Elastin decomposition enzyme

Abstract

To obtain a polypeptide having a novel elastin decomposition activity.SOLUTION: The inventors have found a polypeptide derived from Streptomyces, the polypeptide showing a molecular weight of about 18 kDa, about 14 kDa, or about 11 kDa, and having an optimal reaction pH: pH 7.0-11.0 and an optimal reaction temperature: 50-80°C, the polypeptide having an elastin decomposition enzyme activity.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a polypeptide having an activity of degrading elastin. [Background technology]

[0002] Elastase is a type of proteolytic enzyme that degrades elastin, which is difficult to degrade with normal proteolytic enzymes. Elastin is present in the arteries, dermis, etc. of vertebrates, and is usually present in the body as an insoluble protein with a three-dimensional mesh structure. Water-insoluble elastin is hydrolyzed by acid, alkali treatment, or enzyme treatment to obtain water-soluble elastin. Water-soluble elastin is used in cosmetics, especially as a moisturizing agent, and is also used as a health food together with collagen, etc., as it has beauty effects such as giving elasticity to the skin. Furthermore, water-soluble elastin is also increasingly used in the field of regenerative medicine, such as artificial blood vessels.

[0003] Water-soluble elastin is obtained by hydrolysis using acid or alkali treatment, papain, or various microbial proteases, but the molecular weight of the elastin obtained varies depending on the treatment method and the origin of the elastin. It has been reported that elastin with a molecular weight of 43,000 or more derived from bonito has a platelet aggregation inhibitory effect (Patent Document 2), but when used as a cosmetic or health food, it is unclear what molecular weight of water-soluble elastin is optimal. Therefore, a variety of methods for decomposing elastin is required. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2007-45722 A [Patent Document 2] JP 2011-093872 A Summary of the Invention [Problem to be solved by the invention]

[0005] An objective of the present invention is to obtain a novel elastase. [Means for solving the problem]

[0006] The present inventors discovered that a protein containing a specific amino acid sequence has elastase activity, and thus completed the present invention.

[0007] The present invention relates to (a) A polypeptide having decomposition activity against elastin, said polypeptide having the amino acid sequence of (1) or (2) below: (1) A polypeptide having the same sequence as an amino acid sequence selected from SEQ ID NO:1. (2) A polypeptide comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO:1; (b) The polypeptide of the present invention is derived from the genus Streptomyces; (c) The polypeptide of the present invention is an enzyme having decomposition activity against elastin, and has the following properties: (1) Optimal reaction pH: pH 7.0 to 11.0 (2) Optimum reaction temperature: 50-80℃ (3) Molecular weight of about 18 kDa, about 14 kDa, or about 11 kDa Effect of the Invention

[0008] The elastin-degrading enzyme of the present invention degrades elastin with a wide molecular weight distribution and is an enzyme with high specificity in the degradation of elastin. [Brief description of the drawings]

[0009] [Figure 1] Molecular weight distribution in the examples [Diagram 2]Myofibril degradability [Diagram 3] Elastin degradability [Figure 4] Relative activity by pH [Diagram 5] Relative activity by temperature [Figure 6] temperature stability [Figure 7] Elastin degradability [Figure 8] Amino acid sequence of SEQ ID NO:1 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention relates to a polypeptide having elastinolytic activity.

[0011] The enzyme action of the present invention refers to the activity of hydrolyzing elastin as a substrate. There is no particular limitation on the elastin that can be used as the substrate of the present invention. Elastin is a protein that is widely distributed in elastic tissues such as arteries, ligaments, and skin. Elastin present in the ligamentum nuchalis and arteries of livestock products, and the bulbus arteriosus of fish and shellfish can be used.

[0012] The enzyme of the present invention exhibits a molecular weight of about 18 kDa, about 14 kDa, or about 11 kDa in SDS-polyacrylamide gel electrophoresis and may be a single molecular weight composition, but may also be a composite molecular weight composition of about 18 kDa and about 14 kDa and about 11 kDa, 18 kDa and 14 kDa, 18 kDa and 11 kDa, or 14 kDa and 11 kDa.

