KR102489629B1 - Methylglyoxal synthase from Clostridium difficile 630 strain attatched strep-tag, and method for producing methylglyoxal using thereof - Google Patents

Abstract

The present invention relates to a Clostridium difficile 630 strain-derived methylglyoxal synthetase containing a strep-tag, a polypeptide, a polynucleotide encoding the same, a recombinant vector containing the polynucleotide, and the recombinant vector It relates to a method for producing methylglyoxal using an introduced transformed microorganism and the methylglyoxal synthetase.

Description

Methylglyoxal synthase derived from Clostridium difficile 630 strain containing strep-tag and method for producing methylglyoxal using the same

The present invention relates to a Clostridium difficile 630 strain-derived methylglyoxal synthetase containing a strep-tag, a polypeptide, a polynucleotide encoding the same, a recombinant vector containing the polynucleotide, and the recombinant vector It relates to a method for producing methylglyoxal using an introduced transformed microorganism and the methylglyoxal synthetase.

1,2-propanediol (PDO) is used as a building block in various fields such as antifreeze, polyester resin, pharmaceuticals, cosmetics, and detergents. It is material. Due to this high availability, more than 1 trillion lb/yr are sold in the US alone, and worldwide demand is estimated at more than 3 trillion lb/yr.

Meanwhile, most 1,2-propanediol is produced through petrochemical processes. Propylene, a product of steam cracking, is converted to propylene oxide, and harmful intermediates and by-products are produced through hydrolysis to finally produce 1,2-propylene glycol. In order to replace this process with a more sustainable and environmentally friendly bio process, attempts have been made to produce 1,2-propanediol using various microorganisms.

1,2-propanediol can be produced only when dihydroxy acetone phosphate is converted to methylglyoxal and further reduced by methylglyoxal reductase. Here, methylglyoxal is an important substrate for the production of 1,2-propanediol.

Methylglyoxal is an organic compound having the chemical formula CH ₃ C(O)CHO, and is a reduced derivative of pyruvate. Methylglyoxal is produced industrially by the breakdown of carbohydrates using methylglyoxal synthetase. In organisms, methylglyoxal is formed as a by-product of several metabolic pathways, mainly glycolysis with substrates such as glyceraldehyde-3-phosphate and dehydroxy acetone phosphate. occurs as a by-product of

Methylglyoxal synthase (MGS) is an enzyme that catalyzes a chemical reaction that decomposes dehydroxy acetone phosphate (DHAP) into methylglyoxal and phosphate. The activity of methylglyoxal synthetase is inhibited by phosphoenol pyruvate, 3-phosphoglycerate, pyrophosphate, and phosphoric acid.

Currently, Pseudomonas saccharophila ( Pseudomonas saccharophila ), Proteus vulgaris ( Proteus vulgaris ), Clostridium acetobutylicum ( Clostridium acetobutylicum ), Thermus sp. GH5 strain (Thermus sp. GH5 strain), Escherichia coli ( E. coli ), Bacillus subtilis ( Bacillus subtilis ) Some studies on the properties of methylglyoxal synthetase have been made, but very limited. In particular, studies on specific activity are difficult to find reported except for Bacillus subtilis and E. coli .

Accordingly, the present inventors studied to find the optimal methylglyoxal synthetase for the production of methylglyoxal through comparison of inactivity of methylglyoxal synthetase of various origins, and as a result, Clostridium containing a strep-tag Difficile ( Clostridium difficile ) It was confirmed that the methylglyoxal synthetase derived from strain 630 had a significantly superior methylglyoxal synthase activity compared to synthetases derived from other strains.

Accordingly, an object of the present invention is to provide a methylglyoxal synthetase derived from Clostridium difficile 630 strain containing a strep-tag, which is a polypeptide.

Another object of the present invention is to provide a polynucleotide encoding a methylglyoxal synthetase derived from Clostridium difficile 630 strain including a nucleotide sequence encoding a Strep-tag.

Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a methylglyoxal synthetase derived from Clostridium difficile 630 strain containing a strep-tag.

Another object of the present invention is to provide a transformed microorganism into which a recombinant vector containing a polynucleotide encoding a methylglyoxal synthetase derived from Clostridium difficile 630 strain containing a strep-tag is introduced.

Another object of the present invention is to provide a method for preparing methylglyoxal using a methylglyoxal synthetase derived from Clostridium difficile 630 strain containing a strep-tag.

The present invention relates to a Clostridium difficile 630 strain-derived methylglyoxal synthetase containing a strep-tag, a polypeptide, a polynucleotide encoding the same, a recombinant vector containing the polynucleotide, and the recombinant vector It relates to a method for producing methylglyoxal using an introduced transformed microorganism and the methylglyoxal synthetase.

Hereinafter, the present invention will be described in more detail.

The present inventors attached a nucleotide sequence encoding a strep-tag to the 5'-end or 3'-end of the nucleotide sequence encoding methylglyoxal synthase derived from Clostridium difficile 630 strain, and T7 Introduced into the pET duet-1 vector together with the promoter, Clostridium difficile ( Clostridium difficile ) methylglyoxal synthetase derived from strain 630 containing a strep-tag was purified.

