JP5260941B2 - Novel collagenolytic enzymes and their use - Google Patents

Description

  The present invention relates to a novel collagenolytic enzyme, its gene and use thereof. More specifically, the present invention relates to a novel collagenolytic enzyme having high activity in an alkaline region derived from Alkalamonas spp., Its gene and use thereof.

  Collagen degrading enzymes and gelatin degrading enzymes are enzymes with high industrial utility value. For example, collagen hydrolyzate of collagen has utility as a cosmetic material such as wrinkle removal, and has also found interesting physiological activities such as moisturizing properties, wrinkle formation preventing action, blood pressure lowering action ( For example, refer to Non-Patent Documents 1 and 2), and in fact, peptides with a low molecular weight collagen are used in large quantities in the medical and cosmetic fields (see, for example, Patent Documents 1 to 3). Gelatin is used in photographic emulsions and the like, and gelatinolytic enzymes are used in recycling X-rays and photographic films (for example, see Patent Document 4). Gelatin degrading enzymes are useful for the recovery of photographic resources from waste films.

  In recent years, bioconversion (biological conversion) for effective use of organic waste has been actively attempted. For example, composting garbage is a good example. More than 30% of animal protein is made of collagen, and a large amount of collagen is contained in raw garbage from ordinary households and canteens, and waste discharged in the meat processing industry. Collagen is generally poorly soluble and hardly decomposable due to its special higher order structure, and is a relatively slow degrading protein even in composting. Since most of the unused parts of livestock resources are hardly decomposable, most of them are currently incinerated.

  Therefore, the development of a collagenolytic enzyme capable of degrading collagen and gelatin is very important, and many reports have been made so far about collagenolytic enzymes and collagenolytic enzyme-producing bacteria (for example, see Patent Documents 5 to 8). . However, many of these enzymes exhibit an optimum reaction pH in a neutral to acidic range, and few collagenolytic enzymes exhibiting an optimum reaction pH under alkaline conditions are not well known. Furthermore, many of the collagen-degrading enzymes derived from microorganisms are metalloenzymes called collagenases, and collagen-degrading enzymes classified into serine proteases are not well known (Non-patent Document 3).

JP 2006-151847 A JP 2001-267553 A JP 2005-314265 A JP 2000-231179 A JP 2005-245285 A JP-A-8-70853 JP 2001-178456 A JP 2001-61474 A JP 2006-516889 A Tanaka et al. “Bioindustry” No. 9, pp. 18-23, 2005 Kuhare et al. "J. Immunol" 155,3653-3659,1995 Watanabe “Appl. Microbiol Biotechnol” 63,520-526, 2004 (Kurata et al. “Int. J. Syst. Evol. Microbiol.” 57,1549-53, 2007)

  The present invention has been made in view of the current situation regarding collagen degrading enzymes as described above, and aims to provide a collagen degrading enzyme that is active under alkaline conditions. It shows an optimum reaction pH in alkaline conditions and is classified as a serine protease. It is an object of the present invention to provide a novel collagenolytic enzyme having a low amino acid sequence homology to a known collagenolytic enzyme and a gene encoding the same.

  The inventors of the present invention have conducted extensive research to achieve the above-described object, and as a result of screening collagen-degrading enzyme-producing bacteria from deep-sea soil, they have found a new collagen-degrading enzyme and further encoded this enzyme. The present invention has been completed by cloning the gene to be used.

That is, the present invention is encoded by the polynucleotide of SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of SEQ ID NO: 1 in the sequence listing or an amino acid sequence in which one or more amino acids in this sequence are deleted, substituted or added The present invention provides a novel collagenolytic enzyme obtained through a collagenolytic enzyme precursor having the following physicochemical properties:
(1) Action:
It degrades casein, gelatin, collagen, keratin and elastin proteins, and has a strong degrading action particularly on collagen and gelatin.
Synthetic substrates N -methoxysuccinyl-Ala-Ile-Pro-Met- p -nitroanilide, N -succinyl-Ala-Ala-Pro-Phe- p -nitroanilide, N -succinyl-Ala-Ala-Pro -Met- p -nitroanilide, N -glutaryl-Ala-Ala-Pro-Leu- p -nitroanilide, and N -succinyl-Ala-Ala-Val-Ala- p -nitroanilide N -methoxysuccinyl-Ala-Ala-Pro-Val- p -nitroanilide and N -succinyl-Gly-Pro-Leu-Gly-Pro-4-methylcoumaryl-7-amide are not degraded.
(2) Optimum reaction pH: pH 8.5-9.0
(3) The optimum reaction temperature is 40-50 ° C, and the enzyme activity is stable up to 45 ° C.
(4) Molecular weight:
The molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 50,000 to 70,000.
(5) Inhibitor:
Although it is completely inhibited by phenylmethylsulfonyl fluoride which is an inhibitor of serine protease, ethylenediaminetetraacetic acid which is a chelating agent shows a residual activity of 35% at a concentration of 10 mM.

  The present invention also includes an amino acid sequence represented by SEQ ID NO: 3 in the sequence listing, or an amino acid sequence in which one or more amino acids in this sequence are deleted, substituted, or added, or SEQ ID NO: in the sequence listing. The above-mentioned novel collagenolytic enzyme having an amino acid sequence having 80% or more homology with the amino acid sequence shown in FIG.

  The present invention also provides the above-described novel collagen degradation having the amino acid sequence represented by SEQ ID NO: 5 in the sequence listing, or an amino acid sequence in which one or more amino acids in this sequence are deleted, substituted, or added. An enzyme is provided.

  The present invention also provides the above-described novel collagen degradation having the amino acid sequence represented by SEQ ID NO: 7 in the sequence listing, or an amino acid sequence in which one or more amino acids in the sequence are deleted, substituted, or added. An enzyme is provided.

