KR101106171B1 - Novel 2-Deoxyribose 5-Phosphate Aldolase Derived from Yersinia sp. EA015 - Google Patents

Abstract

The present invention is Yersinia sp. Novel 2-deoxyribose 5-phosphate aldolase (DERA) from EA015 strains, more specifically Yersinia sp . Of a novel 2-deoxyribose 5-phosphate aldolase derived from EA015 strain, a gene encoding the new 2-deoxyribose 5-phosphate aldolase and a bioactive substance using the 2-deoxyribose 5-phosphate aldolase It relates to a manufacturing method.

According to the present invention, two carbon units can be added to a natural product by enzymatic method, and thus, the importance of white biotechnology can be replaced by enzymatic method by various bioactive substances that have been manufactured by chemical methods. .

2-dioxyribose 5-phosphate aldolase, aldol condensation reaction, Yersinia sp.

Description

Ｙｅｒｓｉｎｉ ａ ｓｐ． Novel 2-Deoxyribose 5-Phosphate Aldolase Derived from Yersinia sp. EA015}

The present invention is Yersinia sp. Novel 2-deoxyribose 5-phosphate aldolase (DERA) from EA015 strains, more specifically Yersinia sp . Of a novel 2-deoxyribose 5-phosphate aldolase derived from EA015 strain, a gene encoding the new 2-deoxyribose 5-phosphate aldolase and a bioactive substance using the 2-deoxyribose 5-phosphate aldolase It relates to a manufacturing method.

Aldolase is known as an enzyme that catalyzes a carbon-carbon bond by an aldol condensation reaction and can be used in the synthesis of various compounds having physiological activity. Among aldolases, 2-dioxyribose 5-phosphate aldolase is one of a variety of aldolases and is an acetaldehyde dependent aldolase and is known as the most useful enzyme for carbon-carbon bonds (Jennewein et al ., Biotechnol. J., 1: 537, 2006). In general, 2-deoxyribose 5-phosphate aldolase is involved in the process of reversibly synthesizing deoxyribose 5-phosphate by catalyzing the aldol reaction of acetaldehyde with D-glyceraldehyde-3-phosphate.

2-deoxyribose 5-phosphate aldolase can be used in a variety of acceptor substrates, especially propanal, acetone, fluoroacetone, etc. sugar) can be synthesized.

Synthesis of compounds by conventional chemical methods has limitations in complexity, stability, or synthesis of various compounds, whereas synthesis of enzymatic compounds can not only solve the above problems, but also by using two or more enzymes. Compounds with enhanced function can be synthesized. Carbohydrate-related compounds using 2-dioxyribose 5-phosphate aldolase enzyme and fructose-1,6-diphosphate aldolase derived from rabbit muscle compounds, which can be useful for chiral building blocks or carbohydrate mimetics. In particular, sialic acid, a glycoprotein, is known to inhibit hemagglutination by influenza virus. ) when used with a sialic acid (for synthesizing derivatives of sialic acid) to obtain a synergistic effect (Takayama et al Ann Rev Microbiol 51 :.... 285, 1997).

Recently, antiviral nucleosides, epothilone A, an anticancer agent, and statins, which are used for treating hyperlipidemia, are used to treat 2-dioxyribose 5-phosphate aldolase. It has been reported that each intermediate can be prepared in the course of synthesis (Greenberg et al ., PNAS , 101, 5788-5793, 2004; Sukumaran and Hanefeld, Chem. Soc. Rev. , 34, 530-542, 2005; Horinouchi et al ., Appl. Microbiol. Biotechnol ., 71, 615-621, 2006). In particular, therapeutic agents for hyperlipidemia, such as atorvastatin and rosuvastatin, have a high market share worldwide, and demand is expected to increase gradually. Therefore, novel 2-dioxyribose 5-phosphate aldolase may be important for the production of lactone, an intermediate of statin-based therapeutics. In addition, it is believed that the novel 2-deoxyribose 5-phosphate aldolase may be important for synthesizing bioactive substances.

Recently, in the synthesis of many physiologically active substances, in order to overcome many problems such as economics, stability, and environmental problems existing in the conventional chemical synthesis method, the direction of using an enzymatic synthesis method has been sought.

Accordingly, the present inventors deem that it is necessary to develop 2-deoxyribose 5-phosphate aldolase with improved efficiency in synthesizing the various physiologically active substances exemplified, and 2-dioxyribose 5-phosphate aldolase having excellent activity. Efforts have been made to develop the Yersinia sp. 2-dioxiribose 5-phosphate aldolase having excellent 2-dioxyribose 5-phosphate degrading activity and 2-dioxyribose 5-phosphate synthesis activity was isolated from EA015 strain, and the amino acid sequence of the enzyme was analyzed to identify a novel enzyme. It confirmed and completed this invention.