One example of the present invention relates to a methylglyoxal synthetase derived from Clostridium difficile 630 strain containing a strep-tag.

In the present invention, the methylglyoxal synthetase may include the amino acid sequence of SEQ ID NO: 1, for example, may consist of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

In the present invention, the strep-tag may include the amino acid sequence of SEQ ID NO: 8, for example, may consist of the amino acid sequence of SEQ ID NO: 8, but is not limited thereto.

In the present invention, the strep-tag may be bound to the N-terminus, C-terminus, or both ends of methylglyoxal synthetase, and may be, for example, bound to the N-terminus.

In the present invention, the strep-tag-containing methylglyoxal synthetase may exhibit synthetic activity at pH 4 to 7, pH 5 to 7, and pH 6 to 7, for example, at pH 6 to 7 It may indicate the activity of, but is not limited thereto.

In the present invention, the strep-tag-containing methylglyoxal synthase is 20 to 60 ℃, 30 to 60 ℃, 36 to 60 ℃, 20 to 50 ℃, 30 to 50 ℃, 36 to 50 ℃, 20 to 40 ℃ It may exhibit synthetic activity at 30 to 40 °C and 36 to 40 °C, for example, it may exhibit maximum activity at 36 to 40 °C, but is not limited thereto.

In the present invention, the term "specific activity (specific activity)" refers to the enzyme activity per unit weight of protein, and generally the unit of activity is expressed as a ratio (U / mg or mole / min * g-protein).

Another example of the present invention relates to a polynucleotide encoding a methylglyoxal synthetase derived from Clostridium difficile 630 strain including a nucleotide sequence encoding a strep-tag.

In the present invention, the nucleotide sequence encoding the methylglyoxal synthase derived from Clostridium difficile 630 strain may include the nucleotide sequence of SEQ ID NO: 2, for example, the base sequence of SEQ ID NO: 2 It may consist of a sequence, but is not limited thereto.

In the present invention, the base sequence encoding the strep-tag may include the base sequence of SEQ ID NO: 3, for example, may consist of the base sequence of SEQ ID NO: 3, but is not limited thereto.

In the present invention, the nucleotide sequence encoding the strep-tag is at the 5'-end, 3'-end or both ends of the nucleotide sequence encoding the methylglyoxal synthetase derived from Clostridium difficile 630 strain It may be bonded, for example, it may be bonded to the 5'-end.

Another example of the present invention relates to a recombinant vector including a polynucleotide encoding a methylglyoxal synthetase derived from Clostridium difficile 630 strain including a strep-tag.

In the present invention, the term "vector" means a means for expressing a target gene in a host cell. Examples include viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenoviral vectors, retroviral vectors and adeno-associated viral vectors. Vectors that can be used as recombinant vectors include plasmids often used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series and pUC19, etc.), phages (eg, λgt4λB, λ-Charon, λΔz1, and M13, etc.) or viruses (eg, SV40, etc.) may be engineered, but are limited thereto It is not.

Vectors in the present invention can typically be constructed as vectors for cloning or vectors for expression. As a vector for expression, a conventional vector used in the art for expressing foreign proteins in plants, animals, or microorganisms may be used, but is not limited thereto. The recombinant vector may be constructed through various methods known in the art.

In the present invention, the recombinant vector can be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when the vector used is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter) , T7 promoter, etc.), ribosome binding sites for initiation of translation and transcription/translation termination sequences, but are not limited thereto.

Another example of the present invention is transformation into which a recombinant vector containing a nucleotide sequence encoding a strep-tag and a nucleotide sequence encoding a methylglyoxal synthetase derived from Clostridium difficile 630 strain is introduced It's about microbes.

In the present invention, the transformed microorganism may be obtained by introducing the recombinant vector into an appropriate microorganism, but is not limited thereto.

In the present invention, the microorganism is a cell capable of stably and continuously cloning or expressing the recombinant vector, and any host cell known in the art may be used.

In the present invention, microorganisms may include Escherichia coli, yeast, animal cells, plant cells, or insect cells. For example, prokaryotic cells include E. coli JM109, E. coli BL21, E. coli RR1, and E. . _ _ _ In the case of transformation into eukaryotic cells, yeast ( Saccharomyce cerevisiae ), insect cells, plant cells and animal cells, such as Sp2 / 0, CHO (Chinese hamster ovary) K1 , CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell lines, etc. may be used, but are not limited thereto.

In the present invention, transfer (introduction) of the recombinant vector into the host cell may use a transfer method widely known in the art.

In the present invention, the delivery method may be, for example, a CaCl ₂ method or an electroporation method when the microorganism is a prokaryotic cell, and a microinjection method, a calcium phosphate precipitation method, or an electroporation method when the microorganism is a eukaryotic cell. , Liposome-mediated transfection and gene bombardment may be used, but are not limited thereto.

