KR100313667B1 - THERMOACTINOMYCES SP.) A novel high-potency control protein A of E79 and a DNA encoding the same - Google Patents

Abstract

The present invention relates to a novel catabolite control protein (CcpA) and a DNA encoding the same, and more particularly, to a novel catabolite control protein (CcpA) catabolite repression, and a DNA sequence coding therefor and a strong promoter therefor. The DNA encoding the metabolite regulatory protein A of the present invention is characterized in that the DNA encoding the catabolite repression And thus it is possible to produce a mutant which constitutively produces a protein hydrolyzing enzyme regardless of the sugar composition in the fermentation medium.

Description

Thermoactinomyces sp. E79, a novel catabolic regulatory protein A and DNA encoding it

The present invention relates to novel catabolite control protein A (CcpA) and DNA encoding the same, and more particularly, to a novel catabolite control protein A (CcpA) The present invention relates to a catabolite repression (Catabolite repression) phenomenon and a DNA sequence encoding the same.

In general, protein hydrolytic enzymes, xylanases, amylases, and cellulases are mostly inducible enzymes that are induced in the presence of a substrate. When the enzymes are industrially produced, there are various factors that limit their productivity. On the other hand, when the above-mentioned enzyme is produced, its production is regulated by various biosynthetic metabolic control mechanisms such as catabolite repression or end product feedback inhibition, (1995)). In addition, it is important to reduce the productivity of microorganisms .

Accordingly, various methods have been studied to overcome the above-mentioned problems, and a method of randomly producing mutants using a classical mutagenesis method such as a mutagenesis agent or ultraviolet ray treatment and selecting a high production strain has been used . In addition, methods such as optimization of fermentation conditions, cloning of genes, and construction of a high expression system using a strong promoter have been proposed as a solution to the above problems.

On the other hand, many kinds of industrial enzymes have been produced in Gram-positive bacteria including Bacillus. However, studies on regulation systems such as intracellular regulatory proteins and regulatory factors involved in enzyme production in these industrial strains have not yet been developed yet Because of this, more fundamental enzyme production control has not been achieved.

As is well known, E. coli has a regulatory system that regulates the transcription of a specific gene by a catabolite activator protein and a cAMP complex. On the other hand, in the case of gram-positive bacteria including bacillus, (Hueck, CJ, & H. Wolfgang, & Hughes, R., et al., ≪ RTI ID = 0.0 >Mol. Microbiol., 15, 395-401 (1995)). Among the above studies, there have been active studies on a total regulatory protein such as a cyanide metabolite regulatory protein A (CcpA) and a phosphotransferase system HPr, which are involved in the inhibition of divalent metabolites in Gram-positive bacteria, : ≪ / RTI > Bacillus (Christoph J. et al.Mol. Microbiol., 16, 855-864 (1995)), Staphylococcus(Monterero, V. et al., &Quot; Microbiol. &Quot;, 21, 739-749 (1996)J. Bacteriol., 179, 6657-6664 (1997)).

In the above prior arts, there is disclosed a base sequence and an amino acid sequence by separating an eicosyl metabolite regulatory protein and its gene involved in a biosynthesis regulatory system of various enzymes, and the relation between the eicosanoid regulatory protein and the enzyme biosynthesis control .

On the other hand, conventional studies have mainly focused on mesophilic strains, and the research stage is at a very early stage, and most studies have focused on studies on the regulation of the production of sugar hydrolase, Studies on high temperature bacteria producing alkaline protein hydrolytic enzymes are rarely conducted.

The inventors of the present invention have made extensive efforts to develop a strain capable of producing the above-described protein hydrolyzing enzyme at a high yield. As a result, it has been found that a thermoactinomycetes producing a useful industrial enzyme such as a heat resistant and an alkaline protease ( CcpA ) was isolated from Thermoactinomyces sp. E79, and the encoded metabolite regulatory protein A (CcpA) encoded thereby regulates the biosynthesis of the protein hydrolase Thereby completing the present invention.

Accordingly, an object of the present invention is to provide a DNA encoding a cyanide metabolite regulatory protein A (CcpA).

It is another object of the present invention to provide an eukaryotic metabolite control protein A (CcpA).

It is yet another object of the present invention to provide a method for producing the above-mentioned metabolite controlling protein A (CcpA).