  The present invention also provides the novel collagenolytic enzyme described above, which is derived from Alkaline monas collagena marina.

  The present invention also provides a novel collagenolytic enzyme precursor having an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, or an amino acid sequence in which one or more amino acids in this sequence are deleted, substituted or added. Is to provide.

Furthermore, the present invention relates to a gene encoding the amino acid sequence of the above-described novel collagenolytic enzyme or a precursor thereof, the group consisting of the following (a) to (h):
(A) a polynucleotide encoding a polypeptide having an amino acid sequence constituting the mature enzyme shown in SEQ ID NO: 3,
(B) a polynucleotide encoding a polypeptide having an amino acid sequence in which one or more amino acids in the amino acid sequence constituting the mature enzyme shown in SEQ ID NO: 3 have been deleted, substituted or added;
(C) a polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 5,
(D) a polynucleotide encoding a polypeptide having an amino acid sequence in which one or more amino acids in the amino acid sequence shown in SEQ ID NO: 5 have been deleted, substituted or added;
(E) a polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 7,
(F) a polynucleotide encoding a polypeptide having an amino acid sequence in which one or more amino acids in the amino acid sequence shown in SEQ ID NO: 7 have been deleted, substituted or added;
(G) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 2, or (h) a protein that hybridizes with a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2 under stringent conditions and has a collagenolytic activity A polynucleotide,
The present invention provides a gene encoding a collagenolytic enzyme selected from

  The present invention also provides a recombinant vector containing any one of the above genes and a microorganism transformed with the vector.

  Furthermore, the present invention also relates to a collagen having the physicochemical properties or the amino acid sequence, characterized by culturing the alkaline monas collagena marina or the transformed microorganism and collecting a collagenolytic enzyme from the culture solution. A method for producing a degrading enzyme is provided.

  The novel collagenolytic enzyme of the present invention has a high collagenolytic activity in an alkaline region not found in conventional collagenolytic enzymes, and is classified as a serine protease. Therefore, it has a characteristic property that collagen and gelatin can be decomposed with high activity particularly in an alkaline region.

  The novel collagenolytic enzyme of the present invention (hereinafter sometimes referred to as “the enzyme of the present invention”) has the enzymatic properties defined in the following (1) to (5).

(1) Action:
The enzyme of the present invention degrades casein, gelatin, collagen, keratin and elastin proteins, and particularly has a strong degrading action on collagen and gelatin.
In addition, the enzyme of the present invention includes N -methoxysuccinyl-Ala-Ile-Pro-Met- p -nitroanilide (referred to as “AIPM”), N -succinyl-Ala-Ala-Pro-Phe- p -nitro, which are synthetic substrates. Anilide (referred to as "AAPF"), N -succinyl-Ala-Ala-Pro-Met- p -nitroanilide (referred to as "AAPM"), N -glutaryl-Ala-Ala-Pro-Leu- p -nitroanilide (referred to as "AAPL") ), And N -succinyl-Ala-Ala-Val-Ala- p -nitroanilide (referred to as “AAVA”), but N -methoxysuccinyl-Ala-Ala-Pro-Val- p It has no degrading action on -nitroanilide (referred to as "AAPV") and N -succinyl-Gly-Pro-Leu-Gly-Pro-4-methylcoumaryl-7-amide (referred to as "GPLGP").
(2) The optimum reaction pH of the enzyme of the present invention is pH 8.5 to 9.0.
(3) The optimum reaction temperature of the enzyme of the present invention is 40-50 ° C. The enzyme of the present invention has a stable enzyme activity up to 45 ° C.
(4) Molecular weight:
The molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (referred to as “SDS-PAGE”) is 50,000 to 70,000.
(5) Inhibitor The enzyme of the present invention is completely inhibited by phenylmethylsulfonyl fluoride (hereinafter referred to as “PMSF”), which is an inhibitor of serine protease, but in the case of ethylenediaminetetraacetic acid (hereinafter referred to as “EDTA”) which is a chelating agent. 35% residual activity at 10 mM concentration.

  Such a collagenolytic enzyme of the present invention is not particularly limited to the method shown below, but is found, for example, among enzymes produced by alkaline genus bacteria. As an example of such a microorganism belonging to the genus Alkaline monas, there is preferably Alkaline monas Collageni marina AC40 strain. This strain has already been specified in detail by Kurata et al. And has been proved to be a novel strain. The bacteriological properties and the like of this strain have already been reported in detail in Non-Patent Document 4 (Kurata et al. “Int. J. Syst. Evol. Microbiol.” 57, 1549-53, 2007). The description is quoted here in the book. This strain is also known as Alkalimonas collagenimarina AC40, a microbial strain preservation facility (RICM), 2-1-01 Hirosawa, Wako City, Saitama Prefecture, RIKEN Microbial strain preservation facility) is deposited as JCM14267, and NCIMB Ltd (Ferguson Building Craibstone Estate Bucksburn Aberdeen AB21 9YA UK) as NCIMB14266, which can be obtained from these depository institutions.

  The collagenolytic enzyme of the present invention is a carbon source, nitrogen source or other essential nutrients that can assimilate the above-mentioned collagenolytic enzyme-producing bacteria, for example, microorganisms belonging to the genus Alkalamonas, preferably Alkaline monas Collagenimarina AC40 strain (JCM14267, NCIMB14266). Can be obtained from the culture supernatant.

Assimilar carbon sources necessary for the culture are glucose, maltose, mannose and the like, and assimilar nitrogen sources are fish meat extract, polypeptone, casein, collagen and the like.
The culture temperature is preferably 5 to 37 ° C, particularly 33 ° C, and the pH is preferably 7.0 to 10.5, particularly 8.5 to 10.5. Under these conditions, the culture is usually completed in 1 to 3 days.