In the present invention, the method for selecting a transformed microorganism can be easily performed according to a method widely known in the art using a phenotype expressed by a selection marker. For example, when the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the specific antibiotic.

Another example of the present invention relates to a method for producing methylglyoxal, comprising the following steps:

An enzyme reaction step of synthesizing methylglyoxal by adding a substrate to a methylglyoxal synthetase derived from Clostridium difficile 630 strain containing a strep-tag.

In the present invention, the substrate may be dihydroxyacetone phosphoric acid, but is not limited thereto.

In the present invention, the enzymatic reaction step may be performed at pH 4 to 7, pH 5 to 7, pH 6 to 7, for example, pH 6 to 7, but is not limited thereto. .

In the present invention, the enzyme reaction step is 20 to 60 ℃, 30 to 60 ℃, 36 to 60 ℃, 20 to 50 ℃, 30 to 50 ℃, 36 to 50 ℃, 20 to 40 ℃, 30 to 40 ℃, 36 It may be carried out at 40 ° C to 40 ° C, for example, it may be carried out at 36 to 40 ° C, but is not limited thereto.

In the present invention, a primer set means a primer set including a forward primer and a reverse primer. The forward primer or the reverse primer may each contain a nucleotide sequence encoding a restriction site and/or a strep-tag, and at least one of the forward primer and the reverse primer contains a nucleotide sequence encoding a strep-tag. It may be a primer set.

In the present invention, a primer refers to a single-stranded polynucleotide that can act as an initiation point in a polymerization reaction of nucleotides by a polymerase. For example, the primer is a single strand capable of serving as the starting point of template-directed DNA synthesis under suitable conditions (i.e., the presence of four different nucleoside triphosphates and a polymerase) at a suitable temperature and in a suitable buffer. It may be a polynucleotide of The suitable length of the primer may depend on various factors, such as temperature and use of the primer.

In the present invention, the primer does not have to have a sequence completely complementary to a partial sequence of the template, and it is sufficient to have complementarity within a range capable of hybridizing with the template and performing the specific function of the primer.

In the present invention, restriction sites are 4 to 9 specific nucleotide sequence portions in which a specific portion of a nucleotide sequence is recognized by a restriction enzyme or a restriction endonuclease and the portion or its surroundings are cleaved. means For example, EcoRI recognizes and cuts the 6-base sequence GAATTC as a restriction site, HaeIII recognizes and cuts the 4-base sequence GGCC, KpnI recognizes and cuts the 6-base sequence GGTACC, and NdeI can recognize and cleave CATATG, a six-nucleotide sequence.

The present invention relates to a Clostridium difficile 630 strain-derived methylglyoxal synthetase containing a strep-tag, a polypeptide, a polynucleotide encoding the same, a recombinant vector containing the polynucleotide, and the recombinant vector It relates to a method for producing methylglyoxal using an introduced transformed microorganism and the methylglyoxal synthetase. The methylglyoxal synthetase derived from Clostridium difficile 630 strain containing the strep-tag of the present invention has the highest specific activity at 30 ° C. ( Bacillus subtilis ) derived methylglyoxal synthetase and In comparison, it has about 1.37 times higher specific activity. Therefore, methylglyoxal can be efficiently produced by using the methylglyoxal synthetase derived from Clostridium difficile 630 strain containing the strep-tag of the present invention.

1 is a nucleotide sequence encoding a methylglyoxal synthetase derived from Clostridium difficile 630 strain and a nucleotide sequence including a nucleotide sequence encoding a strep-tag according to an embodiment of the present invention. This is a cleavage map showing introduction to the pET duet-1 vector.
Figure 2 is a calibration curve measuring the culture level of E. coli BL21 strain transformed according to an embodiment of the present invention.
3 is an SDS-PAGE photograph of methylglyoxal synthetase purified from Clostridium difficile 630 strain according to an embodiment of the present invention.
4 is a calibration curve for quantifying methylglyoxal derived from Clostridium difficile 630 strain according to an embodiment of the present invention.
5 is Bacillus subtilis , Clostridium difficile 630 strain, Oceanithermus profundus DSM 14977, Thermus sp. ) GH5, a graph comparing the enzymatic activities of Deinococcus koreensis -derived methylglyoxal synthetase.
6 is a methylglyoxal synthase derived from Bacillus subtilis, a methylglyoxal synthetase derived from Clostridium difficile 630, and a methylglyoxal synthase derived from Oceanic themus Profundus DSM 14977 according to an embodiment of the present invention. , When a strep-tag is attached to the N-terminus or C-terminus of methylglyoxal synthetase derived from thermus species GH5 strain and methylglyoxal synthetase derived from Deinococcus corinsis, graphs showing respective inactivity .
7 is a graph showing the specific activity of Clostridium difficile 630-derived methylglyoxal synthase according to an embodiment of the present invention according to pH.
8 is a graph showing the specific activity of Clostridium difficile 630-derived methylglyoxal synthetase according to an embodiment of the present invention according to temperature.

