KR100358716B1 - Novel Protease and Process for Preparing the Same - Google Patents

Abstract

The present invention is isolated from Thermoanaerobacter yonseii ( KFCC-11116) , which is a high temperature bacterium, and encodes thermicin, which shows high thermostable protease activity, genomic DNA encoding the same, and electric thermini. The present invention relates to a method for producing a recombinant thermini from a microorganism transformed with an expression vector comprising genomic DNA and an electrotransformed microorganism. According to the present invention, culturing a microorganism transformed with an expression vector comprising genomic DNA encoding the acecin can be produced in a large amount by the method comprising the step of obtaining a thecein from it, Since the prepared acecin has stable properties at high temperatures, high temperatures, detergents and organic solvents, the thermini of the present invention can be effectively used in additives, food processing, leather processing, industrial synthesis using collagen, and chemical reaction using reverse reaction. Can be used.

Description

Novel protease and its preparation method {Novel Protease and Process for Preparing the Same}

The present invention relates to a novel protease and a preparation method thereof. More specifically, the present invention is isolated from Thermoanaerobacter yonseii ( KFCC-11116) , which is a high temperature bacterium, thermicin exhibiting high thermostable protease activity, genomic DNA encoding the same, and electrical The present invention relates to a method for producing a recombinant thermini from a microorganism transformed with an expression vector comprising genomic DNA encoding the thermoxy and an electrotransformed microorganism.

Proteases found in various animals, plants or microorganisms are enzymes that break down peptide bonds in proteins. Electroproteinases are not only used as reagents for diagnosis or treatment, but are also widely used in applications such as detergent additives, food processing processes and chemical synthesis using reverse reactions. Moreover, microbial alkaline proteases are one of the commercially important enzymes as components of detergents. Proteolytic enzymes used in these applications are desirable for use with high thermal stability enzymes because of their physicochemical stability.

Currently, proteolytic enzymes mainly used in industrial fields are produced in bacteria of the genus Bacillus, which have relatively high thermal stability, and thermophilic proteolytic enzymes isolated from these strains have high temperature and their resistance to detergents and organic solvents. It has been focused on a lot of attention. The best known alkaline proteolytic enzymes so far are subtilisin BPN isolated from Bacillus amyloliquefaciens and subtilisin Carlsberg isolated from Bacillus licheniformis (subtilisin Carlsberg) (M. Jacobs, et al., Nucl.Acids Res., 13, 8913-8926, 1985). However, the disadvantage has been exposed that the high temperature bacteria-derived protein hydrolase is stable at high temperature but unstable at high pH.

Thus, in general, strongly alkaline serine type protease are Alkalophilic Bacillus even at high alkaline environment (Bacillus) (reference:... K. Horikoshi, Agric Biol Chem, 35, 1407-1414, 1971) and Streptomyces ( Streptomyces ) (MHJ Zuidweg, et al., Biotechnol. Bioeng., 16, 685-714, 1972). Serine-based proteolytic enzymes from electroalkaline microorganisms have the advantage of being stable in an alkaline environment, but have a weakness in their resistance to very high temperatures.

Therefore, there is a constant need to develop proteolytic enzymes with high activity and stability under high temperature and alkaline conditions, which are of great value in various applications such as detergents, production of essential amino acids, processing of leather and industries using collagen. It has emerged.

Therefore, the present inventors have made intensive studies to develop proteolytic enzymes having high activity and stability. As a result, the thermophilic proteolytic enzymes and the genes encoding the same from thermoanaerobacter yonseii (KFCC-11116) Isolating, transforming the microorganism into a vector containing the electric gene and culturing the same, and confirming that the high-temperature-stable protease can be produced by the method comprising the step of obtaining the protease therefrom. The present invention has been completed.

After all, the first object of the present invention is to provide genomic DNA encoding thermoxycin, a protease derived from thermophilic thermoaerobacter Yonsei.

It is a second object of the present invention to provide a thermine having an amino acid sequence inferred from genomic DNA derived from the previous Thermoaerobacter Yonsei.

It is a third object of the present invention to provide an expression vector encoding the thermoxy.

It is a fourth object of the present invention to provide an E. coli transformed with an expression vector encoding the thermoxy.

A fifth object of the present invention is to provide a method for preparing thermini using an electrotransformed microorganism.

1 is a diagram showing the DNA sequence of the protease of the present invention.

2 is a diagram showing the amino acid sequence of the protease of the present invention and the amino acid sequence of the other microorganism-derived serine hydrolase.

