JP5515057B2 - Cryogenic bacteria producing collagen degrading enzyme, collagen degrading enzyme, method for producing the same, and method for producing softened meat using the enzyme - Google Patents

Description

  The present invention relates to a psychrotrophic bacterium that produces a collagenolytic enzyme, a collagenolytic enzyme, a method for producing the same, and a method for producing softened meat using the enzyme.

  Meat is processed from meat of livestock and poultry for food and is used as a major source of protein intake. However, some muscles such as livestock and poultry have low utility value as meat because the meat quality is hard depending on the region and breeding method, such as striped meat and dairy cow meat. If such hard meat can be softened, it can be effectively used as a protein resource, and it becomes possible to develop foods that can be easily chewed, thereby increasing the commercial value of hard meat as meat.

  On the other hand, proteins contained in meat can be broadly classified into myofibrillar proteins such as myosin and actin, connective tissue proteins such as collagen, and myoplasm proteins such as myoglobin. Among these, myosin and actin, which are the main components of myofibrillar proteins, are proteins involved in meat texture (softness, flavor and juiciness). Collagen, which is the main component of connective tissue protein, is a protein involved in meat hardness. Meat hardness is due to the complexity and increase in the cross-linked state between collagen molecules due to aldol condensation and Schiff base formation. Therefore, conventionally, protease treatment has been widely used as a method for softening meat (meat softening method). Plant-derived papain, bromelain, and the like are used as the protease (Non-patent Document 1). However, since the protease has a higher substrate specificity for myofibrillar protein than collagen, it degrades myofibrillar protein preferentially and impairs the texture of meat. In addition, the protease has low enzyme activity at low temperatures and does not act in an environment of 4 ° C. or lower where meat is ripened and stored. Therefore, the use of the protease under low temperature conditions requires a large amount of enzyme, has low efficiency, and deteriorates taste. In addition, the use of the protease in the vicinity of 30 ° C. having enzyme activity is not preferable from the viewpoint of safety because microorganisms are easy to propagate.

Prusa, K .; J. et al. et al. J. et al. Food Sci. 1981, Vol. 46, p. 1684-1686

  Accordingly, an object of the present invention is to produce a collagen-degrading enzyme that has collagen-degrading activity at low temperatures and preferentially degrades collagen, the collagen-degrading enzyme produced, a method for producing the same, and softening using the same It is to provide a method for producing meat.

In order to achieve the object, cold bacterium of the present invention has a trait of the following (1) to (8), produce collagen degradation enzyme shown in the following (9), bacterial names, Shewanella sp & Strain C35 (Shewanella sp. Strain C35),
NITE P-520 is the accession number of the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation .
(1) Motile bacilli (2) Size is about 3μm x about 1μm
(3) Gram negative (4) Cell color at the time of collection is red (5) Range of viable temperature is about 4 to about 37 ° C
(6) The optimal growth temperature range is about 20 to about 33 ° C.
(7) The base sequence of 16S rRNA gene that does not produce hydrogen sulfide under aerobic conditions is the base sequence described in (a) or (b) below: (a) the base sequence described in SEQ ID NO: 1 (B) a base sequence in which one or several bases are substituted, added, inserted or deleted in the base sequence set forth in SEQ ID NO: 1 (9) a molecular weight by SDS-PAGE method is 60 to 72 kDa,
Has collagen degradation activity in the temperature range of about 0 to about 50 ° C., and the optimum temperature is in the range of about 20 to about 40 ° C .;
has collagen degradation activity in the pH range of about 5 to about 9, and the optimum pH is in the range of about pH 6 to about 8,
Collagen degradation activity is inhibited by EDTA, EGTA and 1,10-phenanthroline monohydrate,
Collagen degradation to degrade (7-methoxycoumarin-4-yl) acetyl-prolyl-leucyl-glycyl-leucyl- [N ^β- (2,4-dinitrophenyl) -2,3-diaminopropionyl] -alanyl-argininamide enzyme

The collagenolytic enzyme of the present invention is
The molecular weight by SDS-PAGE method is 60-72 kDa,
Has collagen degradation activity in the temperature range of about 0 to about 50 ° C., and the optimum temperature is in the range of about 20 to about 40 ° C .;
has collagen degradation activity in the pH range of about 5 to about 9, and the optimum pH is in the range of about pH 6 to about 8,
Collagen degradation activity is inhibited by EDTA, EGTA and 1,10-phenanthroline monohydrate,
(7-methoxycoumarin-4-yl) acetyl-prolyl-leucyl-glycyl-leucyl- [N ^β- (2,4-dinitrophenyl) -2,3-diaminopropionyl] -alanyl-argininamide ,
It is a collagenolytic enzyme that does not degrade myosin heavy chain .

  The method for producing a collagenolytic enzyme of the present invention is a method for producing the collagenolytic enzyme of the present invention, and includes a culture step of culturing the psychrophilic bacteria of the present invention to produce the collagenolytic enzyme.

  The method for producing softened meat of the present invention is a method for producing softened meat in which meat is softened by enzyme treatment, and the collagenolytic enzyme of the present invention is used as the enzyme.

  If the psychrophilic bacterium of the present invention is used, the collagenolytic enzyme of the present invention can be easily produced by culturing the bacterium. If the collagenolytic enzyme of the present invention is used, meat can be softened with a small amount of enzyme even under low temperature conditions. Moreover, since the collagen-degrading enzyme of the present invention acts specifically on collagen, the meat can be softened without damaging the texture of the meat. Furthermore, since the manufacturing method of the softened meat using the collagenolytic enzyme of this invention can be implemented on low-temperature conditions, it can suppress the propagation of microorganisms and is excellent in food safety, for example.

  Generally, a psychrophilic bacterium refers to a bacterium that can grow in a low-temperature environment such as Takayama, the deep sea, and a refrigerator. In addition, according to the definition of Morita et al. (Bacteriologic Reviews, 1975, Vol. 39, p. 144-167), psychrophilic bacteria are considered to be psychrophiles having an optimum temperature of 15 ° C. or less and an upper limit temperature of growth of 20 ° C. or less. The growth upper limit temperature is classified as a psychrotrophic bacterium having a growth upper limit temperature of 20 ° C. or higher. In addition, the psychrophilic bacteria in this invention should just have the above-mentioned character.

Low-temperature bacteria of the present invention, bacteria name is a Shewanella sp., Strain C35 (Shewanella sp. Strain C35) , National Institute of Technology and Evaluation accession number of the Patent Microorganisms Depositary Center, at NITE P-520 Oh Ru.