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1. Preparation of reagents

The methylglyoxal synthetase gene (cdMGS) derived from Clostridium difficile 630 was commercially synthesized by Bioneer (Korea) (Genbank ID: AJP10844.1) (see SEQ ID NO: 2). Restriction enzymes (QuickCutTM NdeI, QuickCutTM KpnI) were purchased from Takare (Japan). E. coli DH5a and E. coli BL21 (DE3) were purchased from RBC (Taiwan). pET duet-1 used as a vector for expression was purchased from Novagen (USA).

sequence number denomination order note One cdMGS
amino acid
sequence MNIALVAHDQMKNTMVGFCIGYESILKKYGLYATGTTGKRIMDETELNINRLASGPLGGDQQIGSLIVTQEIDLVIFLRDPLTSQAHETDIQALIRLCDVYHVPIATNLASAEIFIKALDRGELSWREVRKSKSQRI 137aa 2 cdMGS
nucleotide
sequence ATGAATATAGCATTAGTAGCACATGACCAAATGAAAAACACTATGGTCGGATTTTGTATAGGTTATGAATCAATTTTAAAAAAGTATGGACTATATGCAACTGGAACTACAGGAAAAAGAATAATGGATGAAACTGAATTAAATATAAATAGATTAGCATCTGGTCCTTTAGGTGGAGACCAACAAATAGGTTCTTTAATAGTAACACAAGAAATAGATTTAGTTATTTTTTTAAGAGACCCATTGACATCTCAGGCTCATGAAACAGATATACAAGCGTTAATAAGACTTTGTGATGTTTATCATGTTCCAATAGCTACAAATCTAGCTTCAGCAGAAATATTTATAAAAGCTCTTGATAGAGGAGAACTATCATGGAGAGAAGTTAGAAAAAGCAAAAGTCAACGTATTTAA 414bp

Example 2. Amplification of polynucleotides containing base sequences encoding methylglyoxal synthetase derived from Clostridium difficile 630 strain and base sequences encoding strep-tags

For the purification of methylglyoxal synthetase, a strep-tag, which is a polypeptide having little effect on the tertiary structure of methylglyoxal synthetase derived from Clostridium difficile 630 strain, was added to the methylglyoxal synthetase. Overexpression of the recombinant protein added to the N-terminus was designed.

A forward primer containing the nucleotide sequence encoding the strep-tag was prepared (SEQ ID NO: 4). Then, a polynucleotide to which a nucleotide sequence encoding a strep-tag was attached to the 5′-end of a nucleotide sequence encoding methylglyoxal synthetase derived from Clostridium difficile 630 strain was amplified (SEQ ID NO: 6). The nucleotide sequences encoding the strep-tags included in SEQ ID NOs: 4 and 6 are shown underlined.

sequence number denomination Sequence Listing (5`- -> 3`-) note 3 Strep-tag
nucleotide
Sequence TGGAGCCACCCGCAGTTCGAAAAA 24bp 4 Primer
Forward ATAAGAAGGAGATATA CATATG ACAGTCCTGAAAAGGAGCATCACCGATG TGGAGCCACCCGCAGTTCGAAAAA ATGAATATAGCATTAGTAGCACAT 98bp 5 Primer
Reverse TTTCTTTACCAGACTCGAG GGTACC TTAAATACGTTGACTTTTGCTTTT 49bp 6 Strep-tag
cdMGS
nucleotide
Sequence ATG TGGAGCCACCCGCAGTTCGAAAAA ATGAATATAGCATTAGTAGCACATGACCAAATGAAAAACACTATGGTCGGATTTTGTATAGGTTATGAATCAATTTTAAAAAAGTATGGACTATATGCAACTGGAACTACAGGAAAAAGAATAATGGATGAAACTGAATTAAATATAAATAGATTAGCATCTGGTCCTTTAGGTGGAGACCAACAAATAGGTTCTTTAATAGTAACACAAGAAATAGATTTAGTTATTTTTTTAAGAGACCCATTGACATCTCAGGCTCATGAAACAGATATACAAGCGTTAATAAGACTTTGTGATGTTTATCATGTTCCAATAGCTACAAATCTAGCTTCAGCAGAAATATTTATAAAAGCTCTTGATAGAGGAGAACTATCATGGAGAGAAGTTAGAAAAAGCAAAAGTCAACGTATTTAA 441bp

Example 3. Preparation of vectors for expressing methylglyoxal synthetase

The pET duet-1 vector containing the strong T7 promoter was digested using restriction enzymes KpnI and NdeI. A base encoding a strep-tag at the 5′-end of the pET duet-1 vector in which two sites, KpnI and NdeI, among the restriction sites were truncated, and a nucleotide sequence encoding methylglyoxal synthetase derived from Clostridium difficile 630 strain The polynucleotides to which the sequences were attached were combined using infusion (In-fusion, Takara, Japan). Thereafter, heat shock was applied at 42° C. for 2 minutes to transform the E. coli DH5a strain.