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a process diagram showing a method for separating a gene encoding a novel metabolite regulating protein A of the present invention,

Figure 2 shows the cleavage map of the recombinant plasmid pTCA2 containing the gene encoding the novel cyanobacterium metabolite control protein A of the present invention,

Figure 3 shows the amino acid sequences of the novel metabolizing enzyme control A of the present invention and another known metabolizing enzyme controlling protein A,

FIG. 4 shows the cleavage map of the recombinant plasmid pTCAB3 containing the gene encoding the novel cyanobacterium metabolite control protein A of the present invention,

FIG. 5 is an SDS-PAGE photograph showing the purification scheme of the novel EBA metabolizable protein A of the present invention,

6 is a schematic diagram showing a method for producing a cavity-expressing plasmid for examining the interaction between the E79 proteinase gene and the eukaryotic metabolite regulatory protein A,

FIG. 7 is a photograph showing inhibition of the production of protein hydrolyzing enzyme by the eicosyl metabolite regulatory protein A gene.

The present invention is characterized by a DNA encoding a cyanide metabolite regulatory protein A (CcpA) having the nucleotide sequence as set forth in SEQ ID NO: 1 and a promoter thereof.

In addition, the present invention is characterized by a catabolic control protein A (CcpA) having the amino acid sequence of SEQ ID NO: 1.

On the other hand, the present invention includes a DNA encoding a cyanide metabolite regulatory protein A (CcpA) having the nucleotide sequence of SEQ ID NO: 1 and its promoter, and the recombinant plasmid pTCA2 having the cleavage map shown in FIG. 2 Other features.

The present invention relates to a recombinant plasmid pTCAB3 comprising the DNA coding for the cyanide metabolite regulatory protein A (CcpA) having the DNA base sequence set forth in SEQ ID NO: 1 and its promoter and having the cleavage map shown in FIG. 4 do.

The present invention is characterized by a method for producing the above-mentioned metabolite controlling protein A (CcpA).

Hereinafter, the present invention will be described in detail.

In order to confirm the nucleotide sequence of the DNA encoding the metabolite regulatory protein A of the present invention (hereinafter referred to as "CcpA"), firstly, Thermoactinomycetes genus E79 (Lee, JK et al., Biosci. Biotech. Biochem ., 60, 840 ~ 846 (1996); the chromosomal DNA is isolated from KCTC 1045BP).

Next, in order to isolate DNA encoding CcpA, primers corresponding to homologous regions are first prepared by comparing the amino acid sequence homologies of several known CcpAs, and PCR is performed using the primers. The ccpA gene is partially amplified through the PCR experiment, and the amplified DNA fragment is labeled with digoxigenin to isolate the entire ccpA gene.

Then, in order to isolate the entire ccpA gene derived from genus Thermoactinomyces sp. E79, the following procedure is carried out according to the attached strategy shown in Fig. 1: First, the chromosomal DNA of Thermolactinomyces sp. E79 is digested with restriction enzymes After partial cleavage, DNA fragments of 2 to 5 kb are recovered. Next, the DNA fragment is mixed with pUC19 digested with restriction enzymes, ligated to each other using DNA ligase, and introduced into E. coli XL1-Blue. Then, the transformed E. coli is transferred to a nitrocellulose membrane, DNA in the E. coli cells is transferred to a nitrocellulose membrane, the digoxigenin-labeled probe is added, and a colony hybridization reaction is carried out. Finally, the clone having the ccpA gene was isolated by selecting a clone showing a positive reaction for the colony hybridization reaction, and then the plasmid was separated from the transformant having the separated ccpA gene, which was named pTCA2 (see, 2). E. coli XL1-Blue transformed with pTCA2 was designated as E. coli XL1-Blue (pTCA2), which was deposited with the Gene Bank of Korea Institute of Science and Technology as of January 8, 1998 And deposited with the deposit number KCTC 8865P.

Subsequently, the nucleotide sequence of the DNA containing the cloned ccpA gene was determined, and the sequence thereof is shown in SEQ ID NO: 1.

In order to confirm transformation of Bacillus megaterium CcpA mutant by the ccpA gene isolated in the above procedure, pTCAB3 was prepared by inserting it into a Bacillus vector pHV33 (see FIG. 4), and the pHV33 vector Because it has a cloning origin that can be replicated in Bacillus and has a chloramphenicol resistance gene that can be used as a selection marker. Then, the recombinant plasmid pTCAB3 was introduced into a Bacillus megaterium CcpA mutant WH356, transformed, and the restoration of the metabolite-suppressing trait was confirmed.