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel 2-deoxyribose 5-phosphate aldolase having excellent 2-deoxyribose 5-phosphate degrading activity and 2-deoxyribose 5-phosphate synthesizing activity useful for enzymatic synthesis of physiologically active substances. have.

Another object of the present invention is to provide the 2-deoxyribose 5-phosphate aldolase gene, a recombinant vector containing the gene and a recombinant microorganism transformed with the gene.

In order to achieve the above object, the present invention provides a 2-deoxyribose 5-phosphate aldolase having an amino acid sequence of SEQ ID NO: 1.

The present invention also provides a 2-deoxyribose 5-phosphate aldolase gene encoding the amino acid sequence of SEQ ID NO: 1.

The present invention also provides a recombinant vector containing the 2-deoxyribose 5-phosphate aldolase gene.

The present invention also provides a recombinant microorganism in which a 2-deoxyribose 5-phosphate aldolase gene is inserted on a chromosome of a host cell or the recombinant vector is introduced into a host cell.

The present invention also provides a method for producing 2-dioxiribose 5-phosphate aldolase, wherein the recombinant microorganism is cultured.

The present invention also provides a method for preparing a physiologically active substance using D-diglyceraldehyde-3-phosphate as a starting material or an intermediate, wherein the 2-dioxyribose 5-phosphate aldolase is used for the intermediate product. A method is characterized by condensing two carbon units.

According to the present invention, two carbon units can be added to a natural product by enzymatic method, and thus, the importance of white biotechnology can be replaced by enzymatic method by various bioactive substances that have been manufactured by chemical methods. .

In one aspect, the invention relates to 2-dioxyribose 5-phosphate aldolase having the amino acid sequence of SEQ ID NO: 1.

The 2-dioxyribose 5-phosphate aldolase is an enzyme that can be used to synthesize various bioactive substances by adding two carbon units to various natural compounds. The enzyme may be utilized to improve the problems of economy and stability that may occur when the compound is synthesized by a chemical process. In particular, as the importance of environmentally friendly processes is emphasized, they can be used to reduce chemical processes.

In the present invention, the 2-deoxyribose 5-phosphate aldolase is Yersinia sp. It may be characterized by derived from EA015 strain.

Yersinia sp. EA015 strain was isolated from soil, and the separation process is as follows.

First, strains having the ability to decompose 2-dioxyribose were preferentially selected, and then 2-dioxyribose 5 was examined by investigating the activity in the reaction to decompose or decompose 2-dioxyribose 5-phosphate or in the reaction to synthesize. -Strains with phosphate aldolase activity were selected secondarily.

16S rRNA sequences of the selected strains were determined and the results were retrieved from the GenBank database, Yersinia sp. (isolate YEM11A), Yersinia massiliensis strain CCUG 53443 and 99%, strain CCUG 26329 Yersinia bercovieri than determine that it has more than 98% of high homology, and Yersinia sp . Named EA015.

In another aspect, the present invention relates to a 2-deoxyribose 5-phosphate aldolase gene encoding the amino acid sequence of SEQ ID NO: 1.

In the present invention, in order to obtain the gene, by comparing the amino acid sequence of the highly conserved portion from the 2-deoxyribose 5-phosphate aldolase amino acid sequence of various strains, to produce a degenerate primer (degenerate primer), Some nucleotide sequences of the gene encoding the enzyme were amplified. In addition, in order to determine the remaining total nucleotide sequence of the 2-deoxyribose 5-phosphate aldolase gene other than the nucleotide sequence, an inverse PCR primer was prepared from the nucleotide sequences identified above, followed by PCR.

As a result, it was confirmed that the nucleotide sequence of the 2-dioxyribose 5-phosphate aldolase gene was composed of 672 bases, and the amino acid sequence was composed of 223 (24.8 kDa).

As a result of BlastP search of the amino acid sequence, Yersinia intermedia ATCC 29909 (ZP_00832327), Yersinia enterocolitica subsp. enterocolitica 8081 (YP_001005782) and Yersinia frederiksenii ATCC 33641 (ZP_00830328) was identified as novel 2-deoxyribose 5-phosphate aldolase with 94%, 93%, 91% homology, respectively.

In another aspect, the present invention relates to a recombinant vector containing the 2-deoxyribose 5-phosphate aldolase gene and a microorganism transformed with the recombinant vector.