  The collagenolytic enzyme of the present invention is not particularly limited, and may be, for example, the enzyme derived from the psychrophilic bacteria of the present invention.

  The method for producing the collagenolytic enzyme of the present invention is not particularly limited. For example, the method may further comprise a recovery step of recovering the supernatant containing the collagenolytic enzyme from the culture solution of the low-temperature bacteria.

  Although the manufacturing method of the softened meat of this invention is not restrict | limited in particular, For example, it is preferable to perform the said enzyme treatment on 0-37 degreeC temperature conditions.

  The present invention is described in detail below.

<Pyrogen>
The psychrotrophic bacterium of the present invention is a bacterium having the traits of (1) to (8) above and producing the collagenolytic enzyme shown in (9) above, and having the accession number NITE P-520, Shuwanella sp. Strain C35 ( Shewanella sp. Strain C35 ) . As will be described later, this microbial cell is a novel microbial cell isolated by the present inventors, and deposited at the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation, under the accession number NITE P-520. Yes.

In the present invention, the species of the genus Shewanella is not particularly limited, and examples thereof include Shewanella putreffaciens species and Shewanella baltica species. Bacteria of the Shewanella sp (Shewanella) may be a closely related species of the Shewanella sp (Shewanella).

  As described above, the viable temperature of the psychrophilic bacteria of the present invention is in the range of about 4 ° C. to about 37 ° C., but is not limited thereto, and is, for example, in the range of about −5 ° C. to about 50 ° C. May be. In addition, the psychrophilic bacteria of the present invention may be able to grow outside these temperature ranges. Further, as described above, the optimum growth temperature of the psychrophilic bacteria of the present invention is in the range of about 20 ° C. to about 33 ° C., but is not limited thereto, and is, for example, in the range of about 10 to about 40 ° C. May be. Even outside these temperature ranges, the psychrotrophic bacteria of the present invention may exhibit growth optimality.

<Collagen-degrading enzyme>
The collagenolytic enzyme of the present invention is not particularly limited as long as it has the above-mentioned properties, and examples thereof include the enzyme derived from the psychrophilic bacteria of the present invention.

  As described above, the collagenolytic enzyme of the present invention has collagen degrading activity in the temperature range of 0 to 50 ° C. That is, the collagenolytic enzyme of the present invention has collagen degrading activity in a temperature range of 2 to 10 ° C. which is a general refrigeration temperature. In addition, the collagenolytic enzyme of the present invention has collagen degrading activity even within a temperature range of 0 to 2 ° C. that is an ice temperature. The collagenolytic enzyme of the present invention may have activity even outside these temperature ranges.

  In addition, the collagenolytic enzyme of the present invention has, for example, temperature stability at 50 ° C. or less, and may have temperature stability at 60 ° C. or less. For this reason, the collagenolytic enzyme of the present invention still retains the collagenolytic activity, for example, after being maintained in the temperature range for a certain time.

The collagenolytic enzyme of the present invention is preferably reacted, for example, in the presence of a divalent cation. Specific examples include Ca ²⁺ and Mg ²⁺ .

  As described above, the collagenolytic activity of the collagenolytic enzyme of the present invention is inhibited by EDTA, EGTA and 1,10-phenanthroline monohydrate. However, inhibition of the collagen degradation activity may occur with protease inhibitors other than EDTA, EGTA, and 1,10-phenanthroline monohydrate.

Collagenase of the present invention, as described above, (7-methoxy-coumarin-4-yl) acetyl - prolyl - leucyl - glycyl - leucyl - [N ^beta - (2,4-dinitrophenyl) -2,3 Diaminopropionyl] -alanyl-arginine amide. This substrate is represented by, for example, MOCAc-Pro-Leu-Gly-Leu-A ₂ pr (Dnp) -Ala-Arg-NH ₂ (manufactured by Peptide Institute, Inc.), but hereinafter referred to as “PLGL (D) AR " PLGL (D) AR is a fluorescent synthetic substrate, (7-methoxycoumarin-4-yl) acetyl group (MOCAc) is a fluorescent group, N ^β- (2,4-dinitrophenyl) -2,3-diamino The propionyl group (A ₂ pr (Dnp)) is a quenching group. The fluorescent peptide MOCAc is bound to the C-terminal side of the tetrapeptide Pro-Leu-Gly-Leu, and the dipeptide Ala-Arg is bound to the N-terminal side of the tetrapeptide via the quenching group A ₂ pr (Dnp). doing. The reaction rate constant for the PLGL (D) AR collagenolytic enzyme of the present invention (kcat / Km) is not particularly limited, for example, be in the range of 150,000~200,000M ^-1 · ^{s -1} Good.

  The collagenolytic enzyme of the present invention may have, for example, an enzyme activity other than collagenolytic activity. In addition, when used for softening meat as described later, the collagenolytic enzyme of the present invention preferably has a low degrading activity on myofibrillar proteins such as myosin and actin, and the degrading activity of the myofibrillar protein. It is more preferable not to have.

<Method for producing collagen degrading enzyme>
Although the manufacturing method of the collagenolytic enzyme of this invention is not specifically limited, For example, as above-mentioned, it can manufacture by culture | cultivating the low temperature bacteria of this invention, and producing a collagenolytic enzyme.

  The culture method of the psychrophilic bacteria is not particularly limited, and can be appropriately performed based on conventionally known culture methods.

  The form of the medium used for the culture of the psychrophilic bacteria is not particularly limited, and for example, a conventionally known medium such as a solid medium or a liquid medium can be appropriately used. The components contained in the medium are not particularly limited, and examples include nitrogen sources, sugars, and salts. The nitrogen source is not particularly limited, and examples thereof include casein, gelatin, fish-derived protein, and bean-derived protein. Moreover, the said saccharides are not specifically limited, For example, glucose, sucrose, fructose, raffinose etc. are mentioned. The salts are not particularly limited, and examples thereof include sodium chloride, dipotassium hydrogen phosphate, and magnesium chloride. The medium may include a commercially available medium, for example. The commercially available medium is not particularly limited, and for example, tryptic soy broth (manufactured by Difco), Bacterio-N KN, Bacterio-N KS, Bacterio-N SH, Bacterio-N PF (manufactured by Maruha Corporation), polypeptone ( Wako Pure Chemical Industries, Ltd.), gelatin peptone (Nacalai Tesque, Inc.) and the like. One kind of these may be used, or two or more kinds may be used in combination. The pH of the culture medium is not particularly limited, and is, for example, in the range of pH 3-11, preferably in the range of pH 5-8.

