KR20200002183A - A Novel Enzyme for Production of D-threonine, and A Method for Preparing D-threonine Using The Same - Google Patents

Abstract

The present invention relates to a novel D-threonine production enzyme. The present invention provides a D-threonine production enzyme comprising an amino acid sequence having sequence number 1 separated from Alcaligenes microorganisms or an amino acid sequence having 90% or more of homology with the sequence number 1; and a gene coding the same. The D-threonine production enzyme of the present invention can produce D-threonine at a high yield while being environmentally friendly compared to existing enzymes.

Description

A Novel Enzyme for Production of D-threonine, and A Method for Preparing D-threonine Using The Same}

The present invention relates to a novel D-threonine producing enzyme.

Most amino acids in nature have alpha carbon which shows optical activity and are divided into L-amino acid and D-amino acid according to stereospecificity. Most proteins in nature are composed of L-amino acids, except for microorganisms such as peptidoglycan and peptide antibiotics, and bioactive substances of higher plants. have.

According to the research to date, among the above D-amino acids, D-threonine is widely used in food and pharmaceutical fields as an intermediate or precursor for synthesizing bioactive substances such as neurotransmitters, vaccines, synthetic sweeteners, antibiotics and hormones. Therefore, methods for producing D-threonine have been developed.

Methods of producing D-threonine can be broadly divided into chemical synthesis and biological synthesis using enzymes or microorganisms. Since the production of amino acids by chemical synthesis method results in a racemic mixture of D- or L-amino acids, it is difficult to undergo another complicated optical purification process to obtain pure D-threonine. In addition, the chemical synthesis method as described above has a problem that serious environmental pollution is caused by the by-products generated in the process. On the other hand, biological synthesis method can directly produce only optically pure D-threonine in one step, attracting attention as a low pollution clean production technology that can replace the multi-step chemical synthesis method that has the problem of environmental pollution.

Development of related enzymes is essential for such a biological synthesis method, but in the case of the conventionally known D-threonine producing enzyme, its activity and production amount are very small, which is insufficient for industrial use.

Accordingly, research and development of novel D-threonine synthase, which is more active, are needed.

It is an object of the present invention to provide a novel enzyme capable of producing D-threonine and a method for producing D-threonine using the same, which are superior in efficiency to the prior art.

In order to achieve the above object, an aspect of the present invention provides a D-threonine producing enzyme comprising an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 90% homology with SEQ ID NO: 1.

In addition, to achieve the above object, the present invention provides a gene encoding the D-threonine producing enzyme.

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 into which the expression vector is introduced.

In addition, in order to achieve the above object, another aspect of the present invention comprises the step of reacting the composition for producing D-threonine comprising the D-threonine producing enzyme, and the D-threonine producing enzyme with glycine and acetaldehyde It provides a method for producing D-threonine in vitro ( in vitro ) comprising a.

In addition, to achieve the above object, another aspect of the present invention provides a method for producing D-threonine in vivo comprising the step of culturing the transformant in the presence of glycine and acetaldehyde. .

The D-threonine producing enzyme of the present invention not only generates toxic substances or by-products causing environmental pollution in converting glycine and acetaldehyde into D-threonine as substrates, but also known enzymes having specific activity. Remarkably superior to the bar, there is an advantage that can be produced in high yield and environmentally friendly D-threonine.

However, the effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

1 is a photograph showing a protein having an amino acid sequence of SEQ ID NO: 1 overexpressed and purified from a transformant by SDS-PGAE.
Figure 2 is a graph showing the degree of D-threonine conversion by the protein having the amino acid sequence of SEQ ID NO: 1.
3 is a graph confirming the type (A) and concentration (B) of metal cations affecting the enzymatic activity of a protein having the amino acid sequence of SEQ ID NO: 1.
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.
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.
6 is a graph confirming the effect of pyridoxal-5-phosphate on the enzyme activity of the protein having the amino acid sequence of SEQ ID NO: 1.
7 is a graph confirming the effect of the ratio of the substrate on the production amount of D-threonine by the protein having the amino acid sequence of SEQ ID NO: 1.

First, the terms used in the specification of the present invention will be described.

As used herein, the term " D-threonine producing enzyme " refers to an enzyme having activity of producing D-threonine from glycine and acetaldehyde, as shown in the following scheme.

[Scheme]

Hereinafter, the present invention will be described in detail.

 One. New D-threonine Producing Enzymes and Their Genes

One aspect of the invention provides a D-threonine producing enzyme comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having 90% or more homology with SEQ ID NO: 1.

In addition, another aspect of the present invention provides a gene of the D-threonine producing enzyme encoding the D-threonine producing enzyme.

The D-threonine producing enzyme of the present invention may be derived from a microorganism of the genus Filomicrobium , and particularly derived from Filomicrobium marinum .