3 is a graph showing the relationship between the enzyme activity of thermicin and temperature.

4 is a graph showing the relationship between the enzyme activity of pH and the pH.

Fig. 5 is a graph showing the relationship between the enzyme activity and the thermal stability of thermsin.

Fig. 6 is a graph showing the stability of the thermionic enzyme activity against SDS.

7 is a graph showing the stability of acetonitrile to acetonitrile.

8 is a schematic diagram showing the construction of the vector pQEXI.

9 is a diagram showing the amino acid sequence inferred from the DNA base sequence of the protease of the present invention.

Hereinafter, a recombinant protein is prepared from a microorganism transformed with an expression vector including a thermine showing a high heat-stable protease activity, a genomic DNA encoding the same, a genomic DNA encoding the electric thermini, and an electrotransformed microorganism. The method of making a superstition will be described in more detail.

In order to obtain proteolytic enzymes from Thermoanaerobacter yonseii (KFCC-11116), a thermophile collected from volcanic regions, the present inventors first cloned the enzyme gene without information on the primary structure of the enzyme protein. Library screening and cloning methods were employed: polymerized using oligonucleotide primers corresponding to amino acid sites well preserved in serine-based proteolytic enzymes, with the genomic DNA of Thermoaerobacter Yonsei as a template. An enzyme chain reaction (PCR) was carried out, resulting in a PCR product. It was confirmed that the PCR product obtained previously showed 67% and 56% similarity with the genes encoding serine-based protease and subtilisin-like proteinase, respectively, and the PCR product was used as a probe for genome library screening. I decided to use it. The genomic DNA encoding the proteolytic enzyme similar to 2.6 kb of subtilisin was then cloned using the electrical PCR product as a probe from the Genomic DNA library of Thermoaerobacter Yonsei.

The genomic DNA of protease, similar to the electrocloned subtilisin, has an open reading frame of 1239 nucleotides encoding 413 amino acids, and the estimated amino acid sequence is a signal peptide consisting of 13 amino acids, a protease consisting of 89 amino acids. It consists of a serine protease of a mature enzyme consisting of a peptide and 311 amino acids. The protein had an estimated molecular weight of 44.4 kD from the amino acid sequence and was expected to have a molecular weight of 32.9 kD when it became a mature enzyme, and the isoelectric point (pI) was estimated to be 8.51 from the amino acid component.

In addition, from the information search results of the protein, it was confirmed that the serine-based protease has similarity on the amino acid sequence of 61% to 51% with the subtilisin-like proteases of other microorganisms. In addition, the novel protein of the present invention was confirmed that the Asp, His, Ser and the surrounding amino acid sequence constituting the active site of the serine protease is well preserved as known enzymes, the present invention It is expected that the novel protein of has similar proteolytic activity to subtilisin. Thus, the novel protein was named 'Thermicin'.

Subsequently, the activity of the proteolytic enzymes of the thermini was examined for various synthetic substrates and proteins. As a result, the thermini was a substrate of p-nitroanilide (pNA) and Mca (7-Methoxycoumarin-4-yl) substrate. Exhibits hydrolytic activity against L-glycine-L-proline-p-nitroanilide hydrochloride in p-nitroanilide (pNA) substrates. It was confirmed to decompose. In addition, it was also confirmed that thermini hydrolyzes gelatin, collagen types I, II, IV, fibronectin, and casein, and it was found that the thermini of the present invention is a protease that hydrolyzes various proteins.

In the enzymatic activity of the thermionic acid, the proteolytic enzyme, the optimum temperature condition was found, and the thermionic acid exhibited the enzyme activity at 40 to 95 ° C, preferably 80 to 95 ° C, and most preferably at 92.5 ° C. In particular, it was confirmed that even after heat treatment at 80 ° C. for 30 minutes or at 120 ° C. for 120 hours, the activity of the enzyme was 50% or more. In addition, the optimum pH conditions of the enzyme activity of the thermini was confirmed to be active at pH 6.0 to 10.0, preferably pH 8.0 to 9.0, most preferably pH 9.0, the thermine is active at a wide range of temperatures and pH It was found that the protease was stable to high temperature and alkali. In addition, in order to examine the stability of the thermicin in the industrial use aspect of the present invention, the activity of the thermicin in an environment exposed to a large amount of exposure to organic solvents and detergents, as a result, 0.1 ℃ SDS to 1% SDS 80 ℃ The enzyme activity was stable for 3 hours at and was found to be more than 80% of enzyme activity after treatment for 3 hours at 80 ° C. in the presence of 30% acetonitrile. It was found that the enzyme activity was stable even at high temperatures.