Another aspect of the present invention is a method for producing a CcpA protein, Using the strong promoter of the ccpA gene and the thermal stability of the CcpA protein isolated from the above, (i) a step of culturing a strain containing the recombinant plasmid pTCAB3; (Ii) a step of pulverizing the cultured strain to obtain a cell extract; And (iii) heat treating the cell extract at 55-65 < 0 > C. The temperature in the heat treatment step during the purification process is preferably 55 to 65 ° C, and most preferably 60 ° C. If the temperature is lower than 55 캜 in the heat treatment step, excess protein of the host in which the recombinant plasmid of the present invention is introduced remains in excess, and if it exceeds 65 캜, there is a loss of CcpA protein.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1: Isolation of chromosomal DNA from Thermolactinomyces sp. E79

In the isolation of chromosomal DNA of the genus Thermolactinomyces sp. E79 (KCTC 1045BP), it was difficult to dissolve cells, and the conventional method was significantly modified to isolate as follows: First, the cells were cultured in a 1.5 L protein- The cells were cultured for 16 hours in a liquid medium (1.0% sorbitol, 2.0% soluble starch, 0.3% K ₂ HPO ₄ and 0.05% MgSO ₄ , pH 7.2), centrifuged at 250 rpm and the cell precipitates were suspended in TEN buffer Tris-HCl, 1 mM EDTA and 10 mM NaCl, pH 7.6). Then, the washed cells were frozen at -70 ° C. and then dissolved again. The cells were suspended in SET buffer (20% sucrose, 50 mM Tris-HCl and 50 mM EDTA, pH 7.6) supplemented with lysozyme And reacted at 37 DEG C for 1 hour. Next, 20 ml of a 25% SDS solution was added, 2 ml of protein hydrolyzing enzyme K (10 mg / ml) was added to the partially lysed solution, and the mixture was shaken at 60 ° C for 1 hour. Then, Chloroform mixed solution treatment was repeated three times and the mixture was gradually shaken for 2 hours in the presence of a phenol / chloroform mixture at a high temperature of 50 캜. Then, 5 M NaCl was added to the supernatant thus formed to a final concentration of 0.3 M, and 2-fold volume of ethanol was added. The precipitated DNA was wrapped with a glass rod, immersed in 95% ethanol and washed And dissolved in TE buffer solution (10 mM Tris-HCl and 1 mM EDTA, pH 7.8).

Example 2: Partial amplification of the ccpA gene derived from E79-derived thermolactinomycetes by PCR and preparation of probe using DIG labeling

Amplification of the C7pA gene derived from Thermolactinomyces sp. E79 was carried out using PCR as follows. First, an axon degradation primer (SEQ ID NO: 2) was constructed based on a consensus sequence known to have homology as a primer terminal primer: 5'-CTGAATTCATGGCNACNGT-3 ', C-terminal primer: 5'-CTGGATCCGCNCCNATRTCRTA-3'). Next, the chromosomal DNA of the thermotacticinase strain E79 isolated in Example 1 was modeled, PCR was performed using the synthesized primer, and a 0.8 kb DNA fragment was finally amplified. The PCR was performed using a Perkin Elmer 9600 model. The reaction was performed at 94 ° C for 5 minutes, followed by 30 cycles at 94 ° C for 1 minute, at 55 ° C for 1 minute, and at 72 ° C for 1 minute And DNA extension reaction was finally performed at 72 ° C for 10 minutes. Meanwhile, in order to prepare a probe for isolating the entire ccpA gene through colony hybridization, the amplified ccpA gene DNA was labeled with digoxigenin-labeled dUTP using a DIG labeling system (Boehringer Mannheim) .