  The method for cultivating the psychrophilic bacteria is not particularly limited, and examples thereof include a plate culture method and a slope culture method when the solid medium is used. Moreover, when using the said liquid culture medium, it does not specifically limit, For example, a stationary culture method, an aeration culture method, a shaking culture method etc. are mentioned.

  The culture temperature is not particularly limited, and examples thereof include the above-described temperature at which the above-mentioned psychrophilic bacteria can grow and the optimum growth temperature. Specifically, for example, it can be carried out in the range of -5 to 50 ° C, the range of 10 to 40 ° C, and the like.

  The psychrophilic bacteria may be cultured under an aerobic condition or an anaerobic condition, for example. The aerobic condition or the anaerobic condition is not particularly limited, and can be set using a conventionally known method.

  The method for producing a collagenolytic enzyme of the present invention may further comprise, for example, a collagenolytic enzyme recovery step.

  The recovery step is not particularly limited. However, since the psychrotrophic bacterium of the present invention releases the collagenolytic enzyme of the present invention to the outside of the microbial cell, for example, the collagenolytic enzyme of the present invention from the culture medium in which the psychrophilic bacteria are cultured. A step of collecting the supernatant (liquid fraction) containing The method for recovering the supernatant is not limited at all, and can be recovered by removing the cultured bacteria by, for example, centrifugation or filtration. The centrifugation and filtration treatment can be appropriately performed using a conventionally known method and is not particularly limited.

  The recovery step may further include, for example, a step of purifying the collagenolytic enzyme of the present invention. The purification of the enzyme may be, for example, partial purification or complete purification. The method for purifying the collagenolytic enzyme of the present invention is not particularly limited, and a conventionally known method can be adopted. As specific examples, for example, salting-out, ultrafiltration, hydrophobic interaction chromatography, chromatography such as molecular sieve chromatography and affinity chromatography, and other conventionally known purification techniques can be employed. Examples of the method for purifying the collagenolytic enzyme of the present invention include, for example, a method of sequentially performing salting out, hydrophobic interaction chromatography, and gel filtration chromatography which is molecular sieve chromatography. Not limited.

  The microbial cells used in the production method of the present invention are not particularly limited. In addition to the psychrophilic bacteria of the present invention, for example, microorganisms, plant cells, and animal cells transformed by introducing the collagenase gene of the present invention. And so on. The microorganisms and cells to be transformed are not particularly limited, and the transformation method is not particularly limited, and can be appropriately determined according to the type of host into which the gene is introduced.

<Method for producing softened meat>
The method for producing softened meat of the present invention is characterized by using the collagenolytic enzyme of the present invention for enzyme treatment of meat. For this reason, other processes, conditions, etc. are not restrict | limited at all other than using the collagenolytic enzyme of this invention, for example.

  In the method for producing softened meat of the present invention, the form of the collagenolytic enzyme is not particularly limited, and examples thereof include a powder form and a liquid form in which the enzyme is dissolved or dispersed.

  The method for applying the collagen-degrading enzyme to meat is not particularly limited, and examples thereof include coating, pouring, and dipping. The application method is not particularly limited, and for example, the collagenolytic enzyme may be sprinkled and rubbed directly on the meat surface. In addition, the injection method is not particularly limited, and for example, a liquid collagenolytic enzyme may be injected into meat using a syringe or the like. The soaking method is not particularly limited, and for example, the meat may be soaked in a solution containing the collagenolytic enzyme of the present invention. The amount of the collagen degrading enzyme with respect to the meat is not particularly limited.

  Although the temperature of the said enzyme treatment is not specifically limited, For example, it is the range of 0-37 degreeC, Preferably, it is the range of 0-20 degreeC. In addition, the said enzyme process is not restricted to this temperature range, For example, it can process also at 0 degrees C or less, for example, -5-0 degreeC.

  The number of days for the enzyme treatment is not particularly limited, but is, for example, in the range of 1 to 1000 days, and preferably in the range of 1 to 10 days.

  In the present invention, the kind of meat to be enzyme-treated is not particularly limited. For example, edible meat such as cattle, pigs, chickens, sheep, and whales, and organs thereof, and edible fish meat such as squid, shrimp and tuna and organs thereof. Etc. The meat may be, for example, raw meat, frozen meat, or processed meat, and is not particularly limited. The part of the animal meat is not particularly limited, and examples thereof include a neck, shape, rose, fin, ribulose, sirloin, and shin. The site of the organ is not particularly limited, and examples thereof include tongue, heart, diaphragm, Achilles tendon, stomach, intestine, liver, uterus, testis, ovary, egg, and skin. The method for producing softened meat according to the present invention can be used, for example, for softening and improving the quality of processed meat such as roast beef, sheep intestine and pork intestine used for sausage casings, and the like.

  Next, examples of the present invention will be described. However, the present invention is not limited to the following examples.

Example 1
The psychrophilic bacteria of this example were prepared by the following procedure.

(1) Isolation of psychrotrophic bacteria First, a soil sample in a cold region and a food (rice cooked rice, Nanban, bean sprouts, chestnut syrup) stored for a long time in a refrigerator were prepared. After adding 1 mL of physiological saline (0.88 (w / v)% NaCl) and suspending to about 0.5 g of each of the soil and food samples, further adding physiological saline to 1 × 10 ⁴ to 1 × It was diluted 10 ⁶ times. Each diluted solution was smeared on a tryptic soy agar medium (manufactured by Difco) prepared by a conventional method and cultured at 4 ° C. for 10 days. After completion of the culture, the formed colonies were isolated by streak culture.

(2) Screening with collagen gel medium Firstly, it was prepared from pig skin based on the collagen preparation method described in the reference (Journal of Agricultural and Food Chemistry, 2003, Vol. 51, No. 27, p. 8088-8092). Acid-soluble collagen was dissolved in an acetic acid solution (pH 4.0) at a concentration of 10 mg / mL to prepare a collagen solution. A 45 mg / mL tryptic soy broth solution and the collagen solution were mixed in equal amounts in a Petri dish or a 96-well plate, and then incubated at 37 ° C. for 1 hour to prepare a collagen gel medium. The collagen gel medium was sterilized by irradiation with ultraviolet rays (254 nm) for 1 hour. The collagen gel medium is allowed to stand at 4 ° C. overnight, and then a small amount of cells are scraped off from the isolated colonies of cold bacteria, transferred onto the collagen gel medium, and cultured at 4 ° C. for 3 to 5 days. did. After completion of the culture, changes in the collagen gel medium were observed. Bacteria in which the collagen gel medium was dissolved and changed to be transparent were used as the low-temperature bacteria of this example.