The D-threonine producing enzyme of the present invention comprises the amino acid sequence of SEQ ID NO: 1, wherein the D-threonine producing enzyme does not affect the function of the protein, deletion, insertion, substitution or combination of amino acid residues May be variants, or fragments of amino acids having different sequences. Amino acid exchanges at the protein and peptide levels that do not alter the activity of the D-threonine producing enzyme as a whole are known in the art. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, and the like. Accordingly, the present invention includes a protein having an amino acid sequence substantially identical to a protein comprising the amino acid sequence of SEQ ID NO: 1, and a variant thereof or an active fragment thereof. The substantially identical protein means those having homology of 90% or more, preferably 93% or more, most preferably 95% or more amino acid sequences, but are not limited thereto, and have homology of 90% or more amino acid sequences and the same. If it has enzyme activity, it is included in the scope of the present invention.

The gene of the D-threonine producing enzyme is preferably composed of the nucleotide sequence of SEQ ID NO: 2. However, the gene encoding the D-threonine producing enzyme and variants or active fragments thereof of the present invention may be modified in various ways in the coding region within the range that does not change the amino acid sequence of the enzyme and its variants or active fragments expressed from the coding region. This may be made, and various variations may be made within a range that does not affect the expression of the gene even in a portion except the coding region, and such a mutated gene is also included in the scope of the present invention. Therefore, the present invention includes a gene consisting of a base sequence substantially the same as the gene of SEQ ID NO: 2 and fragments of the gene. Genes consisting of the same base sequence means those having sequence homology of at least 80%, preferably at least 90%, most preferably at least 95%, but are not limited thereto, and at least 80% sequence homology. And encoded proteins are included in the present invention if they have the same enzymatic activity. As described above, as long as the gene of 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 included in the scope of the invention.

The amino acid sequence of SEQ ID NO: 1 contained in the D-threonine producing enzyme is preferably encoded by a gene consisting of the nucleotide sequence of SEQ ID NO: 2, but the present invention is not limited thereto, and the protein of the present invention having the same amino acid sequence As long as it can encode, it may be encoded by a gene consisting of another base sequence substantially the same as the base sequence of SEQ ID NO: 2. Such base sequences may be single stranded or double stranded, and may be DNA molecules or RNA molecules.

In a preferred embodiment of the present invention, the inventors isolate the protein having the amino acid sequence of SEQ ID NO: 1 to identify the function of the protein having the amino acid sequence of SEQ ID NO: 1 derived from Filomicrobium marinum And purified (see FIG. 1), and the activity thereof was confirmed that the protein having the amino acid sequence of SEQ ID NO: 1 has activity as a D-threonine producing enzyme (see Table 1 and FIG. 2).

 2. Expression Vectors and Transformants of D-Threonine Producing Enzymes

Another aspect of the present invention provides a recombinant expression vector comprising the gene of the D-threonine producing enzyme of the present invention and a transformant into which the expression vector is introduced.

The recombinant expression vector of the present invention contains the gene of the D-threonine producing enzyme.

The expression vector includes, but is not limited to, plasmid vector, cosmid vector, bacteriophage vector, viral vector, and the like.

The recombinant expression vector is an expression control sequence such as a promoter, a terminator, an enhancer, or a sequence for secretion, etc., depending on the type of host cell to produce the D-threonine producing enzyme of the present invention. Can be combined as appropriate for the purpose.

The expression vector may further comprise a selection marker for selecting a host cell into which the vector has been introduced, and in the case of a replicable expression vector, may comprise a replication origin.

In addition, the recombinant expression vector may include a sequence for facilitating purification of the expression protein, specifically, the gene encoding the separation and purification tag to be operable to the gene encoding the D-threonine production enzyme of the present invention Can be connected. In this case, the separation and purification tag may be used alone, or GST, poly-Arg, FLAG, histidine-tag (His-tag) and c-myc, or two or more of them may be sequentially connected.

The gene encoding the D-threonine producing enzyme can be cloned through a restriction enzyme cleavage site. When a gene encoding a protein cleavage site is used in the vector, the gene encoding the D-threonine producing enzyme is different from the gene of the D-threonine producing enzyme. Linked in frame, the enzyme can be obtained and then cleaved with protein cleavage enzymes so that the original form of the D-threonine producing enzyme can be produced.

In a specific embodiment of the present invention, a recombinant cloning vector was prepared by inserting the D-threonine producing enzyme gene of the present invention into pET28a (+), a plasmid vector. In addition to pET28a (+) used in the preparation of the cloning vector, various prokaryotic vectors or eukaryotic vectors (such as pPIC and pPICZ) are known, and thus, various expression vectors other than the vector may be used depending on the purpose of expression.

The recombinant expression vector of the present invention contains a D-threonine production enzyme gene can be usefully used as a vector capable of producing it.

The transformant of the present invention also incorporates a recombinant expression vector comprising a gene encoding a D-threonine producing enzyme.