The present invention reveals the gene sequence of the enzyme, amplifies the gene by constructing a primer from the sequence, recombination into the Eco RI- Bam HI site of the expression vector pET302 / NT-his and the recombinant vector is introduced Transformed microorganisms were produced.

Recombinant plasmids were introduced into Escherichia coli BL21 (DE3) for overproduction of 2-deoxyribose 5-phosphate aldolase, and the resulting transformants were shaken in LB liquid medium to which 100 μg / ml ampicillin was added. Culture was induced to overexpress the enzyme. The cells obtained in the culture were crushed by ultrasonication, and the supernatant was collected by centrifugation, and the chromatography was performed by passing through a Ni-NTA agarose carrier. The enzyme activity fraction was collected and purified by SDS-PAGE (FIG. 1).

In the present invention, the optimum reaction conditions were investigated to investigate the biochemical properties of the obtained enzyme. The reaction temperature showed high activity at 40 ℃ ~ 60 ℃, the optimum reaction temperature was confirmed that the maximum relative activity at 50 ℃ (A of Figure 2). Optimum pH showed relatively maximum activity at pH 6 and relatively high activity at neutral and alkaline pH (FIG. 2B). And after checking the remaining activity after treatment for 1 hour at each temperature to determine the stability to heat it was found that the enzyme activity is maintained stable at 60 ℃ or less (Fig. 2 C).

In another aspect, the present invention is a method for producing a bioactive material using D-diglyceraldehyde-3-phosphate as a starting material or intermediate, using the 2-dioxyribose 5-phosphate aldolase A method is characterized by condensing two carbon units on an intermediate.

In the present invention, the 2-dioxyribose 5-phosphate aldolase may be characterized by condensing acetaldehyde with D-diglyceraldehyde-3-phosphate.

In the present invention, the bioactive material may be selected from the group consisting of antiviral nucleosides, epothilone A, and statin-based hyperlipidemia therapeutics.

In the present invention, "two carbon unit" means a functional group having two carbons, such as acetaldehyde derivatives such as acetaldehyde, 2-hydroxyacetaldeyhde, chloroacetaldehyde and the like.

In the present invention, the statin-based hyperlipidemia treatment agent may be atorvastatin, rosuvastatin.

In one embodiment of the present invention, in the method for preparing atorvastatin, the 2-dioxyribose 5-phosphate aldolase is a donor substrate of acetaldehyde and an acceptor substrate of aldehyde (residue: H, Cl , OMe, N ₃ ) to synthesize (3R, 5S) -6-chloro-2,4,6-trideoxyhexapyranoside (lactone) to synthesize various statins. Subsequently, (4R, 6R)-[6-Cyanomethyl-2,2-dimethyl (1,3) dioxan-4-yl] acetic acid methyl ester is synthesized through chemical synthesis and finally atorvastatin is synthesized. do.

In another embodiment of the present invention, in the method for preparing rosuvastatin, the 2-dioxyribose 5-phosphate aldolase is an acetaldehyde which is a donor substrate and an aldehyde which is an acceptor substrate (residue: H, Cl, OMe , Lactone is synthesized to synthesize various statins through aldol condensation with N ₃ ). Thereafter, (3R, 5S) -3,5,5-Trihydroxyhexanoic acid is synthesized through chemical synthesis, and finally rosuvastatin is synthesized.

In the present invention, "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. The vector may be a plasmid, phage particles, or simply a potential genomic insert. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors. Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.

After ligation, the vector should be transformed into the appropriate host cell. In the present invention, preferred host cells are prokaryotic cells. Suitable prokaryotic host cells include E. coli DH5α, E. col JM101, E. coli K12, E. coli W3110, E. coli X1776, E. coli XL-1Blue (Stratagene), E. coli BL21, and the like. However, E. coli strains such as FMB101, NM522, NM538 and NM539 and other prokaryotic species and genera may also be used. In addition to E. coli , strains of the genus Agrobacterium, such as Agrobacterium A4, bacilli , such as Bacillus subtilis , Salmonella typhimurium or Serratia marcescens ) is another variety of enteric bacteria and Pseudomonas (Pseudomonas) in the strain, such as may be used as host cells.

Prokaryotic transformation can be readily accomplished using the calcium chloride method described in section 1.82 of Sambrook et al., Supra. Alternatively, electroporation (Neumann et al., EMBO J., 1: 841, 1982) can also be used for transformation of these cells.

In the present invention, a method of inserting a 2-dioxyribose 5-phosphate aldolase gene on a chromosome of a host cell may be a commonly known genetic manipulation method. For example, a retroviral vector, an adenovirus vector, or adeno- And a method using an associated viral vector, a herpes simplex virus vector, a poxvirus vector, a lentiviral vector or a nonviral vector.