Example 3: Cloning of whole gene of ccpA using colony hybridization

To isolate the entire ccp gene from Thermolactinomyces sp. E79, the following procedure was carried out according to the attached strategy shown in Fig. 1: First, the chromosomal DNA (100 쨉 g) of Thermolactinomyces sp. E79 was transferred into 5 units A DNA fragment of 2 to 5 kb was recovered using a sucrose gradient in a sample cut with Sau 3AI. This DNA fragment was mixed with pUC19 (Boehringer Mannheim) digested with 1 unit of Bam HI and 1 unit of T4 DNA The reaction was carried out at 16 ° C for 16 hours using a linking enzyme. Next, Escherichia coli XL1-Blue (Stratagene) was transformed by electrowinning with 2.5 V of electrowinning air (electroporator; BioRad Co., Ltd., Stratagene) by 20 연결 of the above-mentioned coupling reaction mixture and transformed with the nitrocellulose membrane 10 ml of denaturation buffer (0.5 M NaOH and 1.5 M NaCl), 10 ml of regeneration buffer (0.5 M Tris-HCl and 3 M NaCl, pH 7.3) and 10 ml of X20 SSC (pH 7.4) using a vacuum transfer (Hopper, TransVac TE80) (0.3 M sodium citrate and 3 M NaCl, pH 7.0), the DNA was transferred to a nitrocellulose membrane and immobilized using ultraviolet light. Then, 10 ml of hybridization buffer (5X SSC, 1% BSA, N-lauroylsacosin and 0.02% SDS) and 20 μl of the probe prepared in Example 2 were added to the membrane, and 16 Colony hybridization was performed for a period of time. Then, in order to select a clone showing a positive reaction to the colony hybridization reaction, an antibody conjugate (anti-digoxigenin) at a concentration of 150 μg / ml in a buffer (100 mM maleic acid and 150 mM NaCl, pH 7.5) Alkaline phosphatase conjugate) was added, followed by reaction with the membrane for 30 minutes. Finally, NBT (nitro blue tetrazolium salt) and 5-bromo-4-chloro-3-indole phosphate were added to select clones that developed color .

Example 4: Isolation of recombinant plasmid containing ccpA gene

5 ml of the transformant showing a positive result for the colony hybridization of Example 3 was cultured in liquid, and the plasmid was isolated using a Wizard Mini Prep Kit (Promega). Then, the isolated plasmid was digested with Bam HI, Sal I, Sma I, Xho I, Xmn I and Acc I restriction enzymes, and finally, 2.5 kb DNA was inserted into pUC19. A recombinant plasmid was identified and designated pTCA2 (see Figure 2). E. coli XL1-Blue transformed with pTCA2 was designated as E. coli XL1-Blue (pTCA2), which was deposited with the Gene Bank of Korea Institute of Science and Technology as of January 8, 1998 And deposited with the deposit number KCTC 8865P.

Example 5: Determination of nucleotide sequence and analysis of ccpA gene

In order to determine the nucleotide sequence of the cloned gene, a DNA fragment of about 2.5 kb was sequenced using an automatic nucleotide sequencing system (ABI 377 Prism DNA Sequencer, Perkin Elmer), and the result was as shown in SEQ ID NO: Respectively. As shown in SEQ ID NO: 1, a DNA fragment of about 2.5 kb contained a total of 1038 bp open reading frames coding for its own promoter and CcpA. As shown in SEQ ID NO: 1, the amino acid sequence of CcpA was deduced from the above-mentioned nucleotide sequence, and it was found that the protein was composed of 346 amino acids in total.

3, the amino acid sequence of the CcpA protein by the gene isolated from the above is compared with the known CcpA protein derived from other bacteria. As shown in FIG. 3, Ta represents Thermoactinomyces sp. E79, Bs represent Bacillus subtilis , Bm represents Bacillus megaterium , Sx represents Staphylococcus xylosus , and Lc represents Lactobacillus delbruckii , The dashed box represents the helix-turn-helix motif, * represents the case where the sequences of the five kinds of microorganisms are all identical to each other, and. As can be seen from Fig. 3, the CcpA of the genus Thermotactinomycetes E79 was found to be a novel CcpA exhibiting about 60% homology with CcpA of other microorganisms.