A transformant may be prepared by transforming the recombinant expression vector according to the present invention into any one suitable host cell selected from the group consisting of bacteria, yeast, Escherichia coli, fungi, plant cells and animal cells according to expression purposes. . For example, the host cell may be Escherichia coli ( E. coli BL21 (DE3), DH5α, etc.) or yeast cells ( Saccharomyces , Pichia , etc.) and the like. In this case, appropriate culture methods, media conditions and the like can be easily selected by those skilled in the art according to the type of host cell.

As a method of introducing a recombinant expression vector for producing a transformant of the present invention, a known technique, that is, a heat shock method, an electric shock method, or the like may be used.

In a specific embodiment of the present invention, a transformant was prepared by transforming Escherichia coli with a recombinant expression vector comprising a gene encoding the D-threonine producing enzyme of the present invention, as E. coli as a host cell.

Since the protein expressed from the transformant is a protein of a novel sequence having D-threonine producing enzyme, the enzyme activity, mass production of the D-threonine producing enzyme is facilitated by culturing the transformant in large quantities and expressing the gene. It can be done.

 3. Production method of D-threonine producing enzyme

In addition, another aspect of the present invention provides a method for producing a D-threonine producing enzyme using a transformant introduced with a recombinant expression vector comprising a gene encoding the D-threonine producing enzyme of the present invention.

D-threonine production enzyme production method of the present invention comprises the steps of: 1) culturing a transformant introduced with a recombinant expression vector comprising a gene encoding the D-threonine production enzyme of the present invention; 2) inducing expression of a gene encoding the D-threonine producing enzyme in the transformant cultured in step 1); And 3) separating the D-threonine producing enzyme from the culture of the transformant in which the expression of the D-threonine producing enzyme gene is induced in step 2).

The N-terminus of the gene encoding the D-threonine producing enzyme of step 1) may be further linked to a gene or protein cleavage cleavage site encoding the tag for separation and purification, thereby purifying recombinant D-threonine producing enzyme. Alternatively, it may be possible to obtain D-threonine producing enzyme in its original form.

Cultivation of the transformant of step 1) may be performed according to a known method, and conditions such as culture temperature, incubation 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 should suitably meet the requirements of the particular strain.

Separation of the D-threonine producing enzyme from the culture produced by the culture of step 3) can be carried out a method commonly performed in the art, such as centrifugation, filtration. In addition, the D-threonine producing enzyme isolated by the above method can be purified in a conventional manner, for example, salting out (eg ammonium sulfate precipitation, sodium phosphate precipitation), solvent precipitation (acetone, protein using ethanol and the like) The enzyme of the present invention can be purified alone or in combination with techniques such as fractional precipitation), dialysis, gel filtration, ion exchange, chromatography such as reversed phase column chromatography, and ultrafiltration.

In a specific embodiment of the present invention it was confirmed that the protein having the amino acid sequence of SEQ ID NO: 1 purified as described above shows the activity of D-threonine producing enzyme (see Fig. 2). From the above results, it can be seen that the mass production of D-threonine producing enzyme according to the production method of the present invention is possible.

 4. Production method of D-threonine and composition for production of D-threonine

Another aspect of the present invention provides a method for producing D-threonine from glycine and acetaldehyde in vitro using the D-threonine producing enzyme of the present invention and the production of D-threonine in vitro . It provides a composition for producing D-threonine to be used in.

The composition for producing D-threonine of the present invention comprises the D-threonine producing enzyme of the present invention.

In addition, the in vitro production method of D-threonine of the present invention comprises the steps of 1) reacting the D-threonine producing enzyme of the present invention with glycine and acetaldehyde; And 2) recovering D-threonine from the reaction product resulting from the reaction in step 1).

Since the method for producing D-threonine and the composition for producing D-threonine utilize the enzymatic activity of the D-threonine producing enzyme of the present invention in vitro , glycine, the substrate thereof, using the D-threonine producing enzyme And from acetaldehyde to produce D-threonine.

The reaction of step 1) may be performed at a temperature range of 20 ° C to 55 ° C, preferably at a temperature range of 22 ° C to 45 ° C, more preferably at a temperature of 25 ° C to 40 ° C, but is not limited thereto. In addition, the reaction of step 1) may be performed at a pH range of 7 to 10, preferably at a pH range of 8 to 9.5, more preferably at a pH of 8.5 to 9, but is not limited thereto.

In addition, the reaction of step 1) may include a metal cation together with the D-threonine producing enzyme to improve the activity of the D-threonine producing enzyme. The metal cation may be a divalent metal cation, the divalent metal cation may be a manganese cation or a magnesium cation, and is preferably a manganese cation.

In addition, the reaction of step 1) may further include pyridoxal-5-phosphate together with the D-threonine producing enzyme to enhance the activity of the D-threonine producing enzyme.