The method for producing a recombinant thermini of the present invention comprises culturing a microorganism transformed with a vector containing a gene for the propeptide of the thermine, and then obtaining a recombinant thermini from the same. For example, the DNA sequence of a novel subtilisin-like serine protease isolated from thermophilic thermoaerobacter Yonsei may be expressed by an expression system established in recombinant E. coli, yeast, The host is not limited thereto, and transformation using a host such as Bacillus brevis, Lactobacillus, yeast, mold, fungus, animal cell, plant cell, insect cell, and the like is possible, Thermini must be active. In addition, it is preferable that the expressed protein is folded unless it is toxic or has a lethal effect.

On the other hand, the present inventors have found that there are many problems in the amount or activity of the expression of the electric protease in Escherichia coli, which is generally used for mass production, and this is due to the fact that the product generated from the gene is harmful to the host. It was estimated. Therefore, the present inventors have made various attempts to overcome the above problems of expression of proteolytic enzymes. As a result, it has been confirmed that mass production of proteolytic enzymes is possible by attaching a propeptide as an enzyme precursor.

In the present invention, an expression vector pQEXI comprising a gene of a propeptide-expressing propeptide was constructed, and the expression vector pQEXI was introduced into E. coli M15 (pREP4) to finally obtain transformed E. coli M15 (pREP4 + pQEXI). The former was named " E. coli M15 (pREP4 + pQEXI)" and the division was located at 361-221 Hongje 1-dong, Seodaemun-gu, Seoul on July 12, 2000. Deposited KFCC-11182 with the Korean spawn association.

By culturing the electric transformants, it was confirmed that the thermine obtained therefrom exhibits activity at a wide range of temperatures and pHs as described above, is stable to high temperatures and alkalis, and particularly has high thermal stability at 95 ° C. It can be effectively used for mass production of thermini of the present invention, such as reagents for diagnosis or treatment, additives for detergents, food processing, leather processing, chemical synthesis using industrial and reverse reaction with collagen.

Hereinafter, the present invention will be described in detail through examples. The following examples are intended to illustrate 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 Construction of Genomic DNA Library of Thermophile Thermoaerobacter

2 liters of the medium inoculated with Thermoanaerobacter yonseii (KFCC-11116) was incubated at 80 ° C., followed by centrifugation of the culture medium at 5,000 × g to obtain lysozyme and lysozyme. Reacts with proteolytic enzyme K, extracts protein and carbohydrate complexes by adding a 24: 1 (v / v) mixture of the same amount of chloroform and isoamyl alcohol, and centrifuged at 13,000 × g for 20 minutes. Isolation gave a supernatant. Subsequently, a 25: 24: 1 (v / v / v) mixture of the same amount of phenol / chloroform / isoamyl alcohol was added to the supernatant and extracted several times, followed by centrifugation for 20 minutes to give 0.7-fold volume of isopropanol and 3M. Sodium acetate was added and genomic DNA in solution was selected with a sterile Pasteur pipette. Then, the remaining salt was removed by washing with 70% ethanol, dried in a cleanbench, and the DNA pellet was suspended in 1 ml of TE buffer to obtain genomic DNA.

Subsequently, the obtained genomic DNA was partially cut into Sau 3AI restriction enzymes and inserted into lambda shock (ZAP Express, Stratagene, USA), the library was constructed through in vitro packaging, and assayed. It was found that the library contained 10 ⁶ independent plaques.

Example 2 Construction of Probes

To screen subtilisin-like proteases from the genomic DNA library of Example 1, oligonucleotide primers were constructed for amino acid sites that are well conserved in serine-based proteases of microorganisms: ie 5 'primers. : 5'- AAY GGN CAY GGN CAN CAY-3 '(SEQ ID NO: 3) is a degenerate oligonucleotide based on NGHGTH (SEQ ID NO: 1), an amino acid sequence around histidine, one of the active sites of serine protease. 3 'primer: 5'- YGG YGT YGC CAT NGA NGT NCC -3' (SEQ ID NO: 4) is GTSMATP (SEQ ID NO: 2), an amino acid sequence around serine, one of the active sites of serine proteolytic enzymes It was constructed based on. Wherein Y is C or T and N represents each nucleotide of A, G, C or T.

