KR20000025930A - Novel catabolite control protein purified from thermoactinomyces sp. e79 and dna encoding the same - Google Patents

Abstract

PURPOSE: A novel catabolism control protein(CcpA) and DNA encoding the same are provided which induce a mutation discharging a catabolism repression phenotype, so it can be useful to construct the mutant which produces a hydrolysis enzyme regardless of a sugar composite ion in a fermentative medium. CONSTITUTION: Provided are a CcpA, DNA sequence encoding the same and a strong promoter thereof which is an inhibitory factor of enzyme production. DNA encoding the CcpA is described by SEQ ID NO. 1 and purified from Thermoactinomyces sp.. The CcpA is concerned in a catabolism repression as an inhibitory factor of enzyme production and regulates the synthesis of protein hydrolysis enzyme.

Description

Novel catabolic regulatory protein A of the genus Thermo actinomyces E79 and DNA encoding the same

The present invention relates to a novel catabolic control protein A (CcpA) and DNA encoding the same, and more particularly, to inhibit catabolic metabolism, which is one of the inhibitors of enzyme production during industrial production of enzymes. (catabolite repression) relates to catabolic metabolic regulatory protein A and DNA sequences encoding the same.

In general, proteolytic enzymes, xylanase, amylase and cellulase and the like are mostly induced enzymes in the presence of a substrate, there are a number of factors that limit their productivity when industrial production of the enzymes. On the other hand, when the enzyme is produced, its production is regulated by various biosynthetic metabolic regulators such as catabolite repression or end product feedback inhibition, which can produce the enzyme industrially. At the same time, it has become a significant factor in lowering productivity (fixed phase, microbial and industrial , 21, 262-269 (1995)).

Accordingly, various methods have been studied to overcome the above problems, and thus randomly preparing the variants using classical mutation induction methods such as mutation inducing agents or UV treatment, and methods of selecting high-producing strains have been used. . In addition, methods for optimizing fermentation conditions, cloning of genes, and constructing a high expression system using a strong promoter are proposed as solutions to the above problems.

On the other hand, many kinds of industrial enzymes are produced in Gram-positive bacteria including Bacillus, but research on the regulatory system such as intracellular regulatory proteins or regulators involved in enzyme production in these industrial strains is still in its infancy. Therefore, more fundamental enzyme production regulation is not achieved.

As is known, there is a regulatory system in which Escherichia coli controls the transcription of a specific gene between the catabolite activator protein and the cAMP complex. Meanwhile, even in Gram-positive bacteria, including Bacillus, which does not have the regulation mechanism. There is active research on whether there is a global regulatory mechanism that regulates intracellular glucose metabolism (Hueck, CJ, & H. Wolfgang, Mol. Microbiol. , 15, 395-401 (1995). )). Among the studies, active control of total regulatory proteins such as catabolic regulatory protein A (CcpA), which is involved in the suppression of catabolic metabolism in Gram-positive bacteria, and HPr of the phosphotransferase system, are as follows. Bacillus (Christoph J. et al., Mol. Microbiol. , 16, 855-864 (1995)) , Staphylococcus (Egeter, O. et al., Mol. Microbiol., 21, 739-749 (1996) ) And lactic acid bacteria (Monedero, V. et al., J. Bacteriol. , 179, 6657-6664 (1997)).

The above prior arts disclose nucleotide metabolism regulatory proteins involved in the biosynthesis control system of various enzymes and their genes, and disclose nucleotide sequences and amino acid sequences, and the relationship between catabolic regulatory proteins and enzyme biosynthesis control. It is mentioned.

On the other hand, the conventional research mainly on mesophilic strains, the research stage is in a very rudimentary stage, and most of the studies are focused on the study on the production regulation of sugar hydrolase, heat resistance and resistance Very little research has been conducted on thermophilic bacteria producing alkaline proteinases.

The inventors of the present invention intensively researched to develop strains capable of producing the above-described protease in high yields, resulting in the genus Thermoactinomyces producing useful industrial enzymes such as heat- and alkaline-resistant proteinases. ( Thermoactinomyces sp.) By separating the gene encoding catabolic regulatory protein A (CcpA) from E79, and confirming that the catabolic metabolic regulatory protein A (CcpA) encoded thereby regulates the biosynthesis of proteolytic enzymes The present invention has been completed.

