벡터 및 상기 재조합 발현벡터가 도입된 형질전환체를 제공한다.Another aspect of the present invention provides a recombinant expression vector containing the gene of the D-threonine producing enzyme of the present invention and a transformant into which the recombinant expression vector has been introduced.
본 발명의 재조합 발현벡터는 상기 D-트레오닌 생산 효소를 암호화하는 유전자를 포함하며, 이에 따라 상기 D-트레오닌 생산 효소를 제조할 수 있는 벡터로서 유용하게 이용될 수 있다. 상기 D-트레오닌 생산 효소에 관한 설명은 상기 '신규 D-트레오닌 생산 효소 및 그 유전자'에서 설명한 바와 동일하다.The recombinant expression vector of the present invention includes a gene encoding the D-threonine-producing enzyme, and thus can be usefully used as a vector capable of producing the D-threonine-producing enzyme. The description of the D-threonine-producing enzyme is the same as described in the'new D-threonine-producing enzyme and its genes'.
상기 발현벡터는 플라스미드 벡터, 코즈미드 벡터, 박테리오파이지 벡터 및 바이러스 벡터 등을 포함하나 이에 한정되지 않는다. The expression vector includes, but is not limited to, a plasmid vector, a cozmid vector, a bacteriophage vector, and a viral vector.
상기 재조합 발현벡터는 본 발명의 D-트레오닌 생산 효소를 제조하고자하는 숙주세포의 종류에 따라 프로모터(promoter), 터미네이터(terminator), 인핸서(enhancer) 등과 같은 발현조절서열, 또는 분비를 위한 서열 등을 적절히 목적에 따라 조합할 수 있다.The recombinant expression vector contains an expression control sequence such as a promoter, a terminator, or an enhancer, or a sequence for secretion, depending on the type of host cell for which the D-threonine producing enzyme of the present invention is to be produced. It can be combined according to the purpose as appropriate.
상기 발현벡터는 벡터가 도입된 숙주세포를 선택하기 위한 선택 마커를 추가로 포함할 수 있고, 복제 가능한 발현 벡터인 경우 복제 기원을 포함할 수 있다. The expression vector may further include a selection marker for selecting a host cell into which the vector has been introduced, and may include an origin of replication in the case of a replicable expression vector.
또한, 상기 재조합 발현벡터는 발현 단백질의 정제를 용이하게 하기 위한 서열을 포함할 수 있으며, 구체적으로 본 발명의 D-트레오닌 생산 효소를 암호화하는 유전자에 작동 가능하도록 분리정제용 태그를 암호화하는 유전자가 연결될 수 있다. 이때, 상기 분리정제용 태그는 GST, poly-Arg, FLAG, 히스티딘-태그(His-tag), c-myc 등이 단독으로 사용되거나 이들 중 두 개 이상을 순차적으로 연결하여 사용할 수도 있다.In addition, the recombinant expression vector may include a sequence for facilitating purification of the expressed protein, and specifically, the gene encoding the tag for separation and purification so as to be operable to the gene encoding the D-threonine producing enzyme of the present invention is Can be connected. At this time, the tags for separation and purification may be used alone, such as GST, poly-Arg, FLAG, histidine-tag, c-myc, or two or more of them may be sequentially connected.
상기 D-트레오닌 생산 효소를 암호화하는 유전자는 제한효소 절단위치를 통해 클로닝될 수 있으며, 상기 벡터에 단백질 절단효소 인식부위를 암호화하는 유전자가 사용된 경우에는 상기 D-트레오닌 생산 효소의 유전자와 틀이 맞도록(in frame) 연결되어, 상기 효소를 수득한 후 단백질 절단 효소로 절단 시, 원래 형태의 D-트레오닌 생산 효소가 제조될 수 있도록 할 수 있다.The gene encoding the D-threonine-producing enzyme can be cloned through the restriction enzyme cleavage site, and when the gene encoding the protein-cleaving enzyme recognition site is used in the vector, the gene and the frame of the D-threonine-producing enzyme are different. It is connected to fit (in frame), when the enzyme is obtained and then digested with a protein cleavage enzyme, the original form of D-threonine-producing enzyme can be prepared.
상기 재조합 발현벡터는 D-트레오닌 생산 효소를 암호화하는 유전자를 플라스미드 벡터인 pET28a(+)에 삽입함으로써 제조될 수 있으며, 상기의 클로닝 벡터의 제조에 이용된 pET28a(+) 이외에도 다양한 원핵세포용 벡터 또는 진핵세포용(pPIC 및 pPICZ 등) 벡터가 알려져 있으므로 발현의 목적에 따라 상기 벡터 이외의 다양한 발현벡터를 이용할 수 있다.The recombinant expression vector may be prepared by inserting a gene encoding a D-threonine-producing enzyme into a plasmid vector pET28a(+), and in addition to pET28a(+) used in the preparation of the cloning vector, various vectors for prokaryotic cells or Since vectors for eukaryotic cells (pPIC and pPICZ, etc.) are known, various expression vectors other than the above vectors can be used depending on the purpose of expression.
본 발명의 형질전환체는 본 발명의 D-트레오닌 생산 효소를 암호화하는 유전자를 포함하는 상기 재조합 발현벡터가 도입된다. 상기 D-트레오닌 생산 효소 및 이의 유전자에 관한 설명은 상기 '신규 D-트레오닌 생산 효소 및 그 유전자'에서 설명한 바와 동일하다.In the transformant of the present invention, the recombinant expression vector containing the gene encoding the D-threonine producing enzyme of the present invention is introduced. The description of the D-threonine producing enzyme and its gene is the same as described in the'new D-threonine producing enzyme and its gene'.
본 발명에 따른 상기 재조합 발현벡터를 발현 목적에 따라 박테리아, 효모, 대장균, 진균류, 식물 세포 및 동물 세포로 구성된 군으로부터 선택되는 어느 하나의 적절한 숙주 세포에 형질전환시킴으로써 형질전환체를 제조할 수 있다. 예컨대, 상기 숙주세포는 대장균(E. coli BL21(DE3), DH5α등) 또는 효모 세포 (Saccharomyces 속, Pichia 속 등)일 수 있다. 이때, 숙주세포의 종류에 따라 적절한 배양 방법 및 배지 조건 등은 당해 분야의 공지 기술로부터 당업자가 용이하게 선택할 수 있다. A transformant can be prepared by transforming the recombinant expression vector according to the present invention into any suitable host cell selected from the group consisting of bacteria, yeast, E. coli, fungi, plant cells, and animal cells depending on the purpose of expression. . For example, the host cell may be E. coli ( E. coli BL21(DE3), DH5α, etc.) or yeast cells ( Saccharomyces genus, Pichia genus, etc.). At this time, appropriate culture methods and medium conditions, etc., depending on the type of host cell, can be easily selected by a person skilled in the art from known techniques in the art.
본 발명의 형질전환체의 제조를 위한 재조합 발현벡터의 도입 방법은 공지의 기술, 즉 열 충격법, 전기충격법 등을 사용할 수 있다.As a method of introducing the recombinant expression vector for the production of the transformant of the present invention, a known technique, that is, a heat shock method, an electric shock method, or the like can be used.
상기 형질전환체로부터 발현된 단백질은 본 발명의 D-트레오닌 생산 효소이므로, 상기 형질전환체를 대량 배양하여 상기 유전자를 발현시킴으로써 상기 D-트레오닌 생산 효소를 용이하게 대량생산 할 수 있다. Since the protein expressed from the transformant is the D-threonine-producing enzyme of the present invention, the D-threonine-producing enzyme can be easily mass-produced by mass-culturing the transformant to express the gene.
본 발명의 또 다른 측면은 본 발명의 D-트레오닌 생산 효소를 암호화하는 유전자를 포함하는 재조합 발현벡터가 도입된 형질전환체를 이용하여 D-트레오닌 생산 효소를 제조하는 방법을 제공한다.Another aspect of the present invention provides a method for preparing a D-threonine-producing enzyme using a transformant into which a recombinant expression vector containing a gene encoding a D-threonine-producing enzyme of the present invention has been introduced.
본 발명의 D-트레오닌 생산 효소의 제조 방법은 본 발명의 D-트레오닌 생성 효소를 암호화하는 유전자를 포함하는 재조합 발현벡터가 도입된 형질전환체를 배양하는 단계; 및 상기 형질전환체의 배양물로부터 D-트레오닌 생산 효소를 분리하는 단계;를 포함한다.The method for producing a D-threonine-producing enzyme of the present invention comprises the steps of culturing a transformant into which a recombinant expression vector containing a gene encoding the D-threonine-producing enzyme of the present invention has been introduced; And separating the D-threonine-producing enzyme from the culture of the transformant.
상기 D-트레오닌 생산 효소의 제조 방법은 상기 배양된 형질전환체에서 상기 D-트레오닌 생산 효소를 암호화하는 유전자의 발현을 유도하는 단계를 더 포함할 수 있다.The method for preparing the D-threonine-producing enzyme may further include inducing expression of a gene encoding the D-threonine-producing enzyme in the cultured transformant.
상기 D-트레오닌 생산 효소를 암호화하는 유전자의 N-말단에는 분리정제용 태그를 암호화하는 유전자가 추가로 연결될 수 있으며, 이로 인해 D-트레오닌 생산 효소의 수득이 가능할 수 있다. 또한, 상기 N-말단에는 단백질 절단효소 절단위치가 추가로 연결될 수 있고, 이로 인해 재조합된 형태의 D-트레오닌 생산 효소의 정제가 가능할 수 있다.A gene encoding a tag for separation and purification may be additionally linked to the N-terminus of the gene encoding the D-threonine-producing enzyme, thereby making it possible to obtain a D-threonine-producing enzyme. In addition, a protein cleavage enzyme cleavage site may be additionally linked to the N-terminus, and thus, purification of a recombinant D-threonine-producing enzyme may be possible.
상기 형질전환체의 배양은 공지된 방법에 따라서 수행될 수 있고, 배양 온도, 배양 시간 및 배지의 pH 등의 조건은 적절하게 조절될 수 있다. 또한, 배양 방법에는 회분식 배양(batch culture), 연속식 배양(continuous culture) 및 유가식 배양(fed-batch culture)이 포함될 수 있다. 사용되는 배양 배지는 특정한 균주의 요구 조건을 적절하게 충족시켜야 한다. The cultivation of the transformant may be performed according to a known method, and conditions such as cultivation temperature, cultivation time, and pH of the medium may be appropriately adjusted. In addition, the culture method may include batch culture, continuous culture, and fed-batch culture. The culture medium used must adequately meet the requirements of the specific strain.
상기 형질전환체의 배양물로부터 D-트레오닌 생산 효소의 분리는 원심분리, 여과 등 당해 분야에서 통상적으로 수행되는 방법을 통해 실시할 수 있다. 또한, 상기 방법으로 분리된 D-트레오닌 생산 효소는 통상의 방식으로 정제될 수 있으며, 예컨대 염석(예를 들어 황산암모늄 침전, 인산나트륨 침전), 용매 침전(아세톤, 에탄올 등을 이용한 단백질 분획 침전), 투석, 겔 여과, 이온 교환, 역상 칼럼 크로마토그래피와 같은 크로마토그래피, 한외여과 등의 기법을 단독 또는 조합하여 본 발명의 효소를 정제할 수 있다.Separation of the D-threonine-producing enzyme from the culture of the transformant may be performed through a method commonly performed in the art, such as centrifugation and filtration. In addition, the D-threonine-producing enzyme isolated by the above method can be purified in a conventional manner, such as salting out (eg, ammonium sulfate precipitation, sodium phosphate precipitation), solvent precipitation (protein fraction precipitation using acetone, ethanol, etc.) , Dialysis, gel filtration, ion exchange, chromatography such as reverse phase column chromatography, ultrafiltration, and the like may be used alone or in combination to purify the enzyme of the present invention.
3. D-트레오닌 생산용 조성물 및 D-트레오닌의 생산 방법3. Composition for producing D-threonine and method for producing D-threonine
본 발명의 또 다른 측면은 D-트레오닌을 고순도로 생산하기 위한 D-트레오닌 생산용 조성물을 제공한다. Another aspect of the present invention provides a composition for producing D-threonine for producing D-threonine with high purity.
상기 D-트레오닌 생산용 조성물은 본 발명의 D-트레오닌 생산 효소를 유효성분으로 포함한다. 상기 D-트레오닌 생산 효소에 관한 설명은 상기 '신규 D-트레오닌 생산 효소 및 그 유전자'에서 설명한 바와 동일하며, 상기 D-트레오닌 생산 효소는 D-트레오닌을 높은 순도로 생산할 수 있는 활성이 있는바, 상기 효소를 포함하는 조성물을 D-트레오닌의 효율적인 생산을 위한 용도로 이용할 수 있다. The composition for producing D-threonine includes the enzyme for producing D-threonine of the present invention as an active ingredient. The description of the D-threonine-producing enzyme is the same as described in the'new D-threonine-producing enzyme and its gene', and the D-threonine-producing enzyme has an activity capable of producing D-threonine with high purity, A composition containing the enzyme can be used for efficient production of D-threonine.