Using the primers, the genomic DNA of Thermoaerobacter prepared in Example 1 was denatured at 95 ° C. for 2 minutes using Taq DNA polymerase as a template, 30 seconds at 95 ° C., 30 seconds at 45 ° C., After 25 PCR experiments at 72 ° C. for 30 seconds, a PCR product of 585 bp was obtained, and the PCR product was subcloned in plasmid pGEM Teasy (Promega, USA) and sequenced (SEQ ID NO: 5). And an amino acid sequence inferred therefrom (SEQ ID NO: 6). As a result, the amino acid sequence derived from the base sequence of the PCR product was known as a serine protease, that is, an alkaline serine protease and Streptomyces albogriseol isolated from Bacillus subtilis . As a result of 67% and 56% similarity with subtilisin-like protease isolated from Streptomyces albogriseolus , it was decided to use the 585bp PCR product as a probe in screening genomic DNA libraries. .

Example 3 : Screening of Genomic DNA Libraries

The previously prepared genomic DNA library was introduced into E. coli XL-1 Blue and plated to obtain a total of 1.2 × 10 ⁵ plaques, the plaques were transferred to nitrocellulose membranes, and the electronitrocellulose membranes were then subjected to 0.5N NaOH, 1.5M. After treatment for 2 minutes in a modified solution containing NaCl, 5 minutes in a neutralizing solution containing 0.5M Tris-HCl (pH 7.4), 1.5M NaCl, and washed for 30 seconds in 2 × SSC. The membrane was then treated for 2 minutes in a UV crosslinker and then 65 ° C. in a solution containing 6 × SSC, 5 × Denhardt solution, 0.5% SDS, 100 μg / ml single strand salmon sperm DNA. , 3 hours prehybridization. The probe selected in Example 2 was then randomly-labeled with [ ³² P] dCTP and added to the prehybridization solution at a concentration of 1 × 10 ⁶ cpm / ml, followed by 4 hours at 65 ° C. Hybridized during the day. Hybridized membranes were washed twice with 15 minutes at room temperature using a solution containing 2 × SSC, 0.1% SDS; Wash twice at 65 ° C. for 15 min using a solution containing 1 × SSC, 0.1% SDS; And, using a solution containing 0.1 × SSC, 0.1% SDS was washed twice at 65 ℃, 15 minutes, and then exposed to the X-ray film for 14 hours at -70 ℃ under an intensifying screen (intensifying screen). As a result, 14 positive clones in which spots were formed at the same positions on the X-ray film were identified.

In addition, the phage plaques transferred from the same plate to the nitrocellulose membrane were hybridized, then exposed to the X-ray film, plated at a low dilution factor, and the secondary and tertiary screenings were repeated in the same manner as above to separate independent plaques. By the end, genomic clones were finally obtained. Subsequently, the genomic clone was cloned into the BamHI site of the pBK-CMV vector (Stratagene, U.S.A.) and subjected to restriction enzymes. As a result, the genome clone was found to be 1 to 2.6 kb. In addition, as a result of confirming these partial nucleotide sequences, it was found that 14 clones contained a part of the same gene, and among them, the plasmid containing the longest 2.6kb clone was selected and named 'pTAY'.

Example 4 DNA Sequencing and Amino Acid Sequencing

In the base sequence of the previous pTAY, the base sequence of the entire open translation frame region was analyzed using an automated DNA sequencer (ABI 310, PE biosystem, USA), and the base sequence of 2605 bp was determined (see FIG. 1, sequence). Number 7). Figure 1 shows the base sequence of 2605bp in the beginning, the dark letter GTG near 5 'represents the start codon, the dark letter TAA near the 3' represents the stop codon.

In addition, the cloned DNA has an open reading frame of 1239 nucleotides encoding 413 amino acids, and the amino acid sequence estimated therefrom is a signal peptide consisting of 13 amino acids, a propeptide consisting of 89 amino acids, and a maturation consisting of 315 amino acids. It was confirmed that the enzyme is separated into a serine proteolytic enzyme of a nature enzyme. The serine proteolytic enzyme has an estimated molecular weight of 44.4 kD from the amino acid sequence and is expected to have a molecular weight of 32.9 kD when it becomes a mature enzyme. The isoelectric point (pI) was estimated to be 8.51 from the amino acid component. As a result of comparing the DNA sequence (PCR 585) and the amino acid sequence of the PCR product of 585bp obtained in Example 2, it was found that the well-conserved active sites and other nucleotides and the corresponding amino acids were identical.