  Table 1 below shows the screening results of the psychrophilic bacteria of this example. 692 colonies of psychrophilic bacteria were isolated from 55 samples of soil and food collected from a low temperature environment. Among these isolated psychrophilic bacteria, 10 colony psychrophilic bacteria transparently liquefied the collagen gel medium used for the culture and showed collagen degrading activity. Among these, the C35 strain was the psychrophilic bacterium that liquefied the collagen gel medium in the widest range in a short period of time. The fungus color of the collected C35 was red.

(Table 1)
Collection source Number of samples Number of bacteria Number of active bacteria Soil in cold region 1 17 169 8
Soil in cold region 2 10 120 0
Soil in cold regions 3 5 30 0
Soil in cold regions 4 7 233 2
Soil in cold regions 5 11 126 0
Food in refrigerator 5 14 0
Total 55 692 10

(3) Phase-contrast microscope observation In order to observe the C35 strain with a phase-contrast microscope, an agar-coated slide glass was prepared as follows. First, after adding 200 mL of pure water to 2.2 g of agar powder (manufactured by Wako Pure Chemical Industries, Ltd.) and mixing for 10 minutes, the agar powder was allowed to settle and the supernatant was removed to remove water-soluble impurities contained in the agar. . After this removal process was performed three times, 100 mL of pure water was added to the agar powder from which impurities were removed and heated to dissolve the agar. The obtained agar solution was smeared on a slide glass using a pipette and then dried overnight to prepare an agar-coated slide glass. On the other hand, the psychrophilic bacteria of this example were suspended in an aqueous solution to prepare a cell suspension. The bacterial cell suspension was dropped onto the agar-coated slide glass, covered with a cover glass, and observed with an inverted microscope (ECLIPSE TE2000-U, manufactured by Nikon Corporation). The lens magnification used for the observation was 10 times for the eyepiece and 100 times for the objective lens.

  As a result, it was confirmed that the C35 strain is a koji mold having a size of about 3 μm × about 1 μm and exhibits motility.

(4) Gram staining observation Gram staining of the C35 strain was performed as follows. First, crystal violet solution and Lugol solution having the following composition were prepared. Further, safranin was dissolved in ethanol at a concentration of 2.5%, and further diluted 10 times with pure water to prepare a safranin solution.

(Composition of crystal violet solution)
Ingredients Amount
Crystal violet 2g
Ethanol 20mL
0.8g ammonium oxalate
80mL pure water

(Composition of Lugol solution)
Ingredients Amount
Iodine 1g
Potassium iodide 2g
300 mL of pure water

  The cell suspension was applied on the agar-coated slide glass and dried. After drying, the slide glass was passed 2 to 3 times above the flame of the gas burner to evaporate the water of the cell suspension, and the cells were fixed on the slide glass. Then, the crystal violet solution was dropped on the slide glass, and the fixed cells were stained for 1 minute. Next, the slide glass was washed with water, and the Lugor solution was applied to the cells for 1 minute to insolubilize the pigment. Further, the slide glass was washed with water and decolorized using 95% ethanol. After washing again with water and air-drying, the safranin solution was added dropwise and stained for 1 minute. After washing with water and air drying, the Gram-stained cells were observed using an inverted microscope (ECLIPSE TE2000-U, manufactured by Nikon Corporation). The microscope lens magnification was 10 times for the eyepiece and 100 times for the objective lens.

  The results of Gram-staining the C35 strain are shown in FIG. As a result, the C35 strain was stained red and confirmed to be a Gram-negative bacterium.

(5) DHL medium culture DHL agar medium “DAIGO” (manufactured by Nippon Pharmaceutical Co., Ltd.) 64 g and glucose 10 g were dissolved in 1 L of purified water by heating and autoclaved, then dispensed into a Petri dish or 96-well plate and solidified on a flat plate. A DHL gel medium was prepared. A small amount of bacterial cells were scraped from the isolated colonies of psychrophilic bacteria, transferred onto the DHL gel medium, and cultured at 28 ° C. for 2 days. After incubation, the colony color was visually observed to confirm the presence or absence of hydrogen sulfide production.

  The C35 strain cultured using DHL medium formed a red colony. From this result, it was confirmed that the C35 strain does not produce hydrogen sulfide under aerobic conditions.

(6) Analysis of 16S rRNA gene sequence For the psychrophilic bacteria of this example, the 16S rRNA gene sequence was analyzed as follows.

(6-a) Extraction of genomic DNA First, a buffer solution, an RNase solution, and a CTAB / NaCl solution having the following composition were prepared. Further, chloroform and isoamyl alcohol were mixed at a ratio of 24: 1 to prepare a CI solution. Further, 30 g of tryptic soy broth (manufactured by Difco) was dissolved in 1 L of purified water to prepare a tryptic soy broth medium.

(Composition of buffer solution)
Ingredient concentration
Tris-HCl buffer solution (pH 8.0) 10 mM
EDTA 1 mM
NaCl 5M
Sodium acetate solution (pH 5.2) 3M

(Composition of RNase solution)
Ingredient concentration
Tris-HCl buffer solution (pH 7.5) 10 mM
NaCl 15 mM
RNase (Wako Pure Chemical Industries, Ltd.) 10mg / mL

(Composition of CTAB / NaCl solution)
Ingredient concentration
CTAB 10w / v%
NaCl 0.7M

  The psychrophilic bacteria of this example were cultured with shaking in the above tryptic soy broth medium at 28 ° C. and 110 rpm (reciprocating) for 12 hours. After culture, 500 μL of the culture solution was placed in a microtube, and after centrifugation, the supernatant was removed. 1 mL of physiological saline was added to and suspended in the obtained microbial cells, and after centrifugation, the step of removing the supernatant was performed three times to wash the microbial cells. Sterile ultrapure water was added to the washed cells and stored frozen at -20 ° C. 40 μL of the frozen cell suspension was thawed on ice, and 567 μL of the buffer solution was added. Furthermore, 30 μL of 10 w / v% SDS (sodium dodecyl sulfate) and 3 μL of 20 mg / mL proteinase K (manufactured by Wako Pure Chemical Industries, Ltd.) were added, followed by incubation at 37 ° C. for 1 hour after vortexing.