상기 D-트레오닌 생산용 조성물은, D-트레오닌 생산 효소의 기질인 글리신 및 아세트알데히드를 더 포함할 수 있다. 상기 글리신 및 아세트알데히드는 목적하는 D-트레오닌의 양에 맞추어 충분한 양으로 포함될 수 있으며, 상기 글리신과 아세트알데히드는 1:1 내지 10:1의 비율, 예컨대 2:1 내지 8:1의 비율, 3:1 내지 5:1의 비율, 또는 4:1 내지 4.5:1의 비율로 포함될 수 있다. 또한, 상기 조성물에는 상기 D-트레오닌 생산 효소의 활성을 향상시키기 위해, 상기 D-트레오닌 생산 효소와 함께 금속 양이온 및/또는 피리독살-5-인산이 더 포함될 수 있다. 상기 금속 양이온은 2가 금속 양이온일 수 있고, 상기 2가 금속 양이온은 망간 양이온 또는 마그네슘 양이온일 수 있다.The composition for producing D-threonine may further include glycine and acetaldehyde, which are substrates for D-threonine producing enzymes. The glycine and acetaldehyde may be included in a sufficient amount according to the amount of desired D-threonine, and the glycine and acetaldehyde are in a ratio of 1:1 to 10:1, such as a ratio of 2:1 to 8:1, 3 It may be included in a ratio of :1 to 5:1, or in a ratio of 4:1 to 4.5:1. In addition, in order to improve the activity of the D-threonine-producing enzyme, the composition may further contain a metal cation and/or pyridoxal-5-phosphate together with the D-threonine-producing enzyme. The metal cation may be a divalent metal cation, and the divalent metal cation may be a manganese cation or a magnesium cation.
본 발명의 또 다른 측면은 인 비트로(in vitro)에서의 D-트레오닌 생산 방법을 제공한다.Another aspect of the present invention provides a method for producing D-threonine in vitro.
상기 인 비트로(in vitro)에서의 D-트레오닌 생산 방법은 본 발명의 D-트레오닌 생산 효소를 글리신 및 아세트알데히드와 함께 반응시키는 단계를 포함한다. 상기 D-트레오닌 생산 효소에 관한 설명은 상기 '신규 D-트레오닌 생산 효소 및 그 유전자'에서 설명한 바와 동일하다.The method for producing D-threonine in vitro includes the step of reacting the D-threonine producing enzyme of the present invention with glycine and acetaldehyde. The description of the D-threonine-producing enzyme is the same as described in the'new D-threonine-producing enzyme and its genes'.
상기 반응에서, 상기 글리신 및 아세트알데히드는 외부에서 주입되는 것일 수 있고, 상기 글리신 및 아세트알데히드는 목적하는 D-트레오닌의 양에 맞추어 충분한 양이 주입되어야 한다. 상기 글리신 및 아세트알데히드는 1:1 내지 10:1의 비율로 효소와 반응시키는 것일 수 있고, 예컨대 2:1 내지 8:1의 비율, 3:1 내지 5:1의 비율, 또는 4:1 내지 4.5:1의 비율로 반응시킬 수 있다. 또한, 상기 글리신 및 아세트알데히드의 주입은 연속적으로 수행될 수 있다. 특히 상기 글리신 및 아세트알데히드가 연속적으로 주입되는 경우, 상기 D-트레오닌 생산 효소에 의하여 D-트레오닌을 지속적으로 생성할 수 있다.In the above reaction, the glycine and acetaldehyde may be injected from the outside, and the glycine and acetaldehyde must be injected in a sufficient amount according to the amount of the desired D-threonine. The glycine and acetaldehyde may be reacted with the enzyme in a ratio of 1:1 to 10:1, for example, in a ratio of 2:1 to 8:1, a ratio of 3:1 to 5:1, or 4:1 to It can be reacted in a ratio of 4.5:1. In addition, the injection of glycine and acetaldehyde may be performed continuously. In particular, when the glycine and acetaldehyde are continuously injected, D-threonine may be continuously produced by the D-threonine producing enzyme.
상기 D-트레오닌 생산 효소를 글리신 및 아세트알데히드와 함께 반응시키는 단계는, 상기 D-트레오닌 생산 효소의 활성을 향상시키기 위해 상기 D-트레오닌 생산 효소와 함께 금속 양이온 및/또는 피리독살-5-인산을 더 포함하여 반응시키는 것일 수 있다. 상기 금속 양이온은 2가 금속 양이온일 수 있고, 상기 2가 금속 양이온은 망간 양이온 또는 마그네슘 양이온일 수 있다.The step of reacting the D-threonine-producing enzyme with glycine and acetaldehyde includes metal cation and/or pyridoxal-5-phosphate with the D-threonine-producing enzyme to enhance the activity of the D-threonine-producing enzyme. It may be to react by including more. The metal cation may be a divalent metal cation, and the divalent metal cation may be a manganese cation or a magnesium cation.
상기 반응시키는 단계는 20 ℃ 내지 55 ℃의 온도, 예컨대 22 ℃ 내지 45 ℃의 온도, 또는 25 ℃ 내지 40 ℃의 온도에서 수행될 수 있으나, 이에 한정되지 않는다. 또한, 상기 반응은 7 내지 10의 pH, 예컨대 8 내지 9.5의 pH, 또는 8.5 내지 9의 pH에서 수행될 수 있으나, 이에 한정되지 않는다.The reacting step may be performed at a temperature of 20° C. to 55° C., for example, 22° C. to 45° C., or 25° C. to 40° C., but is not limited thereto. In addition, the reaction may be carried out at a pH of 7 to 10, such as a pH of 8 to 9.5, or a pH of 8.5 to 9, but is not limited thereto.
상기 인 비트로(in vitro)에서의 D-트레오닌 생산 방법은 상기 D-트레오닌 생산 효소, 글리신 및 아세트알데히드의 반응 생성물로부터 D-트레오닌을 회수하는 단계를 더 포함할 수 있다.The method for producing D-threonine in vitro may further include recovering D-threonine from the reaction product of the D-threonine producing enzyme, glycine and acetaldehyde.
본 발명의 또 다른 측면은 인 비보(in vivo)에서의 D-트레오닌 생산 방법을 제공한다.Another aspect of the present invention provides a method for producing D-threonine in vivo.
상기 인 비보(in vivo)에서의 D-트레오닌 생산 방법은 본 발명의 상기 형질전환체를 글리신 및 아세트알데히드의 존재 하에서 배양하는 단계를 포함한다. 상기 형질전환체에 관한 설명은 상기 'D-트레오닌 생산 효소의 발현벡터, 형질전환체 및 이를 이용한 D-트레오닌 생산 효소의 제조 방법'에서 설명한 바와 동일하다.The method for producing D-threonine in vivo includes culturing the transformant of the present invention in the presence of glycine and acetaldehyde. The description of the transformant is the same as described in the'expression vector of the D-threonine-producing enzyme, the transformant, and a method for producing a D-threonine-producing enzyme using the same.'
상기 형질전환체를 글리신 및 아세트알데히드의 존재 하에서 배양하는 단계에서, 상기 글리신 및 아세트알데히드는 목적하는 D-트레오닌의 양에 맞추어 충분한 양이 존재해야 하며, 1:1 내지 10:1의 비율로 존재할 수 있고, 예컨대 2:1 내지 8:1의 비율, 3:1 내지 5:1의 비율, 또는 4:1 내지 4.5:1의 비율로 존재할 수 있다. 상기 글리신 및 아세트알데히드는 외부에서 인위적으로 공급해 주는 것일 수 있다. 또한, 상기 형질전환체의 배양에 이용되는 배지 내에 글리신 및 아세트알데히드를 첨가하여 함께 공급해 주는 것일 수도 있고, 상기 글리신 및 아세트알데히드가 생산되는 환경을 제공해 주는 것일 수도 있다. 상기 글리신 및 아세트알데히드가 생산되는 환경은 상기 글리신 및 아세트알데히드의 생산에 관여하는 적어도 하나의 효소를 암호화하는 유전자들을 함께 형질전환시킴으로써 제공될 수 있다.In the step of culturing the transformant in the presence of glycine and acetaldehyde, the glycine and acetaldehyde must be present in a sufficient amount according to the amount of the desired D-threonine, and exist in a ratio of 1:1 to 10:1. And, for example, in a ratio of 2:1 to 8:1, in a ratio of 3:1 to 5:1, or in a ratio of 4:1 to 4.5:1. The glycine and acetaldehyde may be artificially supplied from the outside. In addition, glycine and acetaldehyde may be added and supplied together in a medium used for culturing the transformant, or an environment in which glycine and acetaldehyde are produced may be provided. The environment in which glycine and acetaldehyde are produced may be provided by transforming genes encoding at least one enzyme involved in the production of glycine and acetaldehyde together.
상기와 같은 형질전환체의 배양은 본 기술 분야에 알려진 적당한 배지와 배양 조건에 따라 이루어질 수 있다. 통상의 기술자라면 선택되는 형질전환체의 숙주세포의 종류에 따라 배지 및 배양조건을 용이하게 조정하여 사용할 수 있다. 배양 방법은 회분식, 연속식, 유가식, 또는 이들의 조합 배양을 포함할 수 있다.The cultivation of the transformant as described above may be performed according to an appropriate medium and culture conditions known in the art. Those of ordinary skill in the art can easily adjust and use the medium and culture conditions according to the type of host cell of the selected transformant. The culture method may include a batch type, continuous type, fed-batch type, or a combination culture thereof.
상기 배지는 다양한 탄소원, 질소원 및 미량원소 성분을 포함할 수 있다.The medium may contain various carbon sources, nitrogen sources, and trace element components.
상기 탄소원은, 예를 들면, 포도당, 자당, 유당, 과당, 말토오스, 전분, 셀룰로오스와 같은 탄수화물, 대두유, 해바라기유, 피마자유, 코코넛유와 같은 지방, 팔미트산, 스테아린산, 리놀레산과 같은 지방산, 글리세롤 및 에탄올과 같은 알코올, 아세트산과 같은 유기산, 또는 이들의 조합을 포함할 수 있다. 상기 배양은 글루코스를 탄소원으로 하여 수행될 수 있다. 상기 질소원은, 펩톤, 효모 추출물, 육즙, 맥아 추출물, 옥수수 침지액(CSL), 및 대두밀과 같은 유기 질소원 및 요소, 황산암모늄, 염화암모늄, 인산암모늄, 탄산암모늄 및 질산암모늄과 같은 무기 질소원, 또는 이들의 조합을 포함할 수 있다. 상기 배지는 인의 공급원으로서, 예를 들면, 인산이수소칼륨, 인산수소이칼륨을 포함할 수 있고 이에 상응하는 소듐-함유 염, 황산마그네슘, 황산철과 같은 금속염을 포함할 수 있다.The carbon source is, for example, glucose, sucrose, lactose, fructose, maltose, starch, carbohydrates such as cellulose, soybean oil, sunflower oil, castor oil, fats such as coconut oil, palmitic acid, stearic acid, fatty acids such as linoleic acid, Alcohols such as glycerol and ethanol, organic acids such as acetic acid, or combinations thereof. The cultivation may be performed using glucose as a carbon source. The nitrogen source is an organic nitrogen source and urea such as peptone, yeast extract, broth, malt extract, corn steep liquor (CSL), and soybean meal, an inorganic nitrogen source such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, or Combinations of these may be included. The medium may include, for example, potassium dihydrogen phosphate and dipotassium hydrogen phosphate as a source of phosphorus, and a corresponding metal salt such as sodium-containing salt, magnesium sulfate, and iron sulfate.
또한, 아미노산, 비타민, 및 적절한 전구체 등이 배지에 포함될 수 있다. 상기 배지 또는 개별 성분은 배양액에 회분식 또는 연속식으로 첨가될 수 있다.In addition, amino acids, vitamins, and suitable precursors may be included in the medium. The medium or individual components may be added to the culture medium in a batch or continuous manner.
또한, 배양 중에 지방산 폴리글리콜 에스테르와 같은 소포제를 사용하여 기포 생성을 억제할 수 있다.In addition, the generation of air bubbles can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester during culture.
또한, 형질전환체에 의해 배양되어 제조된 본 발명의 D-트레오닌 생산 효소의 활성을 향상시키기 위해 배지 내에 금속 양이온 및/또는 피리독살-5-인산을 더 포함할 수 있다. 상기 금속 양이온은 2가 금속 양이온일 수 있고, 상기 2가 금속 양이온은 망간 양이온 또는 마그네슘 양이온일 수 있다.In addition, a metal cation and/or pyridoxal-5-phosphate may be further included in the medium to enhance the activity of the D-threonine-producing enzyme of the present invention prepared by culturing by the transformant. The metal cation may be a divalent metal cation, and the divalent metal cation may be a manganese cation or a magnesium cation.
상기와 같은 형질전환체의 배양은 20 ℃ 내지 50 ℃에서 수행될 수 있고, 예컨대 25 ℃ 내지 45 ℃, 또는 30 ℃ 내지 40 ℃에서 수행될 수 있다. 상기 형질전환체의 배양이 20 ℃ 미만 또는 50 ℃ 초과의 온도 범위에서 수행될 경우 충분한 양의 중간 생성물이 생성되지 않아, 결국 최종 생산물인 D-트레오닌의 생산량 또한 충분해지지 못하는 문제가 발생할 수 있다.The cultivation of the transformant as described above may be carried out at 20 °C to 50 °C, for example, it may be carried out at 25 °C to 45 °C, or 30 °C to 40 °C. When the cultivation of the transformant is performed in a temperature range of less than 20° C. or more than 50° C., a sufficient amount of an intermediate product is not produced, and as a result, the production amount of D-threonine, which is a final product, may not be sufficient.