On the other hand, from the results of the information search of the protein using Blast, the electric clone has an identity on 47% to 32% amino acid sequence with subtilisin-like proteolytic enzymes of other known microorganisms, and 51% to 61% amino acid. It was identified as a novel protein with similarity in sequence (see FIG. 2). In addition, the novel protein was found that the amino acid sequence of Asp, His, Ser, and its surroundings, which constitute the active site of the serine protease, is well preserved like the known enzymes. 13). From these results, it could be expected that there will be very similar serine proteolytic enzymes with different amino acid sequences in Thermoaerobacter Yonsei. Figure 2 shows the amino acid sequence of the protease of the present invention and the amino acid sequence (SEQ ID NO: 8) of the other microorganism-derived serine-based hydrolase, the box designation is a conserved amino acid; The asterisk indicates an active site among them. Thus, it was expected that the novel proteins of the present invention would have similar protein activity as subtilisin, and we named the protein with the novel novel amino acid sequence 'Thermicin'.

Example 5 Physical and Chemical Properties of Thermiciin

In order to clarify the physicochemical properties of the thermins obtained previously, the activity of enzyme, optimal conditions of enzyme activity, etc. were investigated.

Example 5-1 Substrate Degradation Activity of Thermoxy

The enzymatic activity of thermini was measured using the synthetic peptide as substrate: 20 μl of 0.1 M CAPS buffer (pH 9.0) containing 10 ng / ml thermini was added 1 μM of 7-methoxycoumarin-. L-arginine-L-proline-L-lysine-L-proline-L-tyrosine-L-alanine-L-2-aminovaleric acid-L-tryptophan-L-methionine-L-lysine-2,4-di Nitrophenyl-amide (Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys (Dnp) -NH ₂ (Mca, (7-Methoxycoumarin-4-yl) acetyl; Nva, L- It was added to 80 µl of 0.1M CAPS buffer solution (pH 9.0) containing 2-aminovaleric acid; Dnp, dinitrophenyl, hereinafter referred to as 'synthetic substrate 1', Bachem, Switzerland) and reacted for 10 minutes at 80 ° C. , 100 µl of 2M sodium acetate was added to stop the reaction, and measured at an excitation wavelength of 325 nm and a fluorescence wavelength of 393 nm to quantify the amount of 7-amino-4-methylcoumarin produced. Against synthetic substrate 1 at 92 ℃ Hydrolytic activity was confirmed that the maximum value.

In addition, L-phenylalanine-p-nitroanilide (L-Phe-p-NA, hereinafter referred to as 'synthesis substrate 2'), L-proline-p-nitroanilide-trifluoroacetate (L-Pro-p -NA trifluoroacetate salt, hereinafter referred to as 'synthetic substrate 3' and L-glycine-L-proline-p-nitroanilide hydrochloride (hereinafter referred to as 'synthetic substrate 4') Degradation activity of thermini using Sigma Chemical Co., USA) as a substrate was measured by using photometric method, that is, 20 μl of the dilute solution of thermini contained 0.3 mM solution of electric synthetic substrate, respectively. 80 μl of 0.1 M CAPS buffer solution (pH 9.0) was added, and the resultant was reacted at 80 ° C. for 20 minutes, and then absorbance at 405 nm was measured to quantify the amount of p-nitroaniline. As a result, it was confirmed that the thermini has a hydrolytic activity for the synthetic substrates 1, 2 and 3 under the conditions of pH 9.0, 90 ℃ (see Table 1).

In addition, it was confirmed that the thermini hydrolyzed the synthetic substrate 1 to produce a fluorescent substance (7-amino-4-methylcoumarin) (see Table 1).

Substrate Specificity of Thermsin p-nitroanilide substrate (pNA) Vmax (mmol / min) Km (mmol) Vmax / Km (1 / min) Drainage L-Pro-p-NAL-Gly-Pro-p-NAL-Phe-p-NA 0.2638 1.7743 0.1487 0.19 0.4170 0.5254 0.7937 1.00 0.1127 0.3632 0.3103 0.39 MCA substrate Vmax (μmol / min) Km (μmol) Vmax / Km (1 / min) Drainage (Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys (Dnp) -NH ₂ 1.3690 12.5334 0.1092 0.14

As shown in Table 1, it was found that the thermini hydrolyzes L-Glysine-L-proline-p-nitroanilide hydrochloride (L-Gly-Pro-p-NA hydrochloride) most effectively.