Then, after stabilization for 1 hour by adding LB medium at 37 ° C., the cells were plated on LB medium supplemented with ampicillin at a concentration of 20 ug/ml. After culturing the E. coli DH5a strain in an incubator at 37 ° C for 16 hours, the obtained colonies of the E. coli DH5a strain were pre-cultured in LB medium supplemented with 3 ml of ampicillin (20 ug/ml) to extract the recombinant vector. proceeded. Then, the extracted recombinant vector was selected through sequencing.

1 is a cleavage map of a recombinant vector including a nucleotide sequence encoding a strep-tag prepared according to Example 3 and a nucleotide sequence encoding a methylglyoxal synthetase derived from Clostridium difficile 630 strain. to be.

Example 4. Transformation and selection of E. coli BL 21 strain containing methylglyoxal synthetase expression vector

Transform E. coli BL21 strain (RBC, Taiwan) with a recombinant vector containing a nucleotide sequence encoding a strep-tag and a nucleotide sequence encoding a methylglyoxal synthetase derived from Clostridium difficile 630 strain converted. They were plated on LB medium supplemented with ampicillin at a rate of 20 ug/ml. Colony PCR was performed using forward primer (SEQ ID NO: 4) and reverse primer (SEQ ID NO: 5) on colonies obtained after culturing in an incubator set at 37 ° C. for 16 hours. After confirming the results by electrophoresis of the colony PCR reaction solution, transformants were selected.

Example 5. Overexpression and purification of strep-tagged methylglyoxal synthetase

The transformed E. coli BL21 was cultured in LB medium supplemented with 100 ug/ml of ampicillin at 37° C. until an OD (600 nm) of 0.5 was reached, and the results are shown in FIG. 2 and Table 3.

BSA (ug/ul) Absorbance (595 nm) 1.430 0.492 0.715 0.249 0.358 0.147 0.179 0.068 0.089 0.042 0.045 0.016

As can be seen in FIG. 2 and Table 3, the R ³ value was 0.9976 and was used as a standard calibration curve for protein quantification. Thereafter, 80 uM IPTG was induced at 20° C. and 200 rpm for 19 hours. The cultured cells were centrifuged at 4000 X g for 10 minutes, then the supernatant was removed and the cells were recovered. The recovered cells were suspended in a solution for cell disruption (50 mM sodium phosphate buffer, 300 mM sodium chloride, pH 8, 10 mM imidazole), and then disrupted using an ultrasonicator. The disrupted total protein extract was filtered through Strep- ^{tactin ® resin} to purify strep-tagged methylglyoxal synthetase. Protein purity was determined by SDS-PAGE using a 4-12% Tris gel (Bio-Rad, USA). The enzyme concentration was measured using the Bradford assay (Bradford, 1976) and is shown in FIG. 3 .

As can be seen in FIG. 3, most of the methylglyoxal synthetase derived from Clostridium difficile 630 strain containing the strep-tag was soluble and purified with high purity.

The amino acid sequence of methylglyoxal synthetase derived from strain 630 of Clostridium difficile containing a strep-tag at the N-terminus is shown in SEQ ID NO: 7, and the amino acid sequence of the strep-tag is shown in SEQ ID NO: 8. indicated (see Table 4). In SEQ ID NO: 7, the amino acid sequence corresponding to the strep-tag is underlined.

sequence number denomination order note 7 Strep-tag
cdMGS
amino acid sequences WSHPQFEK MNIALVAHDQMKNTMVGFCIGYESILKKYGLYATGTTGKRIMDETELNINRLASGPLGGDQQIGSLIVTQEIDLVIFLRDPLTSQAHETDIQALIRLCDVYHVPIATNLASAEIFIKALDRGELSWREVRKSKSQRI 145aa 8 Strep-tag
amino acid sequences WSHPQFEK 8aa

Example 6. Measurement of specific activity of methylglyoxal synthase derived from various strains

6-1. Measurement of methylglyoxal conversion rate

In order to analyze the in vitro activity of the enzyme, the enzyme reaction was carried out with dihydroxyacetone phosphate as a precursor at 30 ° C, and 2,4-dinitrophenylhydrazine having a chromophore was added to the reaction solution to enzymatically Methyl glyoxal prepared by the reaction was detected after derivatization.

The production of methylglyoxal was measured for a certain period of time using a 555nm UV spectrophotometer. Clostridium difficile containing a strep-tag ( Clostridium difficile ) 630 strain-derived methylglyoxal synthase measures the rate at which dihydroxyacetone phosphate is converted to methylglyoxal, and the results are shown in FIG. 4 and Table 5 shown in

Methylglyoxal concentration (mM) Absorbance (555 nm) 1.500 1.956 0.750 0.935 0.375 0.515 0.188 0.322 0.000 0.050

As can be seen in Figure 4 and Table 5, the production of methylglyoxal was quantified based on the R ³ value of 0.9977.