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

Another object of the present invention is to provide a catabolic regulatory protein A (CcpA).

Still another object of the present invention is to provide a method for preparing the catabolic metabolism regulatory protein A (CcpA).

1 is a process diagram showing a method of separating a gene encoding a novel catabolic regulatory protein A of the present invention,

2 is a cleavage map of the recombinant plasmid pTCA2 comprising a gene encoding a novel metabolism regulatory protein A of the present invention,

Figure 3 is the amino acid sequence of the novel catabolic regulatory protein A and other known metabolic regulatory protein A of the present invention,

4 is a cleavage map of the recombinant plasmid pTCAB3 comprising a gene encoding a novel catabolic regulatory protein A of the present invention,

5 is a SDS-PAGE photograph showing a purification degree of the novel catabolic regulatory protein A of the present invention,

6 is a schematic diagram showing a method for preparing a co-expression plasmid for investigating the interaction between E79 protease gene and catabolic regulatory protein A;

Figure 7 is a photograph showing the inhibition of proteolytic enzyme production by catabolic regulatory protein A gene.

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

In another aspect, the present invention is characterized by a catabolic regulatory protein A (CcpA) having the amino acid sequence set forth in SEQ ID NO: 1.

On the other hand, the present invention comprises a DNA encoding a catabolic metabolic regulatory protein A (CcpA) having a DNA sequence of SEQ ID NO: 1 and a promoter thereof, and further comprising a recombinant plasmid pTCA2 having a cleavage map shown in FIG. It is another feature.

The present invention is characterized by a recombinant plasmid pTCAB3 having a DNA encoding the catabolic metabolism regulatory protein A (CcpA) having a DNA sequence set forth in SEQ ID NO: 1 and a promoter thereof and having a cleavage map shown in FIG. do.

The present invention is characterized by a method for producing the catabolic metabolic regulatory protein A (CcpA).

The present invention is described in detail as follows:

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

Subsequently, in order to separate DNA encoding CcpA, a primer corresponding to the homology region is prepared by first comparing the amino acid sequence homology of several previously known CcpAs, and PCR is performed using the same. Partial amplification of the ccpA gene is performed through the PCR experiment, and the amplified DNA fragment is labeled with digoxigenin to isolate the entire ccpA gene.

Then, to isolate the entire ccpA gene derived from Thermoactinomyces sp. E79, the following strategy is carried out as follows: First, the chromosomal DNA of Thermoactinomyces sp. After partial cleavage, DNA fragments of 2 to 5 kb are recovered. Subsequently, the DNA fragments are mixed with pUC19 digested with restriction enzymes, linked to each other using DNA ligase, and then introduced into E. coli XL1-Blue. Then, the transformed Escherichia coli is transferred to a nitrocellulose membrane, and the DNA in the Escherichia coli cell is transferred to a nitrocellulose membrane, and then a probe labeled with digoxigenin is added and colony hybridization is performed. Finally, clones carrying the ccpA gene were isolated by selecting clones that tested positive for the colony hybridization, followed by plasmid isolation from transformants carrying the isolated ccpA gene, which was named pTCA2 (see: 2). Thus, E. coli XL1-Blue transformed with pTCA2 was named E. coli XL1-Blue (pTCA2), which was assigned to the Korea Institute of Science and Technology Gene Bank on January 8, 1998. And deposited with accession number KCTC 8865P.

Then, the base sequence of the DNA containing the cloned ccpA gene was determined, the sequence is the same as SEQ ID NO: 1 of the Sequence Listing.

Meanwhile, in order to confirm the transformation of Bacillus megaterium CcpA mutant strain by the ccpA gene isolated in the above process, pTCAB3 was prepared by inserting into Bacillus vector pHV33 (see FIG. 4). The reason for using is because it has a replication origin that can be replicated in Bacillus and has a chloramphenicol resistant gene that can be used as a selection marker. Subsequently, the recombinant plasmid pTCAB3 is introduced into Bacillus megaterium CcpA mutant strain WH356 and transformed to confirm recovery of catabolic inhibitory trait.