Example 6: Preparation of recombinant plasmid for transformation of Bacillus megaterium CcpA mutant

To introduce the ccpA gene into the Bacillus megaterium CcpA mutant WH356, 1.6 kb of the ccpA gene purified by digesting the plasmid pTCA2 with 1 unit of Bam HI and the Bacillus vector pHV33 (ATCC 39217) cut with the same restriction enzyme To prepare a recombinant plasmid, which was named pTCAB3 (see Fig. 4). To introduce the recombinant plasmid pTCAB3 into the Bacillus megaterium CcpA mutant WH356, protoplast transformation was used as follows: Bacillus megaterium WH356 was inoculated into 5 ml of LB medium and incubated overnight at 37 ° C with shaking Respectively. Then, when the absorbance at 550 nm was 0.5, the cells were centrifuged at 2,700 x g for 10 minutes. The precipitated cells were washed with 5 ml of SMM (2 × SMM (0.5 M sucrose, 0.02 M maleate and 0.02 M MgCl2, pH 6.5) and 4X PAB medium) and allowed to stand for 1 hour at 37 ° C. After confirming the degree of protoplast formation under a phase contrast microscope, The resulting cells were washed twice with the SMMP and dissolved in 2 ml of SMMP to be used as protoplast cells. Then, 1 ml of the protoplast cells was mixed with an equal volume of 2X SMM to be transformed After mixing with the DNA, 3 ml of 40% PEG 6000 was immediately added and left at room temperature for 2 minutes. Next, 10 ml of SMMP was added, followed by centrifugation at 2,700 x g for 15 minutes to remove the cells in 1 ml of SMMP Suspended and cultured at 37 DEG C for 90 minutes with shaking (100 ml 1M sodium succinate, 70 ml 10% yeast extract, 5% kanamycinose, 0.8% agar, 20 ml 3.5% K2HPO4 / 1.5% KH2PO4, 5 ml 20% glucose, 10 ml / 4 ml of 1 M MgCl 2 and 1 ml of 2% BSA, pH 7.3). After transforming, transformants having the ccpA gene introduced into the transformed cells in the regeneration medium were selected using a M9 minimal medium (75 g (0.5%) was added to a plate medium supplemented with xylose (0.5%) in a buffer solution (0.5 g / l Na2HPO4, 30 g / l KH2PO4, 5 g / l NaCl, 10 g / l NH4Cl, 0.21 g / , And xylose (0.5%), and cultured for 16 hours at 37 ° C. At this time, the mutant strain in which the ccpA gene was not inserted and the ccpA gene was mutated was a strain having a mutation in the ccpA gene There is no catabolite repression due to glucose, Blue colonies were formed in the medium. On the other hand, in the Bacillus megaterium CcpA mutant in which pTCAB3 was introduced, the inhibition of cyanobacterial metabolism was restored by the introduced ccpA gene, whereas in the medium supplemented with xylose only blue colonies were observed. On the medium supplemented with xylose and glucose, Colony. Thus, by confirming the formation of white colonies, Bacillus megaterium transformants selected by pTCAB3 were selected.

Example 7: Restoration of the eicin metabolite inhibitory properties of the Bacillus megaterium CcpA mutant by the ccpA gene

The reproductive phenotype of the Bacillus megaterium CcpA mutant WH356 by the ccpA gene isolated using β-galactosidase as a reporter enzyme was measured as follows: First, 0.5% of xylose and glucose The plasmid pHV33 was introduced into wild-type Bacillus megaterium WH331, Bacillus megaterium CcpA mutant WH356 and Bacillus megaterium CcpA mutant WH356 in the M9 minimal medium of Example 6 and the M9 minimal medium supplemented with xylose alone, respectively Plasmids transformed with the plasmid pTCAB3 were transfected for 5 hours in the transformant and Bacillus megaterium CcpA mutant WH356, and then 50 쨉 l of cells were added with the same amount of chloroform and shaken vigorously for 10 seconds. Then, 0.2 ml of ONPG (2-nitrophenyl-β-D-galactopyranoside, 4 mg / ml) was added as a substrate to the mixture and reacted for 10 minutes. Then, an enzyme activity (unit) was calculated by measuring an OD ₄₂₀ value , And the results are shown in Table 1 below. In Table 1 below, the inhibition ratio is the ratio of the activity of the unredunded? -Galactosidase to that of the inhibited? -Galactosidase.