In the reaction of step 1), the glycine and acetaldehyde are preferably injected externally, and the glycine and acetaldehyde should be injected in a sufficient amount in accordance with the desired amount of D-threonine. The glycine and acetaldehyde may be supplied in a ratio of 0.5: 1 to 30: 1, preferably in a ratio of 1: 1 to 20: 1, and more preferably in a ratio of 2.5: 1 to 12.5; 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 can be continuously produced by the D-threonine producing enzyme.

In a specific embodiment of the present invention, it was confirmed that D-threonine is produced from glycine and acetaldehyde by the D-threonine producing enzyme as described above (see FIG. 2). By addition, the reaction is carried out at a temperature close to 35 ° C., the reaction is performed at a pH close to 8.5-9.0, the pyridoxal-5-phosphate is added, and glycine and acetaldehyde are added at a ratio of 10: 1. It was confirmed that the more improved (see FIGS. 3 to 7).

Another aspect of the present invention provides a method for producing D-threonine in vivo using the transformant of the present invention.

In addition, the in vivo production method of D-threonine of the present invention comprises the steps of 1) culturing the transformant under an environment in which glycine and acetaldehyde are supplied; And 2) recovering D-threonine from the culture in step 1).

The environment in which the glycine and acetaldehyde is supplied may be one that supplies glycine and acetaldehyde artificially from the outside or adds glycine and acetaldehyde to the medium used for culture, and the environment in which the glycine and acetaldehyde is produced. It may be to provide a. The environment in which the glycine and acetaldehyde are produced may be provided by transforming genes encoding at least one enzyme involved in the production of glycine and acetaldehyde.

Cultivation of such a transformant can be made according to suitable media and culture conditions known in the art. Those skilled in the art can easily use the medium and culture conditions according to the type of host cell of the transformant to be selected. Culture methods can include batch, continuous, fed-batch, or combination cultures thereof.

The medium may comprise various carbon sources, nitrogen sources and trace element components.

The carbon source may be, for example, glucose, sucrose, lactose, fructose, maltose, starch, carbohydrates such as cellulose, soybean oil, sunflower oil, castor oil, fats such as coconut oil, fatty acids such as palmitic acid, stearic acid, linoleic acid, Alcohols such as glycerol and ethanol, organic acids such as acetic acid, or combinations thereof. The culturing can be performed using glucose as a carbon source. The nitrogen source may be an organic nitrogen source and urea such as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL), and soybean wheat, an inorganic nitrogen source such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, or Combinations thereof. The medium may comprise, as a source of phosphorus, metal salts such as, for example, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and the corresponding sodium-containing salts, magnesium sulfate or iron sulfate.

In addition, amino acids, vitamins, appropriate precursors, and the like may be included in the medium. The medium or individual components may be added batchwise or continuously to the culture.

In addition, anti-foaming agents such as fatty acid polyglycol esters can be used during the culture to suppress bubble generation.

Cultivation of the transformant as described above may be carried out at 20 ° C to 50 ° C, preferably 25 ° C to 45 ° C, more preferably 30 ° C to 40 ° C. If the culturing of the transformant is carried out at a temperature range of less than 20 ° C or more than 50 ° C, a sufficient amount of intermediate product is not produced, resulting in a problem in that the yield or inadequate production of the final product D-threonine occurs.

In addition, the culturing of the transformant as described above may be performed at pH 5 to pH 10, preferably at pH 6 to pH 9, more preferably at pH 6.5 to pH 8, but is not limited thereto. No. The culture pH conditions of the transformant 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 of the transformant is out of the above range, it is difficult to grow the transformant, and thus there is a problem in that D-threonine is not easily expressed, and thus, D-threonine is not efficiently synthesized.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are intended to specifically illustrate the present invention, the content of the present invention is not limited by the following examples.

Isolation of a Protein Having the Amino Acid Sequence of SEQ ID NO: 1

[1-1] Cloning and Transformation of Proteins Having the Amino Acid Sequence of SEQ ID NO: 1

Filomicrobium In order to obtain a protein having an amino acid sequence of SEQ ID NO: 1 derived from marinum ), primers of SEQ ID NOs: 3 to 6 were designed based on the nucleotide sequence of SEQ ID NO: 2, as shown in Table 1 below.

SEQ ID NO: Primer pair Sequence (5 '-> 3') 3 Forward primer 5'-tggtgccgcgcggcagccatatgcgcgcaccagcacggct-3 ' 4 Reverse primer 5'-ggtggtggtggtgctcgagctagaacacacaacctcgcgc-3 ' 5 Forward primer 5'-gcgcgaggttgtgtgttctagctcgagcaccaccaccacc-3 ' 6 Reverse primer 5'-agccgtgctggtgcgcgcatatggctgccgcgcggcacca-3 '

The gene of the nucleotide sequence of SEQ ID NO: 2 was synthesized by BIONIA Co., Ltd., and PCR was performed using the primer pairs of SEQ ID NOs 3 and 4 designed as described above using the synthesized gene as a template. The base sequence was amplified. The plasmid vector pET28a (+) (amplified by PCR using a primer pair of SEQ ID NOS: 5 and 6), using a Gibson assembly (New England Biolabs, USA), amplified as described above using a primer pair of SEQ ID NOs: 5, 6 The recombinant expression vector was prepared by inserting into multiple cloning sites of Novagen, USA, and confirmed that the nucleotide sequence of SEQ ID NO: 2 was correctly inserted through sequencing (Macrogen), which was E. coli C2566 strain (Novagen). , USA). The transformants obtained as described above were stored frozen by adding 20% glycerin solution before use.