6-2. inactivity measurement

30 mM dihydroxyacetone in 0.44 ml of 40 mM imidazole buffer (pH 7.5) to which 0.1 ug of methylglyoxal synthetase derived from Clostridium difficile 630 strain was added containing a purified strep-tag Using 50 ul of phosphoric acid as a substrate, a methylglyoxal synthetase reaction was performed at 30 °C for 10 minutes. The specific activity was measured, and the results are shown in FIG. 5 and Table 6.

Methylglyoxal synthase derived from various strains Inactivity (mole/min*g-protein) Bacillus subtilis 0.150 Clostridium difficile (cdMGS) 0.205 Oceanithermus profundus DSM 14977 (opMGS) 0.074 Thermus sp. GH5 (tMGS) 0.015 Deinococcus koreensis (dcMGS) 0

As can be seen in Figure 5 and Table 6, methylglyoxal synthase derived from Clostridium difficile 630 strain containing a strep-tag, except for methylglyoxal synthetase (dcMGS) derived from Deinococcus corinsis ( cdMGS), methylglyoxal synthetase (opMGS) derived from Oceanic themus Profundus DSM 14977, and methylglyoxal synthetase (tMGS) derived from GH5 of the genus Thermus, respectively, showed inactivity with respect to substrates. Strep-tag Clostridium difficile 630 strain-derived methylglyoxal synthetase (cdMGS), which contains Bacillus subtilis-derived methylglyoxal synthetase (cdMGS), which has the highest specific activity at 30 ° C. It was measured to have a specific activity 1.37 times higher.

Example 7. Comparison of activities of methylglyoxal synthetase derived from various strains according to the attachment position of the strep-tag

Strep-tagged methylglyoxal synthase (cdMGS) derived from Clostridium difficile 630 strain, methylglyoxal synthetase (opMGS) derived from Oceanic themus Profundus DSM 14977, methylglyoxal derived from GH5 of the genus Thermus The specific activities when attached to the N-terminus or C-terminus of Sal synthase (tMGS) and Deinococcus corinsis-derived methylglyoxal synthetase (dcMGS) were compared, respectively.

The specific activity was measured using 3 mM dihydroxyacetone phosphate as a substrate in 40 mM imidazole buffer (pH 7.5) to which 0.5 ug of methylglyoxal synthetase derived from various purified strains was added. 6 and Table 7.

Methylglyoxal synthase derived from various strains Inactivity (mole/min*g-protein) N-terminus C-terminus Bacillus subtilis 0 0 Clostridium difficile (cdMGS) 0.094793 0.053063 Oceanithermus profundus DSM 14977 (opMGS) 0 0.017314 Thermus sp. GH5 (tMGS) 0.015586 - Deinococcus koreensis (dcMGS) 0 0 Escherichia coli 0.006283 0.047787

As can be seen in FIG. 6 and Table 7, the specific activity of methylglyoxal synthetase derived from various strains was measured differently depending on the location of the strep-tag. In particular, Clostridium difficile 630 strain-derived methylglyoxal synthase (cdMGS) has a specific activity when a strep-tag is attached to the N-terminus (specific activity ) was confirmed to be improved by about 80%.

Example 8. Specific activity analysis according to the pH of the enzyme

The specific activity of methylglyoxal synthetase (cdMGS) derived from Clostridium difficile 630 strain containing a purified strep-tag was measured according to pH conditions. pH 4 to pH 6 is 40 mM succinic acid/sodium hydroxide buffer, pH 7 is imidazole/hydrochloric acid buffer, pH 8 is tris(hydroxymethyl)aminomethane buffer, pH 9 is glycine/sodium hydroxide Adjusted with a buffer solution. The effect of the pH of the buffer solution on the specific activity of methylglyoxal synthetase (cdMGS) derived from Clostridium difficile 630 strain containing a strep-tag was investigated, and the results are shown in FIG. 7 and Table 8.

pH Inactivity (mole/min*g-protein) 4.000 0.049 5.000 0.139 6.000 0.145 7.000 0.178 8.000 0.024 9.000 0.000

As can be seen in FIG. 7 and Table 8, methylglyoxal synthase (cdMGS) derived from Clostridium difficile 630 strain containing a strep-tag showed maximum activity at pH 6 to 7.

Example 9. Analysis of enzyme inactivity according to temperature

The temperature of the reaction solution was divided from 10 ° C. to 90 ° C., and the effect of temperature on the specific activity of Clostridium difficile 630 strain-derived methylglyoxal synthetase (cdMGS) containing a strep-tag was investigated, and the results were It is shown in Figure 8 and Table 9.

Temperature Inactivity (mole/min*g-protein) 10 0.000 20 0.063 30 0.205 36 0.226 40 0.245 50 0.209 60 0.119 65 0.068 70 0.024 75 0.008 80 0.000 90 0.000

As can be seen in FIG. 8 and Table 9, methylglyoxal synthetase (cdMGS) derived from Clostridium difficile 630 strain containing a strep-tag showed maximum activity at 36 to 40 °C.