Bacillus megaterium mutant The introduced plasmid Culture conditions ? -Galactosidase activity (unit) Inhibition ratio WH331 Xylose 742.2 ± 5.5 18.1 Xylose + glucose 41.0 ± 2.5 WH356 Xylose 735.4 + - 7.3 1.1 Xylose + glucose 660.5 ± 5.2 WH356 pHV33 Xylose 517.4 ± 8.7 1.5 Xylose + glucose 337.6 ± 9.8 WH356 pTCAB3 Xylose 437.7 + - 6.8 10.1 Xylose + glucose 43.4 ± 3.1

As shown in Table 1, in the case of Bacillus megaterium CcpA mutant WH356, the activity of β-galactosidase was not inhibited even in the presence of glucose in the M9 minimal medium due to the mutation of CcpA, but the isolated ccpA gene In the case of WH356, the activity of beta -galactosidase was remarkably inhibited as shown in wild-type Bacillus megaterium WH331, indicating that the growth inhibitory property of glucose metabolized by WH356 was recovered there was.

Example 8: Partial purification of CcpA protein

First, E. coli transformants containing 5 ml of the recombinant plasmid pTCAB3 cultured overnight at 37 ° C were centrifuged, suspended in 0.5 ml of buffer (50 mM Tri-HCl, pH 8.0), and the cells were cultured using a sonicator The cell extract was obtained by destroying. Next, 0.1 ml of the cell extract was heat-treated at a high temperature and subjected to SDS-PAGE to confirm purification. The results are shown in FIG. 5 attached hereto. In FIG. 5, lane 1 indicates the molecular weight marker, 2 lane indicates the cell extract without heat treatment (control group), 3 lane indicates heat treatment at 55 캜 for 30 minutes, 4 lane indicates heat treatment at 60 캜 for 15 minutes, Is a heat treatment at 60 占 폚 for 30 minutes.

As can be seen in FIG. 5, the bands representing the CcpA protein in the two lanes loaded with the untreated cell extract were very strong, indicating that CcpA was highly expressed in E. coli by a strong promoter located above the ccpA structural gene , And the promoter is expected to have applicability to high expression of other useful proteins.

As can be seen from FIG. 5, CcpA of the present invention has relatively stable thermostability, so that unlike the protein derived from Escherichia coli used as a host, most of the CcpA remains stable even after heat treatment at 60 ° C for 30 minutes, and partial purification using heat treatment is possible .

Example 9 Coexpression of E79 Protease and CcpA

To investigate whether CcpA of the present invention is involved in the regulation of the biosynthesis of the E79 protein hydrolase, a co-expression plasmid was prepared as follows (see FIG. 6): First, the protein encoding the E79 proteinase kb of BamHI DNA fragment was inserted into the Bam H1 site of pHV33 to prepare a recombinant plasmid pTAP5. In addition, the recombinant plasmid pTCAB3 having the ccpA gene of the present invention inserted into the plasmid vector pHV33 was digested with Eco RI and then treated with 2 units of Crana fragments to smooth the restriction enzyme-treated ends. At this position, a 1.6 kb BamHI E79 protein hydrolase gene having a restriction enzyme-treated end smoothed with 2 units of Cranow fragment was inserted through a blunt end ligation to finally produce a 10.1 kb co-expression plasmid pTCO5 Respectively.

Next, E. coli XL1-Blue (Stratagene) was transformed into E. coli with 100 ng of the two kinds of recombinant plasmids using an electroporator (GenePulser) of Example 3, The activity of the protease expressed by the transformant containing the recombinant plasmid of the present invention was measured as follows. The results are shown in Table 2. First, the E. coli transformant was suspended in 0.5 ml of LB medium , And then cell extracts were obtained in the same manner as in Example 8, followed by centrifugation, and the supernatant was used as a source of a protein hydrolyzing enzyme. Subsequently, 3 ml of a substrate solution (10 mM Gly-KCl-KOH and 0.6% hemasuthene casein, pH 10.0) was added to 0.5 ml of the enzyme solution, reacted at 55 ° C for 2 hours and then 3 ml of TCA solution M TCA, 0.22 M sodium acetate and 0.33 M acetic acid) was added to stop the reaction. Then, the undifferentiated casein was allowed to stand for 10 minutes at room temperature to precipitate, and the absorbance was measured at 275 nm. On the other hand, the enzyme reverse transcriptase was determined by using 1 unit of enzyme to increase the OD ₂₇₅ value by 0.01 per minute.

The specific growth rate in the following Table 2 was determined by measuring OD ₆₀₀ at 1 hour intervals at 37 캜 for 20 hours.