Example 5-2 Gelatin Degradation Activity of Thermoxy

In order to determine the gelatin degrading activity of thermcin, gelatin digestion of the enzyme was performed on SDS polyacrylamide gel as follows: 2.5 µl of sample buffer (50 mM Tris-HCl, 5% SDS, 0.005% bromophenol blue, 50% glycerol, pH 7.5), and then added to 100V at room temperature using 10% polyacrylamide gel with 0.1% SDS containing 0.05% gelatin. It was electrophoresed for hours. Subsequently, the gel was immersed in 50 mM phosphate buffer (pH 7.0) containing Triton X-100 at room temperature for 1 hour, left at 60 ° C for 12 hours, and then the gel was stained with a solution (2.5% Coomasibrillant Blue). Brilliant Blue) R-250, 25% ethanol and 10% acetic acid) for 30 minutes and washed in 7% acetic acid solution for 4 hours to remove excess dye. As a result, the thermini hydrolyzed the gelatin into a peptide having a size sufficient to diffuse out of the gel, so that the degraded gelatin site was not stained with Couma Brilliant Blue. Therefore, it was confirmed that the thermini of the present invention has gelatin hydrolytic activity at 60 ℃.

Example 5-3 Casein Degradation Activity of Thermoxy

To determine whether the thermini has a casein hydrolytic activity: 100 μl of the auxin dilution solution corresponding to 50 ng / ml of thermini, 0.1M potassium phosphate buffer containing 0.2% casein (pH 7.0 ) Was added to 900 μl and reacted at 90 ° C. for 20 minutes. The reaction was then stopped by addition of 500 μl 15% trichloroacetic acid and the amount of acid-soluble short chain polypeptide contained in the supernatant obtained by centrifugation was determined by absorbance at 420 nm. As a result, it was confirmed that the thermini has a casein hydrolytic activity at 90 ℃.

In addition, it was confirmed by SDS-PAGE that thermini hydrolyzes not only gelatin but also collagen types I, II, IV, fibronectin and casein to produce short chain polypeptides.

Example 5-4 Optimal Temperature of Thermsin Enzyme Activity

Except for changing the temperature, the enzyme activity of the thermoxy was examined using the same method as in Example 5-1 (see Fig. 3). 3 is a graph showing the relationship between the enzyme activity of the thermoxy and the temperature. As shown in Figure 3, the thermini showed activity at 40 to 95 ℃ under the measurement conditions of pH 9.0, the optimum temperature was confirmed to be 92,5 ℃.

Example 5-5 Optimal pH of thermsin enzyme activity

A solution of Synthetic Substrate 1 was prepared using buffers with various pHs, while 100 mM HEPES buffer was used as a buffer in the range of pH 6.0 to 8, 100 mM Tris-HCl was used at pH 8.0, and 100 mM CAPS buffer was used at pH The enzymatic activity of the thermoxy was measured according to pH using in the range of 9.0 to 11.0 (see Figure 4). 4 is a graph showing the relationship between the enzyme activity of pH and the pH. As shown in Figure 4, the thermini shows activity at pH 6.0 to 10.0, it was confirmed that the optimum pH is 9.0.

Example 5-6 Thermal Stability of Thermsin

The thermini was left in a 100 mM CAPS buffer at pH 9.0 for various periods of time at 60 ° C. and 80 ° C., and then their enzyme activity was measured using the method of Example 5-1. Compared with the enzyme activity (see Fig. 5). 5 is a graph showing the relationship between the enzyme activity and the thermal stability of thermoxy, (◆) represents the enzyme activity at 60 ℃, (■) represents the enzyme activity at 80 ℃. As shown in Fig. 5, the thermini shows an amazing thermal stability having more than 50% of the activity even after heat treatment at 80 ℃ 30 hours, and at least 50% of the activity even after heat treatment at 60 ℃ 120 hours.

Example 5-7 Stability of Thermiciin to Detergent

Therminicin was activated by heat treatment at 80 ° C. for 30 minutes, SDS was added at a concentration of 0.1% or 1%, 50 μl of the electrolytic solution was reacted at 80 ° C. for various times, and the activity of the enzyme was reacted. It measured by the method of 5-1 (refer FIG. 6). Figure 6 is a graph showing the stability of the thermionic enzyme activity against SDS, (◆) shows the enzyme activity in the presence of 0.1% SDS, (■) shows the enzyme activity in the presence of 1% SDS. As shown in Figure 6, the auxin was maintained at 60% activity in the presence of SDS for 3 hours at 80 ℃, it can be seen that the thermini has a high stability in the presence of SDS.