<110> SOGANG UNIVERSITY RESEARCH FOUNDATION <120> Methylglyoxal synthase from Clostridium difficile 630 strain attached strep-tag, and method for producing methylglyoxal using its <130> PN200052 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 137 <212> PRT <213> unknown <220> <223> Clostridium difficile 630 methylglyoxal synthase amino acid sequence <400> 1 Met Asn Ile Ala Leu Val Ala His Asp Gln Met Lys Asn Thr Met Val 1 5 10 15 Gly Phe Cys Ile Gly Tyr Glu Ser Ile Leu Lys Lys Tyr Gly Leu Tyr 20 25 30 Ala Thr Gly Thr Thr Gly Lys Arg Ile Met Asp Glu Thr Glu Leu Asn 35 40 45 Ile Asn Arg Leu Ala Ser Gly Pro Leu Gly Gly Asp Gln Gln Ile Gly 50 55 60 Ser Leu Ile Val Thr Gln Glu Ile Asp Leu Val Ile Phe Leu Arg Asp 65 70 75 80 Pro Leu Thr Ser Gln Ala His Glu Thr Asp Ile Gln Ala Leu Ile Arg 85 90 95 Leu Cys Asp Val Tyr His Val Pro Ile Ala Thr Asn Leu Ala Ser Ala 100 105 110 Glu Ile Phe Ile Lys Ala Leu Asp Arg Gly Glu Leu Ser Trp Arg Glu 115 120 125 Val Arg Lys Ser Lys Ser Gln Arg Ile 130 135 <210> 2 <211> 414 <212> DNA <213> unknown <220> <223> Clostridium difficile 630 methyglyoxal synthase nucleotide sequence <400> 2 atgaatatag cattagtagc acatgaccaa atgaaaaaca ctatggtcgg attttgtata 60 ggttatgaat caattttaaa aaagtatgga ctatatgcaa ctggaactac aggaaaaaga 120 ataatggatg aaactgaatt aaatataaat agattagcat ctggtccttt aggtggagac 180 caacaaatag gttctttaat agtaacacaa gaaatagatt tagttatttt tttaagagac 240 ccattgacat ctcaggctca tgaaacagat atacaagcgt taataagact ttgtgatgtt 300 tatcatgttc caatagctac aaatctagct tcagcagaaa tatttataaa agctcttgat 360 agaggagaac tatcatggag agaagttaga aaaagcaaaa gtcaacgtat ttaa 414 <210> 3 <211> 441 <212> DNA <213> unknown <220> <223> Strep-tag amino nucleotide sequence <400> 3 atgtggagcc acccgcagtt cgaaaaaatg aatatagcat tagtagcaca tgaccaaatg 60 aaaaacacta tggtcggatt ttgtataggt tatgaatcaa ttttaaaaaa gtatggacta 120 tatgcaactg gaactacagg aaaaagaata atggatgaaa ctgaattaaa tataaataga 180 ttagcatctg gtcctttagg tggagaccaa caaataggtt ctttaatagt aacacaagaa 240 atagatttag ttattttttt aagagaccca ttgacatctc aggctcatga aacagatata 300 caagcgttaa taagactttg tgatgtttat catgttccaa tagctacaaa tctagcttca 360 gcagaaatat ttataaaagc tcttgataga ggagaactat catggagaga agttagaaaa 420 agcaaaagtc aacgtattta a 441 <210> 4 <211> 98 <212> DNA <213> artificial sequence <220> <223> Primer forward <400> 4 ataagaagga gatacatacata tgacagtcct gaaaaggagc atcaccgatg tggagccacc 60 cgcagttcga aaaaatgaat atagcattag tagcacat 98 <210> 5 <211> 49 <212> DNA <213> artificial sequence <220> <223> Primer reverse <400> 5 tttctttacc agactcgagg gtaccttaaa tacgttgact tttgctttt 49 <210> 6 <211> 441 <212> DNA <213> unknown <220> <223> Strep-tag Clostridium difficile 630 methylglyoxal synthase nucleotide sequence <400> 6 atgtggagcc acccgcagtt cgaaaaaatg aatatagcat tagtagcaca tgaccaaatg 60 aaaaacacta tggtcggatt ttgtataggt tatgaatcaa ttttaaaaaa gtatggacta 120 tatgcaactg gaactacagg aaaaagaata atggatgaaa ctgaattaaa tataaataga 180 ttagcatctg gtcctttagg tggagaccaa caaataggtt ctttaatagt aacacaagaa 240 atagatttag ttattttttt aagagaccca ttgacatctc aggctcatga aacagatata 300 caagcgttaa taagactttg tgatgtttat catgttccaa tagctacaaa tctagcttca 360 gcagaaatat ttataaaagc tcttgataga ggagaactat catggagaga agttagaaaa 420 agcaaaagtc aacgtattta a 441 <210> 7 <211> 145 <212> PRT <213> unknown <220> <223> Strep-tag Clostridium difficile 630 methylglyoxal synthase amino acid sequence <400> 7 Trp Ser His Pro Gln Phe Glu Lys Met Asn Ile Ala Leu Val Ala His 1 5 10 15 Asp Gln Met Lys Asn Thr Met Val Gly Phe Cys Ile Gly Tyr Glu Ser 20 25 30 Ile Leu Lys Lys Tyr Gly Leu Tyr Ala Thr Gly Thr Thr Gly Lys Arg 35 40 45 Ile Met Asp Glu Thr Glu Leu Asn Ile Asn Arg Leu Ala Ser Gly Pro 50 55 60 Leu Gly Gly Asp Gln Gln Ile Gly Ser Leu Ile Val Thr Gln Glu Ile 65 70 75 80 Asp Leu Val Ile Phe Leu Arg Asp Pro Leu Thr Ser Gln Ala His Glu 85 90 95 Thr Asp Ile Gln Ala Leu Ile Arg Leu Cys Asp Val Tyr His Val Pro 100 105 110 Ile Ala Thr Asn Leu Ala Ser Ala Glu Ile Phe Ile Lys Ala Leu Asp 115 120 125 Arg Gly Glu Leu Ser Trp Arg Glu Val Arg Lys Ser Lys Ser Gln Arg 130 135 140 Ile 145 <210> 8 <211> 8 <212> PRT <213> unknown <220> <223> Strep-tag amino acid sequence <400> 8 Trp Ser His Pro Gln Phe Glu Lys 1 5