As the vector used for overexpression of the gene according to the present invention, an expression vector known in the art may be used, and it is preferable to use a pET family vector (Novagen). When the cloning is performed using the pET family vector, histidine groups are bound to the ends of the expressed protein, and thus the protein can be effectively purified. In order to separate the expressed protein from the cloned gene, a general method known in the art may be used, and specifically, it may be separated by a chromatographic method using Ni-NTA His-binding resin (Novagen). .

The expression "expression control sequence" in the present invention means a DNA sequence essential for the expression of a coding sequence operably linked in a particular host organism. Such regulatory sequences include promoters for performing transcription, any operator sequence for regulating such transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. For example, suitable control sequences for prokaryotes include promoters, optionally operator sequences, and ribosomal binding sites. Eukaryotic cells include promoters, polyadenylation signals, and enhancers. The factor that most influences the amount of gene expression in the plasmid is the promoter. As the promoter for high expression, an SRα promoter, a promoter derived from a cytomegalovirus, and the like are preferably used. To express the DNA sequences of the invention, any of a wide variety of expression control sequences can be used in the vector. Useful expression control sequences include, for example, early and late promoters of SV40 or adenovirus, lac system, trp system, TAC or TRC system, T3 and T7 promoters, major operator and promoter regions of phage lambda, fd code protein Regulatory region of, promoter for 3-phosphoglycerate kinase or other glycolysis enzymes, promoters of the phosphatase such as Pho5, promoter of yeast alpha-crossing system and gene expression of prokaryotic or eukaryotic cells or viruses Other sequences known to modulate and various combinations thereof. The T7 promoter can be usefully used to express the proteins of the invention in E. coli.

Nucleic acids are "operably linked" when placed in a functional relationship with other nucleic acid sequences. This may be genes and regulatory sequence (s) linked in such a way as to allow gene expression when appropriate molecules (eg, transcriptional activating proteins) bind to regulatory sequence (s). For example, the DNA for a pre-sequence or secretion leader is operably linked to the DNA for the polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, "operably linked" means that the linked DNA sequence is in contact, and in the case of a secretory leader, is in contact and present within the reading frame. However, enhancers do not need to touch. Linking of these sequences is performed by ligation (linking) at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.

As used herein, the term “expression vector” generally refers to a fragment of DNA that is generally double stranded as a recombinant carrier into which fragments of heterologous DNA have been inserted. Here, heterologous DNA refers to heterologous DNA, which is DNA not naturally found in host cells. Once expression vectors are within a host cell, they can replicate independently of the host chromosomal DNA and several copies of the vector and their inserted (heterologous) DNA can be produced.

As is well known in the art, to raise the expression level of a transfected gene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host. Preferably, the expression control sequence and the gene of interest are included in one expression vector including the bacterial selection marker and the replication origin. If the host cell is a eukaryotic cell, the expression vector must further comprise an expression marker useful in the eukaryotic expression host.

Host cells transformed or transfected with the expression vectors described above constitute another aspect of the present invention. As used herein, the term “transformation” means introducing DNA into a host so that the DNA is replicable as an extrachromosomal factor or by chromosomal integration. As used herein, the term "transfection" means that the expression vector is accepted by the host cell whether or not any coding sequence is actually expressed.

Of course, it should be understood that not all vectors and expression control sequences function equally in expressing the DNA sequences of the present invention. Likewise not all hosts function equally for the same expression system. However, those skilled in the art can make appropriate choices among various vectors, expression control sequences and hosts without departing from the scope of the present invention without undue experimental burden. For example, in selecting a vector, the host must be considered, since the vector must be replicated in it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered. In selecting expression control sequences, several factors must be considered. For example, the relative strength of the sequence, its controllability, and its compatibility with the DNA sequences of the present invention should be considered, particularly with regard to possible secondary structures. Single cell hosts may be selected from a host for the selected vector, the toxicity of the product encoded by the DNA sequence of the invention, the secretory properties, the ability to accurately fold the protein, the culture and fermentation requirements, the product encoded by the DNA sequence of the invention from the host. It should be selected in consideration of factors such as the ease of purification. Within the scope of these variables, one skilled in the art can select a variety of vector / expression control sequence / host combinations capable of expressing the DNA sequences of the invention in fermentation or large scale animal culture. As a screening method when cloning the cDNA of a protein according to the present invention by expression cloning, a binding method (binding method), a panning method, a film emulsion method and the like can be applied.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1 Isolation of a Strain Having 2-Deoxyribose 5-Phosphate Aldolase Activity

In order to isolate strains having 2-deoxyribose 5-phosphate aldolase activity, soil samples were collected from Naejangsan and Gangwon-do. 1 g of the collected soil sample is suspended in 9 ml of saline solution and diluted 10 ^-4 to 10 ^-6 times to 100 μl of solid medium (Max1000 15g / L, Bacto peptone 5g / L, yeast extracts 1g / L, K ² HPO ₄ 1 g / L, NaH ₂ PO ₄ .2H ₂ O 0.6 g / L, 0.5 g / L MgSO ₄ · 7H ₂ O, agar 20 g / L) and incubated at 30 ° C. for 2-3 days.