In another aspect, the present invention provides a method for producing a CcpA protein, wherein the preparation method utilizes the strong promoter of the ccpA gene isolated above and the thermal stability of the CcpA protein, and (i) the strain containing the recombinant plasmid pTCAB3. Incubation step; (Ii) grinding the cultured strains to obtain cell extracts; And (iii) a heat treatment step of the cell extract at 55-65 ° C. In the heat treatment step of the purification process, the temperature is preferably 55 to 65 ℃, most preferably 60 ℃. If the temperature in the heat treatment step is less than 55 ℃ excess of the other protein of the host into which the recombinant plasmid of the present invention is introduced, and if it exceeds 65 ℃ there is a loss of the CcpA protein.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1 Isolation of Chromosome DNA from Thermoactinomyces Genus E79

Digestion of chromosomal DNA of E79 (KCTC 1045BP) genus Thermoactinomyces is difficult for cell lysis, and the conventional method is considerably modified and separated as follows: First, 1.5 L protein hydrolase production medium at 50 ° C. (1.0% Soyton, 2.0% Soluble Starch, 0.3% K ₂ HPO ₄ and 0.05% MgSO ₄ , pH 7.2) 16 hours liquid culture, centrifuged at 250 rpm and the cell precipitate was TEN buffer (10 mM) Tris-HCl, 1 mM EDTA and 10 mM NaCl, pH 7.6). Then, the washed cells were frozen at −70 ° C. and re-dissolved twice, and then suspended in a SET buffer solution (20% sucrose, 50 mM Tris-HCl and 50 mM EDTA, pH 7.6) containing lysozyme. The reaction was carried out at 37 ° C. for 1 hour. Then, 20 ml of 25% SDS solution was added and 2 ml of protease K (10 mg / ml) was added to the partially lysed solution, and shaken at 60 ° C. for 1 hour, followed by the same amount of phenol / The chloroform mixture solution treatment was repeated three times, and gently shaken for 2 hours in the presence of the phenol / chloroform mixture solution at a high temperature of 50 ° C. Then, centrifugation was performed and 5 M NaCl was added to a final concentration of 0.3 M to the formed supernatant, followed by adding two volumes of ethanol. At this time, the precipitated DNA was rolled up with a glass rod and immersed in 95% ethanol and washed. It was dissolved in TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 7.8).

Example 2: Partial amplification of ccpA gene derived from E79 genus Thermoactinomyces using PCR and preparation of probe using DIG labeling

Amplification of the CcpA gene derived from E79 genus Thermoactinomyces using PCR was performed as follows: First, a degenerate primer (consensus sequence) based on a consensus sequence known to have homology as a primer was used. degenerate primer) (N-terminal primer: 5'-CTGAATTCATGGCNACNGT-3 ', C-terminal primer: 5'-CTGGATCCGCNCCNATRTCRTA-3'). Subsequently, the chromosomal DNA of the genus E79 isolated from Example 1 was modeled, PCR was performed using the synthesized primers, and finally, 0.8 kb of DNA fragment was amplified. The PCR process was carried out using a 9600 model of Perkin Elmer, wherein the reaction was denatured at 94 ° C. for 5 minutes, followed by 30 cycles of 1 minute at 94 ° C., 1 minute at 55 ° C. and 1 minute at 72 ° C. It was repeated and finally DNA extension reaction was performed for 10 minutes at 72 ℃. On the other hand, the ccpA gene DNA amplified in the above process was labeled with a digoxigenin-labeled dUTP using the DIG labeling system (Boehringer Mannheim) to prepare a probe for isolating the entire ccpA gene through colony hybridization. .