또한, 상기와 같은 형질전환체의 배양은 pH 5 내지 pH 10에서 수행될 수 있고, 예컨대 pH 6 내지 pH 9, 또는 pH 6.5 내지 pH 8에서 수행될 수 있으나, 이에 한정하지 않는다. 상기와 같은 형질전환체 배양의 pH 조건은 형질전환체의 배양 배지에 수산화암모늄, 수산화칼륨, 암모니아, 인산 및 황산과 같은 화합물을 첨가함으로써 조정할 수 있다. 형질전환체의 배양 pH 조건이 상기 범위를 벗어나는 경우 형질전환체의 생장이 저해되므로 D-트레오닌 생산 효소의 발현이 감소되고, 이에 따른 D-트레오닌의 생산이 감소되는 문제점이 발생할 수 있다.In addition, cultivation of the transformant as described above may be performed at pH 5 to pH 10, for example, pH 6 to pH 9, or pH 6.5 to pH 8, but is not limited thereto. The pH conditions for culturing the transformants as described above can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the culture medium of the transformant. When the culture pH condition of the transformant is out of the above range, the growth of the transformant is inhibited, so that the expression of the D-threonine-producing enzyme decreases, and thus the production of D-threonine may be reduced.
상기 인 비보(in vivo)에서의 D-트레오닌 생산 방법은 상기 배양된 형질전환체에서 상기 D-트레오닌 생산 효소를 암호화하는 유전자의 발현을 유도하는 단계를 더 포함할 수 있다. The method for producing D-threonine in vivo may further include inducing expression of a gene encoding the D-threonine-producing enzyme in the cultured transformant.
상기 인 비보(in vivo)에서의 D-트레오닌 생산 방법은 상기 형질전환체의 배양물로부터 D-트레오닌을 회수하는 단계를 더 포함할 수 있다.The method for producing D-threonine in vivo may further include recovering D-threonine from the culture of the transformant.
이하, 본 발명을 실시예에 의하여 상세히 설명한다.Hereinafter, the present invention will be described in detail by examples.
[실시예 1][Example 1]
서열번호 1의 아미노산 서열을 갖는 단백질의 분리Isolation of a protein having the amino acid sequence of SEQ ID NO: 1
[1-1] 서열번호 1의 아미노산 서열을 갖는 단백질의 클로닝 및 형질전환[1-1] Cloning and transformation of a protein having the amino acid sequence of SEQ ID NO: 1
필로마이크로비움 마리넘(Filomicrobium marinum)에서 유래한 서열번호 1의 아미노산 서열을 갖는 단백질을 얻기 위하여, 서열번호 2의 염기 서열을 기초로, 하기와 같은 프라이머를 제작하였다.In order to obtain a protein having the amino acid sequence of SEQ ID NO: 1 derived from Filomicrobium marinum, the following primers were prepared based on the nucleotide sequence of SEQ ID NO: 2.
㈜바이오니아에 의뢰하여 서열번호 2의 염기 서열의 유전자를 합성하였고, 상기 합성된 유전자를 주형으로 하여 D-트레오닌 서열 증폭을 위해 프라이머를 설계하였다. 정방향 프라이머(5'-tggtgccgcgcggcagccatatgcgcgcaccagcacggct-3'), 역방향 프라이머(5'-ggtggtggtggtgctcgagctagaacacacaacctcgcgc-3') 쌍을 이용하여 PCR을 수행함으로써, 상기 서열번호 2의 염기 서열을 증폭하였다. Gibson assembly(New England Biolabs, 미국)를 이용하여 상기와 같이 증폭된 서열번호 2의 염기 서열을 포함하는 PCR 산물을, 벡터 증폭용 정방향 서열(5'-gcgcgaggttgtgtgttctagctcgagcaccaccaccacc-3')과 역방향 서열(5'-agccgtgctggtgcgcgcatatggctgccgcgcggcacca-3')을 이용하여 증폭한 플라스미드 벡터 pET28a(+)(Novagen, 미국)의 다중 클로닝 자리에 삽입하였다. 염기 서열 분석(sequencing, ㈜마크로젠)을 통해 상기 서열번호 2의 염기 서열이 제대로 삽입되었음을 확인하였으며, 이를 대장균 C2566 균주(Novagen, 미국)에 형질전환하였다. 상기와 같이 얻어진 형질전환체는 이용하기 전에 20% 글리세린 용액을 첨가하여 냉동 보관하였다.The gene of the nucleotide sequence of SEQ ID NO: 2 was synthesized by requesting Bioneer, and a primer was designed for amplifying the D-threonine sequence using the synthesized gene as a template. The nucleotide sequence of SEQ ID NO: 2 was amplified by performing PCR using a pair of forward primers (5'-tggtgccgcgcggcagccatatgcgcgcaccagcacggct-3') and reverse primers (5'-ggtggtggtggtgctcgagctagaacacacaacctcgcgc-3'). The PCR product containing the nucleotide sequence of SEQ ID NO: 2 amplified as described above using Gibson assembly (New England Biolabs, USA) was used for vector amplification forward sequence (5'-gcgcgaggttgtgtgttctagctcgagcaccaccaccacc-3') and reverse sequence (5' -agccgtgctggtgcgcgcatatggctgccgcgcggcacca-3') was inserted into the multiple cloning site of the amplified plasmid vector pET28a(+) (Novagen, USA). It was confirmed that the nucleotide sequence of SEQ ID NO: 2 was properly inserted through nucleotide sequence analysis (sequencing, Macrogen, Inc.), and this was transformed into E. coli C2566 strain (Novagen, USA). The transformant obtained as described above was stored frozen by adding a 20% glycerin solution before use.
[1-2] 서열번호 1의 아미노산 서열을 갖는 단백질의 과발현 및 정제[1-2] Overexpression and purification of a protein having the amino acid sequence of SEQ ID NO: 1
상기 실시예 [1-1]에서 냉동 보관된 형질전환체를 3 ㎖의 LK 고체 배지(LB 배지 + 2 μg/㎖ 카나마이신)에 선조접종하고 37 ℃에서 10시간 이상 배양하였다. 고체 배지에 나타나는 하나의 콜로니를, 단집락 분리를 통해 3 ㎖의 LK 고체 배지가 포함된 시험관(test tube)에 접종하고 37 ℃의 진탕 배양기로 12시간 동안 종균 배양을 실시하였다. 그런 다음, 상기 종균 배양된 배양액 2 ㎖를 200 ㎖의 LK 배지가 포함된 1,000 ㎖ 플라스크에 첨가하여 본 배양을 실시하였고, 600 ㎚에서의 흡광도가 0.6이 될 때의 최종 농도가 0.1 mM이 되도록 IPTG(isopropyl-1-thio-β-D-galactopyranoside)를 첨가하여 상기 서열번호 1의 아미노산 서열을 갖는 단백질의 과발현을 유도하였다. 상기와 같이 서열번호 1의 아미노산 서열을 갖는 단백질의 과발현을 유도하는 과정에서, 교반 속도는 200 rpm, 배양 온도는 37 ℃가 유지되도록 조정하였고, IPTG를 첨가 후에는 교반 속도를 150 rpm으로, 배양 온도를 18 ℃로 조정하여 20시간 동안 배양하였다.The transformants stored frozen in Example [1-1] were pre-inoculated in 3 ml of LK solid medium (LB medium + 2 μg/ml kanamycin) and cultured at 37° C. for 10 hours or longer. One colony appearing in the solid medium was inoculated into a test tube containing 3 ml of LK solid medium through colony separation, and seed culture was performed for 12 hours in a shaking incubator at 37°C. Then, 2 ml of the seed cultured culture solution was added to a 1,000 ml flask containing 200 ml of LK medium to perform main culture, and the final concentration when the absorbance at 600 nm was 0.6 was 0.1 mM. (isopropyl-1-thio-β-D-galactopyranoside) was added to induce overexpression of the protein having the amino acid sequence of SEQ ID NO: 1. In the process of inducing overexpression of the protein having the amino acid sequence of SEQ ID NO: 1 as described above, the stirring speed was adjusted to maintain 200 rpm and the culture temperature at 37°C, and after the addition of IPTG, the stirring speed was set to 150 rpm, and culture The temperature was adjusted to 18° C. and incubated for 20 hours.
또한, 상기와 같이 서열번호 1의 아미노산 서열을 갖는 단백질의 과발현이 유도된 형질전환체의 배양액을 50 ㎖의 코니칼 튜브(conical tube, SPL Life Sciences Co., Ltd., 한국)에 분주하고, 4 ℃에서 3,000 rpm으로 20분 동안 원심분리하여 상층액을 분리해 낸 펠렛에 프로피니아 1X 용해 완충용액(Profinia 1X Lysis buffer, Bio-Rad Laboratories, 미국) 10 ㎖를 첨가하고, 소니케이션(sonication) 방법으로 세포를 파쇄함으로써, 형질전환체의 세포 용해물(cell lysate)을 수득하였다.In addition, as described above, the culture medium of the transformant in which the overexpression of the protein having the amino acid sequence of SEQ ID NO: 1 is induced was dispensed into 50 ml of a conical tube (SPL Life Sciences Co., Ltd., Korea), and To the pellet from which the supernatant was separated by centrifugation at 3,000 rpm for 20 minutes at 4°C, 10 ml of Profinia 1X Lysis buffer (Bio-Rad Laboratories, USA) was added, and sonication (sonication) By disrupting the cells by the method, a cell lysate of the transformant was obtained.
상기와 같이 수득된 세포 용해물을 4 ℃에서 14,000 rpm으로 20분 동안 다시 원심분리하여 상층액을 수득하였고, IMAC Kit® His tag 흡착 컬럼(Bio-Rad Laboratories, 미국)이 장착된 고속 단백질 액체 크로마토그래피(Fast Protein Liquid Chromatography, Bio-Rad Laboratories, 미국)를 이용하여 상기와 같이 수득된 상층액으로부터 과발현된 서열번호 1의 아미노산 서열을 갖는 단백질을 분리하였다.The cell lysate obtained as described above was centrifuged again at 14,000 rpm for 20 minutes at 4° C. to obtain a supernatant, and high-speed protein liquid chromatography equipped with an IMAC Kit ® His tag adsorption column (Bio-Rad Laboratories, USA) A protein having an overexpressed amino acid sequence of SEQ ID NO: 1 was isolated from the supernatant obtained as described above using Fast Protein Liquid Chromatography (Bio-Rad Laboratories, USA).
이렇게 분리된 단백질을, 2-머캅토에탄올(2-mercaptoethanol) 100 ㎕ 및 Laemmli Sample Buffer(#1610747, Bio-Rad Laboratories, 미국) 900 ㎕를 혼합한 4X 단백질 염색약과 3:1의 비율로 혼합하고, 끓는 물에 넣어 10분 동안 가열하였다. 이 과정에서 단백질은 변성되어 선형의 아미노산 서열을 가지므로 SDS-PAGE를 통해 분자량에 따라 분리될 수 있다. 상기와 같은 일련의 과정에 의하여 His-Tag이 붙어 있는 40.88 kDa의 서열번호 1의 아미노산 서열을 갖는 단백질이 과발현되고, 형질전환체의 세포 용해물로부터 정제 및 분리되었음을 확인하였다(도 1의 A).The thus-separated protein was mixed with a 4X protein dye mixed with 100 µl of 2-mercaptoethanol and 900 µl of Laemmli Sample Buffer (#1610747, Bio-Rad Laboratories, USA) at a ratio of 3:1. , Put in boiling water and heated for 10 minutes. In this process, the protein is denatured and has a linear amino acid sequence, so it can be separated according to its molecular weight through SDS-PAGE. It was confirmed that the protein having the amino acid sequence of SEQ ID NO: 1 of 40.88 kDa to which His-Tag is attached was overexpressed by the above-described series of processes, and purified and separated from the cell lysate of the transformant (Fig. 1A). .
한편, 과발현된 단백질이 변성되지 않는 자연 상태를 유지했을 때의 분자량을 측정하기 위해, 젤-여과 컬럼(Zorbax GF-250, Agilent, 미국)과 HPLC (Agilent Infinity 1220, Agilent, 미국)를 이용하였고, 분석 조건은 130 mM 염화나트륨 / 20 mM 인산 수소 나트륨(pH 7.0)의 이동상을 1.0 ㎖/분의 속도로 흘려주었으며, 검출 파장은 210 ㎚ 로 하여 샘플 20 ㎕을 분석하였다. 이 때, 스탠다드 단백질 마커(MWGF1000-1KT, Sigmaaldrich, 미국)는 티로글로불린 (669 kDa), 아포단백질 (443 kDa), 베타-아밀라아제 (200 kDa), 알코올 탈수소효소 (150 kDa), 탄산무수화효소 (29 kDa)를 사용하였고, D-트레오닌 알돌레이즈와 단백질 마커를 상기와 같이 분석한 결과, 접힌 구조의 D-트레오닌 알돌레이즈의 크기는 87 kDa으로 확인되었다(도 1의 B).On the other hand, in order to measure the molecular weight when the overexpressed protein remains in its natural state not denatured, a gel-filtration column (Zorbax GF-250, Agilent, USA) and HPLC (Agilent Infinity 1220, Agilent, USA) were used. , As for the analysis conditions, a mobile phase of 130 mM sodium chloride / 20 mM sodium hydrogen phosphate (pH 7.0) was flowed at a rate of 1.0 ml/min, and 20 µl of a sample was analyzed with a detection wavelength of 210 nm. At this time, standard protein markers (MWGF1000-1KT, Sigmaaldrich, USA) are thyroglobulin (669 kDa), apoprotein (443 kDa), beta-amylase (200 kDa), alcohol dehydrogenase (150 kDa), carbonic anhydrase (29 kDa) was used, and as a result of analyzing the D-threonine aldolase and the protein marker as described above, the size of the folded structure of D-threonine aldolase was confirmed to be 87 kDa (FIG. 1B).