In order to select strains having the activity of 2-deoxyribose 5-phosphate aldolase enzyme, liquid medium (Max1000 15g / L, Bacto peptone 5g / L, yeast extracts 1g / L, K ₂ HPO ₄ 1g / L, NaH ₂ PO ₄ · 2H ₂ O 0.6g / L, 0.5g / L MgSO ₄ · 7H ₂ O) and strain-cultured at 200 rpm for 3 days at 30 ° C for the first time to strain 2-deoxyribose Were selected.

Specifically, 100 μl of 0.1MK ₂ HPO ₄ (pH 7.0) buffer containing 50 mM _2- dioxyribose was added to the cells obtained by centrifugation, and reacted in a shaker at 30 ° C. for 24 hours. The decomposing capacity of 2-deoxyribose was confirmed by recovering the thin layer chromatography.

Thin layer chromatography was carried out as follows. 2-Deoxyribose was used as standard reagent and the supernatant was spotted onto a plate for chromatography (TLC aluminum sheets silicagel 60 F254, Merck co.). This was developed once in a reagent in which n-butanol, acetic acid and distilled water were mixed at a ratio of 3: 1: 1 (v / v / v). Thereafter, 0.5 ml of p-anisaldehyde was dissolved in 50 ml of acetic acid, sprayed with a coloring reagent containing 1 ml of sulfuric acid, and heated. The product was confirmed by Rf value and color development (US 2004/0038351).

After the primary strain was shaken for 3 days in a liquid medium, the culture medium was a DR medium containing 0.5% (w / v) 2-deoxyribose (0.5% 2-deoxyribose) , 0.2% ammonium chloride, 0.1% KH ₂ PO ₄ , 0.1% K ₂ HPO ₄ , 0.03% MgSO ₄ ˙8H ₂ O, and 0.01% yeast extract, pH7.0) at 120 rpm for 3 days at 30 ° C. Strains with 2-deoxyribose 5-phosphate aldolase activity were selected secondaryly after shaking culture. In order to secondaryly select strains having 2-deoxyribose 5-phosphate aldolase activity, deoxyribose 5-phosphate (DR5P) was decomposed and synthesized.

Specifically, for the production of deoxyribose 5-phosphate, the reaction solution was 200 µl, 100 mM fructose 1,6-diphosphate (Fructose 1,6-diphosphate, FDP), 200 mM acetaldehyde, 200 mM KH ₂ PO ₄ (pH 7.0), 15 mM MgSO ₄ 7H ₂ O, enzyme extract was reacted in a shaker at 125 ℃ for 3 hours at 30 ℃. Thereafter, the produced deoxyribose 5-phosphate was confirmed by the Cysteine-sulfate method (Stumpf, PK, JBC , 169: 367, 1947).

The deoxyribose 5-phosphate digestion reaction was performed with 100 mM Tris-Cl (pH8.8), 1 mM DR5P (deoxyribose 5-phosphate), and 10 µl of an appropriately diluted enzyme solution. The reaction was carried out and ice stopped. Thereafter, 0.3 mM NADH, 10 U alcohol dehydrogenase (ADH) was added thereto, and enzyme activity was measured by confirming a decrease in NADH at 340 nm through an absorbance meter (Horinouchi et. al ., Appl . Env . Microbiol ., 69: 3791, 2003).

Strains selected by the above method were requested to determine 16S rRNA sequences, and the results were retrieved from the GenBank database. As a result, Yersinia sp . (isolate YEM11A), Yersinia massiliensis strain CCUG 53443 contains 99%, Yersinia bercovieri strain CCUG 26329 Was confirmed to have a high homology of more than 98%, this strain was Yersinia sp. Named EA015.

Example 2: Yersinia sp. Isolation of a New 2-deoxyribose 5-phosphate Aldolase Gene from the EA015 Strain

Yersinia sp. The EA015 strain was shaken in a liquid medium used in Example 1, and chromosomal DNA was isolated from the recovered cells. Encoding 2-deoxyribose 5-phosphate aldolase Some sequences of the gene ( deoC ) were amplified using degenerate PCR.