  The enzyme of the present invention is accumulated in the culture supernatant, and the remaining culture solution from which the bacterial cells have been separated can be used as a crude enzyme solution. Furthermore, these crude enzymes can be purified using a conventional purification method such as ion exchange chromatography or gel filtration chromatography. If necessary, it can be recovered by means such as ultrafiltration or precipitation, and pulverized using an appropriate method.

  Separation / purification of this crude enzyme solution is more specifically, for example, if necessary, separation means such as salting-out method, precipitation method, ultrafiltration method, such as ion exchange chromatography, isoelectric point chromatography, What was further separated and purified by combining known methods such as hydrophobic chromatography, gel filtration chromatography, adsorption chromatography, affinity chromatography, and reverse phase chromatography can also be used.

  The collagenolytic enzyme of the present invention is a protein comprising the amino acid sequence shown in SEQ ID NO: 3, or a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 3. The gene of the present invention is a DNA encoding the protein as shown in SEQ ID NO: 4.

  Here, in the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequence in which one or more amino acids are deleted, substituted, or added means an amino acid sequence equivalent to the amino acid sequence of SEQ ID NO: 3, respectively. It is an amino acid sequence in which a plurality of amino acids, preferably 1 to 10 amino acids have been deleted, substituted or added, and still retains collagenase activity. For addition, 1 to several amino acids are added to both ends. Addition of the amino acids. In addition, the said equivalent amino acid sequence can be performed by comparing amino acid sequences using well-known algorithms, such as Lipman-Pearson method (Science, 227, 1435, 1985), for example. Specifically, the homology can be obtained using a search homology or maximum matching program of GENENTYX-Mac (software development).

  The collagenolytic enzyme precursor of the present invention is a protein comprising the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1. The gene of the present invention is DNA encoding the protein shown in SEQ ID NO: 2.

The collagenolytic enzyme precursor of the present invention is composed of the first to 760th amino acids of the amino acid shown in SEQ ID NO: 1. This can be encoded by a DNA consisting of the 2283th base from the 1st base of the base sequence shown in SEQ ID NO: 2. As a result of searching the homology of the amino acid sequence of SEQ ID NO: 1, it showed 47% homology with cold-active alkaline serine protease (YP269576) produced by Colwellia psychrerythraea 34H strain. In addition, in Japanese Patent Application Publication No. 2006-516889 (Patent Document 9), which is a prior patent application, a novel protease (Proteases) having an amino acid sequence similar to the amino acid sequence shown in SEQ ID NO: 1 was described. This showed 57% homology, but there is a description as a proteolytic enzyme, but there is no description that it has collagen degradation activity.
The homology with other known proteases and collagen degrading enzymes was extremely low, suggesting that the collagen degrading enzyme precursor of the present invention is a novel enzyme precursor. Accordingly, when appropriately aligned with the amino acid sequence shown in SEQ ID NO: 1, it preferably has a homology of 80% or more, more preferably 90% or more, and still more preferably 95% or more. Are encompassed by the present invention.

  In addition, the collagenolytic enzyme of the present invention shown in SEQ ID NO: 3 is produced by removing the prepropeptide from Alkaline monas collagena strain AC40. That is, in the collagenolytic enzyme precursor shown in SEQ ID NO: 1, amino acids 1 to 124 (corresponding to nucleotides 1 to 372 in SEQ ID NO: 2) are removed as prepeptides during the production of the enzyme of the present invention. Therefore, the enzyme of the present invention composed of the 636th amino acid from the 1st amino acid of the amino acid shown in SEQ ID NO: 3 is obtained as the mature collagenolytic enzyme from which the prepropeptide has been removed. This can be encoded by DNA consisting of the first base to the 1911th base of the base sequence shown in SEQ ID NO: 4.

  As a result of searching the homology of the amino acid sequence of SEQ ID NO: 3, it showed 52% homology with the cold-active alkaline serine protease (YP269576) produced by the Colwellia psychrerythraea 34H strain. The homology with other known proteases and collagen degrading enzymes was extremely low, suggesting that mature collagen degrading enzymes are novel enzymes. Accordingly, when appropriate alignment is made with the amino acid sequence consisting of the first to 636th amino acids of the amino acid shown in SEQ ID NO: 3, preferably 80% or more, more preferably 85% or more, and still more preferably 90% or more. Collagen-degrading enzymes having the homology of 1 and satisfying the enzymatic characteristics of 1 to 4 are included in the present invention.

  Generally, alkaline protease genes have a long prepro sequence. The pre-sequence is necessary for secretion of the enzyme out of the microbial cell, and the pro-sequence is a sequence necessary for forming an active three-dimensional structure of the enzyme. The present inventors have found the entire gene sequence encoding a collagenolytic enzyme precursor containing the prepro sequence represented by SEQ ID NO: 2 in the sequence listing, and found the amino acid sequence of the precursor represented by SEQ ID NO: 1. Furthermore, from this collagenolytic enzyme precursor, the amino acid sequence of the collagenolytic enzyme of the present invention produced outside the cells represented by SEQ ID NO: 3, that is, the amino acid sequence of the mature enzyme was found.

The collagenolytic enzyme of the present invention can be cloned from, for example, the chromosomal DNA of Alkaline monas collagena AC40 strain and produced in large quantities using an appropriate vector and host fungus. Furthermore, it is possible to produce two types of miniaturized collagenolytic enzymes having a molecular weight smaller than that of mature collagenolytic enzymes. This miniaturized enzyme has an amino acid deficiency on the C-terminal side compared to the mature enzyme.
These two types of miniaturized collagenolytic enzymes A and B are amino acids 441 to 636 of the amino acid sequence shown in SEQ ID NO: 3 (corresponding to the 1321 to 1908th nucleotides of SEQ ID NO: 4), and SEQ ID NO: The 551st to 636th amino acids (corresponding to the 1654th to 1908th nucleotides of SEQ ID NO: 4) of the amino acid sequence shown in Fig. 3 are missing.