Example 3: Cloning of the ccpA Whole Gene Using Colony Hybridization

In order to isolate the entire ccp gene derived from Thermoactinomyces genus E79, the following strategy was carried out as follows: First, chromosomal DNA (100 μg) of Thermoactinomyces sp. A sample of 2-5 kb of DNA fragment was recovered from the sample partially cut with Sau 3AI, and mixed with pUC19 (Boehringer Mannheim) cut with 1 unit of Bam HI. The reaction was carried out at 16 ° C. for 16 hours using a ligase. Subsequently, 20 μl of the ligation reaction mixture and an electroporator (BioRad, GenePulser) were used to transform the E. coli XL1-Blue (Stratagene) at 2.5 kV, followed by transformation of the transformants with the nitrocellulose membrane. Transfer to 10 ml of denatured buffer (0.5 M NaOH and 1.5 M NaCl), a regeneration buffer (0.5 M Tris-HCl and 3 M NaCl, pH 7.3) using a vacuum transfer (TransVac TE80, Hopper, Inc.) and X20 SSC. 10 ml (0.3 M sodium citrate and 3 M NaCl, pH 7.0) were used to denature and neutralize the DNA, transfer the DNA to the nitrocellulose membrane, and immobilize using ultraviolet light. Then, 10 ml of hybridization buffer solution (5X SSC, 1% BSA, N-lauroyl sacosin, and 0.02% SDS) and 20 µl of the modified probe prepared in Example 2 were added to the membrane, and 16 at 42 ° C. Colony hybridization was performed for a period of time. The antibody conjugate (anti-digoxigenin) was then added at a concentration of 150 μg / ml in buffer (100 mM maleic acid and 150 mM NaCl, pH 7.5) to screen for clones positive for the colony hybridization. Alkaline phosphatase conjugate) was added, followed by reaction with the membrane for 30 minutes, and finally, clones were developed by adding nitro blue tetrazolium salt (NBT) and 5-bromo-4-chloro-3-indoleyl phosphate. .

Example 4 Isolation of Recombinant Plasmids Containing the ccpA Gene

5 ml liquid culture of the transformant showing positive results for the colony hybridization of Example 3 was carried out to isolate the plasmid using Wizard miniprep kit (Promega). Subsequently, 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 of DNA was inserted into pUC19. Recombinant plasmids were identified and named pTCA2 (see FIG. 2). Thus, E. coli XL1-Blue transformed with pTCA2 was named E. coli XL1-Blue (pTCA2), which was assigned to the Korea Institute of Science and Technology Gene Bank on January 8, 1998. And deposited with accession number KCTC 8865P.

Example 5 Determination and Analysis of Sequence of ccpA Gene

In order to determine the base sequence of the cloned gene, a sequence of about 2.5 kb of DNA fragment was determined using an automatic sequencing system (ABI 377 Prism DNA Sequencer, Perkin Elmer). Shown in As can be seen in SEQ ID NO: 1, there was a total of 1038 bp open reading frame encoding its promoter and CcpA in the DNA fragment of about 2.5 kb. From the base sequence described above, the amino acid sequence of CcpA could be deduced as shown in SEQ ID NO: 1 and it was found that the protein was composed of a total of 346 amino acids.

On the other hand, the amino acid sequence of the CcpA protein by the gene isolated above is shown in Figure 3 compared to the CcpA protein derived from other known bacteria, in Figure 3 Ta is Thermoactinomyces sp. E79, Bs represents Bacillus subtilis , Bm represents Bacillus megaterium , Sx represents Staphylococcus xylosus and Lc represents Lactobacillus delbruckii . , Dotted line boxes indicate helix-turn-helix motifs, * denotes cases where the sequences of the five kinds of microorganisms all match, and. As can be seen in Figure 3, CcpA of the genus E79 of the thermoactinomyces was found to be a novel CcpA showing about 60% homology with CcpA of other microorganisms.

Example 6 Preparation of Recombinant Plasmids for Transformation of Bacillus Megaterium CcpA Mutant