[0013] The polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 1. The polypeptide of the present invention comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0014] The polypeptide of the present invention is a polypeptide having a molecular weight of about 18 kDa, about 14 kDa, or about 11 kDa, and comprising an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. The sequence identity is, for example, a value calculated by appropriately aligning a query sequence (sequence to be evaluated) with the sequence of SEQ ID NO: 1. Specifically, in the present application, the sequence identity is a value calculated by the CLUSTAL algorithm. In addition, the amino acid sequences of the polypeptides having molecular weights of about 14 kDa and about 11 kDa are unknown, but the polypeptides are those in which the N-terminal side or C-terminal side of the amino acid sequence of SEQ ID NO: 1 has been truncated.

[0015] The polypeptide of the present invention having elastin decomposition activity can be produced by genetic engineering. The gene encoding the polypeptide of the present invention can be selected and used from a nucleotide sequence encoding SEQ ID NO: 1, or a nucleotide sequence encoding an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 1. The sequence identity of the nucleotide sequence is a value calculated using the CLUSTA L algorithm.

[0016] The polypeptide having elastin decomposition activity of the present invention can be produced using a carrier for expression of the polypeptide, for example, using an actinomycete such as Streptomyces as a host. The vector used can be any vector for actinomycetes, such as the vector described in JP 2014-207898 A. The host actinomycete can be any commonly available actinomycete, but it is particularly preferable to use an actinomycete belonging to the Streptomyces genus. Specific examples include Streptomyces erythraeus, Streptomyces griseus, Streptomyces omiyaensis, Streptomyces fradiae, Streptomyces roseoflavus, Streptomyces septatus, Streptomyces lividans, Streptomyces lavendulae, Streptomyces virginia, and Streptomyces coelicolor. In the production of the recombinant polypeptide of the present invention, the medium composition, medium pH, culture temperature, culture time, and the like can be appropriately determined to be optimal conditions for efficient production.

[0017] In addition to recombinant genetic production, the polypeptide of the present invention can be isolated from microorganisms of the genus Streptomyces, such as Streptomyces erythraeus, Streptomyces griseus, Streptomyces omiyaensis, Streptomyces fradiae, Streptomyces roseoflavus, etc. General proteins are separated from the microorganism, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, or a polypeptide comprising an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1, and having a molecular weight of about 18 kDa, about 14 kDa, or about 11 kDa is isolated.

[0018] The medium used in the culture step may be a medium containing a carbon source, a nitrogen source, and inorganic salts that can be assimilated by the cells used in the present invention. Either a natural medium or a synthetic medium may be used.

[0019] As carbon sources, glucose, acetic acid, ethanol, glycerol, molasses, sulfite pulp waste liquor, etc. are used, and as nitrogen sources, urea, ammonia, ammonium sulfate, ammonium chloride, nitrates, etc. are used. As sources of phosphate, potassium, and magnesium, normal industrial raw materials such as superphosphate, ammonium phosphate, potassium chloride, potassium hydroxide, magnesium sulfate, magnesium chloride, etc. may be used, and inorganic salts such as zinc, copper, manganese, and iron ions are added. As for other substances, vitamins, amino acids, nucleic acid-related substances, etc., can be cultured without using them, but these may be added. Organic substances such as casein, yeast extract, meat extract, and peptone may also be added.

[0020] The culture conditions may be appropriately selected depending on the type of medium, the culture method, etc., and are not particularly limited as long as the cells can grow and produce the enzyme of the present invention. In general, the culture is carried out in a liquid medium under aerobic conditions such as shaking culture or aeration and agitation culture at 25 to 35°C, preferably 27 to 30°C, for 6 to 96 hours. The pH is adjusted to 3.0 to 9.0, preferably 5.0 to 8.5, more preferably 6.0 to 8.0, and further preferably 6.0 to 7.0, and cultured. The pH may be adjusted at the start of culture to a constant pH, or may be adjusted at regular intervals after culture adjustment. The culture pH can be adjusted using an inorganic or organic acid, an alkaline solution, etc.