That is, the downsized collagenase A is composed of a protein having the amino acid sequence shown in SEQ ID NO: 5. This can be encoded by DNA having the base sequence shown in SEQ ID NO: 6.
As a result of searching for the homology of the amino acid sequence of SEQ ID NO: 5, it showed 57% homology with the cold-active alkaline serine protease (YP929185) produced by Shewanella amazonensis SB2B strain. The homology with other known proteases and collagen degrading enzymes is extremely low, suggesting that miniaturized collagen degrading enzyme A is a novel enzyme. Therefore, when appropriately aligned with the amino acid sequence of the protein having the amino acid sequence shown in SEQ ID NO: 5, preferably has a homology of 80% or more, more preferably 85% or more, and still more preferably 90% or more, Collagen-degrading enzymes that satisfy enzymological properties 1 to 4 are included in the present invention.

On the other hand, miniaturized collagenase B is composed of a protein having the amino acid sequence shown in SEQ ID NO: 7. This can be encoded by DNA having the base sequence shown in SEQ ID NO: 8.
As a result of searching for the homology of the amino acid sequence of SEQ ID NO: 7, it showed 53% homology with the cold-active alkaline serine protease (YP269576) produced by the Colwellia psychrerythraea 34H strain. The homology with other known proteases and collagen degradation enzymes is extremely low, suggesting that miniaturized collagenase B is a novel enzyme. Therefore, when appropriately aligned with the amino acid sequence of the protein having the amino acid sequence shown in SEQ ID NO: 7, preferably has a homology of 80% or more, more preferably 85% or more, and still more preferably 90% or more, Collagen-degrading enzymes that satisfy enzymological properties 1 to 4 are included in the present invention.

  In addition, the gene encoding the collagenolytic enzyme of the present invention is obtained from the above-mentioned collagenolytic enzyme-producing bacterium, for example, a microorganism belonging to the genus Alkaline monas, preferably Alkaline monas collagenae AC40 strain, for example, shotgun cloning or using a specific primer. The gene of the present invention can be obtained by PCR amplification or the like.

In order to produce a collagen degrading enzyme using the gene of the present invention, the collagen degrading enzyme gene is incorporated into any vector suitable for expressing the gene in the target host, and the recombinant vector is used as a host. The obtained transformant is cultured, and the collagenolytic enzyme is collected from the culture solution.
That is, a method of obtaining a gene encoding the amino acid sequence of the enzyme of the present invention from the above-mentioned Alkaline monas Collagenimarina AC40 strain, producing a recombinant microorganism using a genetic engineering technique, and culturing the recombinant microorganism. Specifically, a nucleotide sequence encoding the amino acid sequence of the enzyme of the present invention is obtained from the above strain, this nucleotide sequence is then incorporated into an appropriate vector, and a host such as Escherichia coli is further transformed with this vector. The enzyme of the present invention may be produced by culturing, and the enzyme of the present invention may be collected from the culture.

  The culture may be carried out by inoculating a medium containing a carbon source, a nitrogen source and other essential nutrients that can assimilate microorganisms, and following a conventional method. Glucose, maltose, mannose, etc. can be used as a carbon source capable of assimilating microorganisms, and fish extract, polypeptone, casein, collagen, etc. can be used as nitrogen sources capable of assimilating microorganisms. .

Therefore, specifically, the gene encoding the amino acid sequence of the enzyme of the present invention or its precursor includes a gene selected from the group consisting of the following (a) to (h).
(A) a polynucleotide encoding a polypeptide having an amino acid sequence constituting the mature enzyme shown in SEQ ID NO: 3,
(B) a polynucleotide encoding a polypeptide having an amino acid sequence in which one or more amino acids in the amino acid sequence constituting the mature enzyme shown in SEQ ID NO: 3 have been deleted, substituted or added;
(C) a polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 5,
(D) a polynucleotide encoding a polypeptide having an amino acid sequence in which one or more amino acids in the amino acid sequence shown in SEQ ID NO: 5 have been deleted, substituted or added;
(E) a polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 7,
(F) a polynucleotide encoding a polypeptide having an amino acid sequence in which one or more amino acids in the amino acid sequence shown in SEQ ID NO: 7 have been deleted, substituted or added;
(G) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 2, or (h) a protein that hybridizes with a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2 under stringent conditions and has a collagenolytic activity A polynucleotide,
Is a gene selected from

  The amino acid sequence in which one or a plurality of amino acids are deleted, substituted, or added herein means a sequence equivalent to the original sequence, and one or a plurality, preferably 1 to An amino acid sequence in which 10 amino acids have been deleted, substituted, or added, and still retains collagenolytic activity. The addition includes addition of one to several amino acids at both ends. .

  In addition, “stringent conditions” referred to here are, for example, “Molecular Cloning: A Laboratory Manual 2nd ed.” (Edited by T. Maniatis et al., Cold. And the conditions described in Spring Harbor Laboratory (1989). Specifically, as stringent conditions, for example, 6 × SSC (composition of 1 × SSC: 0.15M NaCl, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5 × A condition is that the denhart is stored in a solution containing 100 μg / mL heat-denatured herring sperm DNA overnight and hybridized with the probe at a temperature of 50 to 65 ° C.

  The base sequence encoding the equivalent amino acid sequence can be prepared using a known technique such as site-directed mutagenesis. For example, the mutation may be introduced using a mutation introduction kit (Mutan-super Express Km kit; manufactured by Takara) using site-directed mutagenesis.