To introduce the ccpA gene into Bacillus megaterium CcpA mutant strain WH356, the plasmid pTCA2 was digested with 1 unit of Bam HI and purified to 1.6 Vb ccpA gene and Bacillus vector pHV33 (ATCC 39217) digested with the same restriction enzyme. Recombinant plasmids were prepared by insertion, which was named pTCAB3 (see FIG. 4). Protoplast transformation was used to introduce the recombinant plasmid pTCAB3 into Bacillus megaterium CcpA mutant WH356 as follows: Bacillus megaterium WH356 was inoculated in 5 ml of LB medium and shaken overnight at 37 ° C. It was. Then, when the absorbance was 0.5 at 550 nm, the cells were centrifuged at 2,700 × g for 10 minutes, and the precipitated cells were treated with 5 ml of SMMP ((2 × SMM (0.5 M sucrose, 0.02) with 2 mg / ml of lysozyme). Suspended in M maleate and 0.02 M MgCl 2, pH 6.5) and 4X PAB medium), and then left for 1 hour at 37 ° C. Then, the degree of protoplasting was confirmed by retardation microscopy, and then 15 at 2,700 × g. After centrifugation for a minute, the obtained cells were washed twice with SMMP and lysed in 2 ml of SMMP to be used as protoplast cells, and then 1 ml of the protoplast cells were mixed with an equal amount of 2X SMM for transformation. After mixing with the prepared DNA, 3 ml of 40% PEG 6000 was immediately added and allowed to stand for 2 minutes at room temperature. Then, 10 ml of SMMP was added, followed by centrifugation at 2,700 × g for 15 minutes to transfer the cells into 1 ml of SMMP. Suspension and shake incubation for 90 minutes at 37 ℃ DM3 medium with 10 mg / ml chloramphenicol (100 ml 1M sodium succinate, 70 ml 10% yeast extract, 5% kazamino acid, 0.8% agar, 20 ml 3.5% K2HPO4 / 1.5% KH2PO4, 5 ml 20% glucose, 4 ml 1 M MgCl 2 and 1 ml 2% BSA, pH 7.3) M9 minimal medium (75 g) for screening transformants with ccpA genes in transformants which appeared in regenerated medium after transformation. / l Na2HPO4, 30 g / l KH2PO4, 5 g / l NaCl, 10 g / l NH4Cl, 0.21 g / l MgSO4, pH 7.4) Plate medium containing only xylose (0.5%), and glucose (0.5%) And toss-picked each plate medium to which both xylose (0.5%) was added and incubated for 16 hours at 37 ° C. At this time, because the mutant strain which did not introduce pTCAB3 to which the ccpA gene was linked was a strain in the ccpA gene. Glucose-catabolite repression does not appear, which is why Blue colonies were formed in the medium. In the Bacillus megaterium CcpA mutant to which pTCAB3 was introduced, catabolic inhibition was restored by the introduced ccpA gene, indicating blue colonies in xylose-only medium, but white in medium containing both xylose and glucose. Colonies were shown. Therefore, Bacillus megaterium transformants with pTCAB3 were selected by confirming white colony formation.

Example 7 Recovery of Catabolic Metabolism Inhibitory Traits of Bacillus Megaterium CcpA Mutant by ccpA Gene

Catabolism-inhibited transfection of Bacillus megaterium CcpA mutant WH356 by ccpA gene isolated using β-galactosidase as a reporter enzyme was measured as follows: First, 0.5% of xylose and glucose were measured. The plasmid pHV33 was introduced into the wild type Bacillus megaterium WH331, the Bacillus megaterium CcpA mutant WH356, and the Bacillus megaterium CcpA mutant WH356 in each of the M9 minimal medium of Example 6 and M9 minimal medium to which only xylose was added. After transforming the transformants and transformants introduced with the plasmid pTCAB3 into the Bacillus megaterium CcpA mutant strain WH356 for 5 hours, 50 μl of cells were added with the same amount of chloroform and shaken vigorously for 10 seconds. Subsequently, 0.2 ml of ONPG (2-nitrophenyl-β-D-galactopyranoside, 4 mg / ml) was added to the mixed solution for 10 minutes, and the enzyme activity (unit) was calculated by measuring the OD ₄₂₀ value. The results are shown in Table 1 below. In Table 1 below, the inhibition ratio represents the ratio of the activity of the inhibited β-galactosidase to the activity of the inhibited β-galactosidase.

Bacillus Megaterium Mutants Introduced plasmid Culture condition β-galactosidase activity (unit) Restraining 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 can be seen in Table 1, in the case of Bacillus megaterium CcpA mutant strain WH356, β-galactosidase activity was not inhibited in the presence of glucose in M9 minimal medium due to CcpA mutation, but the isolated ccpA gene In the case of WH356 introduced with glucose, the activity of β-galactosidase was markedly inhibited as shown in wild-type Bacillus megaterium WH331 when it was grown in a medium containing glucose. there was.