Example 5-8 Stability to Organic Solvents

In order to determine the stability of therminicin to organic solvents, acetonitrile was examined as follows: 50 μl of each acetonitrile solution containing acetonitrile at a final concentration of 30% at 80 ° C. for various times. And the enzymatic activity of the thermine obtained therefrom was measured by the method of Example 5-1 (see Fig. 7). 7 is a graph showing the stability of acetonitrile to acetonitrile. As shown in FIG. 7, the thermicin was found to be more than 80% of enzyme activity even after treatment at 80 ° C. for 3 hours in the presence of 30% acetonitrile.

Example 5-9 : Effect of Various Reagents on Enzyme Activity of Thermiciin

Various reagents of various concentrations in the auxin were treated for 30 minutes at 37 ° C., and the enzyme activity of the obtained aminosine was measured by the method of Example 5-1, and compared with the enzyme activity of the control group without adding the reagent. (See Table 2).

Enzymatic Activity of Thermine by Various Reagents Type of reagent Concentration (mM) % Of residual thecinin activity Control 100.0 Cyanimide 0.1110 122.792.193.2 DTNB 0.1110 124.619.46.5 EDC 0.1110 102.6110.3112.2 N-acetylimidazole 0.1110 103.4121.0123.2 NEM 0.1110 95.0127.395.3 PCMB 0.1110 90.6130.599.3 Phenylglyoxal 0.1110 93.992.538.1 PMSF 0.1110 101.880.09.5 Salicylaldehyde 0.1110 101.880.09.5 CaCl ₂ 0.11 104.599.3 ZnCl ₂ 0.11 76.730.7 CuSO ₄ 0.11 55.526.4 MnCl ₂ 0.11 96.591.7 MgCl ₂ 0.11 101.0113.5

As shown in Table 2, when treated with phenylmethanesulfonyl fluide (PMSF, phenylmehthansulfonyl fluride), DTNB, salicylaldehyde, phenylglyoxal, ZnCl ₂ and CuSO ₄ , it was confirmed that the enzyme activity of the thermoxy is reduced .

Example 6 Construction of a Transformant

In order to construct a vector containing a gene for a precursor of an enzyme expressing the thermoxy, a subclonal pTAY containing 2.6 kb DNA encoding the gene of the thermoxy was used as a template, followed by a primer (ProEX-IF). 5'-CTC AAA AGG ATC C AA G GA AAT CTA TAC-3 '(SEQ ID NO: 9) and Primer (ProEX-001R) 5'-ACA GAG CTC TTA PCR was performed using AAC TTT TTT TAA AGC-3 '(SEQ ID NO: 10). Specifically, 50 μl of the total reaction solution containing 50 ng of template, 10 pmol of primer, and 0.25 units of DNA polymerase was denatured at 95 ° C. for 2 minutes. The reaction was repeated 25 times for 30 seconds at 95 ° C, 50 seconds at 55 ° C, and 1 minute 20 seconds at 72 ° C to obtain a PCR product. At this time, AAG of the primer (ProEX-IF) represents the start codon for the proenzyme gene, and TTA of the primer (ProEX-001R) represents the complementary sequence of the stop codon for the gene.

The nucleotide sequence of the fragment obtained by inserting the PCR product into the pGEM T-easy vector and digested with restriction enzymes BamH I and Sac I was confirmed using a DNA sequence analyzer. In addition, the electrical fragment was inserted into the overexpression vector pQE30, which was named "pQEX1" (see Figure 8). Then, the transformant constructed by inserting the electric vector into E. coli M15 (pREP4) ( E. coli M15 (pREP4)) was named E. coli M15 (pREP4 + pQEXI) ( E. coli M15 (pREP4 + pQEXI)). It was deposited on July 12, 2000, with the deposit number KFCC-11182, to the Korean spawn association of corporation, 361-221 Hongje 1-dong, Seodaemun-gu, Seoul.

Example 7 Cultivation of Transformant and Obtaining Thermsin

The transformants were inoculated in five 3 ml LB medium containing 50 µg / ml ampicillin and 10 µg / ml kanamycin, incubated at 37 ° C. for 12 hours, and then in five 200 ml media of the same component. Inoculated again and incubated for 3 hours at 37 ℃. Then, isopropylthiogalactoside (IPTG) was added to the medium to 1 mM, and incubated at 25 ° C. for 5 hours.