  Collection and purification of mature collagenolytic enzyme and miniaturized collagenolytic enzyme from the thus obtained culture can be performed according to a general method. That is, the cells can be removed from the culture by centrifugation or filtration, and the target enzyme can be concentrated from the obtained culture supernatant by conventional means. The enzyme solution or dry powder thus obtained can be used as it is, but can be further crystallized or granulated by a known method. The collagenolytic enzyme of the present invention prepared as described above is an enzyme that can be used industrially, such as production of collagen peptides in the fields of medicine, cosmetics, and food, and recovery of silver ions from photographic films, particularly X-ray photographic films. Useful.

  Next, the present invention will be described in more detail with reference to examples. In the examples, unless otherwise noted, “%” is mass%.

  In the following examples, the activity of collagenase was determined by the following method.

After keeping 0.09 mL of 100 mM glycine-sodium hydroxide buffer (pH 9) containing 2.5% (w / v) collagen (Sigma Aldrich) and 1 mM calcium chloride at 45 ° C. for 2 minutes, 0.01 mL of enzyme solution And reacted at 45 ° C. for 25 minutes. The reaction was stopped by adding 0.25 mL of a 100% trichloroacetic acid solution (TCA solution), left on ice for 15 minutes, and then centrifuged (20,400 × g, 10 minutes) to precipitate the acid-denatured protein. . The soluble protein in the obtained supernatant was quantified using a DC protein assay kit (BioRad).
On the other hand, what added 100 mM glycine-sodium hydroxide buffer (pH 10) instead of the enzyme solution was used for the blank. One unit of enzyme (P.U) was defined as the amount of enzyme that liberates acid-soluble proteolysate corresponding to 1 mmol of tyrosine per minute under the above reaction conditions.

Example 1 Screening for Collagen-Degrading Enzyme-Producing Bacteria Samples collected from the seabed off Torishima were smeared on a 1.0% collagen-containing alkaline agar medium, followed by stationary culture at 4 ° C. for 1 month. The grown bacteria were selected and purified, then inoculated into a liquid medium, cultured at 15 ° C. for 2 days with shaking, and the collagen degradation activity in the supernatant was measured. The liquid medium composition for screening is shown in the following Table 1, and collagen (type 1, sigma aldrich) 1.0% was added thereto. From the obtained culture broth, Alkalamonas Collageni marina AC40 strain was selected as a strain with high production of collagenase.

Example 2: Production and purification of collagen degrading enzyme, determination of N-terminal and internal amino acid sequence Alkaline Monas Collagenimarina strain AC40 was inoculated into a 500 mL liquid medium having the composition shown in Table 1 above, and cultured at 15 ° C for 3 days with shaking. Went.
The culture solution was centrifuged (7,500 × g, 30 minutes, 4 ° C.), and ammonium sulfate was gradually added to 80% saturation to the obtained supernatant solution (480 mL), followed by centrifugation (9,000 × g). , 30 minutes, 4 ° C.), and the precipitated protein was recovered. The recovered protein was dissolved in 100 mM glycine-sodium hydroxide buffer (pH 10.0), and then desalted and concentrated by ultrafiltration. The dissolved protein was applied to a DEAE Toyopearl column that had been equilibrated in advance with 100 mM glycine hydroxide-sodium buffer (pH 10.0), and the adsorbed protein having collagenolytic activity was eluted with 0.22 M sodium chloride.
Next, after adsorbing to a Butyl Toyopearl column equilibrated with 100 mM glycine-sodium hydroxide buffer (pH 10) containing 1 M ammonium sulfate in advance, the adsorbed protein was eluted by a linear concentration gradient method of 1 M to 0.5 M ammonium sulfate. It was. Collagen-degrading active fractions were collected and concentrated and desalted by ultrafiltration. When the concentrate was subjected to SDS-PAGE, two protein bands were confirmed around a molecular weight of 60 kDa. These bands were blotted on a PVDF membrane (manufactured by Bio-Rad), and the amino acid sequence from the N-terminal was determined with an amino acid sequencer (476A type, manufactured by Applied Biosystems).
The amino acid sequences of both bands are identical and the Ala-Gln-Gln-Thr-Pro-Tyr-Gly-Tyr-Thr-Met-Val-Gln-Ala-Asp-Gln-Val-Ser-Asp-Gln A 19 amino acid sequence was determined. Therefore, both bands were judged to be the same collagenolytic enzyme. The band having a low molecular weight was considered to be a deletion of the C-terminal portion of the band having a high molecular weight.
Next, the purified collagenolytic enzyme was partially cleaved using Achromobacter Protease I (Residue-specific Proteases Kit, Takara Bio), and the resulting peptide was separated by SDS-PAGE, and then the amino acid sequence of the band was analyzed. . As a result, the 20 amino acid sequence of Thr-Leu-Ala-Gly-Phe-Ser-Gln-Arg-Asn-Ala-Gln-Val-Glu-Leu-Ala-Gly-Pro-Gly-Val-Asp was determined. .

Example 3 Properties of Collagen Degrading Enzyme The properties of the purified enzyme obtained in Example 2 (enzyme of the present invention) were examined.
(1) Optimal reaction pH:
Collagen degradation reaction with the enzyme of the present invention was carried out using the following various buffers containing 2.5% (w / v) collagen (Sigma Aldrich) and having a pH of 100 mM in the range of 6.0 to 13.0. .
pH 6.0-8.0 ... morpholinopropanesulfonic acid-sodium hydroxide
(MOPS), (●)
pH 7.1-8.9 Tris-hydrochloric acid, (◯)
pH 8.0-10.0 boric acid-potassium chloride-sodium hydroxide, (Δ)
pH 8.5-11.1 glycine-sodium hydroxide, (■)
pH 10.9-13.0 .. Potassium chloride-sodium hydroxide, (▲)
The result is shown in FIG. As can be seen from this figure, the enzyme of the present invention had the highest reaction rate in the glycine-sodium hydroxide buffer (pH 8.5-9.0).