[1-2] Overexpression and Purification of Proteins Having the Amino Acid Sequence of SEQ ID NO: 1

The transformants stored frozen in Example [1-1] were pre-inoculated with 3 ml of LK solid medium (LB medium + 25 µg / ml kanamycin) and incubated at 37 ° C. for at least 10 hours. One colony appearing in the solid medium was inoculated into a test tube containing 3 ml of LK solid medium by a single colony separation process, and the seed culture was performed for 18 hours using a shaking incubator at 37 ° C. Then, 2 ml of the seed cultured medium was added to a 1000 ml flask containing 200 ml of LK medium, and the main culture was performed. When the absorbance at 600 nm was 0.6, IPTG (isopropyl) was added to a final concentration of 0.1 mM. -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 200 rpm, the culture temperature was maintained to 37 ℃, after adding the IPTG stirring speed to 150 rpm, The culture temperature was adjusted to 18 ° C. and incubated for 20 hours.

In addition, the culture medium of the transformant induced overexpression of the protein having the amino acid sequence of SEQ ID NO: 1 is dispensed into 50 ml conical tube (SPL Life Sciences Co., Ltd., Korea), 10 ml of Profinia 1X Lysis buffer (Bio-Rad Laboratories, USA) was added to the pellet from which the supernatant was separated by centrifugation at 4 ° C. at 4000 rpm for 20 minutes, followed by sonication. By crushing the cells, cell lysates of the transformants were obtained.

The cell lysate obtained above was centrifuged again at ¹⁴⁰⁰⁰ rpm for 20 minutes at 4 ° C. to obtain a supernatant, and a high-speed protein liquid chromatography equipped with an IMAC Kit ^ㄾ His tag adsorption column (Bio-Rad Laboratories, USA). Fast Protein Liquid Chromatography (Bio-Rad Laboratories, USA) was used to separate the protein having the amino acid sequence of SEQ ID NO: 1 from the supernatant obtained as above.

As a result of confirming the separated protein through SDS-PAGE, a protein having an amino acid sequence of SEQ ID NO: 1 of 42.22kDa having His-Tag attached thereto was overexpressed by the above-described series of procedures, and the cell lysate of the transformant Confirmed purification and separation from (Fig. 1).

Confirmation of Activity of Protein Having Amino Acid Sequence of SEQ ID NO: 1

The inventors anticipated that the protein having the amino acid sequence of SEQ ID NO: 1 had activity as a 'D-threonine producing enzyme' as shown in the following scheme, and confirmed this.

[Scheme]

Specifically, 0.25 mg / ml of the protein having the amino acid sequence of SEQ ID NO: 1 purified and isolated in Example [1-2] together with 100 mM glycine and 100 mM acetaldehyde, 100 μM pyridoxal-5-phosphate (pyridoxal) -5-phosphate, PLP) and 50 mM HEPES buffer solution (pH 7.5) containing 1 mM MnCl ₂ , and reacted at 37 ℃ for 10 minutes, and then the reaction was terminated by boiling in 100 ℃ for 10 minutes After the reaction, the concentration of D-threonine was measured in the buffer solution.

The produced D-threonine was analyzed by HPLC after OPA / NAC pretreatment (OPA / NAC derivatization). The pretreatment was performed by dissolving 8 mg of OPA ( o -phthalaldelhyde) and 10 mg of NAC ( N -acetylcysteine) in 1 ml of methanol to make an OPA / NAC solution, and 25 µl of the sample to be analyzed. And 175 μl of 0.4 M sodium borate buffer (pH 10.4) were added to the mixture and filtered through a 0.2 μm filter to prepare a pretreated sample.

20 μl of the pretreated sample prepared as 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 ㅧ 150 mm, 3.5 μm) (Agilent). Injected into the graphics, the concentration of D-threonine was measured. The mobile phase used in performing such high pressure liquid chromatography consists 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. It was flowed for a while, and the mobile phase A and the mobile phase B at a ratio of 3: 7 for 0 to 3 minutes, and the mobile phase A and the mobile phase B at a ratio of 7: 3 for 3 to 10 minutes. And commercially available D-threonine (Sigma-aldrich, USA) and D- allo-threonine (TOKYO CHEMICAL INDUSTRY CO., LTD., Japan) were used as standard to quantify the produced D-threonine.