Oligonucleotide primers were selected by comparing the amino acid sequences of 2-deoxyribose 5-phosphate aldolase of various strains, and amplified by PCR using the following primers.

SEQ ID NO: 5'-GTIATHGGITTYCCIYTIGGIGC-3 '(forward)

SEQ ID NO: 5'-RAANCCNGTNWSNGTYTTNACRAA-3 '(reverse)

Where R = A + G, Y = C + T, S = G + C, H = A + T + C, N = A + G + C + T

The reaction solution for PCR was composed of 100ng chromosomal DNA, 5uM primer, 200nM dNTP, 2U Taq polymerase, 1x Taq DNA polymerase (including MgCl ₂ ) buffer in 20µl and used for PCR amplification. The PCR reaction was denatured at 95 ° C. for 3 minutes, followed by 30 seconds of denaturation at 95 ° C., 1 minute annealing at 48 ° C., and 1 minute extension at 72 ° C. for 30 times. Finally a 10 minute extension was performed at 72 ° C. and maintained at 4 ° C. The obtained amplified fragment was cloned into pGEM-Teasy plasmid to request sequencing, and it was confirmed that it was a partial sequence of 2-deoxyribose 5-phosphate aldolase gene.

In order to determine the overall sequence of the 2-deoxyribose 5-phosphate aldolase gene, an inverse PCR method was used. Primers were prepared from the 2-deoxyribose 5-phosphate aldolase gene sequence identified from the PCR.

SEQ ID NO: 5'-ACCGTTGAAGGTAATATTAGAA-3 (forward)

SEQ ID NO: 5'-TGATAACCATATCAATTTCTTG-3 '(reverse)

For PCR amplification, chromosomal DNA was treated with various restriction enzymes, and 500 ng of restriction enzyme-treated chromosomal DNA was used for self-ligation. The reaction solution for PCR was composed to 50ng in 25ng chromosome DNA, 0.5uM primer, 200nM dNTP, 2U EF Taq polymerase, 1x Taq DNA polymerase (including MgCl2) buffer was used for PCR amplification. The PCR reaction was denatured at 94 ° C. for 3 minutes, followed by 30 seconds of denaturation at 94 ° C., annealing at 50 ° C. for 30 seconds, and 2 minutes extension at 72 ° C. for 35 times. Finally a 10 minute extension was performed at 72 ° C. and maintained at 4 ° C.

The obtained amplified fragment was cloned into pGEM-Teasy plasmid to request nucleotide sequence determination, and the entire sequence of 2-deoxyribose 5-phosphate aldolase gene was confirmed. The amino acid sequence translated from the 2-deoxyribose 5-phosphate aldolase gene sequence is shown in SEQ ID NO: 1.

Example 3: Expression and Purification of 2-Deoxyribose 5-phosphate Aldolase Produced in Recombinant Escherichia Coli

Yersinia sp. Eco RI- of pET302 / NT-his, an expression vector of 2-deoxyribose 5-phosphate aldolase gene ( deoC ), for overexpressing 2-deoxyribose 5-phosphate aldolase from EA015 strain in E. coli (DH5α) The recombinant plasmid pET302EA001D was prepared by insertion into the Bam HI site.

2-deoxyribose 5-phosphate aldolase gene was prepared primers from the gene sequence identified above, Yersinia sp. Obtained using chromosomal DNA of EA015 strain.

SEQ ID NO: 5'-GAGAATTCGATGACCATCAATTACGCGAATT-3 (forward)

SEQ ID NO: 5'-TGGGATCCTTAGTAGCTTGATGCGGCC-3 '(reverse)

PCR amplification solution for amplifying the gene is composed of 100ng chromosomal DNA, 0.2uM primer, 200nM dNTP, 2U Taq polymerase, 1x Taq DNA polymerase (including MgCl ₂ ) buffer in 20µl and used for PCR amplification. It was. The PCR reaction was denatured at 95 ° C. for 3 minutes, followed by 30 seconds of denaturation at 95 ° C., 45 seconds of annealing at 48 ° C., and 1 minute extension at 72 ° C. for 25 times. Finally a 10 minute extension was performed at 72 ° C. and maintained at 4 ° C.