(2) Optimal reaction temperature Standard reaction conditions {2.5% (w / v) collagen (Sigma Aldrich), 0.09 mL of 100 mM glycine-sodium hydroxide buffer (pH 9) containing 1 mM calcium chloride, enzyme fraction 0. 01 mL}, the reaction temperature was variously changed to 5 to 70 ° C., and the collagen decomposition reaction with the enzyme of the present invention was performed. The result is shown in FIG. As can be seen from the results, the enzyme of the present invention showed the highest reaction rate at 40 to 50 ° C., particularly the highest reaction rate at 45 ° C.

(3) Thermostability The enzyme of the present invention was added to 100 mM Tris-HCl buffer (pH 8) and heat-treated at each temperature of 5 to 55 ° C. for 15 minutes, and the standard reaction conditions {2.5% (w / v) Residual activity was determined using collagen (Sigma Aldrich), 0.09 mL of 100 mM glycine-sodium hydroxide buffer solution (pH 9) containing 1 mM calcium chloride, 0.01 mL of enzyme fraction}. FIG. 3 shows the residual activity after the heat treatment, assuming that the activity before heating is 100%. It has been found that the collagenolytic enzyme of the present invention has stable and excellent activity up to 45 ° C. Further, when the same test was performed by adding calcium chloride (1 mM), the stability could be slightly increased by adding calcium chloride.

(4) Substrate specificity Similarly, various proteins (casein, keratin, gelatin, elastin) were added in place of collagen under the standard reaction conditions, and the degradation reaction of these proteins was performed. The result is shown in FIG. The activity with respect to gelatin was the highest, and this was defined as 100%, and the reactivity with respect to other substrates was defined as relative activity. As can be seen from the results, the enzyme of the present invention showed a large degrading activity especially on collagen and gelatin.

(5) Action on Synthetic Substrate The reactivity of the collagenolytic enzyme of the present invention to various synthetic substrates was examined using the synthetic oligopeptide substrate shown in Table 2 or the following.
After 0.005 mL of 10 mM synthetic substrate solution is mixed with 0.085 mL of 100 mM glycine-sodium hydroxide buffer solution (pH 9) containing 1 mM calcium chloride, and incubated at 45 ° C. for 2 minutes, 0.01 mL of the enzyme fraction is added and 5 mL is added. The reaction was performed for a minute. 405 using a spectrophotometer
Absorbance at nm was measured every 20 seconds to determine the amount of p-nitroaniline released per minute.
The activity against N -methoxysuccinyl-Ala-Ile-Pro-Met- p -nitroanilide (AIPM) is the highest, and the reactivity with respect to other synthetic substrates is shown in Table 2 as relative activity.

Where AIPM is N -methoxysuccinyl-Ala-Ile-Pro-Met- p -nitroanilide, AAPF is N -succinyl-Ala-Ala-Pro-Phe- p -nitroanilide, and AAPM is N -succinyl-Ala- Ala-Pro-Met- p -nitroanilide, AAPL is N -glutaryl-Ala-Ala-Pro-Leu- p -nitroanilide, AAVA is N -succinyl-Ala-Ala-Val-Ala- p -nitroanilide . The enzyme of the present invention showed excellent activity against AIPM and AAPF, showed some activity with AAPM and AAPL, and showed a slight activity against AAVA, but AAPV ( N -methoxysuccinyl-Ala-Ala -Pro-Val- p -nitroanilide) showed no activity.
Next, the reactivity to N -succinyl-Gly-Pro-Leu-Gly-Pro-4-methylcoumaryl-7-amide (GPLGP) as a synthetic oligopeptide substrate for collagenase was examined by the same method. The reaction composition and conditions were the same as above, and 7-amino-4-methylcoumarin liberated by decomposition was measured at 370 nm using a spectrophotometer. As a result, the enzyme of the present invention did not degrade GPLGP and showed no activity.

(4) Molecular weight The molecular weight of the enzyme of the present invention was measured by 10% SDS-PAGE. Precision plus dual standard (Bio-Rad) was used as the molecular weight marker. The result is shown in FIG. From this result, the molecular weight of the enzyme of the present invention was 50 to 70 kDa.

(5) Inhibitor Various inhibitors shown in Table 3 were added to a 100 mM Tris-HCl buffer (pH 8) so as to have a predetermined concentration, the enzyme of the present invention was added, and the mixture was incubated at 15 ° C. for 15 minutes. Thereafter, under standard reaction conditions {2.5% (w / v) collagen (Sigma Aldrich), 0.09 mL of 100 mM glycine-sodium hydroxide buffer (pH 9) containing 1 mM calcium chloride, 0.01 mL of enzyme fraction} Residual activity was measured. The results are shown in Table 3.

Here, PMSF is phenylmethylsulfonyl fluoride, and EDTA is ethylenediaminetetraacetic acid.
As shown in Table 3, the collagenolytic enzyme of the present invention was completely inhibited by PMSF, which is an inhibitor of serine protease, and the activity became 35% by EDTA, which is a chelating agent.