Example 8: Partial Purification of CcpA Proteins

First, E. coli transformants containing 5 ml of the recombinant plasmid pTCAB3 incubated overnight at 37 ° C were centrifuged and suspended in 0.5 ml of buffer (50 mM Tri-HCl, pH 8.0), and the cells were sonicated using an acoustic wave crusher. The cell extract was obtained by breaking. Subsequently, 0.1 mL of the cell extract was heat-treated at high temperature, and then purified by SDS-PAGE. The results are shown in FIG. 5. In Figure 5, 1 lane is a molecular weight marker, 2 lanes are heat-extracted cell extract (control), 3 lanes are heat-treated at 55 ℃ for 30 minutes, 4 lanes are heat-treated at 60 ℃ for 15 minutes, and 5 lane Is the case of heat treatment at 60 ° C. for 30 minutes.

As can be seen in Figure 5, the band showing the CcpA protein was very strong in two lanes loaded with cell extracts that were not heat treated, which is a high expression of CcpA in E. coli by a strong promoter on the top of the ccpA structural gene. The promoter is expected to have applicability to high expression of other useful proteins.

In addition, as can be seen in Figure 5 CcpA of the present invention has a relatively heat-stable characteristics, unlike the E. coli-derived protein used as a host, most of the remaining stable even after heat treatment at 60 ℃ 30 minutes it was possible partial purification using heat treatment .

Example 9: Coexpression of E79 Protease and CcpA

To investigate whether CcpA of the present invention is involved in the biosynthesis regulation of E79 protease, co-expression plasmids were prepared as follows (see FIG. 6): First, 1.6 encoding E79 protease Recombinant plasmid pTAP5 was prepared by inserting the kb BamHI DNA fragment into the Bam H1 position of pHV33. In addition, the recombinant plasmid pTCAB3 in which the ccpA gene of the present invention was inserted into the plasmid vector pHV33 was digested with Eco RI, and then treated with two units of Cranau fragments to smooth the restriction enzyme treatment ends. In this position, 1.6 kb of BamHI E79 protease gene, which also blunted the restriction enzyme-treated end with two units of Cranau fragment, was inserted through brunch end ligation to finally produce a 10.1 kb co-expression plasmid pTCO5. It was.

Subsequently, 100 ng of the two kinds of recombinant plasmids were transformed into Escherichia coli using E. coli XL1-Blue (Stratagene) using the electroporator (Biorad, GenePulser) of Example 3, respectively. The activity of the proteolytic enzymes represented by the transformants containing the recombinant plasmid was measured as follows, and the results are shown in Table 2 below. First, the E. coli transformants were prepared in 0.5 ml of LB medium. Incubated overnight, and then cell extracts were obtained in the same manner as in Example 8, followed by centrifugation, and the supernatant thereof was used as a proteolytic enzyme source. Then, 3 ml of substrate solution (10 mM Gly-KCl-KOH and 0.6% hemasten casein, pH 10.0) was added to 0.5 ml of the enzyme solution, and reacted at 55 ° C. for 2 hours, followed by 3 ml of TCA solution (0.11 M TCA, 0.22 M sodium acetate and 0.33 M acetic acid) were added to stop the reaction. Then, undigested casein was allowed to stand at room temperature for 10 minutes to precipitate, and then the absorbance was measured at 275 nm. On the other hand, the titer of the enzyme was determined by the amount of enzyme that increases the OD ₂₇₅ value by 0.01 to 1 unit.

In Table 2, the specific growth rate was determined by measuring OD ₆₀₀ at 1 hour intervals for 20 hours at 37 ° C.

Plasmid Protease Activity (Unit / mg) Specific growth rate (h ^-1 ) pTAP5 (pHV33 / apr ) 81 0.43 pTCO5 (pHV33 / apr / ccpA ) 30 0.54

As can be seen in Table 2, the extract of the cells transformed with pTCO5 was found to be about 2.7 times less activity of protease than the extract of cells transformed with pTAP5, which is expressed by pTCO5 This is because catabolic inhibitory traits were recovered by CcpA.