Recombinant plasmids were introduced into Escherichia coli BL21 (DE3) for overproduction of 2-deoxyribose 5-phosphate aldolase, and the resulting transformants were transferred to LB liquid medium containing 100 μg / ml ampicillin. Shake culture at 200 rpm for 16 hours at ℃. Thereafter, when the absorbance value was 0.5 at 600 nm, IPTG was added to the final concentration of 0.2 mM to induce overexpression of the enzyme. The culture was centrifuged to recover the precipitated cells and washed twice with 0.85% NaCl. 100 mM Tris-Cl (pH8.0) added with 1 mM PMSF, protease inhibitor cocktail (Roche) and 10 mM imidazoe was added to the recovered cells and disrupted by ultrasonication. The supernatant was recovered by centrifugation and used to purify the enzyme. The supernatant was chromatographed by passing through a Ni-NTA agarose carrier. When the enzyme was eluted, the enzyme activity fraction was taken with an eluate containing 250 mM imidazole, 300 mM NaCl, and 20 mM Tris-Cl (pH 8.0) to confirm purification by SDS-PAGE (FIG. 1).

The purified enzyme was concentrated in Amicon Ultra-15 (Millipore, 10K NMWL device) and then placed in 100mM Tris-Cl (pH8.0) solution containing 20% glycerol for dialysis at 4 ° C for 12 hours. Was performed.

Pure purified 2-dioxyribose 5-phosphate aldolase activity was determined by adding 100 mM Tris-Cl (pH8.8), 1 mM DR5P (2-deoxyribose 5-phosphate), 0.3 mM NADH, and 10 μl of a properly diluted enzyme solution. The total amount was 100 μl, the reaction was carried out at 30 ° C. for 10 minutes, and ice stopped. After storing the reaction solution at 25 ° C. for 3 minutes, 10U alcohol dehydrogenase (ADH) was added thereto, and enzyme activity was measured by confirming a decrease in NADH at 340 nm through an absorbance meter (Table 1). The unit (U) of the enzyme represents the umole number of acetaldhyde produced per minute by the decomposition of 2-deoxyribose 5-phosphate (DR5P).

Yersinia sp. EA015 Activity and Recovery from Purification of 2-Deoxyribose 5-phosphate Aldolase Total volume
(ml) Total protein
(mg) Total active
(U) Unit activity
(U / mg) Refined (pear) yield
(%) Culture supernatant 2 8.4 476.3 56.4 One 100 Ni-NTA Affinity Chromatography 0.8 1.4 203.3 137.4 2.4 43

Example  4: 2-deoxyribose 5- Phosphate Aldolase  Biochemical properties

The effects of reaction temperature and pH were investigated to determine the optimal reaction conditions for purified 2-dioxyribose 5-phosphate aldolase. Relative activity was measured at intervals of 10 ° C. in the range of 20-80 ° C. using the purified enzyme. As shown in FIG.

To determine the optimal pH of enzyme activity, 100 mM citrate buffer (pH 3 ~ 6), 100 mM sodium phosphate buffer (pH 6 ~ 7), Tris-Cl buffer (pH 7 ~ 10), glycine / NaOH buffer (pH 10 ~ 11) ) Was used. As a result of investigating the relative activity in each pH buffer, it showed maximum activity at pH 6 and was found to be relatively stable at neutral and alkaline pH (FIG. 2B).

In order to investigate the stability to heat, the residual activity was measured at 5 ° C intervals in the range of 35 to 65 ° C. As shown in C of FIG. 2, the residual activity was confirmed after 1 hour at each designated temperature, indicating that the residual activity remained high at 50 ° C. or lower.

Example 5 Identification of Kinetic Parameters of 2-Deoxyribose 5-Phosphate Aldolase

2-Deoxyribose 5-phosphate Aldolase 2-deoxyribose 5-phosphate was used as a substrate to determine the kinetic parameters, was determined using the Lineweaver-Burk plot method. The Km and Vmax values of 2-dioxyribose 5-phosphate aldolase were found to be 9.108 mM and 0.009 umole / min, respectively.

1 shows the results of the Coomassie staining after electrophoresis of 2-deoxyribose 5-phosphate aldolase purified from recombinant Escherichia coli, lane m is a molecular weight marker, lane 1 is a whole cell, lane 2 is whole Cell overexpression is shown, lane 3 is the cell extract and lane 4 is the result of Ni-NTA agarose chromatography.