100 μL of 5M NaCl was added to the incubated cell suspension, and vortexed to mix. Further, 80 μL of the CTNB / NaCl solution was added and mixed, and incubated at 65 ° C. for 10 minutes. After incubation, add an equal amount of the CI solution, vortex for 20 seconds, and centrifuge for 5 minutes at 25 ° C. and 20000 × g (g represents acceleration of gravity (9.80665 m / sec ² ), the same applies hereinafter). did. The upper aqueous layer was collected, an equal amount of the CI solution was added, and the mixture was further centrifuged for 5 minutes under the above conditions. The upper aqueous layer was collected, 1.5 times the volume of isopropanol was added, and the mixture was allowed to stand at room temperature for 10 minutes. After standing, the mixture was centrifuged for 10 minutes under the above conditions. The supernatant was removed, and 1 mL of 70 w / w% ethanol was added to the precipitated DNA. After mixing, the mixture was further centrifuged for 5 minutes at 4 ° C. and 20000 × g. The resulting precipitate was dried using a centrifugal freeze dryer to prepare genomic DNA. The genomic DNA was dissolved in 100 μL of the buffer solution, 1 μL of the RNase solution was added, and incubated at 37 ° C. for 1 hour. Further, an equal amount of CI solution was added, vortexed for 20 seconds, and then centrifuged for 15 seconds at 25 ° C. and 20000 × g. After centrifugation, the upper aqueous layer was collected and transferred to another tube. 100 μL of the buffer solution was added to the lower layer precipitate, vortexed for 20 seconds, and then centrifuged at 25 ° C. and 20000 × g for 15 seconds. After centrifugation, the upper aqueous layer was collected and added to the aqueous layer that had been transferred to the other tube. To 200 μL of the obtained aqueous layer, 20 μL of 3M sodium acetate solution and 500 μL of 100% ethanol were added and mixed, and allowed to stand at room temperature for 10 minutes. After standing, the mixture was centrifuged at 4 ° C. and 20000 × g for 10 minutes, and the supernatant was removed. 1 mL of 70 w / w% ethanol was added to the resulting precipitate and mixed, followed by centrifugation at 4 ° C. and 20000 × g for 5 minutes. The supernatant was removed, and the resulting precipitate was dried and dissolved in 100 μL of the buffer solution to prepare a DNA solution. The DNA solution was stored frozen at −20 ° C. until use.

(6-b) Agarose gel electrophoresis First, an electrophoresis buffer and a loading buffer having the following composition were prepared.

(Composition of electrophoresis buffer (× 50 TAE))
Ingredients Amount
Tris 242g
Acetic acid 57.1 mL
EDTA · 2Na (2H ₂ O) 18.6 g
The above three components were mixed and made up to 1000 mL with pure water.

(Composition of loading buffer)
Ingredients Amount
Bromophenol blue 25mg
Xylene cyanol FF 25mg
Glycerol 3mL
0.5 M EDTA aqueous solution (pH 8.0) 100 μL
The above four components were mixed and made up to 10 mL with pure water.

  The electrophoresis buffer was added to agarose (Nacalai Tesque) and heated to prepare a 0.6% agarose gel solution. The agarose gel solution was cooled to 50 to 60 ° C. and then poured into a gel forming tray on which a gel tray and a comb were set. After the agarose gel was solidified, the comb was removed, and the agarose gel was set in an electrophoresis tank (product name “Genius”, manufactured by SK Bio International). The buffer solution was added to the electrophoresis tank to such an extent that the agarose gel surface was immersed. Meanwhile, the DNA solution and the loading buffer were mixed at a ratio of 5: 1 to prepare an electrophoresis sample. After the electrophoresis sample was applied to the well, electrophoresis was performed at a voltage of 100 V until bromophenol blue moved about 70% of the gel length.

  After electrophoresis, the agarose gel was immersed in a 0.5 μg / mL ethidium bromide solution for 30 minutes. After immersion, the agarose gel was irradiated with 302 nm ultraviolet rays using an ultraviolet irradiation device and photographed with a digital camera.

(6-c) Amplification of 16S rRNA gene For the psychrophilic bacteria of this example, the base sequence of the 16S rRNA gene was analyzed as follows.

  First, PCR reaction was performed using the extracted DNA solution. In the PCR reaction, TaKaRa Ex Taq (registered trademark, manufactured by Takara Bio Inc.) was used as a DNA polymerase, and the following primer sets (SEQ ID NOs: 2 to 7) were used as primers. Moreover, PCR reaction was performed on the following PCR conditions using the thermal cycler.

(Primer set 1)
Forward primer 16s F1-1: 5′-agagtttgatcctggctcag-3 ′ (SEQ ID NO: 2)
Reverse primer 16s r3: 5'-ttaccgcggctgctggc-3 '(SEQ ID NO: 3)

(Primer set 2)
Forward primer 16s F3: 5'-gccagcagccgcggtaa-3 '(SEQ ID NO: 4)
Reverse primer 16s r4: 5'-ccgtcaattcctttgagttt-3 '(SEQ ID NO: 5)

(Primer set 3)
Forward primer 16s F4: 5'-aaactcaaaggaattgacgg-3 '(SEQ ID NO: 6)
Reverse primer 16s r1: 5'-aaggaggtgatccagcc-3 '(SEQ ID NO: 7)

(Primer set 4)
Forward primer 16s F1-1: 5′-agagtttgatcctggctcag-3 ′ (SEQ ID NO: 2)
Reverse primer 16s r1: 5'-aaggaggtgatccagcc-3 '(SEQ ID NO: 7)

(PCR conditions: composition of reaction solution)
Compounding amount
10 × Reaction buffer 2.5μL
dNTP mixture (2.5 mM each) 2.0 μL
Forward primer (10 pM) 1.25 μL
Reverse primer (10 pM) 1.25 μL
DNA solution (0.2875 μg / mL) 0.35 μL
Sterile ultrapure water 15.45μL
Ex taq DNA polymerase (5 U / μL) 0.2 μL
MgCl ₂ (25 mM) 2.0 μL

(PCR conditions: reaction conditions)
Cycle 1: Denaturation at 94 ° C. for 3 minutes, annealing at 55 ° C. for 1 minute, extension at 72 ° C. for 2 minutes Cycle 2: denaturation at 94 ° C. for 1 minute, annealing at 55 ° C. for 1 minute, elongation at 72 ° C. for 2 minutes 3:94 PCR was performed in the order of denaturation at 1 ° C. for 1 minute, annealing at 55 ° C. for 1 minute, and extension at 72 ° C. for 10 minutes.