앞서 확인했던 SDS-PAGE 결과 및 젤-여과 크로마토그래피의 데이터를 종합하여 비교했을 때, 자연 상태의 D-트레오닌은 이합체 형태임을 확인할 수 있었다.When comparing the SDS-PAGE results and gel-filtration chromatography data previously confirmed, it was confirmed that D-threonine in its natural state was in the form of a dimer.
[실시예 2][Example 2]
서열번호 1의 아미노산 서열을 갖는 단백질의 활성 확인Identification of the activity of the protein having the amino acid sequence of SEQ ID NO: 1
상기 실시예 1을 통해 정제 및 분리한 상기 서열번호 1의 아미노산 서열을 갖는 단백질은 하기 반응식과 같은 ‘D-트레오닌 생산 효소’로서의 활성을 가지는바, 글리신 및 아세트알데히드를 반응시켜 상기 활성을 확인하였다.The protein having the amino acid sequence of SEQ ID NO: 1 purified and isolated through Example 1 has an activity as a'D-threonine producing enzyme' as shown in the following scheme, and the activity was confirmed by reacting glycine and acetaldehyde. .
[반응식][Reaction Scheme]
Figure PCTKR2020014292-appb-I000002
Figure PCTKR2020014292-appb-I000002
구체적으로, 상기 실시예 [1-2]에서 정제 및 분리한 서열번호 1의 아미노산 서열을 갖는 단백질 0.01 ㎎/㎖를 100 mM의 글리신 및 100 mM의 아세트알데히드와 함께, 100 μM의 피리독살-5-인산(pyridoxal-5-phosphate, PLP)과 1 mM의 MnCl2가 포함된 50 mM의 CHES 완충용액(pH 9.0)에 첨가하고, 37 ℃에서 10분 동안 반응시킨 다음, 100 ℃에서 10분 동안 중탕하여 상기 반응을 종료시킨 다음, 반응이 종료된 완충 용액에서 D-트레오닌의 농도를 측정하였다.Specifically, 0.01 mg/ml of the protein having the amino acid sequence of SEQ ID NO: 1 purified and isolated in Example [1-2] was mixed with 100 mM glycine and 100 mM acetaldehyde, and 100 μM pyridoxal-5. -Added to 50 mM CHES buffer (pH 9.0) containing phosphoric acid (pyridoxal-5-phosphate, PLP) and 1 mM MnCl 2 , reacted at 37° C. for 10 minutes, and then at 100° C. for 10 minutes. After the reaction was terminated by boiling water, the concentration of D-threonine was measured in the buffer solution at which the reaction was completed.
상기 생성된 D-트레오닌은 OPA/NAC 전처리(OPA/NAC derivatization)한 후 HPLC를 통해 분석하였다. 상기 전처리 과정은 8 ㎎의 OPA(o-phthalaldelhyde)와 10 ㎎의 NAC(N-acetylcysteine)를 1 ㎖의 메탄올에 녹여 OPA/NAC 용액을 만들고, 25 ㎕의 분석할 샘플을 OPA/NAC 용액 50 ㎕와 0.4 M의 붕산나트륨 완충용액(sodium borate buffer, pH10.4) 175 ㎕의 혼합물에 넣고 0.2 ㎛ 필터로 필터링하여 전처리된 샘플을 준비하였다.The generated D-threonine was analyzed through HPLC after OPA/NAC pretreatment (OPA/NAC derivatization). In the pretreatment process, 8 mg of OPA (o-phthalaldelhyde) and 10 mg of NAC (N-acetylcysteine) were dissolved in 1 ml of methanol to prepare an OPA/NAC solution, and 25 μl of the sample to be analyzed was prepared with 50 μl of an OPA/NAC solution. And 0.4 M sodium borate buffer (pH 10.4) were added to a mixture of 175 μl and filtered through a 0.2 μm filter to prepare a pretreated sample.
상기와 같이 준비된 전처리된 샘플 20 ㎕을, VWD(Variable wavelength detector)와 역상 C18 칼럼(reverse-phase C18 column, Eclipse XDB-C18, 4.6×150 ㎜, 3.5 ㎛, Agilent)이 장착된 고압 액체 크로마토그래피에 주입하여, D-트레오닌의 농도를 측정하였다. 상기와 같은 고압 액체 크로마토그래피를 수행함에 이용된 이동상은 메탄올(이동상 A)과 50 mM의 아세트산나트륨(sodium acetate, pH 5.9)(이동상 B)으로 구성되고, 유속 1 ㎖/분의 유속으로 10분 동안 흘려주었는데, 0~3분 동안은 이동상 A와 이동상 B를 3:7의 비율로, 3~10분 동안은 이동상 A와 이동상 B를 7:3의 비율로 유지하였다. 그리고 생성된 D-트레오닌을 정량화하기 위해 시판되는 D-트레오닌(Sigma-aldrich, 미국)과 D-알로트레오닌(TOKYO CHEMICAL INDUSTRY CO., LTD., 일본)을 스탠다드로 사용하였다.20 µl of the pretreated sample prepared as described above was subjected to high pressure liquid chromatography equipped with a variable wavelength detector (VWD) and a reverse-phase C18 column (Eclipse XDB-C18, 4.6×150 mm, 3.5 µm, Agilent). Was injected into, and the concentration of D-threonine was measured. The mobile phase used to perform the high-pressure liquid chromatography as described above is composed of methanol (mobile phase A) and 50 mM sodium acetate (pH 5.9) (mobile phase B), and flow rate is 10 minutes at a flow rate of 1 ml/min. For 0 to 3 minutes, mobile phase A and mobile phase B were maintained at a ratio of 3:7, and for 3 to 10 minutes, mobile phase A and mobile phase B were maintained at a ratio of 7:3. In order to quantify the generated D-threonine, commercially available D-threonine (Sigma-aldrich, USA) and D-allothreonine (TOKYO CHEMICAL INDUSTRY CO., LTD., Japan) were used as standards.
그 결과, 상기와 같이 반응이 종료된 완충 용액에서는 D-트레오닌과 D-알로트레오닌이 모두 검출되었고(도 2), 애초에 존재하지 않았던 D-트레오닌이 상기 반응에 의해 생성되었는바, 상기 실시예 [1-2]에서 정제 및 분리한 서열번호 1의 아미노산 서열을 갖는 단백질이 글리신과 아세트알데히드로부터 D-트레오닌을 생성하는, D-트레오닌 생산 효소로서의 활성을 가짐을 확인하였다.As a result, in the buffer solution in which the reaction was terminated as described above, both D-threonine and D-allothreonine were detected (FIG. 2), and D-threonine, which was not present in the first place, was produced by the reaction. It was confirmed that the protein having the amino acid sequence of SEQ ID NO: 1 purified and isolated in 1-2] has an activity as a D-threonine-producing enzyme, producing D-threonine from glycine and acetaldehyde.
[실시예 3][Example 3]
서열번호 1의 아미노산 서열을 갖는 단백질의 효소 활성에 영향을 미치는 조건 확인Confirmation of conditions affecting the enzyme activity of the protein having the amino acid sequence of SEQ ID NO: 1
상기 실시예 2에서 확인된 바와 같이, 서열번호 1의 아미노산 서열을 갖는 단백질이 D-트레오닌 생산 효소로서 작용하므로, 그 활성에 영향을 미치는 조건들을 확인하여 상기 서열번호 1의 아미노산 서열을 갖는 단백질의 최적 활성 조건을 도출하였다.As confirmed in Example 2, since the protein having the amino acid sequence of SEQ ID NO: 1 acts as a D-threonine-producing enzyme, conditions affecting its activity were identified, and the protein having the amino acid sequence of SEQ ID NO: 1 Optimal activity conditions were derived.
[3-1] 금속 양이온의 영향[3-1] Influence of metal cations
상기 서열번호 1의 아미노산 서열을 갖는 단백질의 효소의 활성에 금속 양이온의 종류가 미치는 영향을 확인하기 위하여, 상기 실시예 [1-2]에서 정제 및 분리한 서열번호 1의 아미노산 서열을 갖는 단백질 0.01 ㎎/㎖, 글리신 100 mM, 아세트알데히드 100 mM 및 피리독살-5-인산 100 μM이 포함된 50 mM의 CHES 완충용액(pH 9.0)에 1 mM의 EDTA 또는 금속 양이온(Mn2+, Mg2+, Co2+, Zn2+)과 함께 첨가하여 35 ℃에서 10분 동안 반응시키고, 상기 반응을 종료시키기 위해 100 ℃에서 10분 동안 중탕하였다. 그리고 상기 실시예 2에서와 같은 방법으로, 상기와 같이 반응이 종료된 완충 용액에서 D-트레오닌과 D-알로트레오닌의 농도를 측정하여 그 상대값을 비교하였다.To confirm the effect of the type of metal cation on the enzyme activity of the protein having the amino acid sequence of SEQ ID NO: 1, protein 0.01 having the amino acid sequence of SEQ ID NO: 1 purified and isolated in Example [1-2] Mg/ml, 100 mM glycine, 100 mM acetaldehyde, and 100 μM pyridoxal-5-phosphate in 50 mM CHES buffer (pH 9.0) containing 1 mM EDTA or metal cations (Mn 2+ , Mg 2+ ). , Co 2+ , Zn 2+ ) and reacted at 35° C. for 10 minutes, followed by bathing at 100° C. for 10 minutes to terminate the reaction. And in the same manner as in Example 2, the concentrations of D-threonine and D-allothreonine were measured in the buffer solution in which the reaction was terminated as described above, and their relative values were compared.
그 결과, 상기 서열번호 1의 아미노산 서열을 갖는 단백질은, 여러 종류의 금속 양이온 중 망간 양이온과 마그네슘 양이온이 첨가된 경우에 D-트레오닌 생산 효소의 활성이 향상되었고, 그 중에서도 망간 양이온을 이용한 경우에 D-트레오닌 생산 효소의 활성이 가장 우수한 것으로 확인되었다(도 3의 A).As a result, for the protein having the amino acid sequence of SEQ ID NO: 1, the activity of the D-threonine-producing enzyme was improved when manganese cation and magnesium cation were added among various types of metal cations, and among them, when manganese cation was used. It was confirmed that the activity of the D-threonine-producing enzyme was the most excellent (Fig. 3A).
상기 서열번호 1의 아미노산 서열을 갖는 단백질의 효소 활성에 금속 양이온의 농도가 미치는 영향을 확인하기 위하여, 상기 실시예 [1-2]에서 정제 및 분리한 서열번호 1의 아미노산 서열을 갖는 단백질 0.01 ㎎/㎖, 글리신 100 mM, 아세트알데히드 100 mM 및 피리독살-5-인산 100 μM이 포함된 50 mM의 CHES 완충용액(pH 9.0)에 망간 양이온을 0 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1 mM, 2.5 mM 및 5 mM의 함량으로 각각 첨가하여 35 ℃에서 10분 동안 반응시키고, 상기 반응을 종료시키기 위해 100 ℃에서 10분 동안 중탕하였다. 그리고 상기 실시예 2에서와 같은 방법으로, 상기와 같이 반응이 종료된 완충 용액에서 D-트레오닌과 D-알로트레오닌의 농도를 측정하여 그 상대값을 비교하였다.In order to confirm the effect of the concentration of metal cation on the enzyme activity of the protein having the amino acid sequence of SEQ ID NO: 1, 0.01 mg of the protein having the amino acid sequence of SEQ ID NO: 1 purified and isolated in Example [1-2] /Ml, 100 mM glycine, 100 mM acetaldehyde, and 100 μM pyridoxal-5-phosphate in 50 mM CHES buffer solution (pH 9.0) with manganese cations 0 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1 Mm, 2.5 mM, and 5 mM were added respectively and reacted at 35° C. for 10 minutes, followed by bathing at 100° C. for 10 minutes to terminate the reaction. And in the same manner as in Example 2, the concentrations of D-threonine and D-allothreonine were measured in the buffer solution in which the reaction was terminated as described above, and their relative values were compared.
그 결과, 상기 서열번호 1의 아미노산 서열을 갖는 단백질은 망간 양이온이 존재하는 환경에서만 D-트레오닌 생산 효소의 활성을 나타내는 것으로 확인되었다(도 3의 B).As a result, it was confirmed that the protein having the amino acid sequence of SEQ ID NO: 1 exhibits the activity of the D-threonine-producing enzyme only in an environment in which a manganese cation is present (FIG. 3B).
[3-2] 온도의 영향[3-2] Influence of temperature
상기 서열번호 1의 아미노산 서열을 갖는 단백질의 효소 활성에 온도가 미치는 영향을 확인하기 위하여, 상기 실시예 [1-2]에서 정제 및 분리한 서열번호 1의 아미노산 서열을 갖는 단백질 0.01 ㎎/㎖, 글리신 100 mM 및 아세트알데히드 100 mM를, 100 μM의 피리독살-5-인산 및 1 mM의 Mn2+가 포함된 50 mM의 CHES 완충용액 (pH 9.0)에 첨가한 다음, 25 ℃ 내지 55 ℃의 온도에서 각각 10분 동안 반응시키고, 상기 반응을 종료시키기 위해 100 ℃에서 10분 동안 중탕하였다. 그리고, 상기 실시예 2에서와 같은 방법으로, 상기와 같이 반응이 종료된 완충 용액에서 D-트레오닌과 D-알로트레오닌의 농도를 측정하여 그 상대값을 비교하였다.In order to confirm the effect of temperature on the enzyme activity of the protein having the amino acid sequence of SEQ ID NO: 1, 0.01 mg/ml of the protein having the amino acid sequence of SEQ ID NO: 1 purified and isolated in Example [1-2], 100 mM of glycine and 100 mM of acetaldehyde were added to 50 mM CHES buffer (pH 9.0) containing 100 μM of pyridoxal-5-phosphoric acid and 1 mM of Mn 2+, and then at 25°C to 55°C. The reaction was performed at a temperature for 10 minutes, and a bath was performed at 100° C. for 10 minutes to terminate the reaction. And, in the same manner as in Example 2, the concentrations of D-threonine and D-allotreonine were measured in the buffer solution in which the reaction was terminated as described above, and their relative values were compared.