As a result, in the buffer solution in which the reaction was completed as described above, both D-threonine and D-allo-threonine were detected (FIG. 2), and D-threonine, which was not present in the first place, was generated from the above reaction result. 1-2] confirmed that the protein having the amino acid sequence of SEQ ID NO: 1 purified and separated as a D-threonine production enzyme, producing D-threonine from glycine and acetaldehyde .

A Study on the Conditions Affecting the Enzyme Activity of Proteins Having the Amino Acid Sequence of SEQ ID NO: 1

The inventors of the present invention, as confirmed in Example 2, conducted a study on the conditions affecting the activity of the protein having the amino acid sequence of SEQ ID NO: 1 as D-threonine production enzyme, from the sequence Optimal activity conditions of the protein having the amino acid sequence of No. 1 were derived.

[3-1] Influence of metal cations

In order to confirm the effect of the type of metal cation on the activity of the enzyme of the protein having the amino acid sequence of SEQ ID NO: 1, the protein having the amino acid sequence of SEQ ID NO: 1 purified and separated in Example [1-2] ㎎ / ㎖, 100mM glycine, acetaldehyde and pyridoxal -5- 100mM CHES buffer solution of phosphoric acid containing 10μM 50mM (pH 9.0) 1mM of EDTA or a metal cation (Mn ^{+ 2,} Mg ^{+ 2,} Co ^{+ 2,} was added with Zn ^{+ 2)} is reacted at 35 ℃ for 10 min, water bath at 100 ℃ to terminate the reaction for 10 minutes. And in the same manner as in Example 2, the concentration of D-threonine and D- allo-threonine in the buffer solution in which the reaction was completed as described above was measured and the relative values were compared.

As a result, the protein having the amino acid sequence of SEQ ID NO: 1 improved the activity of D-threonine producing enzyme when the manganese cation and the magnesium cation were added among various kinds of metal cations. The activity of the D-threonine producing enzyme was confirmed to be the best (Fig. 3 (A)).

In order to confirm the effect of the metal cation concentration on the activity of the enzyme of the protein having the amino acid sequence of SEQ ID NO: 1, the protein having the amino acid sequence of SEQ ID NO: 1 purified and separated in Example [1-2] Manganese cations were added to 0 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1 mM, 2.5 mM and 50 mM CHES buffer solution (pH 9.0) containing mg / ml, 100 mM glycine, 100 mM acetaldehyde and 10 μM pyridoxal-5-phosphate. Each was added in a content of 5 mM and reacted at 35 ° C. for 10 minutes, followed by bathing at 100 ° C. for 10 minutes to complete the reaction. In the same manner as in Example 2, the concentrations of D-threonine and D-allo-threonine were measured in the buffer solution in which the reaction was completed as described above, and their relative values were compared.

As a result, it was confirmed that the protein having the amino acid sequence of SEQ ID NO: 1 exhibited the activity of D-threonine producing enzyme only in the presence of manganese cations (FIG. 3B).

[3-2] Influence of temperature

In order to confirm the influence 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 separated in Example [1-2], glycine 100mM and 100mM acetaldehyde, was added to a HEPES buffer solution (pH 7.5) that contains the of pyridoxal -5- phosphate of 10μM and 1mM Mn ^{+ 2} 50mM, then each 10 minutes at a temperature of 25 ℃ to 55 ℃ The reaction was carried out for 10 minutes at 100 ° C. to terminate the reaction. In the same manner as in Example 2, the concentrations of D-threonine and D-allo-threonine in the buffer solution in which the reaction was completed as described above were measured and their relative values were compared.

As a result, the enzymatic activity of the protein having the amino acid sequence of SEQ ID NO: 1 was found to be increasingly excellent closer to 35 ℃ (Fig. 4).

[3-3] pH influence

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, 0.01mg / ml protein having the amino acid sequence of SEQ ID NO: 1 purified and separated in Example [1-2], 100 mM glycine and 100 mM acetaldehyde together with 10 μM pyridoxal-5-phosphate and Mn ^{2 +} 1 mM, MOPS buffers at pH 6.5 to 7.5, HEPES buffer solutions at pH 7.5 to 8.0, EPPS buffer solutions at pH 8.0 to 8.5, respectively And it was added to the CHES buffer solution of pH 8.5-10, respectively, and reacted in the range of pH 6.5-10, respectively. The enzymatic reaction was carried out at a temperature of 35 ° C. for 10 minutes and then bathed at 100 ° C. for 10 minutes to terminate the reaction. In the same manner as in Example 2, the concentrations of D-threonine and D-allo-threonine were measured in the buffer solution in which the reaction was completed as described above, and their relative values were compared.

As a result, it was confirmed that the enzymatic activity of the protein having the amino acid sequence of SEQ ID NO: 1 is getting better as the pH is closer to 8.5 ~ 9.0 (Fig. 5).