(6-d) Purification of 16S rRNA gene The DNA fragment amplified by the PCR reaction was electrophoresed on a 0.6% agarose gel. Agarose gel electrophoresis was performed as described above except that the agarose gel concentration was 0.6%. The agarose gel after electrophoresis was immersed in a 0.5 μg / mL ethidium bromide solution for 30 minutes, and then the gel containing the target band was cut out while irradiating with 302 nm ultraviolet rays. The excised gel was crushed using Parafilm (registered trademark, manufactured by Pechiny Plastic Packaging), and the DNA solution was recovered. The CI solution was added in an amount equal to the recovered DNA solution and vortexed, followed by centrifugation at 4 ° C. and 20000 × g for 1 minute. The upper aqueous layer was separated into another tube, the same amount of the buffer solution was added to the remaining lower layer, and the mixture was centrifuged for 1 minute under the above conditions. The obtained upper aqueous layer was collected and added to the previously collected aqueous layer. Further, 1/10 of the total amount of 3M sodium acetate and 3 times the amount of 100% ethanol were added, left standing at −60 ° C. for 15 minutes, and then centrifuged for 15 minutes under the above conditions. After centrifugation, the resulting precipitate was dried with a centrifugal freeze dryer and dissolved in 8 μL of the buffer solution. 1 μL of the DNA lysate was electrophoresed on a 2% agarose gel to confirm the purity. The agarose gel electrophoresis was performed as described above except that the agarose gel concentration was 2%.

(6-e) Direct sequence reaction Using the DNA solution and the following reaction solution, a direct sequence reaction was performed on the purified C35 strain-derived DNA. For the sequencing reaction, BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Japan Co., Ltd.) was used. As a primer in the reaction solution, the primer set 1 is used for the DNA amplified using the primer set 1, the primer set 2 is used for the DNA amplified using the primer set 2, and the primer The primer set 3 was used for the DNA amplified using set 3. The sequence reaction was performed using a thermal cycler under the following reaction conditions.

(Reaction solution composition)
Compounding amount
DNA solution 3μL
Primer (10 pM) 2 μL
Pre-Mix * 2.5μL
Sterile ultrapure water 2.5μL
* Big Dye Terminator v3.1

(Reaction conditions)
The reaction solution was treated (denatured) at 96 ° C. for 1 minute, and then set at 35 ° C. for 30 seconds (denaturation), 50 ° C. for 15 seconds (annealing), and 60 ° C. for 4 minutes (extension) for a total of 35 sets. Repeatedly, further cooling at 4 ° C.

  After the sequence reaction, 1 μL of a 20 mg / mL glycogen solution was added, ethanol was further added to a concentration of 70% of the total amount, and the mixture was allowed to stand at −20 ° C. overnight. After centrifugation at 4 ° C. and 20000 × g for 15 minutes, 1 mL of 70% ethanol was added to the collected precipitate, mixed gently, and centrifuged again under the above conditions. The obtained precipitate was dried with a centrifugal freeze dryer, and the base sequence was examined with an auto sequencer (ABI PRISM (registered trademark) 3100-Avant Genetic Analyzer, Applied Biosystems Japan).

  The results of agarose gel electrophoresis of 16S rRNA gene DNA extracted from the C35 strain are shown in FIG. In the figure, lane 1 is the extracted genomic DNA, lane 2 is the DNA fragment amplified using the primer set 1, lane 3 is the DNA fragment amplified using the primer set 2, and lane 4 is The DNA fragment amplified using the primer set 3 and lane 5 are the results of electrophoresis of the DNA fragment amplified using the primer set 4. Further, lanes 6 to 9 are results of electrophoresis of purified DNA of the amplified DNA fragment. Lane 6 is the result of electrophoresis of the purified DNA of Lane 2, Lane 7 is the purified DNA of Lane 3, Lane 8 is the purified DNA of Lane 4, and Lane 9 is the result of electrophoresis of the purified DNA of Lane 5. is there.

  The purified DNAs in the lanes 6 to 8 were directly sequenced using the primer sets 1 to 3, and the partial sequence of the 16S rRNA gene (SEQ ID NO: 1) was determined. The base length of the partial sequence was 1536 bases.

FIG. 3 shows the result of BLAST search for the partial sequence of the 16S rRNA gene (SEQ ID NO: 1) based on the information of the Japan DNA data bank and the phylogenetic analysis. The bar indicated by 0.01 in the figure indicates an error of 1% of the total base sequence. As shown in the figure, the C35 strain was classified as Shewanella sp (Shewanella), Shewanella script referencing tumefaciens (S.putrefaciens), Shewanella W. 3-18-1 (S.W3-18-1) It was a related species of S. baltica . The C35 strain was named Shewanella sp. Strain C35 .

(Example 2)
In this example, the optimum growth temperature of the C35 strain was confirmed.

(culture)
The C35 strain was cultured with shaking in the tryptic soy broth medium at 28 ° C. and 110 rpm (reciprocating) for 12 hours. The obtained C35 strain culture solution was added to 25 mL of the tryptic soy broth medium, and the absorbance (OD ₆₀₀ ) at ₆₀₀ nm of the medium was adjusted to 0.360. Six C35 strain culture solutions with adjusted absorbance were prepared, and each was cultured with shaking at a predetermined temperature (4, 10, 20, 28, 33, 37 ° C.) and 110 rpm (reciprocating) for 4 hours.

(Measurement of growth rate)
The OD ₆₀₀ of each C35 strain culture solution cultured at a predetermined temperature (4, 10, 20, 28, 33, 37 ° C.) was measured. The relative growth rate (%) under each temperature condition was calculated with the growth rate under the temperature condition having the highest OD ₆₀₀ value as 100%.

  The results of the relative growth rate of the C35 strain under each temperature condition are shown in Table 2 and FIG. The horizontal axis of the graph is the culture temperature (° C.) of each culture solution, and the vertical axis is the relative growth rate (%). As shown in the graph, the optimum growth temperature for the C35 strain was 28 ° C. The C35 strain was able to grow under temperature conditions of 4 ° C to 37 ° C.

(Example 3)
In this example, the C35 strain was cultured using various media, and the growth rate was confirmed.