Plasmid Activity of protein hydrolyzing enzyme (unit / mg) Non-growth rate (h ^-1 ) pTAP5 (pHV33 / apr ) 81 0.43 pTCO5 (pHV33 / apr / ccpA ) 30 0.54

As shown in Table 2, the activity of the protease was reduced by about 2.7 times as compared with that of the extract of cells transformed with pTCO5, which was expressed by pTCO5 CcpA has restored the metabolism inhibition trait.

The recovery of the EA metabolism inhibitory trait was also confirmed by the growth rate of E. coli transformants having the respective recombinant plasmids. In the case of the cells transfected with pTAP5, the E79 protein hydrolase Because the cells were damaged, normal growth did not occur and significantly decreased. However, the growth rate of the cells containing the co-expressing plasmid pTCO5 was found to be faster due to less damage to the cells due to decreased activity of the proteolytic enzymes.

On the other hand, the transformed Escherichia coli was inoculated into LB plate medium (1% tryptone, 0.5% yeast extract, 0.8% (W / V) skim milk) to inhibit production of protein hydrolyzing enzyme by CcpA And 1% NaCl, pH 7.0) at 37 ° C for 20 hours, and the resulting transparent ring was observed at 55 ° C for 2 hours. The results are shown in FIG. 7 attached hereto.

As shown in FIG. 7, in the case of pTAP5 in which the E79 protein hydrolase gene is expressed alone, it forms relatively large halo in the plate medium supplemented with skim milk, whereas it is separated from the E79 protein hydrolase gene In the case of pTCO5, in which a ccpA gene is simultaneously expressed, the production of protein hydrolytic enzymes was markedly reduced and almost no halo was formed.

As a result, it was confirmed that the CcpA protein isolated by the above results inhibits the expression of the E79 protein hydrolase gene, thereby inhibiting the production of the enzyme.

As described and demonstrated in detail above, the present invention provides a DNA encoding a cyanide metabolite regulatory protein A (CcpA) having the nucleotide sequence of SEQ ID NO: 1 and a DNA encoding the cyanide metabolite regulatory protein A (CcpA ). In addition, the present invention provides an effective method for producing the above-mentioned metabolite controlling protein A (CcpA).

The amino acid of the control protein A of the present invention and the DNA encoding the same are not only new but also the first sugar metabolism regulating genes in thermophilic bacteria. Meanwhile, the ccpA gene isolated by the present invention can be used to induce mutations such as the disruption of the metabolism inhibitory property of eicosapentaenoic acid. Thus, it is possible to produce a mutant that constitutively produces a protein hydrolyzing enzyme regardless of the sugar composition in the fermentation medium Can be manufactured.

Sequence List

SEQ ID NO: 1

Length of sequence: 2459

Sequence type: nucleic acid

Number of chains: 2

Topology: Linear

Molecular type: genomic DNA

origin

Name of species: Thermoactinomyces sp.

Title: E79

Characteristics of sequence

Symbol to characterize: -10 signal

Present location: 907..912

How to determine features: S

Symbols used to indicate characteristics: -35 signal

Present location: 886..891

How to determine features: S

Symbol to characterize: CDS

Present location: 1018..2055

How to determine features: S

Claims (8)

A DNA encoding a cyanide metabolite regulatory protein A (CcpA) having the DNA base sequence set forth in SEQ ID NO: 1 and a promoter therefor. (CcpA) having an amino acid sequence as set forth in SEQ ID NO: 2. A DNA encoding the amino acid sequence of SEQ ID NO: 2 or a functional equivalent thereof. 1. A recombinant plasmid pTCA2 comprising the DNA coding for the metabolite hydrolysis control protein A (CcpA) of claim 1 and its promoter, and having the cleavage map shown in Fig. Escherichia coli XL1-Blue (pTCA2) (KCTC 8865P) transformed with the recombinant plasmid pTCA2 of claim 4. A recombinant plasmid pTCAB3 comprising the DNA coding for the metabolomic control protein A (CcpA) of claim 1 and its promoter, and having the cleavage map shown in Fig. A method for producing an amide metabolism controlling protein A (CcpA) comprising the steps of: (I) culturing a strain containing the recombinant plasmid pTCAB3 of claim 6; (Ii) a step of pulverizing the cultured strain to obtain a cell extract; And (Iii) heat-treating the cell extract at 55 to 65 ° C. A metabolite regulatory protein A (CcpA) produced by the method of claim 7.