[3-4] Influence of pyridoxal-5-phosphate

In order to confirm the effect of the presence and concentration of pyridoxal-5-phosphate on the enzymatic 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] Pyridoxal-5-phosphate was added in 50 mM CHES buffer (pH 9.0) containing 0.01 mg / ml protein having sequence, 100 mM glycine, 100 mM acetaldehyde, and Mn ^{2 +} 1 mM, and 0 μM, 1.0 μM, 1.25 μM, 2.5 μM , 5 μM and 10 μM, respectively, were added and reacted at 35 ° C. for 10 minutes, followed by bathing at 100 ° C. for 10 minutes to complete the reaction. In the same manner as in Example 2, the concentrations of D-threonine and D-allo-threonine were measured in the buffer solution in which the reaction was completed as described above, and their relative values were compared.

As a result, the enzymatic activity of the protein having the amino acid sequence of SEQ ID NO: 1 was found to be better in an environment in which pyridoxal-5-phosphate is present and to reach maximum activity at a concentration of 1.0 μM (FIG. 6).

[3-5] Influence of the proportion of the substrate

In order to confirm the effect of the presence and concentration of pyridoxal-5-phosphate on the enzymatic 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] Glycine was added in 50 mM CHES buffer (pH 9.0) containing 0.01 mg / ml of the sequence-containing protein, 100 mM acetaldehyde, 10 μM of pyridoxal-5-phosphate and Mn ^{2 +} 1 mM, 50 mM, 75 mM, 100 mM, 125 mM, 250 mM, 500 mM, 750 mM, 1000 mM and 1250 mM, respectively, were added to react at 35 ° C. for 10 minutes, followed by bathing at 100 ° C. for 10 minutes to complete the reaction. In the same manner as in Example 2, the concentrations of D-threonine and D-allo-threonine were measured in the buffer solution in which the reaction was completed as described above, and their relative values were compared.

As a result, D-threonine specific synthesis of the protein having the amino acid sequence of SEQ ID NO: 1 was found to be the best when the ratio of glycine and acetaldehyde is 10: 1 (Fig. 7).

In the above described exemplary embodiments of the present invention by way of example, the scope of the present invention is not limited only to the specific embodiments as described above, those skilled in the art to the scope described in the claims of the present invention It will be possible to change accordingly.