(Medium composition)
Ingredients Amount
Basic medium 20.0g
Glucose 2.5g
NaCl 5.0g
K ₂ HPO ₄ 2.5g
1000mL pure water

(Basic medium)
Basic medium (abbreviation, manufacturing company) Origin of nitrogen source (protein) Tryptic soy broth (TSB, Difco) Casein / animal polypeptone (PP, Wako Pure Chemical Industries, Ltd.) Casein gelatin peptone (GP, Nacalai Tesque) Company) Gelatin Bacterio-N KS (KS, Maruha Corporation) Tuna Bacterio-N KN (KN, Maruha Corporation) Skipjack Tuna Bacterio-N SH (SH, Maruha Corporation) Soybean Bacterio-N PF (PF, Maruha Corporation) Company) Pea

(culture)
Using the medium shown in the table, the C35 strain was cultured with shaking at 28 ° C. and 110 rpm (reciprocating) for 12 hours. The obtained C35 strain culture solution was adjusted using the medium so that the absorbance at 600 nm (OD ₆₀₀ ) was 1.000. 50 μL of the C35 strain culture solution with adjusted absorbance was added to 25 mL of the medium, and cultured with shaking at 4 ° C. and 110 rpm (reciprocating) for 3 days.

(Measurement of growth rate)
For each of the obtained C35 strain culture _was measured _{OD 600.} The relative growth rate (%) of each example was calculated by setting the OD ₆₀₀ value when TSB was used as the basic medium as the growth rate of 100%.

  The calculation results of the relative growth rate (%) of each culture solution are shown in Table 3 and FIG. In the figure, each bar of the graph indicates the type of basic medium, and the vertical axis indicates the relative growth rate (%) of each culture solution. As shown in the figure, when GP, KS, and KN were used, respectively, the growth rate was more than twice as high as the growth rate (100%) when TSB was used as the basic medium. On the other hand, when PP, SH, and PF were each used as the basic medium, their growth rate was about 20% or less when TSB was used. From these results, it was found that preferred basic media for growth of the C35 strain were TSB, GP, KS and KN.

Example 4
In this example, the C35 strain was cultured using various media, and collagen degradation activity was confirmed.

(culture)
The C35 strain was cultured using a medium containing TSB, PP, GP, KS, KN, SH or PF in the same manner as in Example 3 except that the culture period was 9 days.

(Measurement of collagen degradation activity)
Centrifuge each obtained C35 strain culture solution. Each supernatant was collected, and collagen degradation activity was measured by the following procedure.

  First, collagen substrate solutions and SDS solutions having the compositions shown in Tables 4 and 5 below were prepared. The following collagen matrix solution was prepared using a collagen solution prepared in the same manner as in Example 1 (2). 10 μL of the supernatant was added to 30 μL of the collagen substrate solution and incubated at 25 ° C. for 3 hours. Furthermore, 8 μL of the SDS solution and 2 μL of mercaptoethanol were added and heated at 100 ° C. for 2 minutes to prepare an SDS-PAGE sample. As a control, a sample without the supernatant was prepared.

(Table 4) Collagen matrix solution
Ingredients Amount
10 mg / mL collagen solution 10 μL
20 mM 10 mM Tris-HCl buffer (pH 7.5) containing 10 mM CaCl ₂

(Table 5) SDS solution
Ingredients Amount
SDS 2.5g
Glycerol 15mL
0.2% bromophenol blue 10mL
50 mM Tris-HCl buffer (pH 6.7) 10 mL
TEMED (Tetramethylethylenediamine) 0.046mL
Pure water Specified amount
50mL total

  On the other hand, a polyacrylamide gel having an upper layer gel concentration of 4% and a lower layer gel concentration of 11% was prepared. The control and the SDS-PAGE sample were applied to the polyacrylamide gel and electrophoresed at 12 mA for 120 minutes by the Laemmli method. After completion of the electrophoresis, the polyacrylamide gel was stained by the CBB staining method.

  In FIG. 6, the result of the electrophoresis showing the collagen degradation activity of each C35 strain culture solution is shown. In the figure, Ct is the control, TSB, PP, GP, KS, KN, SH and PF are the results for the supernatant of the culture solution using each basal medium. The three thick bands seen in the control lane are collagen γ chain, β chain and α chain in order from the top. When the C35 strain culture medium has collagen degrading activity, the three chains are degraded, so the color development of the band at each position becomes weak, and another band appears on the low molecule side (lower side in the figure). It is confirmed. As shown in the figure, the collagen degradation activity was particularly high in the order of TSB and KN. When other media were used, collagen degradation activity was observed in KS, PF, PP, GP, and SH in descending order of activity.

(Example 5)
In this example, the collagenolytic enzyme produced by the C35 strain was purified. The enzyme sample obtained at each purification step was measured for protein concentration and collagen degradation activity according to the following procedure.

(Measurement of protein concentration)
Protein concentration was measured using the Lowry method. Bovine serum albumin was used as the reference protein. From the obtained protein concentration and the amount of each enzyme solution, the total protein amount (mg) of the enzyme sample obtained in each purification step was calculated.

(Measurement of collagen degradation activity)
The enzyme sample was subjected to polyacrylamide gel electrophoresis by SDS-PAGE as follows. 8 μL of the SDS buffer was added to 10 μL of the enzyme sample and heated at 100 ° C. for 2 minutes to prepare an SDS-PAGE sample. In the same manner as in Example 4, electrophoresis and CBB staining were performed on the sample. The obtained gel was dried, the gel was scanned, and the collagen band area was measured using Image Quant (manufactured by GE Healthcare Biosciences). In addition, the SDS-PAGE sample to which no enzyme sample was added was used as a control, and the measurement was performed in the same manner. The amount of enzyme that reduces the β-chain band area of collagen by 1% was defined as collagen degradation activity 1 U, and was calculated based on the following formula (1). The specific activity was calculated based on the following formula (2).

(1) Culture of C35 strain The C35 strain was cultured with shaking using the TSB medium at 28 ° C. and 110 rpm (reciprocating) for 12 hours. 0.5 mL of the obtained C35 strain culture solution was added to 250 mL of the TSB medium, and cultured with shaking at 4 ° C. and 110 rpm (reciprocating) for 84 hours. After culture, the mixture was centrifuged for 15 minutes at 10,000C and 4 ° C to obtain a culture supernatant. With respect to the culture supernatant, protein concentration and collagen degradation activity were measured using the above-described methods.