그 결과, 서열번호 1의 아미노산 서열을 갖는 단백질의 효소 활성은 35 ℃에 가까울수록 점점 우수해지는 것으로 확인되었다(도 4).As a result, it was confirmed that the enzyme activity of the protein having the amino acid sequence of SEQ ID NO: 1 becomes more and more excellent as it approaches 35°C (FIG. 4).
[3-3] pH의 영향[3-3] Effect of pH
상기 서열번호 1의 아미노산 서열을 갖는 단백질의 효소 활성에 pH가 미치는 영향을 확인하기 위하여, 상기 실시예 [1-2]에서 정제 및 분리한 서열번호 1의 아미노산 서열을 갖는 단백질 0.01 ㎎/㎖, 글리신 100 mM 및 아세트알데히드 100 mM를, 피리독살-5-인산 100 μM 및 Mn2+ 1 mM과 함께, 각각 pH 6.5 내지 7.5의 MOPS 완충용액, pH 7.5 내지 8.0의 HEPES 완충 용액, pH 8.0 내지 8.5의 EPPS 완충 용액 및 pH 8.5 내지 10의 CHES 완충 용액에 각각 첨가하여, pH 6.5 내지 10의 범위에서 각각 반응시켰다. 상기 효소 반응은 35 ℃의 온도에서 10분 동안 수행한 뒤, 상기 반응을 종료시키기 위해 100 ℃에서 10분 동안 중탕하였다. 그리고 상기 실시예 2에서와 같은 방법으로, 상기와 같이 반응이 종료된 완충 용액에서 D-트레오닌과 D-알로트레오닌의 농도를 측정하여 그 상대값을 비교하였다.In order to confirm the effect of pH on the enzyme activity of the protein having the amino acid sequence of SEQ ID NO: 1, 0.01 mg/ml of the protein having the amino acid sequence of SEQ ID NO: 1 purified and isolated in Example [1-2], 100 mM glycine and 100 mM acetaldehyde, together with 100 μM of pyridoxal-5-phosphate and Mn 2+ 1 mM, respectively, in a MOPS buffer solution of pH 6.5 to 7.5, HEPES buffer solution of pH 7.5 to 8.0, pH 8.0 to 8.5 Was added to the EPPS buffer solution and the CHES buffer solution of pH 8.5 to 10, respectively, and reacted in the range of pH 6.5 to 10, respectively. The enzymatic reaction was performed at a temperature of 35° C. for 10 minutes, and then bathed at 100° C. for 10 minutes to terminate the reaction. And in the same manner as in Example 2, the concentrations of D-threonine and D-allothreonine were measured in the buffer solution in which the reaction was terminated as described above, and their relative values were compared.
그 결과, 서열번호 1의 아미노산 서열을 갖는 단백질의 효소 활성은 pH가 8.5~9.0에 가까울수록 점점 우수해지는 것으로 확인되었다(도 5).As a result, it was confirmed that the enzyme activity of the protein having the amino acid sequence of SEQ ID NO: 1 becomes more and more excellent as the pH approaches 8.5-9.0 (FIG. 5).
[3-4] 피리독살-5-인산의 영향[3-4] Effect of pyridoxal-5-phosphate
상기 서열번호 1의 아미노산 서열을 갖는 단백질의 효소 활성에 피리독살-5-인산의 존부 및 농도가 미치는 영향을 확인하기 위하여, 상기 실시예 [1-2]에서 정제 및 분리한 서열번호 1의 아미노산 서열을 갖는 단백질 0.01 ㎎/㎖, 글리신 100 mM, 아세트알데히드 100 mM 및 Mn2+ 1 mM가 포함된 50 mM의 CHES 완충용액(pH 9.0)에 피리독살-5-인산을 0 μM, 0.1 μM, 0.5 μM, 2.5 μM, 5 μM, 25 μM, 50 μM, 100 μM 및 200 μM의 함량으로 각각 첨가하여 35 ℃에서 10분 동안 반응시키고, 상기 반응을 종료시키기 위해 100 ℃에서 10분 동안 중탕하였다. 그리고 상기 실시예 2에서와 같은 방법으로, 상기와 같이 반응이 종료된 완충 용액에서 D-트레오닌과 D-알로트레오닌의 농도를 측정하여 그 상대값을 비교하였다.In order to confirm the effect of the presence and concentration of pyridoxal-5-phosphate on the enzyme activity of the protein having the amino acid sequence of SEQ ID NO: 1, the amino acid of SEQ ID NO: 1 purified and isolated in Example [1-2] 0 μM, 0.1 μM of pyridoxal-5-phosphate in 50 mM CHES buffer solution (pH 9.0) containing 0.01 mg/ml of protein having the sequence, 100 mM glycine, 100 mM acetaldehyde, and 1 mM Mn 2+, 0.5 μM, 2.5 μM, 5 μM, 25 μM, 50 μM, 100 μM, and 200 μM were added respectively to react at 35° C. for 10 minutes, followed by bathing at 100° C. for 10 minutes to terminate the reaction. And in the same manner as in Example 2, the concentrations of D-threonine and D-allothreonine were measured in the buffer solution in which the reaction was terminated as described above, and their relative values were compared.
그 결과, 서열번호 1의 아미노산 서열을 갖는 단백질의 효소 활성은 피리독살-5-인산이 존재하는 환경에서 더욱 우수해지고, 100 μM 수준의 농도에서 최대 활성에 도달하는 것으로 확인되었다(도 6).As a result, it was confirmed that the enzymatic activity of the protein having the amino acid sequence of SEQ ID NO: 1 became more excellent in the environment in the presence of pyridoxal-5-phosphate, and reached the maximum activity at a concentration of 100 μM (FIG. 6).
[실시예 4][Example 4]
서열번호 1의 아미노산 서열을 갖는 단백질 효소의 부분 입체 이성질체 특이성 향상을 위한 효소 개량Enzyme improvement to improve the diastereoisomeric specificity of protein enzymes having the amino acid sequence of SEQ ID NO: 1
상기 실시예 3에서 확인된 바와 같이, 서열번호 1의 아미노산 서열을 갖는 단백질이 D-트레오닌 생산 효소로서 고활성을 가지는 것을 확인함에 따라 D-트레오닌을 특이적으로 합성하기 위한 목적으로 효소 개량을 진행하였다.As confirmed in Example 3, as it was confirmed that the protein having the amino acid sequence of SEQ ID NO: 1 has high activity as a D-threonine-producing enzyme, the enzyme was improved for the purpose of specifically synthesizing D-threonine. I did.
먼저, 상기 서열번호 1의 아미노산 서열을 갖는 단백질 효소의 결정구조에서 활성 부위 내의 부분입체 이성질체 변화에 관여할 것으로 예측되는 잔기들을 선별하였고, 해당 잔기들을 알라닌으로 치환함으로써 그 활성과 부분입체 이성질체의 비율 변화를 분석하였다. 그 결과, D-트레오닌에 대한 입체특이성을 향상시키는 잔기들을 도출하였고, D-트레오닌으로의 입체 특이성이 99% 이상으로 향상된 것을 확인하였다. First, residues predicted to be involved in diastereomer changes in the active site in the crystal structure of the protein enzyme having the amino acid sequence of SEQ ID NO: 1 were selected, and the ratio of the activity to the diastereomer by substituting the residues with alanine Changes were analyzed. As a result, residues that improve stereospecificity for D-threonine were derived, and it was confirmed that the stereospecificity for D-threonine was improved to 99% or more.
[4-1] 부분입체 이성질체 비율 변화에 관여하는 아미노산 잔기 선별과 해당 단백질들의 클로닝 및 형질전환[4-1] Selection of amino acid residues involved in diastereomer ratio change, and cloning and transformation of the corresponding proteins
상기 서열번호 1의 아미노산 서열을 갖는 단백질 구조는 기존에 보고된 알칼리제너스 자일로속시단스(Alcaligenes xylosoxidans) 유래의 D-트레오닌 알돌레이즈 X-ray 결정구조를 기반으로 확보하였고(도 7), 결정구조의 활성부위에서 기질(글리신 및 아세트알데히드), 피리독살-5-인산 및 망간 이온과 4 옴스트롱 거리에서 상호작용하는 아미노산 잔기들을 선별하였다. 상기와 같이 선별된 잔기들을 알라닌(alanine) 등으로 치환하여 G145Q, S312A, G179A, S312N, G145L, R147A, S90A, S242A, Q71A, H47A, Y250A, T223A, D313A, Q175A, Y177A, H288L, D294E, D341A, H339A, H183 및 K49A의 단일 치환 단백질을 제작하기 위하여, 서열번호 2의 염기 서열을 기초로 프라이머를 각각 설계하고 ㈜마크로젠에 의뢰하여 합성하였다. The protein structure having the amino acid sequence of SEQ ID NO: 1 was obtained based on the previously reported D-threonine aldolase X-ray crystal structure derived from Alcaligenes xylosoxidans (FIG. 7), and was determined. At the active site of the structure, amino acid residues that interact with substrates (glycine and acetaldehyde), pyridoxal-5-phosphate and manganese ions at a distance of 4 angstroms were selected. G145Q, S312A, G179A, S312N, G145L, R147A, S90A, S242A, Q71A, H47A, Y250A, T223A, D313A, Q175A, Y177A, H288L, D294E, D341A , H339A, H183, and K49A, each primer was designed based on the nucleotide sequence of SEQ ID NO: 2 and synthesized by requesting Macrogen.
상기 서열번호 2의 염기 서열을 포함하는 DNA를 주형(template)으로 하고, 상기 프라이머 쌍 각각을 이용하여 PCR을 수행함으로써 증폭된 각각의 PCR 산물을 Phusion, Q5, KOD 중합효소들을 이용하여 라이게이션/카이네이션 방법으로 염기 서열 말단부위를 각각 연결하였고, 염기 서열 분석(sequencing, ㈜마크로젠)을 통해, 해당 잔기들이 알라닌 등의 아미노산을 암호화하는 염기 서열로 정확히 치환되었음을 확인하였다. 해당 염기 서열들은 각각 대장균 C2566 균주(Novagen, 미국)에 형질전환하였고, 20% 글리세린 용액을 첨가하여 냉동 보관하였다.The DNA containing the nucleotide sequence of SEQ ID NO: 2 is used as a template, and each PCR product amplified by performing PCR using each of the primer pairs is ligated using Phusion, Q5, and KOD polymerases. Each of the ends of the nucleotide sequence was ligated by the kination method, and through sequencing (Macrogen, Inc.), it was confirmed that the corresponding residues were accurately substituted with nucleotide sequences encoding amino acids such as alanine. The corresponding nucleotide sequences were transformed into E. coli C2566 strain (Novagen, USA), respectively, and stored frozen by adding a 20% glycerin solution.
[4-2] 단일 치환 단백질 돌연변이들의 과발현 및 정제[4-2] Overexpression and Purification of Single Substitute Protein Mutations
상기 실시예 [1-2]와 동일한 방법으로 실시예 [4-1]에서 냉동 보관된 형질전환체를 3 ㎖의 LK 고체 배지(LB 배지+카나마이신 25 μg/㎖)에 선조접종하고 37 ℃에서 10시간 이상 배양하였다. 고체 배지에 나타나는 하나의 콜로니를 단집락 분리 과정으로 3 ㎖의 LK 고체 배지가 포함된 시험관(test tube)에 접종하고 37 ℃의 진탕 배양기로 12시간 동안 종균 배양을 실시하였다. 그런 다음, 상기 종균 배양된 배양액 2 ㎖를, 200 ㎖의 LK 배지가 포함된 1,000 ㎖ 플라스크에 첨가하여 본 배양을 실시하였고, 600 ㎚에서의 흡광도가 0.6이 될 때의 최종 농도가 0.1 mM이 되도록 IPTG(isopropyl-1-thio-β-D-galactopyranoside)를 첨가하여 G145Q, S312A, G179A, S312N, G145L, R147A, S90A, S242A, Q71A, H47A, Y250A, T223A, D313A, Q175A, Y177A, H288L, D294E, D341A, H339A, H183A 및 K49A 각각의 단일 치환 단백질의 과발현을 유도하였다. 과발현을 유도하는 과정에서, 교반 속도는 200 rpm, 배양 온도는 37 ℃가 유지되도록 조정하였고, IPTG를 첨가 후에는 교반 속도를 150 rpm으로, 배양 온도를 18 ℃로 조정하여 20시간 동안 배양하였다.In the same manner as in Example [1-2], the transformants frozen in Example [4-1] were pre-inoculated in 3 ml of LK solid medium (LB medium + kanamycin 25 μg/ml) and at 37°C. Incubated for more than 10 hours. One colony appearing in the solid medium was inoculated into a test tube containing 3 ml of LK solid medium in the process of separating a single colony, and seed culture was performed for 12 hours in a shaking incubator at 37°C. Then, 2 ml of the seed cultured culture solution was added to a 1,000 ml flask containing 200 ml of LK medium to perform main culture, and the final concentration when the absorbance at 600 nm was 0.6 was 0.1 mM. IPTG (isopropyl-1-thio-β-D-galactopyranoside) was added to G145Q, S312A, G179A, S312N, G145L, R147A, S90A, S242A, Q71A, H47A, Y250A, T223A, D313A, Q175A, Y177A, H288L, D294E , D341A, H339A, H183A and K49A respectively induced overexpression of single substitution proteins. In the process of inducing overexpression, the stirring speed was adjusted to maintain 200 rpm and the culture temperature at 37°C, and after the addition of IPTG, the stirring speed was adjusted to 150 rpm and the culture temperature to 18°C, followed by incubation for 20 hours.