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> A Novel Enzyme for Production of D-threonine, and A Method for          Preparing D-threonine Using The Same <130> 2018-DPA-2626 <160> 6 <170> KopatentIn 3.0 <210> 1 <211> 376 <212> PRT <213> Filomicrobium marinum <400> 1 Met Arg Ala Pro Ala Arg Leu Gly Ile Pro Leu Ala Asp Val Asp Thr   1 5 10 15 Pro Ala Leu Ile Leu Asp Leu Asp Ala Phe Glu Arg Asn Leu Gln Thr              20 25 30 Met Ala Asp Trp Ala Lys Ser Lys Asn Val Arg Leu Arg Pro His Ala          35 40 45 Lys Ser His Lys Cys Pro Ala Ile Ala His Arg Gln Met Ala Leu Gly      50 55 60 Ala Val Gly Val Cys Cys Gln Lys Val Ser Glu Ala Glu Val Met Val  65 70 75 80 Asp Ala Gly Ile Thr Asn Val Leu Ile Ser Asn Glu Val Val Gly Arg                  85 90 95 Thr Lys Leu Glu Ala Leu Ala Gln Leu Ala Leu Arg Ala Arg Met Gly             100 105 110 Val Cys Val Asp Asp Ile Arg Gln Ile Arg Asp Leu Ser Glu Ala Met         115 120 125 His Ala Ala Gly Ala Thr Ile Asp Val Leu Val Glu Ile Asn Ile Gly     130 135 140 Gly Asn Arg Cys Gly Val Glu Pro Gly Asp Pro Ala Val Arg Leu Gly 145 150 155 160 Ala Ala Val Ala Gln Ala Asp Gly Leu Arg Phe Ala Gly Leu Gln Ser                 165 170 175 Tyr Asp Gly Ile Thr Gln His Val Arg Asp Pro Asp Glu Arg Lys Ala             180 185 190 Arg Ala Ala Arg Ala Gly Asp Val Thr Ala Gln Thr Ile Ala Ala Leu         195 200 205 Arg Asp Ile Gly Leu Glu Cys Glu Thr Val Gly Gly Ala Gly Thr Gly     210 215 220 Ser Phe Ala Phe Asp Gly Met Ser Gly Val Trp Asn Glu Leu Gln Pro 225 230 235 240 Gly Ser Tyr Ala Phe Met Asp Ala Asp Tyr Ala Arg Asn Thr Pro Ala                 245 250 255 Gly Gly Asp Val Pro Lys Phe Glu His Ala Met Phe Val Trp Ala Ile             260 265 270 Val Met Ser Arg Thr Ser Val Gly Gln Ala Val Val Asp Ala Gly His         275 280 285 Lys Val Leu Pro Ile Asp Ser Gly Met Pro Val Pro Phe Asp Arg Pro     290 295 300 Gly Val Arg Tyr Glu Arg Pro Ser Asp Glu His Gly Cys Leu Val Ala 305 310 315 320 Glu Leu Asp Ser Ala Leu Pro Asp Leu Gly Glu Lys Ile Leu Ile Val                 325 330 335 Pro Ser His Val Asp Pro Thr Ala Asn Gln His Asp Phe Phe Ile Gly             340 345 350 Val Arg Gly Met Ala Glu Gly Asp Gly Thr Val Gln Glu Ile Trp Pro         355 360 365 Val Thr Ala Arg Gly Cys Val Phe     370 375 <210> 2 <211> 1131 <212> DNA <213> Filomicrobium marinum <400> 2 atgcgcgcac cagcacggct tggtattccg cttgcagacg tcgatacccc tgctttgatc 60 ctcgatctcg atgcgtttga gcgcaatctc cagacgatgg cggattgggc caaaagcaag 120 aacgtgcgat tgcgcccaca tgcaaaatct cacaaatgcc ccgcgatcgc gcatcgacaa 180 atggcgctcg gagccgtcgg tgtctgctgc cagaaggtct cggaagccga ggtgatggtg 240 gatgctggca taactaatgt gctgatcagc aacgaggtcg tcgggcgcac gaaactagag 300 gcgctggcgc aattggcgct acgagcgcgt atgggtgtgt gtgtcgacga tatccggcaa 360 atcagagatt tgtctgaggc gatgcatgct gctggcgcaa caatagatgt gcttgttgaa 420 atcaatatcg gcgggaaccg ttgcggtgtt gagccgggcg accctgcagt tcggctgggt 480 gcagctgttg cgcaggcgga tggcctgcgt tttgcgggct tacagtctta tgacggaatt 540 acgcagcatg tccgcgatcc ggatgaacgc aaggcgcgtg cggcgcgtgc gggtgatgtt 600 accgcgcaga ccattgcggc gcttcgcgat attgggcttg agtgcgaaac tgtcgggggc 660 gccggaacgg gttccttcgc attcgacggt atgagcggcg tgtggaatga gctgcaaccg 720 gggtcctacg cttttatgga tgccgactat gctcgcaata ccccggcggg tggcgatgtt 780 cccaaatttg agcacgccat gtttgtttgg gcgatcgtca tgagccgtac cagcgttggt 840 caggctgtgg tggatgccgg gcataaagtg ctgcctatcg actcgggtat gccggtcccg 900 tttgatcgtc cgggtgttcg ctatgagcgg ccttcagacg agcacggttg cctggttgcg 960 gaactggact cggcccttcc tgacctaggt gagaaaattc tgatcgtgcc cagtcatgtc 1020 gatccaacgg ccaaccagca cgactttttt atcggtgtgc gtgggatggc cgagggcgat 1080 ggcacagttc aagagatctg gccagttacg gcgcgaggtt gtgtgttcta g 1131 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for SEQ ID No. 2 <400> 3 tggtgccgcg cggcagccat atgcgcgcac cagcacggct 40 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <220> Reverse primer for SEQ ID No. 2 <400> 4 ggtggtggtg gtgctcgagc tagaacacac aacctcgcgc 40 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for pET28a (+) <400> 5 gcgcgaggtt gtgtgttcta gctcgagcac caccaccacc 40 <210> 6 <211> 40 <212> DNA <213> Artificial Sequence <220> Reverse primer for pET28a (+) <400> 6 agccgtgctg gtgcgcgcat atggctgccg cgcggcacca 40

Claims (14)

A D-threonine producing enzyme comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 90% or more homology with SEQ ID NO: 1. The method according to claim 1,
The D-threonine producing enzyme is a D-threonine producing enzyme that is based on glycine and acetaldehyde. The method according to claim 2,
The glycine and acetaldehyde is D-threonine production enzyme that is in the ratio of 0.5: 1 to 30: 1. Gene encoding the D-threonine producing enzyme of claim 1. The method according to claim 3,
The gene is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 2. Recombinant expression vector comprising the gene of claim 4. A transformant wherein the recombinant expression vector of claim 6 is introduced into a host cell. The method according to claim 7,
The host cell is E. coli transformants. D-threonine production composition comprising the D-threonine production enzyme of claim 1 as an active ingredient. Culturing the transformant of claim 7; And
Separating the D-threonine producing enzyme from the culture of the transformant. A method of producing D-threonine in vitro comprising reacting the D-threonine producing enzyme of claim 1 with glycine and acetaldehyde. The method according to claim 11,
Recovering D-threonine from the reaction product of the D-threonine producing enzyme, glycine and acetaldehyde; D-threonine production method in vitro further comprising. A method of producing D-threonine in vivo , comprising: culturing the transformant of claim 7 in the presence of glycine and acetaldehyde. The method according to claim 13,
Recovering D-threonine from the transformed culture; in vivo (D-threonine production method in vivo ) further comprising.

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