[0021] After the culture, a culture supernatant is obtained by separating the cells from the medium components. The separation method may be a common method such as centrifugation or filter press. The culture supernatant can be used as the enzyme solution, but the enzyme solution may also be obtained by purification through UF concentration, hydrophobic chromatography, ion exchange chromatography, gel filtration chromatography, etc.

[0022] The enzyme solution can be used as it is as an elastin-degrading enzyme, but it may be dried. There are no limitations on the drying method, but an appropriate drying method that does not impair the enzyme activity should be selected.

[0023] The optimum reaction pH of the present invention is 7.0 to 11.0. The optimum reaction pH of the present invention is evaluated as a relative value when the activity value at pH 8.0 is taken as 100%, and refers to a pH at which 80% or more of activity is maintained.

[0024] The optimum temperature in the present invention is 50 to 80° C. The optimum temperature in the present invention refers to a temperature at which the activity is maintained at 80% or more, evaluated as a relative value when the activity value at 70° C. is taken as 100%.

[0025] The pH stability of the enzyme of the present invention is equal to or lower than pH 7.0. The pH stability of the present invention is a pH at which 90% or more of the activity before the reaction is maintained even after being left at a certain pH for 24 hours.

[0026] The temperature stability of the present invention is 5 to 60° C. The temperature stability is a temperature at which 70% or more of the activity before incubation is maintained even after being left at a constant temperature and pH 7.5 for 10 minutes.

[0027] The polypeptide of the present invention obtained by the method described above is different from the elastipeptides obtained by conventional methods for degrading elastin, and therefore exhibits a degrading activity different from that of conventional methods. EXAMPLES

[0028] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. EXAMPLES

[0029] (Enzyme production) (1) Spore suspension of Streptomyces nanshensis (JCM 16226) (10 7A platinum loop of the bacteria (more than 100 cells / ml) was inoculated into 20 ml of seed medium (soluble corn starch 30 g / l, corn steep liquor 30 g / l, ammonium sulfate 1 g / l, magnesium sulfate 0.5 g / l, calcium carbonate 3 g / l, pH 7.0) and cultured in a 200 ml Erlenmeyer flask at 28°C and 200 rpm for 12 hours to obtain a completed seed culture liquid. (2) 1 ml of the resulting seed culture solution was transferred to 2000 ml of main medium (soluble corn starch 300 g / l, corn steep liquor 150 g / l, defatted soy flour 250 g / l, ammonium sulfate 10 g / l, magnesium sulfate 5 g / l, calcium carbonate 30 g / l, pH 7.0) and cultured in a 10-liter flask at 28°C and 200 rpm for 40 hours to obtain a main culture solution. (3) The enzyme-treated liquid was filtered through a filter press to remove solid matter such as bacteria, and then treated with a UF membrane with a molecular weight cutoff of 6000. The concentrated liquid fraction was recovered and spray-dried to obtain a polypeptide.

[0030] (Enzyme activity) The purified enzyme was examined for its molecular weight by SDS-polyacrylamide gel electrophoresis. After electrophoresis, it was stained with Coomassie Brilliant Blue (Sigma). The results are shown in Figure 1. Bands were confirmed at approximately 18 kDa, 14 kDa, and 11 kDa.

[0031] (Expression of elastase activity) The internal amino acid sequences and N-terminal amino acid sequences of the approximately 18 kDa, 14 kDa, and 11 kDa proteins were analyzed by standard methods, and it was found that all of the proteins were identical but had been shortened by processing from the N-terminus. Furthermore, the full length of the elastase gene was determined by standard methods, and it was found to be a gene consisting of a 726 bp base sequence. The base sequences corresponding to the approximately 18 kDa, 14 kDa, and 11 kDa proteins were cloned into the multicloning site of pNCMO2 or pNY326 (manufactured by Takara Bio Inc.), and expression analysis was performed using the Brevibacillus expression system (manufactured by Takara Bio Inc.). As a result, elastin decomposition activity was confirmed for all three proteins.