또한, 각각의 상기 단일 치환 단백질의 과발현이 유도된 형질전환체의 배양액을 50 ㎖의 코니칼 튜브(conical tube, SPL Life Sciences Co., Ltd., 한국)에 분주하고, 4 ℃에서 3,000 rpm으로 20분 동안 원심분리하여 상층액을 분리해 낸 펠렛에 프로피니아 1X 용해 완충용액(Profinia 1X Lysis buffer, Bio-Rad Laboratories, 미국) 10 ㎖를 첨가하고, 소니케이션(sonication) 방법으로 세포를 파쇄함으로써, 형질전환체의 세포 용해물(cell lysate)을 수득하였다.In addition, the culture medium of the transformant in which the overexpression of each of the single substituted proteins was induced was dispensed into a 50 ml conical tube (SPL Life Sciences Co., Ltd., Korea), and then at 4° C. at 3,000 rpm. By centrifuging for 20 minutes to separate the supernatant, 10 ml of Profinia 1X Lysis buffer (Bio-Rad Laboratories, USA) was added, and the cells were disrupted by sonication. , To obtain a cell lysate of the transformant (cell lysate).
상기와 같이 수득된 세포 용해물을 4 ℃에서 14,000 rpm으로 20분 동안 다시 원심분리하여 상층액을 수득하였고, IMAC Kit® His tag 흡착 컬럼(Bio-Rad Laboratories, 미국)이 장착된 고속 단백질 액체 크로마토그래피(Fast Protein Liquid Chromatography, Bio-Rad Laboratories, 미국)를 이용하여 상기 상층액으로부터 과발현된 상기 단일 치환 단백질들을 분리하였다.The cell lysate obtained as described above was centrifuged again at 14,000 rpm for 20 minutes at 4° C. to obtain a supernatant, and high-speed protein liquid chromatography equipped with an IMAC Kit ® His tag adsorption column (Bio-Rad Laboratories, USA) The single substituted proteins overexpressed were isolated from the supernatant using Fast Protein Liquid Chromatography (Bio-Rad Laboratories, USA).
이렇게 분리된 각각의 단백질을 SDS-PAGE를 통해 확인한 결과, 상기와 같은 일련의 과정에 의하여 His-Tag이 붙어 있는 40.88 kDa의 단일 치환된 단백질이 과발현되고, 형질전환체의 세포 용해물로부터 정제 및 분리되었음을 확인하였다(도 8).As a result of confirming each of the separated proteins through SDS-PAGE, a 40.88 kDa single substituted protein with His-Tag attached was overexpressed by a series of processes as described above, and purified from the cell lysate of the transformant. It was confirmed that it was separated (FIG. 8).
[4-3] 단일 치환 단백질들의 활성 및 부분 입체 이성질체 비율 변화 분석[4-3] Analysis of changes in activity and diastereomer ratio of single substituted proteins
상기 실시예 [4-1]에서 확보된 각각의 상기 단일 치환 단백질들의 활성, 그리고 이들에 의해 촉매되는 반응 생성물의 부분입체 이성질체 비율 변화를 확인하였다. 상기 실시예 2와 동일한 방법으로 실시예 [4-1]에서 확보된 0.01 ㎎/㎖의 단백질들을 100 mM의 글리신 및 100 mM의 아세트알데히드과 함께, 100 μM의 피리독살-5-인산(pyridoxal-5-phosphate, PLP) 및 1 mM의 MnCl2가 포함된 50 mM의 CHES 완충용액(pH 9.0)에 첨가하고, 37 ℃에서 10분 동안 반응시켰다. 100 ℃에서 10분 동안 중탕하여 반응을 종료시킨 다음, 반응이 종료된 완충 용액에서 D-트레오닌의 농도를 측정하였다.The activity of each of the single substituted proteins obtained in Example [4-1] and the diastereomer ratio change of the reaction product catalyzed by them were confirmed. In the same manner as in Example 2, 0.01 mg/ml of the proteins obtained in Example [4-1] were mixed with 100 mM glycine and 100 mM acetaldehyde, and 100 μM of pyridoxal-5-phosphate (pyridoxal-5). -phosphate, PLP) and 1 mM MnCl 2 were added to 50 mM CHES buffer (pH 9.0), and reacted at 37° C. for 10 minutes. After the reaction was terminated by bathing at 100° C. for 10 minutes, the concentration of D-threonine was measured in the buffer solution at which the reaction was completed.
상기 D-트레오닌의 생성량은 OPA/NAC 전처리(OPA/NAC derivatization)한 후 HPLC를 통해 분석하였다. 상기 전처리 과정은 8 ㎎의 OPA(o-phthalaldelhyde)와 10 ㎎의 NAC(N-acetylcysteine)를 1 ㎖의 메탄올에 녹여 OPA/NAC 용액을 만들고, 25 ㎕의 분석할 샘플을 OPA/NAC 용액 50 ㎕와 0.4 M의 붕산나트륨 완충용액(sodium borate buffer, pH 10.4) 175 ㎕의 혼합물에 넣고 0.2 ㎛ 필터로 필터링함으로써 전처리된 샘플을 준비하였다.The amount of D-threonine produced was analyzed through HPLC after OPA/NAC pretreatment (OPA/NAC derivatization). In the pretreatment process, 8 mg of OPA (o-phthalaldelhyde) and 10 mg of NAC (N-acetylcysteine) were dissolved in 1 ml of methanol to prepare an OPA/NAC solution, and 25 μl of the sample to be analyzed was prepared with 50 μl of an OPA/NAC solution. And 0.4 M sodium borate buffer (pH 10.4) in a mixture of 175 μl and filtered through a 0.2 μm filter to prepare a pretreated sample.
상기와 같이 준비된 전처리된 샘플 20 ㎕을, VWD(Variable wavelength detector)와 역상 C18 칼럼(reverse-phase C18 column, Eclipse XDB-C18, 4.6×150 ㎜, 3.5 ㎛, Agilent)이 장착된 고압 액체 크로마토그래피에 주입하여, D-트레오닌의 농도를 측정하였다. 상기와 같은 고압 액체 크로마토그래피를 수행함에 이용된 이동상은 메탄올(이동상 A)과 50 mM의 아세트산나트륨(sodium acetate, pH 5.9)(이동상 B)으로 구성되고, 유속 1 ㎖/분의 유속으로 10분 동안 흘려주었는데, 0 내지 3분 동안은 이동상 A와 이동상 B를 3:7의 비율로, 3 내지 10분 동안은 이동상 A와 이동상 B를 7:3의 비율로 유지하였다. 그리고 생성된 D-트레오닌을 정량화하기 위해, 시판되는 D-트레오닌(Sigma-aldrich, 미국)과 D-알로트레오닌(TOKYO CHEMICAL INDUSTRY CO., LTD., 일본)을 이용하여 정량선을 작성하여 이용하였다.20 µl of the pretreated sample prepared as described above was subjected to high pressure liquid chromatography equipped with a variable wavelength detector (VWD) and a reverse-phase C18 column (Eclipse XDB-C18, 4.6×150 mm, 3.5 µm, Agilent). Was injected into, and the concentration of D-threonine was measured. The mobile phase used to perform the high-pressure liquid chromatography as described above is composed of methanol (mobile phase A) and 50 mM sodium acetate (pH 5.9) (mobile phase B), and flow rate is 10 minutes at a flow rate of 1 ml/min. During the flow, the mobile phase A and the mobile phase B were maintained at a ratio of 3:7 for 0 to 3 minutes, and the mobile phase A and mobile phase B at a ratio of 7:3 for 3 to 10 minutes. And, in order to quantify the generated D-threonine, commercially available D-threonine (Sigma-aldrich, USA) and D-allotreonine (TOKYO CHEMICAL INDUSTRY CO., LTD., Japan) were used to prepare and use a quantitative line. .
그 결과, 상기와 같이 반응이 종료된 반응액에서는 D-트레오닌과 D-알로트레오닌의 비율 및 활성에 대한 변화가 관찰되었다(도 9). 부분입체 이성질체 과잉률(Diastereomeric excess) 분석에서, 서열번호 1의 아미노산 서열을 가지는 야생형 단백질 효소보다 부분입체 이성질체 과량 비율이 향상된 치환 단백질 돌연변이체를 확인하였다(도 9). 서열번호 5, 4, 3의 아미노산 서열을 갖는 R147A, G179A, S312A는 서열번호 1의 아미노산 서열을 가지는 야생형에 비해 특이 활성도(Specific activity)가 낮지만, 부분입체 이성질체 과잉률(d.e.)이 야생형 55.4%에 비해 각각 79.5%, 93.2%, 79.5%로 월등히 향상된 것을 확인하였다. 따라서, R147, G179, S312가 부분 입체 이성질체 비율 변화에 관여하는 잔기임을 확인하였다.As a result, a change in the ratio and activity of D-threonine and D-allothreonine was observed in the reaction solution for which the reaction was completed as described above (FIG. 9). In the diastereomeric excess analysis, a substituted protein mutant having an improved diastereomeric excess ratio than that of the wild-type protein enzyme having the amino acid sequence of SEQ ID NO: 1 was confirmed (FIG. 9). R147A, G179A, and S312A having the amino acid sequence of SEQ ID NO: 5, 4, and 3 have lower specific activity compared to the wild-type having the amino acid sequence of SEQ ID NO: 1, but the diastereomer excess (de) is wild-type 55.4 Compared to %, it was confirmed that it was significantly improved to 79.5%, 93.2%, and 79.5%, respectively. Therefore, it was confirmed that R147, G179, and S312 are residues involved in the diastereomer ratio change.
[4-4] 알라닌 외 다른 아미노산 치환 단백질들의 활성 및 부분 입체 이성질체 비율 변화에 대한 연구[4-4] Studies on the activity and diastereoisomer ratio change of amino acid substitution proteins other than alanine
상기 실시예 [4-3]에서 R147, G179, S312 아미노산 잔기를 알라닌으로 치환한 경우, 부분입체 이성질체 특이성이 향상된 것을 확인하였다. 이에, 해당 잔기들을 크기와 전하에 따라 다른 종류의 아미노산으로 치환함으로써, D-트레오닌 합성에 대한 활성 및 부분입체 이성질체 비율 변화에 대해 더 개선된 효과가 나타나는지 여부를 확인하였다.When the amino acid residues R147, G179, and S312 were substituted with alanine in Example [4-3], it was confirmed that the diastereomer specificity was improved. Accordingly, it was confirmed whether or not an improved effect on the activity of D-threonine synthesis and the diastereomer ratio change appears by substituting the corresponding residues with different kinds of amino acids depending on the size and charge.
상기 위치의 해당 잔기들을 알라닌 외 아미노산 크기 및 전하에 따라 다른 아미노산으로 치환하여 효소 활성 및 부분입체 이성질체 비율 변화 연구를 진행하였다. 서열번호 5, 4, 3의 아미노산 서열을 가지는 R147A, G179A, S312A에서 알라닌을 각각 다른 아미노산으로 치환하여 R147K, R147Q, R147V, G179L, G179V, G179Y, S312K, S312Q, S312T 및 S312V의 치환 단백질을 제작하기 위하여, 서열번호 2의 염기 서열을 기초로 프라이머를 각각 설계하고 ㈜마크로젠에 의뢰하여 합성하였다.The corresponding residues at the above positions were substituted with amino acids other than alanine depending on the size and charge of amino acids to study changes in enzyme activity and diastereomer ratio. Substituting different amino acids for alanine in R147A, G179A, and S312A having the amino acid sequences of SEQ ID NOs: 5, 4, 3, respectively, to prepare substitution proteins of R147K, R147Q, R147V, G179L, G179V, G179Y, S312K, S312Q, S312T and S312V To do this, primers were designed based on the nucleotide sequence of SEQ ID NO: 2, and were synthesized by requesting Macrogen.
상기 서열번호 2의 염기 서열을 포함하는 DNA를 주형(template)으로 하고, 상기 프라이머 쌍 각각을 이용하여 PCR을 수행함으로써 증폭된 각각의 PCR 산물을 Phusion, Q5, KOD 중합효소들을 이용하여 라이게이션/카이네이션 방법으로 염기 서열 말단부위를 각각 연결하였고, 염기 서열 분석(sequencing, ㈜마크로젠)을 통해, 해당 잔기들이 리신, 글루타민, 발린, 류신, 트레오닌의 아미노산을 암호화하는 염기 서열로 정확히 치환되었음을 확인하였다. 해당 염기 서열들은 각각 대장균 C2566 균주(Novagen, 미국)에 형질전환하였고, 20% 글리세린 용액을 첨가하여 냉동 보관하였다.The DNA containing the nucleotide sequence of SEQ ID NO: 2 is used as a template, and each PCR product amplified by performing PCR using each of the primer pairs is ligated using Phusion, Q5, and KOD polymerases. Each end of the nucleotide sequence was ligated by the kination method, and through sequencing (Macrogen, Inc.), it was confirmed that the corresponding residues were correctly substituted with nucleotide sequences encoding amino acids of lysine, glutamine, valine, leucine, and threonine. . The corresponding nucleotide sequences were transformed into E. coli C2566 strain (Novagen, USA), respectively, and stored frozen by adding a 20% glycerin solution.