Claims (22)

In the methylglyoxal synthase derived from Clostridium difficile 630 strain containing a strep-tag,
The strep-tag is a synthetase that is bound to the N-terminus of Clostridium difficile 630 strain-derived methylglyoxal synthetase. The methylglyoxal synthetase according to claim 1, wherein the methylglyoxal synthetase comprises the amino acid sequence of SEQ ID NO: 1. The methylglyoxal synthetase according to claim 1, wherein the strep-tag comprises the amino acid sequence of SEQ ID NO: 8. delete The methylglyoxal synthetase according to claim 1, wherein the methylglyoxal synthetase has a specific activity of 0.139 U/mg or more at pH 5 to 7. The methylglyoxal synthetase according to claim 1, wherein the methylglyoxal synthetase has a specific activity of 0.063 U/mg or more at 20 to 65 °C. In a polynucleotide comprising a nucleotide sequence encoding a strep-tag and a nucleotide sequence encoding a methylglyoxal synthetase derived from Clostridium difficile 630 strain,
The nucleotide sequence encoding the strep-tag is bound to the 5′-end of the nucleotide sequence encoding methylglyoxal synthetase derived from Clostridium difficile 630 strain, polynucleotide. The polynucleotide according to claim 7, wherein the nucleotide sequence encoding the methylglyoxal synthetase derived from Clostridium difficile 630 strain comprises the nucleotide sequence of SEQ ID NO: 2. The polynucleotide according to claim 7, wherein the nucleotide sequence encoding the strep-tag comprises the nucleotide sequence of SEQ ID NO: 3. delete Includes a nucleotide sequence encoding a strep-tag and a nucleotide sequence encoding a methylglyoxal synthetase derived from Clostridium difficile 630 strain,
The nucleotide sequence encoding the strep-tag is linked to the 5′-end of the nucleotide sequence encoding methylglyoxal synthetase, the recombinant vector. The recombinant vector according to claim 11, wherein the nucleotide sequence encoding the strep-tag comprises the nucleotide sequence of SEQ ID NO: 3. The recombinant vector according to claim 11, wherein the nucleotide sequence encoding the methylglyoxal synthase includes the nucleotide sequence of SEQ ID NO: 2. delete Includes a nucleotide sequence encoding a strep-tag and a nucleotide sequence encoding a methylglyoxal synthetase derived from Clostridium difficile 630 strain,
The nucleotide sequence encoding the strep-tag is a transformed microorganism into which a recombinant vector coupled to the 5′-end of the nucleotide sequence encoding methylglyoxal synthetase is introduced. The transformed microorganism according to claim 15, wherein the nucleotide sequence encoding the strep-tag comprises the nucleotide sequence of SEQ ID NO: 3. The transformed microorganism according to claim 15, wherein the nucleotide sequence encoding the methylglyoxal synthetase comprises the nucleotide sequence of SEQ ID NO: 2. delete An enzyme reaction step of synthesizing methylglyoxal by adding a substrate to a methylglyoxal synthetase derived from Clostridium difficile 630 strain including a strep-tag;
The strep-tag is a method for producing methylglyoxal, which is bound to the N-terminus of methylglyoxal synthase derived from Clostridium difficile 630 strain. The method of claim 19, wherein the substrate is dihydroxyacetone phosphoric acid. The method of claim 19, wherein the enzymatic reaction step is performed under conditions of pH 5 to 7. The method of claim 19, wherein the enzymatic reaction step is performed at a temperature of 20 to 65 °C.

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