Example 4 Cloning of Collagen Degrading Enzyme Gene In order to extract chromosomal DNA, Alkaline monas Collageni marina AC40 strain was inoculated into a 10 mL liquid medium having the composition shown in Table 1 and cultured with shaking at 33 ° C. for 1 day. Subsequently, chromosomal DNA was extracted from the cells using Genomic-Tip100 / G (Qiagen).
Based on the N-terminal 19 amino acid sequence and the internal 20 amino acid sequence obtained in Example 2, primer 1 (5′-TAY GGN TAY ACN ATG GT-3 ′, where N is GATC, Y is CT, R is AG.) Primer 2 shown in SEQ ID NO: 10 (5′-TCN ACY TGN GCR TTN CKY TG-3 ′, where K is GT) was designed. Using the chromosomal DNA purified above as a template, PCR was performed using primer 1 (SEQ ID NO: 9), primer 2 (SEQ ID NO: 10), and EX Taq (Takara Bio). The PCR reaction conditions were denaturation at 94 ° C. for 4 minutes, followed by 30 cycles of 94 ° C. for 1 minute, 51.2 ° C. for 30 seconds, and 72 ° C. for 30 seconds. As a result, a PCR amplified fragment of about 0.5 kb was obtained. The obtained DNA fragment was inserted into pCR4-Topo (TOPO TA PCR cloning kit, Invitrogen), M13 forward primer (Invitrogen) and M13 reverse primer (Invitrogen), and Big Dye Teminator Cycle Sequencing kit (Applied Biosystems) The partial base sequence of the collagenase gene was determined. Primers are synthesized based on the obtained amplified fragments, and base sequences of further upstream and downstream regions are determined by the inverse PCR method and LA PCR in vitro Cloning kit (Takara Bio), and collagenase containing prepro sequences in them. An open reading frame consisting of 2283 base pairs (SEQ ID NO: 2) was found. The amino acid sequence shown in SEQ ID NO: 1 was determined from this base sequence. In the determined amino sequence, the N-terminal amino acid sequence of the purified enzyme (amino acids 1 to 19 of SEQ ID NO: 3) and the amino acid sequence of the peptide fragment obtained by partial digestion with Achromobacter Protease I (183 of SEQ ID NO: 3) -201st amino acid) was confirmed.

Example 5 Production of Recombinant Enzyme Primer 3 (SEQ ID NO: 11, 5′-GGA ATT C CA TAT G TC CAA) that anneals to the 5 ′ end (1st to 19th nucleotides of SEQ ID NO: 2) of the collagenase gene ACC ACA TTT CT-3 ′, the underlined portion indicates the newly added NdeI site, and primer 4 (SEQ ID NO: 12, 5′−) that anneals to the 3 ′ end (nucleotides 2265 to 2283 of SEQ ID NO: 2) CCG GAA TTC TTA CTC GTA GCT AAC TTT C-3 ′, underlined indicates newly added Eco RI site), Pyrobest DNA polymerase (Takara Bio), using chromosomal DNA of AC40 strain as a template, collagenase Nde to both sides of the encoding gene (SEQ ID NO: 2 I and Eco The newly added genes) of I site was amplified by PCR. The resulting PCR amplified fragment was decomposed with Nde I and Eco RI, and introduced into had been similarly treated vector pRSET-a (Invitrogen) to prepare a recombinant plasmid (pRSET-AcpII). E. coli TOP10 strain (Invitrogen) was transformed with this recombinant plasmid. This transformant was grown on an LB plate [1% tryptone (Difco), 0.5% yeast extract (Difco), 1% sodium chloride, 1.5% agar] containing 2% skim milk and 100 μg / mL ampicillin. However, skim milk dissolution spots were observed around the colony.

Subsequently, a recombinant plasmid for producing miniaturized collagenolytic enzyme A (SEQ ID NO: 5) and miniaturized collagenolytic enzyme B (SEQ ID NO: 7) was prepared. Miniaturized collagenolytic enzyme A (SEQ ID NO: 5) and miniaturized collagenolytic enzyme B (SEQ ID NO: 7) are amino acids 441 to 636 of SEQ ID NO: 3 (nucleotides 1321 to 1908 of SEQ ID NO: 4), 551, respectively. ˜636th amino acid (nucleotides 1654 to 1908 of SEQ ID NO: 4) is deleted. Primer 6 (SEQ ID NO: 14, 5′-ACC GCT GTC GCC AGC CAG-3 ′, anneals to nucleotides 1675 to 1692 of SEQ ID NO: 2) and primer 7 (SEQ ID NO: 15, 5′-ACG ACG AGC AGC AAT GCT GAT AT-3 ′, anneals to nucleotides 2006-2025 of SEQ ID NO: 2), primer 5 (SEQ ID NO: 13, 5′-TAA GAA TTC GAA GCT TGA TCC-3 ′, stop codon of collagenase And anneal to the pRSET-a (Invitrogen) part in the downstream region). Miniaturization using primer 5 (SEQ ID NO: 13) and primer 6 (SEQ ID NO: 14) by PCR using Pyrobest DNA polymerase (Takara Bio) using a recombinant plasmid for collagenase production (pRSET-AcpII) as a template Collagen-degrading enzyme A (SEQ ID NO: 5) and primers 5 (SEQ ID NO: 13) and primer 7 (SEQ ID NO: 15) were used to prepare DNA fragments encoding miniaturized collagenolytic enzyme B (SEQ ID NO: 7), respectively. PCR conditions were as follows: both the miniaturized collagenolytic enzyme A (SEQ ID NO: 5) and the miniaturized collagenolytic enzyme B (SEQ ID NO: 7) were denatured at 96 ° C. for 1 minute, then 96 ° C. for 30 seconds, 56 ° C. for 30 seconds, 72 This was carried out for 30 cycles, with 1 minute at 1 ° C as one cycle. Thereafter, in order to circulate each DNA fragment in a molecule to obtain a miniaturized collagenolytic enzyme expression plasmid, the 5 ′ end of the DNA fragment was phosphorylated and ligated. The nucleotide sequences of miniaturized collagenolytic enzyme A (SEQ ID NO: 5) and miniaturizing enzyme B (SEQ ID NO: 7) correspond to SEQ ID NO: 6 and SEQ ID NO: 8, respectively.
Each of the obtained expression plasmids was used to transform E. coli BL21 (DE3) pLysS strain (Invitrogen), and the mixture was successfully treated overnight at 30 ° C. in LB medium containing 100 μg / mL ampicillin and 35 μg / mL chloramphenicol. As a result of agitation culture, the production amounts of miniaturized collagenolytic enzyme A (SEQ ID NO: 5) and miniaturized collagenolytic enzyme B (SEQ ID NO: 7) in the culture broth were 0.44 p. U / L, 0.61 p. U / L.