상기 방법으로 확보한 알라닌 외 다른 아미노산으로 단일 치환된 각각의 단백질들의 활성과 부분입체 이성질체 비율 변화를 확인하기 위해, 실시예 [4-2], [4-3]과 동일한 방법으로 단백질의 발현 및 정제를 확인하였고(도 10), 효소 반응과 부분입체 이성질체 비율의 분석을 진행하였다. 그 결과, 알라닌 외 다른 아미노산으로의 치환의 경우, 효소 활성과 부분 입체 이성질체 비율이 향상된 것은 확인되지 않았다(도 11). 따라서, R147. G179, S312의 아미노산이 각각 알라닌으로 치환된 경우에 부분입체 이성질체 비율이 향상되는 활성이 나타남을 확인할 수 있었다.In order to confirm the change in the activity and diastereomer ratio of each protein single substituted with an amino acid other than alanine obtained by the above method, expression of the protein and the same method as in Examples [4-2] and [4-3] Purification was confirmed (FIG. 10), and analysis of the enzymatic reaction and diastereomer ratio was carried out. As a result, in the case of substitution with amino acids other than alanine, it was not confirmed that the enzyme activity and the diastereomer ratio were improved (FIG. 11). Thus, R147. When the amino acids of G179 and S312 were respectively substituted with alanine, it was confirmed that the activity of improving the diastereomer ratio was shown.
[4-5] 서열번호 9 내지 11의 아미노산 서열을 가지는 알라닌 다중 치환 단백질들의 활성 및 부분 입체 이성질체 비율 변화에 대한 연구[4-5] Study on the activity and diastereomer ratio change of alanine multiple substitution proteins having amino acid sequences of SEQ ID NOs: 9 to 11
도 9 및 도 11의 결과에 따라 서열번호 5, 4, 3의 아미노산 서열을 갖는 R147A, G179A, S312A에서 D-트레오닌 입체 특이성이 향상된 것을 확인하였으므로, 해당 잔기들 간의 다중 알라닌 치환 도입을 통해 부분입체 이성질체 비율이 더 향상되는지 여부를 추가로 확인하였다.According to the results of FIGS. 9 and 11, it was confirmed that the D-threonine stereospecificity was improved in R147A, G179A, and S312A having amino acid sequences of SEQ ID NOs: 5, 4, and 3. It was further checked whether the isomer ratio was further improved.
상기 실시예 [4-1]에서 확보한 R147A, G179A, S312A 단백질을 암호화하는 서열번호 8, 7, 6의 염기 서열을 포함하는 DNA를 주형으로 하고, 상기에서 설계하여 합성한 프라이머 쌍을 이용하여 PCR을 진행함으로써, 해당 잔기들 간에 알라닌이 다중 치환된 PCR 산물을 확보하였다. 상기와 같이 PCR을 통해 증폭시킨 PCR 산물을 Phusion, Q5, KOD 중합효소들을 이용하여 라이게이션/카이네이션 방법으로 염기 서열 말단부위를 각각 연결하였고, 염기 서열 분석(sequencing, ㈜마크로젠)을 통해, 해당 잔기들이 다중의 알라닌을 암호화하는 염기 서열로 정확히 치환되었음을 확인하였다. 해당 염기 서열들은 각각 대장균 C2566 균주(Novagen, 미국)에 형질전환하였고, 20% 글리세린 용액을 첨가하여 냉동 보관하였다.Using the DNA containing the nucleotide sequences of SEQ ID NOs: 8, 7, 6 encoding the R147A, G179A, and S312A proteins obtained in Example [4-1] as a template, and using a primer pair designed and synthesized above By performing PCR, a PCR product in which alanine was multiplexed between the corresponding residues was obtained. The PCR products amplified through PCR as described above were linked to the ends of the nucleotide sequence by ligation/kination method using Phusion, Q5, and KOD polymerases, respectively, and through nucleotide sequencing (Macrogen Co., Ltd.), the corresponding It was confirmed that the residues were correctly substituted with nucleotide sequences encoding multiple alanines. The corresponding nucleotide sequences were transformed into E. coli C2566 strain (Novagen, USA), respectively, and stored frozen by adding a 20% glycerin solution.
상기 방법으로 확보한 서열번호 9 내지 11의 아미노산 서열을 갖는 각각의 단백질들의 활성과 부분입체 이성질체 비율 변화를 확인하기 위해, 실시예 [4-2], [4-3]과 동일한 방법으로 단백질의 발현 및 정제를 확인하였고(도 12), 효소 반응과 분석을 진행하여 특이 활성도(Specific activity)로 비교하였다. 그 결과, 두개의 아미노산 잔기 G179와 S312를 알라닌으로 모두 치환한 경우에서 부분입체 이성질체 과잉률(d.e.)이 99.4%까지 증가하여 부분입체 이성질체 특이성이 향상됨을 확인하였다(도 13). 따라서, G179와 S312 아미노산이 모두 알라닌으로 치환된 경우에 D-트레오닌 특이적인 생산 활성이 향상됨을 확인할 수 있었다.In order to confirm the change in the activity and diastereomer ratio of each of the proteins having the amino acid sequences of SEQ ID NOs: 9 to 11 obtained by the above method, the protein was prepared in the same manner as in Examples [4-2] and [4-3]. Expression and purification were confirmed (FIG. 12), and enzyme reactions and analysis were performed and compared with specific activity. As a result, it was confirmed that when both amino acid residues G179 and S312 were substituted with alanine, the diastereomer excess rate (d.e.) increased to 99.4%, thereby improving the diastereomer specificity (FIG. 13). Therefore, when both G179 and S312 amino acids were substituted with alanine, it was confirmed that D-threonine-specific production activity was improved.
[4-6] 부분 입체 이성질체 비율 변화에 관여하는 잔기들의 동역학 연구[4-6] Study on the kinetics of residues involved in diastereomer ratio change
R147, G179, S312의 아미노산 알라닌 치환이 효소의 D-트레오닌 입체특이성에 미치는 영향을 확인하기 위하여, 반응의 생성물인 D-트레오닌 및 D-알로트레오닌을 역반응의 기질로 이용하여 기질 동역학 연구를 진행하였다.In order to confirm the effect of amino acid alanine substitution of R147, G179, S312 on the D-threonine stereospecificity of the enzyme, a substrate kinetics study was conducted using D-threonine and D-allothreonine, which are products of the reaction, as substrates for the reverse reaction. .
상기 실시예 [4-3]과 동일한 방법으로 각각의 정제된 서열번호 1, 5, 4, 3의 아미노산 서열을 갖는 0.01 ㎎/㎖의 야생형, R147A, G179A, S312A 효소를 50 mM CHES(pH 9.0), 0.1 mM 피리독살-5-인산, 1 mM 망간이온, 35 ℃의 조건으로 반응시켰으며, 0, 1, 2.5, 5, 10, 25, 50, 및 100 mM의 D-트레오닌 또는 D-알로트레오닌과 10분 동안 반응하였을 때, 이들의 분해 정도를 HPLC로 측정하였다.In the same manner as in Example [4-3], 0.01 mg/ml of wild-type, R147A, G179A, and S312A enzymes each having the purified amino acid sequence of SEQ ID NOs: 1, 5, 4, 3 were added to 50 mM CHES (pH 9.0. ), 0.1 mM pyridoxal-5-phosphate, 1 mM manganese ion, and reacted under the conditions of 35° C., and 0, 1, 2.5, 5, 10, 25, 50, and 100 mM of D-threonine or D-allo When reacted with threonine for 10 minutes, the degree of decomposition thereof was measured by HPLC.
그 결과, 서열번호 5, 4, 3의 아미노산 서열을 갖는 R147A, G179A, S312A의 경우, 기질의 전환 정도를 나타내는 지표인 kcat 수치가 D-알로트레오닌에 대해 그 감소량이 큰 것을 확인하였다(하기 표 1). 이는 R147A, G179A, S312A의 D-알로트레오닌에 대한 입체특이성이 감소하여 나타나는 결과로, D-알로트레오닌으로의 전환량이 감소하면서 상대적으로 D-트레오닌의 비율이 증가된 특징이 있는 것임을 확인할 수 있었다.As a result, in the case of R147A, G179A, and S312A having the amino acid sequences of SEQ ID NOs: 5, 4, 3, it was confirmed that the k cat value, an index indicating the degree of conversion of the substrate, was large compared to D-allotreonine (below Table 1). This is a result of a decrease in the stereospecificity of R147A, G179A, and S312A for D-allothreonine, and it was confirmed that there is a characteristic that the ratio of D-threonine is relatively increased while the amount of conversion to D-allothreonine is decreased.
단백질protein 기질temperament Km(mM)K m (mM) k cat (sec-1) k cat (sec -1 ) k cat / Km(sec-1mM-1) k cat / K m (sec -1 mM -1 )
야생형Wild type D-트레오닌D-threonine 17.4 ± 0.3917.4 ± 0.39 239.0 ± 2.83239.0 ± 2.83 13.7 ± 0.6413.7 ± 0.64
D-알로트레오닌D-Allothreonine 23.1 ± 2.0223.1 ± 2.02 206.0 ± 8.30206.0 ± 8.30 8.9 ± 0.868.9 ± 0.86
R147AR147A D-트레오닌D-threonine 12.0 ± 0.5512.0 ± 0.55 152.9 ± 2.18152.9 ± 2.18 12.8 ± 0.6212.8 ± 0.62
D-알로트레오닌D-Allothreonine 3.6 ± 0.243.6 ± 0.24 50.0 ± 0.9050.0 ± 0.90 14.0 ± 0.9714.0 ± 0.97
G179AG179A D-트레오닌D-threonine 91.2 ± 9.0491.2 ± 9.04 72.4 ± 4.1672.4 ± 4.16 0.8 ± 0.090.8 ± 0.09
D-알로트레오닌D-Allothreonine 17.5 ± 2.8117.5 ± 2.81 6.1 ± 0.536.1 ± 0.53 0.4 ± 0.060.4 ± 0.06
S312AS312A D-트레오닌D-threonine 13.4 ± 0.7613.4 ± 0.76 175.1 ± 3.84175.1 ± 3.84 13.1 ± 0.8013.1 ± 0.80
D-알로트레오닌D-Allothreonine 2.6 ± 0.312.6 ± 0.31 43.1 ± 1.8243.1 ± 1.82 16.3 ± 2.0216.3 ± 2.02
[실시예 5][Example 5]
부분 입체 특이성이 개량된 효소를 이용한 D-트레오닌 특이적 생산D-threonine specific production using enzymes with improved partial stereospecificity
상기 실시예 4에서 확인된 바와 같이, 구조 기반의 효소 개량을 통해 D-트레오닌으로의 입체특이성을 향상시키는 잔기를 확인하였고, 그 중에서도 D-트레오닌을 특이적으로 합성하는 서열번호 4의 아미노산 서열을 갖는 개량 효소(G179A)와 서열번호 10의 아미노산 서열을 갖는 개량 효소(G179A+S312A)를 확보하였고, 상기 확보된 개량 효소를 이용한 D-트레오닌의 특이적 생산을 확인하기 위해, 최적의 기질 조건 및 시간에 따른 생산량을 확인하였다. As confirmed in Example 4, residues that improve stereospecificity to D-threonine were identified through structure-based enzyme improvement, and among them, the amino acid sequence of SEQ ID NO: 4 for specifically synthesizing D-threonine The improved enzyme (G179A) and the improved enzyme (G179A+S312A) having the amino acid sequence of SEQ ID NO: 10 were obtained, and in order to confirm the specific production of D-threonine using the obtained improved enzyme, the optimal substrate conditions and The production volume over time was checked.
[5-1] 글리신 비율의 영향[5-1] Influence of glycine ratio
서열번호 4의 아미노산 서열을 갖는 효소(G179A)와 서열번호 10의 아미노산 서열을 갖는 효소(G179A+S312A) 활성에, 기질인 글리신의 비율이 미치는 영향을 확인하기 위해 상기 실시예 [4-2]와 동일한 방법으로 정제 및 분리한 야생형 효소 0.01 ㎎/㎖, G179A 효소 0.1 ㎎/㎖, G179A+S312A 효소 0.25 ㎎/㎖와 아세트알데히드 100 mM, 피리독살-5-인산 100 μM 및 Mn2+ 1 mM가 포함된 50 mM의 CHES 완충용액(pH 9.0)에 글리신을 0, 50, 75, 100, 125, 250, 및 500 mM로 각각 첨가하여 35 ℃에서 10분 동안 반응시켰다. 상기 반응을 종료시키기 위해 100 ℃에서 10분 동안 중탕하였고 상기 실시예 [4-3]과 동일한 방법인 OPA/NAC 유도체화를 경유하는 HPLC 분석을 통해, 상기 반응액에서 D-트레오닌과 D-알로트레오닌의 농도를 측정하여 그 상대값을 비교하였다.To confirm the effect of the ratio of the substrate glycine on the activity of the enzyme having the amino acid sequence of SEQ ID NO: 4 (G179A) and the enzyme having the amino acid sequence of SEQ ID NO: 10 (G179A+S312A), Example [4-2] Wild-type enzyme 0.01 mg/ml, G179A enzyme 0.1 mg/ml, G179A+S312A enzyme 0.25 mg/ml and acetaldehyde 100 mM, pyridoxal-5-phosphate 100 μM and Mn 2+ 1 mM purified and isolated in the same manner Glycine was added at 0, 50, 75, 100, 125, 250, and 500 mM respectively to 50 mM CHES buffer solution (pH 9.0) and reacted at 35° C. for 10 minutes. In order to terminate the reaction, the reaction mixture was bathed at 100° C. for 10 minutes, and through HPLC analysis via OPA/NAC derivatization, which is the same method as in Example [4-3], D-threonine and D-allo The concentration of threonine was measured and its relative value was compared.