2 is Yersinia sp. A graph showing the biochemical properties of 2-deoxyribose 5-phosphate aldolase isolated from EA015 strain. The optimum temperature of 2-deoxyribose 5-phosphate aldolase was confirmed at 10 to 80 ° C at 10 ° C intervals, B was to investigate the optimal pH (closed rectangles: pH3-6, open rectangles: pH6-7, closed triangles). : pH 7 ~ 10, open triangle: pH 10 ~ 11), C is to confirm the thermal stability of 2-deoxyribose 5-phosphate aldolase, and to confirm the remaining activity after treatment for 1 hour at each temperature.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Novel 2-Deoxyribose 5-Phosphate Aldolase Derived from Yersinia          sp. EA015 <130> P08-B176 <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 223 <212> PRT <213> Yersinia sp. <400> 1 Met Thr Ile Asn Tyr Ala Asn Tyr Ile Asp His Thr Leu Leu Ala Met   1 5 10 15 Asp Ala Thr Glu Ala Gln Ile Ile Lys Leu Cys Glu Glu Ala Lys Gln              20 25 30 His His Phe Tyr Ala Val Cys Val Asn Ser Gly Tyr Val Pro Leu Ala          35 40 45 Ala Gln Gln Leu Ala Gly Ser Ser Val Lys Val Cys Ser Val Ile Gly      50 55 60 Phe Pro Leu Gly Ala Gly Leu Thr Glu Ala Lys Ala Phe Glu Ala Gln  65 70 75 80 Ala Ala Ile Asn Ala Gly Ala Gln Glu Ile Asp Met Val Ile Asn Val                  85 90 95 Gly Trp Leu Lys Ser Gly Lys Val Ala Glu Val Lys Ala Asp Ile Gln             100 105 110 Ala Val Arg Asp Asn Cys Ala Ser Thr Pro Leu Lys Val Ile Leu Glu         115 120 125 Thr Cys Leu Leu Ser Asp Ala Gln Ile Val Gln Val Cys Glu Met Cys     130 135 140 Arg Glu Leu Asp Val Ala Phe Val Lys Thr Ser Thr Gly Phe Ser Thr 145 150 155 160 Gly Gly Ala Lys Glu Glu His Val Lys Leu Met Arg Ser Thr Val Gly                 165 170 175 Pro Ala Met Gly Val Lys Ala Ser Gly Ala Val Arg Asp Gln Ala Thr             180 185 190 Ala Glu Lys Met Ile Leu Ala Gly Ala Thr Arg Ile Gly Thr Ser Ser         195 200 205 Gly Val Thr Ile Val Ser Gly Gln Gln Ala Ala Ala Ser Ser Tyr     210 215 220 <210> 2 <211> 672 <212> DNA <213> Yersinia sp. <400> 2 atgaccatca attacgcgaa ttatatcgac cataccttac tggcaatgga tgcgaccgaa 60 gcacaaatta tcaaactgtg tgaagaggct aaacagcatc atttctatgc agtatgcgtg 120 aactcaggtt atgttccttt ggctgcacaa cagctagcgg gtagttcagt gaaagtatgt 180 tcagtcattg gttttccgtt gggtgcaggc ctaacagaag caaaagcgtt tgaagctcaa 240 gctgccatca acgctggcgc gcaagaaatt gatatggtta tcaatgttgg ctggctaaaa 300 agcggcaaag tggctgaagt taaagctgac attcaggccg tgcgtgataa ttgcgcctct 360 acaccgttga aggtaatatt agaaacttgc ctactcagtg atgcacaaat cgtccaagtt 420 tgtgagatgt gccgtgagtt ggacgtcgct tttgtgaaaa cttcaacagg tttcagcacc 480 ggcggcgcaa aagaagaaca tgtaaaattg atgcgctcca ccgttggccc agccatgggt 540 gtcaaagcat ccggcgcagt tcgtgaccaa gccactgcgg agaaaatgat tctcgcaggc 600 gccactcgga ttggcactag ctctggggtc actatcgttt cgggccaaca agcggccgca 660 tcaagctact aa 672 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gtnathggnt tyccnytngg ngc 23 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 raanccngtn wsngtyttna craa 24 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gtggatatgg ttattaacat tg 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ccttcgtttc aaatgctttc gt 22 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gagaattcga tgaacatcgc agcattaat 29 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tgggatcctt agtaggaaga tgtagactgc 30  

Claims (10)

2-Deoxyribose 5-phosphate aldolase having the amino acid sequence of SEQ ID NO: 1 and having thermal stability even at 50 ° C. The method of claim 1, wherein Yersinia sp . 2-Deoxyribose 5-phosphate aldolase, which is derived from EA015 strain. 2-Deoxyribose 5-phosphate aldolase gene encoding the amino acid sequence of SEQ ID NO: 1. The gene according to claim 3, which has a nucleotide sequence of SEQ ID NO: 2. Recombinant vector containing the gene of claim 3. A recombinant microorganism in which the gene of claim 3 is inserted on a chromosome of a host cell, or the recombinant vector of claim 5 is introduced into a host cell. A method for producing 2-deoxyribose 5-phosphate aldolase, comprising culturing the recombinant microorganism of claim 6. delete delete delete

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