  The collagenolytic enzyme of the present invention is a novel enzyme having an amino acid sequence different from that of a conventional proteolytic enzyme, and particularly has a high degrading activity for collagen and gelatin. Therefore, collagen and gelatin can be decomposed to reduce the molecular weight, and the food industry that requires such low molecular weight collagen and low molecular weight gelatin, the cosmetics field that uses moisturizing and anti-wrinkle properties, or various medical treatments Useful in the field. In addition, by utilizing the gene sequences of these enzymes obtained by the present invention, such enzymes can be easily produced using genetic engineering techniques, and low molecular weight collagen and low molecular weight gelatin in the industrial fields described above can be used. Useful for.

It is a figure which shows the relationship between the enzyme activity of this invention enzyme, and pH. It is a figure which shows the relationship between the enzyme activity and temperature for evaluating the temperature dependence of this invention enzyme. It is a figure which shows the relationship between enzyme activity and temperature for evaluating the thermostability of this invention enzyme. It is a figure which shows the enzyme activity with respect to various proteins of this invention enzyme. It is a figure which shows the result of SDS-PAGE of the enzyme of the present invention.

Claims (10)

Collagenase precursor having the amino acid sequence of SEQ ID NO: 1 in the sequence listing or the amino acid sequence of SEQ ID NO: 1 in the sequence listing or an amino acid sequence in which one or more amino acids in this sequence have been deleted, substituted or added A collagenolytic enzyme obtained through the body and having the following physicochemical properties.
(1)
Action:
It degrades casein, gelatin, collagen, keratin and elastin proteins, and has a strong degrading action particularly on collagen and gelatin.
Synthetic substrates N -methoxysuccinyl-Ala-Ile-Pro-Met- p -nitroanilide, N -succinyl-Ala-Ala-Pro-Phe- p -nitroanilide, N -succinyl-Ala-Ala-Pro -Met- p -nitroanilide, N -glutaryl-Ala-Ala-Pro-Leu- p -nitroanilide, and N -succinyl-Ala-Ala-Val-Ala- p -nitroanilide N -methoxysuccinyl-
No degrading action on Ala-Ala-Pro-Val- p -nitroanilide and N -succinyl-Gly-Pro-Leu-Gly-Pro-4-methylcoumaryl-7-amide.
(2)
Optimum reaction pH: pH 8.5-9.0
(3)
Optimum reaction temperature and thermal stability:
The optimum reaction temperature is 40-50 ° C., and the enzyme activity is stable up to 45 ° C.
(Four)
Molecular weight:
The molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 50,000 to 70,000.
(Five)
Inhibitors:
Although it is completely inhibited by phenylmethylsulfonyl fluoride which is an inhibitor of serine protease, ethylenediaminetetraacetic acid which is a chelating agent shows a residual activity of 35% at a concentration of 10 mM.   2. The collagenolytic enzyme according to claim 1, which has the amino acid sequence represented by SEQ ID NO: 3 in the sequence listing, or an amino acid sequence in which one or more amino acids in this sequence are deleted, substituted, or added.   A collagenolytic enzyme having the amino acid sequence represented by SEQ ID NO: 5 in the sequence listing, or an amino acid sequence in which one or more amino acids in this sequence are deleted, substituted, or added.   A collagenolytic enzyme having the amino acid sequence represented by SEQ ID NO: 7 in the sequence listing, or an amino acid sequence in which one or more amino acids in the sequence are deleted, substituted, or added. The collagenolytic enzyme according to any one of claims 1 to 4 , which is derived from alkaline Monascola marine AC40 .   A collagenolytic enzyme precursor having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, or an amino acid sequence in which one or more amino acids in this sequence are deleted, substituted or added. A gene encoding the amino acid sequence of the collagenolytic enzyme according to any one of claims 1 to 6 or a precursor thereof, the group consisting of the following (a) to (g) :
(A) a polynucleotide encoding a polypeptide having an amino acid sequence constituting the mature enzyme shown in SEQ ID NO: 3,
(B) a polynucleotide encoding a polypeptide having an amino acid sequence in which one or more amino acids in the amino acid sequence constituting the mature enzyme shown in SEQ ID NO: 3 have been deleted, substituted or added;
(C) a polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 5,
(D) a polynucleotide encoding a polypeptide having an amino acid sequence in which one or more amino acids in the amino acid sequence shown in SEQ ID NO: 5 have been deleted, substituted or added;
(E) a polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 7,
(F) a polynucleotide encoding a polypeptide having an amino acid sequence in which one or more amino acids in the amino acid sequence shown in SEQ ID NO: 7 have been deleted, substituted or added, or
(G) a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 2 ,
A gene encoding a collagen degrading enzyme selected from . A recombinant vector containing any of the genes according to claim 7 . A microorganism transformed with the recombinant vector according to claim 8 . It is encoded by the polynucleotide of SEQ ID NO: 2 in the sequence listing , characterized by culturing the alkaline monascola marina AC40 or the transformed microorganism according to claim 9 and collecting a collagenolytic enzyme from the culture solution. And is obtained through a collagenolytic enzyme precursor having an amino acid sequence of SEQ ID NO: 1 in the sequence listing or an amino acid sequence in which one or more amino acids in this sequence are deleted, substituted or added. A method for producing a collagenolytic enzyme having physicochemical properties or a collagenolytic enzyme having an amino acid sequence defined in claim 2 .