Subsequently, 1 L of the overexpressed culture solution was centrifuged at 4,000 x g for 10 minutes to obtain cells, and then, 4 g of binding buffer (binding buffer, 5 mM imidazole, 0.5 M NaCl, 20 mM Tri-HCl pH 7.9) per 1 g of cells was obtained. After mixing, and treating the electric cell mixture with an ultrasonic grinder five times for 30 seconds each, the supernatant obtained by centrifugation for 20 minutes at 14,000 × g was used as a cell extract. Then, 10 ml of 10 mg / ml cell extract was introduced into a His-tag affinity column equilibrated with binding buffer (0.5 x 1.2 cm), 10 ml of binding buffer and 10 ml of washing buffer ( washing buffer, 60 mM imidazole, 0.5 M NaCl, 20 mM Tri-HCl pH 7.9), followed by elution with 3 ml of elution buffer (1 M imidazole, 0.5 M NaCl, 40 mM Tris-HCl pH 7.9). And fractionated 500 μl. Subsequently, each fraction was introduced into a gel column (gel filtration column, Sephadex G-25, Bio-Rad, USA) to remove the salts contained in each fraction, followed by SDS-PAGE, 44kDa, 35kDa, 12kDa and 10kDa. The band corresponding to the molecular weight of was shown. The molecular weight of these bands was confirmed by the original size of the thermyn, the size converted to the active form by heat treatment for SDS-PAGE, the size of the His peptide attached to the propeptide and the size of the propeptide. Subsequently, as a result of confirming the enzymatic activity of the recombinant themisin, the same result as the effect of the thermine demonstrated in Example 5 was obtained. Thus, the transformant of the present invention can be effectively used to produce the same recombinant thermine. It could be confirmed.

Example 8 Determination of Molecular Weight and N-Terminal Amino Acid Sequence

After the SDS-PAGE of the previously obtained recombinant thermini, it was transferred to a PVDF membrane (Schleicher Schuell, Germany), and the membrane was stained with 50% methanol containing 1% coumabrilliant blue R-250 and then 60% methanol The solution was bleached so that only the portion containing the protein was stained. The N-terminal amino acid sequence of the recombinant thermini was determined with the membrane portion containing the former protein portion.

On the other hand, Figure 9 shows the amino acid sequence inferred from the DNA nucleotide sequence of the thermoxy, the dark part shows the sequence of the mature thermoxy. The determined N-terminal amino acid sequence is consistent with the amino acid sequence of 103 to 106 of the amino acid sequence of Figure 9, from which it can be seen that the mature thermoxy is an enzyme consisting of a polypeptide including after the 103 amino acid sequence.

As described and demonstrated in detail above, the present invention is isolated from Thermoanaerobacter yonseii ( KFCC-11116) , which is a high-temperature bacterium, and shows thermicin, a genome encoding the thermostable protease activity. Provided are a method for producing a recombinant thermini from a microorganism transformed with an expression vector comprising DNA, genomic DNA encoding the electric thermini and an electrotransformed microorganism. According to the present invention, culturing a microorganism transformed with an expression vector comprising genomic DNA encoding the acecin can be produced in a large amount by the method comprising the step of obtaining a thecein from it, Since the prepared acecin has stable properties at high temperatures, high temperatures, detergents and organic solvents, the thermini of the present invention can be effectively used in additives, food processing, leather processing, industrial synthesis using collagen, and chemical reaction using reverse reaction. Can be used.

Claims (6)

A genomic DNA encoding thermicin, a proteolytic enzyme derived from Thermoanaerobacter yonseii (KFCC-11116), represented by the nucleotide sequence of SEQ ID NO. Thermicin, which is a protease derived from the genomic DNA of claim 1 and represented by the amino acid sequence of SEQ ID NO: 8. An expression vector pQEXI comprising genomic DNA of thermoxy of claim 1. E. coli M15 (pREP4 + pQEXI) transformed with the expression vector pQEXI of claim 3 ( E. coli M15 (pREP4 + pQEXI), KFCC-11182). The method of preparing the thermini comprising culturing the microorganism transformed with the expression vector comprising the genomic DNA of the thermoxy of claim 1, and then obtaining the thecin. The method of claim 5, Transformed microorganism was Escherichia coli M15 (pREP4 + pQEXI) ( E. coli M15 (pREP4 + pQEXI), KFCC-11182) characterized in that Method of making therminicin.