The recovery of catabolic inhibitory traits was also found through the growth rate of the E. coli transformants carrying the respective recombinant plasmids. In the case of cells transformed with pTAP5, E79 proteinase was expressed. Because the cells were damaged, normal growth did not occur and was significantly reduced. However, the growth rate of the cells containing the co-expression plasmid pTCO5 was confirmed that the growth rate is recovered and accelerated because the cells are less damaged by the activity of the reduced protease.

Meanwhile, in order to confirm the inhibition of proteolytic enzyme production by CcpA on the medium, LB plate medium (1% tryptone, 0.5% yeast extract) containing 0.8% (W / V) skim milk of the transformed Escherichia coli was identified. And 1% NaCl, pH 7.0) was grown for 20 hours at 37 ℃ incubator using a 2 ℃ incubator was observed for 2 hours in a 55 ℃ thermostat, the results are shown in Figure 7 attached.

As can be seen in FIG. 7, pTAP5, in which the E79 protease gene is expressed alone, forms a relatively large halo in a plate medium supplemented with skim milk, whereas it is separated from the E79 protease gene. In the case of pTCO5 in which one ccpA gene is expressed at the same time, the production of proteolytic enzymes was markedly reduced, resulting in almost no halo.

From the above results, it was confirmed that the isolated CcpA protein inhibited the production of the enzyme by inhibiting the expression of the E79 protease gene.

As described and demonstrated in detail above, the present invention provides a DNA encoding a catabolic metabolic regulatory protein A (CcpA) having a DNA sequence as set out in SEQ ID NO: 1 and a catabolic regulatory protein A having an amino acid sequence as set out in SEQ ID NO: 1 (CcpA). ). In addition, the present invention provides an effective method for producing the catabolic metabolic regulatory protein A (CcpA).

The amino acids of the catabolic metabolism regulatory protein A of the present invention and the DNA encoding the same are novel as well as the first sugar metabolism regulatory genes isolated from thermophilic bacteria. On the other hand, the ccpA gene isolated by the present invention can be used to induce mutations such as release of catabolic inhibitory traits, thereby producing variants that constitutively produce proteolytic enzymes regardless of the sugar composition in the fermentation medium. It can manufacture.

Sequence Listing

SEQ ID NO: 1

Length of sequence: 2459

Type of sequence: nucleic acid

Number of chains: two prints

Topology: Linear

Type of molecule: genomic DNA

origin

Biometric: Thermoactinomyces sp.

State: E79

Characteristic of the sequence

Characteristic symbol: -10 signal

Location: 907..912

How to determine the feature: S

Characteristic symbol: -35 signal

Location: 886..891

How to determine the feature: S

Characteristic symbol: CDS

Location: 1018..2055

How to determine the feature: S

Claims (8)

A DNA encoding a catabolic regulatory protein A (CcpA) and a promoter thereof, which have a DNA sequence set forth in SEQ ID NO: 1. Catabolic regulatory protein A (CcpA) which has the amino acid sequence of SEQ ID NO: 2. DNA encoding the amino acid sequence set forth in SEQ ID NO: 2 or a functional equivalent thereof. Recombinant plasmid pTCA2 comprising DNA encoding the catabolic metabolism regulatory protein A (CcpA) of claim 1 and a promoter thereof and having a cleavage map shown in FIG. 2. Escherichia coli XL1-Blue (pTCA2) (KCTC 8865P) transformed with the recombinant plasmid pTCA2 of claim 4. Recombinant plasmid pTCAB3 comprising DNA encoding the catabolic metabolism regulatory protein A (CcpA) of claim 1 and a promoter thereof and having a cleavage map shown in FIG. Method for preparing catabolic metabolism regulatory protein A (CcpA) comprising the following steps: (Iii) culturing the strain containing the recombinant plasmid pTCAB3 of claim 6; (Ii) grinding the cultured strains to obtain cell extracts; And (Iii) heat treatment of the cell extract at 55-65 ° C. Catabolic metabolism regulatory protein A (CcpA) prepared by the purification method of claim 7.