[0032] (Examination of substrate characteristics) The decomposition of myofibrils and elastin was examined using the enzyme prepared in Example 1. The elastin decomposition activity was determined by using Elastin Congo Red (manufactured by Nacalai Tesque) as a substrate, and the enzyme activity that changes the absorbance at 495 nm by 1 in 1 hour was defined as 1U. The decomposition of myofibrils was determined by adding 100μl of an enzyme solution prepared at 1U / ml to 900μl of a 1% myofibrils solution prepared from beef shoulder meat, and reacting at pH 8.0 and 25℃ for 6 hours. As a result, as shown in Figure 2, it was revealed that the decomposition of myofibrils was weaker than four commercially available neutral and alkaline proteases (Biophrase OP, Bioprase SP-20FG (both manufactured by Nagase Chemtex), Alcalase (manufactured by Novozymes), and Protin SD-AY10 (manufactured by Amano Enzyme)). The decomposition of elastin was investigated by adding 100 μl of 2 U / ml enzyme solution to 900 μl of 0.6% bovine ligament-derived elastin and reacting for 8 hours at pH 8.0 and 25°C. As a result, as shown in Figure 3, it was revealed that the decomposition of elastin by this enzyme was significantly stronger than that of commercially available enzymes.

[0033] (Study of optimum temperature, optimum pH and temperature stability) Similar to the investigation of substrate properties, various temperatures, pH, and temperature stability were measured. Elastin decomposition activity was measured under the optimum pH conditions of pH 5.0-7.0 (phosphate buffer), pH 7.0-8.5 (Tris-HCl buffer), and pH 8.5-11.0 (carbonate buffer). The activity was evaluated relative to the activity value at pH 8.0, which was taken as 100%, and showed high enzyme activity over a wide pH range from pH 7.0 to 11.0, with more than 80% activity being retained (Figure 4). The optimum pH was also 8.0. The elastin decomposition activity was measured under various conditions with the optimum temperature ranging from 4° C. to 80° C. As a result, activity was observed over a wide temperature range from 4° C. to 80° C., with the optimum temperature being 70° C. (FIG. 5). Temperature resistance was evaluated based on the remaining activity after treatment for 10 minutes at temperatures ranging from 30° C. to 70° C. As a result, the enzyme had high remaining activity at temperatures ranging from 30° C. to 60° C. (FIG. 6).

[0034] (Explanation of decomposition activity) The decomposition activity of elastin contained in the bulbus arteriosus of skipjack tuna was examined. The polypeptide obtained in the Production Example was used to decompose elastin using the method described in JP-A-2005-343851, and elastin peptides were compared. Furthermore, the polypeptide obtained in the Production Example was used to process elastin obtained from the bulbus arteriosus of yellowfin tuna and albacore tuna. The results are shown in Figure 7. [Industrial Applicability]

[0035] As described above, the enzyme of the present invention can produce elastin peptides with a different decomposition pattern compared to conventional elastin-decomposing enzymes. Furthermore, although the enzyme of the present invention has high elastin decomposition activity, it does not decompose red meat, so it can be used as an enzyme to decompose meat tendons and the like and to tenderize meat.

Claims

1. A polypeptide having specific decomposition activity against elastin, said polypeptide having the same amino acid sequence as the amino acid sequence represented by sequence number 1, and having a molecular weight of 18 kDa as shown by SDS-polyacrylamide gel electrophoresis.

2. 2. The polypeptide of claim 1, which is derived from the genus Streptomyces.

3. 3. A polypeptide having decomposition activity against the elastin described in claim 1 or 2, which is an enzyme having the following properties: (1) Optimum reaction pH: pH 7.0 to 11.0 (2) Optimum reaction temperature: 50 to 80° C.

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