그 결과, D-트레오닌의 특이적 합성은 글리신과 아세트알데히드의 비율이 4:1인 경우에 가장 우수한 것으로 확인되었다(도 14).As a result, it was confirmed that the specific synthesis of D-threonine was best when the ratio of glycine and acetaldehyde was 4:1 (FIG. 14).
[5-2] 아세트알데히드 비율의 영향[5-2] Influence of acetaldehyde ratio
서열번호 4의 아미노산 서열을 갖는 효소(G179A)와 서열번호 10의 아미노산 서열을 갖는 효소(G179A+S312A) 활성에, 기질인 아세트알데히드의 비율이 미치는 영향을 확인하기 위해 상기 실시예 [4-2]와 동일한 방법으로 정제 및 분리한 야생형 효소 0.01 ㎎/㎖, G179A 효소 0.1 ㎎/㎖, G179A+S312A 효소 0.25 ㎎/㎖와 글리신 400 mM, 피리독살-5-인산 100 μM 및 Mn2+ 1 mM가 포함된 50 mM의 CHES 완충용액(pH 9.0)에 아세트알데히드를 0, 50, 100, 200, 300, 및 400 mM로 각각 첨가하여 35 ℃에서 10분 동안 반응시켰다. 상기 반응을 종료시키기 위해 100 ℃에서 10분 동안 중탕하였고 상기 실시예 [4-3]과 동일한 방법인 OPA/NAC 유도체화를 경유하는 HPLC 분석을 통해, 상기 반응액에서 D-트레오닌과 D-알로트레오닌의 농도를 측정하여 그 상대값을 비교하였다.To confirm the effect of the ratio of acetaldehyde as a substrate on the activity of the enzyme having the amino acid sequence of SEQ ID NO: 4 (G179A) and the enzyme having the amino acid sequence of SEQ ID NO: 10 (G179A+S312A), Example [4-2 ] Wild-type enzyme 0.01 mg/ml, G179A enzyme 0.1 mg/ml, G179A+S312A enzyme 0.25 mg/ml and glycine 400 mM, pyridoxal-5-phosphate 100 μM and Mn 2+ 1 mM Acetaldehyde was added at 0, 50, 100, 200, 300, and 400 mM respectively to 50 mM CHES buffer solution (pH 9.0) containing and reacted at 35° C. for 10 minutes. In order to terminate the reaction, the reaction mixture was bathed at 100° C. for 10 minutes, and through HPLC analysis via OPA/NAC derivatization, which is the same method as in Example [4-3], D-threonine and D-allo The concentration of threonine was measured and its relative value was compared.
그 결과, D-트레오닌의 특이적 합성은 글리신과 아세트알데히드의 비율이 4:1인 경우에 가장 우수한 것으로 확인되었다(도 15).As a result, it was confirmed that the specific synthesis of D-threonine was best when the ratio of glycine and acetaldehyde was 4:1 (FIG. 15).
[5-3] 시간에 따른 D-트레오닌 생산량 변화[5-3] Changes in D-threonine production over time
도 14 및 도 15의 결과를 통해 확인한 최적의 기질 비율 조건을 이용하여, 서열번호 52, 132 아미노산 서열을 갖는 G179A 및 G179A+S312A 효소의 D-트레오닌의 특이적 생산을 비교하기 위해, 시간에 따른 D-트레오닌의 전환량 및 부분입체 이성질체 과잉률을 확인하였다. 상기 실시예 [4-2]와 동일한 방법으로 정제 및 분리한 야생형 효소 0.01 ㎎/㎖, G179A 효소 0.1 ㎎/㎖, G179A+S312A 효소 0.25 ㎎/㎖와 글리신 400 mM, 아세트알데히드 100 mM, 피리독살-5-인산 100 μM 및 Mn2+ 1 mM가 포함된 50 mM의 CHES 완충용액(pH 9.0)을 35℃에서 15, 30, 60, 90, 120, 150, 180분 동안 반응시켰다. 그리고 상기 반응을 종료시키기 위해 100 ℃에서 10분 동안 중탕하였고 상기 실시예 [4-3]과 동일한 방법인 OPA/NAC 유도체화를 경유하는 HPLC 분석을 통해, 상기 반응액에서 D-트레오닌과 D-알로트레오닌의 농도를 측정하여 그 상대값을 비교하였다.In order to compare the specific production of D-threonine of the enzymes G179A and G179A + S312A having SEQ ID NOs: 52 and 132 amino acid sequences, using the optimal substrate ratio conditions identified through the results of FIGS. 14 and 15, The conversion amount and diastereomer excess of D-threonine were confirmed. Wild-type enzyme 0.01 mg/ml, G179A enzyme 0.1 mg/ml, G179A+S312A enzyme 0.25 mg/ml, glycine 400 mM, acetaldehyde 100 mM, pyridoxal purified and isolated in the same manner as in Example [4-2] 50 mM CHES buffer solution (pH 9.0) containing 100 μM of -5-phosphate and 1 mM Mn 2+ was reacted at 35° C. for 15, 30, 60, 90, 120, 150, and 180 minutes. And in order to terminate the reaction, the reaction solution was bathed at 100° C. for 10 minutes, and through HPLC analysis via OPA/NAC derivatization, which is the same method as in Example [4-3], D-threonine and D- The concentration of allothreonine was measured and its relative values were compared.
그 결과, 서열번호 1의 아미노산 서열을 갖는 야생형에 비해 서열번호 4 및 10의 아미노산 서열을 갖는 G179A 효소 및 G179A+S312A 효소에서 D-트레오닌을 특이적으로 생산하는 것을 확인하였고, 그 중에서도 서열번호 10의 아미노산 서열을 갖는 G179A+S312A 효소는 60분만에 95%의 부분입체 이성질체 과잉률(Diastereomeric excess)의 비율로 46 mM의 D-트레오닌을 생성하는 것을 확인하였다(도 16). As a result, it was confirmed that D-threonine was specifically produced in the enzyme G179A and the enzyme G179A+S312A having the amino acid sequences of SEQ ID NOs: 4 and 10 compared to the wild type having the amino acid sequence of SEQ ID NO: 1. It was confirmed that the enzyme G179A + S312A having the amino acid sequence of produced 46 mM D-threonine at a ratio of 95% diastereomeric excess in 60 minutes (FIG. 16).
상기와 같은 실험 결과들로 볼 때, 본 발명의 서열번호 4, 10의 아미노산 서열을 갖는 상기 효소들은 10분만에 각각 16.4 mM, 18.7 mM의 D-트레오닌을, 각각 95%, 98%의 부분입체 이성질체 과잉률로 특이적으로 합성할 수 있다. 그 중에서도 서열번호 10의 아미노산 서열을 갖는 효소(G179A+S312A)는 시중에 판매하는 D-트레오닌과 동일한 수준의 순도로 D-트레오닌을 합성할 수 있는 것이며, 이는 기존에 보고된 어떤 효소들보다도 D-트레오닌에 대한 특이성이 가장 우수한 것이다.From the above experimental results, the enzymes having the amino acid sequences of SEQ ID NOs: 4 and 10 of the present invention each produced 16.4 mM and 18.7 mM D-threonine in 10 minutes, respectively, 95% and 98% diastereomers. It can be specifically synthesized with an excess of isomers. Among them, the enzyme having the amino acid sequence of SEQ ID NO: 10 (G179A+S312A) is capable of synthesizing D-threonine with the same level of purity as commercially available D-threonine. -It has the best specificity for threonine.
상기에서는 본 발명의 바람직한 실시예를 예시적으로 설명하였으나, 본 발명의 범위는 상기와 같은 특정 실시예에만 한정되지 아니하며, 해당 분야에서 통상의 지식을 가진 자라면 본 발명의 특허청구범위에 기재된 범주 내에서 적절하게 변경이 가능할 것이다.In the above, preferred embodiments of the present invention have been exemplarily described, but the scope of the present invention is not limited to the specific embodiments as described above, and those of ordinary skill in the relevant field have the scope described in the claims of the present invention. It will be possible to make appropriate changes within it.

Claims (16)

  1. 필로마이크로비움 마리넘(Filomicrobium marinum) 유래 D-트레오닌 알돌라아제(D-threonine aldolase)의 147 번째 아르기닌(arginine), 179 번째 글리신(glycine) 및 312 번째 세린(serine)으로 이루어진 군으로부터 선택되는 하나 이상의 아미노산이 알라닌(alanine)으로 치환된, D-트레오닌 생산 효소.One selected from the group consisting of 147th arginine, 179th glycine and 312th serine of D-threonine aldolase derived from Filomicrobium marinum D-threonine-producing enzyme in which the above amino acids are substituted with alanine.
  2. 청구항 1에 있어서,The method according to claim 1,
    상기 D-트레오닌 생산 효소는 글리신 및 아세트알데히드를 기질로 하는 것인, D-트레오닌 생산 효소.The D-threonine-producing enzyme is that of glycine and acetaldehyde as substrates, D-threonine-producing enzyme.
  3. 청구항 1에 있어서,The method according to claim 1,
    상기 D-트레오닌 알돌라아제는 서열번호 1의 아미노산 서열을 포함하는 것인, D-트레오닌 생산 효소.The D-threonine aldolase will comprise the amino acid sequence of SEQ ID NO: 1, D-threonine producing enzyme.
  4. 청구항 1에 있어서,The method according to claim 1,
    상기 D-트레오닌 생산 효소는 서열번호 3, 서열번호 4, 서열번호 5, 서열번호 9, 서열번호 10 및 서열번호 11로 이루어진 군으로부터 선택되는 어느 하나의 아미노산 서열을 포함하는 것인, D-트레오닌 생산 효소.The D-threonine producing enzyme comprises any one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, D-threonine Production enzymes.
  5. 청구항 1 내지 청구항 4 중 어느 한 항에 있어서,The method according to any one of claims 1 to 4,
    상기 D-트레오닌 생산 효소는 D-트레오닌 및 D-알로트레오닌을 생산하고, 상기 반응 생성물 중 D-트레오닌의 비율이 85% 이상인 것인, D-트레오닌 생산 효소.The D-threonine producing enzyme produces D-threonine and D-allothreonine, and the proportion of D-threonine in the reaction product is 85% or more, D-threonine producing enzyme.
  6. 청구항 1의 D-트레오닌 생산 효소를 암호화하는 유전자.The gene encoding the D-threonine producing enzyme of claim 1.
  7. 청구항 6에 있어서,The method of claim 6,
    상기 유전자는 서열번호 6, 서열번호 7, 서열번호 8, 서열번호 12, 서열번호 13 및 서열번호 14로 이루어진 군으로부터 선택되는 어느 하나의 염기 서열로 이루어진 것인, 유전자.The gene is a gene consisting of any one nucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14.
  8. 청구항 6 또는 청구항 7의 유전자를 포함하는 재조합 발현벡터.Recombinant expression vector comprising the gene of claim 6 or 7.
  9. 청구항 8의 재조합 발현벡터가 숙주세포에 도입된 형질전환체.A transformant in which the recombinant expression vector of claim 8 is introduced into a host cell.
  10. 청구항 9에 있어서,The method of claim 9,
    상기 숙주세포는 대장균인, 형질전환체.The host cell is E. coli, a transformant.
  11. 청구항 9의 형질전환체를 배양하는 단계; 및Culturing the transformant of claim 9; And
    상기 형질전환체의 배양물로부터 D-트레오닌 생산 효소를 분리하는 단계;를 포함하는 D-트레오닌 생산 효소의 제조 방법.Separating the D-threonine producing enzyme from the culture of the transformant; D- threonine producing enzyme production method comprising a.
  12. 청구항 1 내지 청구항 4 중 어느 한 항의 D-트레오닌 생산 효소를 유효성분으로 포함하는 D-트레오닌 생산용 조성물.A composition for producing D-threonine comprising the enzyme for producing D-threonine according to any one of claims 1 to 4 as an active ingredient.
  13. 청구항 1 내지 청구항 4 중 어느 한 항의 D-트레오닌 생산 효소를 글리신 및 아세트알데히드와 함께 반응시키는 단계;를 포함하는, 인 비트로(in vitro)에서의 D-트레오닌 생산 방법.The step of reacting the D-threonine producing enzyme of any one of claims 1 to 4 together with glycine and acetaldehyde; containing, in vitro D-threonine production method.
  14. 청구항 13에 있어서,The method of claim 13,
    상기 글리신 및 아세트알데히드는 1:1 내지 10:1의 비율로 효소와 반응시키는 것인, 인 비트로(in vitro)에서의 D-트레오닌 생산 방법.The glycine and acetaldehyde are reacted with the enzyme in a ratio of 1:1 to 10:1, in vitro D-threonine production method.
  15. 청구항 9의 형질전환체를 글리신 및 아세트알데히드의 존재 하에서 배양하는 단계;를 포함하는, 인 비보(in vivo)에서의 D-트레오닌 생산 방법.Cultivating the transformant of claim 9 in the presence of glycine and acetaldehyde; containing, in vivo D-threonine production method.
  16. 청구항 15에 있어서,The method of claim 15,
    상기 글리신 및 아세트알데히드는 1:1 내지 10:1의 비율로 존재하는 것인, 인 비보(in vivo)에서의 D-트레오닌 생산 방법.The glycine and acetaldehyde are present in a ratio of 1:1 to 10:1, in vivo D-threonine production method.
PCT/KR2020/014292 2019-10-23 2020-10-20 Novel enzyme for producing d-threonine, and method for stereospecifically producing d-threonine by using same WO2021080277A1 (en)

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