  The culture supernatant had a total protein amount of 13543.549 mg and a specific activity of 4 U / mg.

(2) Fractionation with ammonium sulfate Ammonium sulfate was added to the culture supernatant to a concentration of 40% to remove 0% -40% saturated precipitate, and then ammonium sulfate was further added to a concentration of 45%. Added salting out. Centrifugation was performed at 4 ° C. and 20000 × g for 15 minutes to recover a salted out precipitate (hereinafter referred to as “40 to 45% salted out product”). The 40-45% salted-out product was dissolved in 10 mM Tris-HCl buffer (pH 7.5) containing 10 mM CaCl _2, and the solution was dialyzed against the CaCl ₂ -containing Tris-HCl buffer. After dialysis, ammonium sulfate is added to the obtained dialysate so as to have a concentration of 30%, 0% -30% saturated precipitate is removed, and then ammonium sulfate is further added until the concentration reaches 40% to salt out. I let you. Centrifugation was performed at 4 ° C. and 20000 × g for 15 minutes to recover a salted out precipitate (hereinafter referred to as “30-40% salted out product”). The 30-40% salted, dissolved in the CaCl ₂ containing Tris-HCl buffer to obtain a crude enzyme solution. About this crude enzyme liquid, the protein concentration and the collagen degradation activity were measured using the above-mentioned method. Similarly, the protein concentration and collagen degradation activity of the 40-45% salted-out product were measured.

  As a result of salting out, the 40-45% salted-out product had a total protein amount of 10.847 mg and a specific activity of 439 U / mg. Further, the crude enzyme solution obtained from the salted-out 30-40% salted out further had collagen degradation activity with a total protein amount of 1.761 mg and a specific activity of 974 U / mg.

(3) Purification by hydrophobic interaction chromatography Hydrophobic interaction chromatography using HiTrap Butyl-S FF (manufactured by GE Healthcare Biosciences), adding ammonium sulfate to the crude enzyme solution to a concentration of 1M. Further purification was performed by chromatography. In the hydrophobic interaction chromatography, protein elution operation, absorbance measurement at 280 nm, and conductivity measurement were performed using AKTA explorer 10S (manufactured by GE Healthcare Biosciences). The protein was eluted using the following elution buffer and elution conditions.

(Elution buffer)
(A): 10 mM Tris-HCl buffer (pH 7.5) containing 1 M ammonium sulfate and 10 mM CaCl ₂
(B): 10 mM Tris-HCl buffer (pH 7.5) containing 10 mM CaCl ₂

(Elution conditions)
Column size (gel volume): 1 cm ³
Concentration gradient: gradient from elution buffer (A) 100% to elution buffer (B) 100%, total 10 mL, elution flow rate: 1 ml / min
Fraction volume: 1 mL

(Confirmation of protein purification)
About each obtained fraction, the refinement | purification degree of protein was confirmed using SDS-PAGE method. First, 8 μL of the SDS buffer was added to 10 μL of the concentrated fraction obtained by concentrating 100 μL of each fraction with a centrifugal concentrator, and heated at 100 ° C. for 2 minutes to prepare an SDS-PAGE sample. Then, the sample was electrophoresed in the same manner as in Example 4. After completion of the electrophoresis, the gel was stained by a silver staining method.

(Measurement of protein concentration and collagen degradation activity)
About each obtained fraction, the protein concentration and the collagen degradation activity were measured using the above-mentioned method.

FIG. 7 shows a protein elution pattern by hydrophobic interaction chromatography and a collagen degradation activity chart of each fraction. In the same figure, the plot of Δ is the absorbance of the fraction at 280 nm and shows the amount of the eluted protein, the plot of ○ is the collagenolytic activity, and the plot of □ is the conductivity of the elution buffer. As a result, as shown in FIG. 7, collagen decomposition activity was observed in fractions 2 to 8 among fractions 1 to 8. Moreover, the result of SDS-PAGE which shows the protein purification degree of the fractions 1-8 is shown in FIG. In the figure, lane M is a molecular weight marker, and lanes 1 to 8 show the electrophoresis results of fractions 1 to 8 in order. As a result, as shown in FIG. 8, a band common to the fractions 2 to 8 was observed at a molecular weight of 70 kDa. Furthermore, the purification degree of fractions 5 to 8 was higher than other fractions. Therefore, the fractions 5 to 8 were collected and used for the next gel filtration chromatography. The fractions of fractions 5 to 8 had a total protein amount of 0.316 mg and a specific activity of 1.72 × 10 ³ U / mg.

Although not shown, the 40-45% salted-out product obtained during the salting-out step is subjected to hydrophobic interaction chromatography in the same manner as the crude enzyme solution (30-40% salted-out product). It was. As a result, the protein elution position moved to the ammonium sulfate low concentration side (low conductivity side) of the elution buffer as compared with the result of FIG. 8 of the crude enzyme solution. This movement is considered to be caused by the fact that the 40 to 45% salted-out product does not contain Ca ²⁺ . That is, in the purification of the crude enzyme solution (30-40% salted-out product), it is presumed that Ca ²⁺ acts on the enzyme and the hydrophobicity of the molecular surface is increased. In the presence of Ca ²⁺ , the collagenolytic enzyme showed stable activity. Matrix metalloproteinase (MMP), which is a collagenolytic enzyme present in the living body of mammals, is known to enhance the enzyme activity by maintaining the three-dimensional structure of the enzyme by Ca ²⁺ and promoting binding with the substrate (Archive of Biochemistry and). Biophysics, 1976, Vol. 173, p.355-361). That is, the behavior of the C35 strain-derived collagenolytic enzyme with respect to Ca ²⁺ is similar to that of the matrix metalloproteinase (MMP).

(4) Purification by gel filtration chromatography Fractions 5-8 collected in the previous step and having collagen degradation activity are concentrated by an ultra filter unit (product name “USY-1”, manufactured by Tosoh Corporation) having a molecular weight cut off of 1000. 200 μL of concentrated enzyme solution was obtained. The concentrated enzyme solution was purified by gel filtration chromatography using TSKgel (registered trademark) SuperSW3000 (manufactured by Tosoh Corporation). In the gel filtration chromatography, protein elution was performed using HPLC-system (manufactured by JASCO Corporation) under the following conditions. About each obtained fraction, the light absorbency in 280 nm was measured.

(Elution conditions)
Column size (gel capacity): 20 cm ³
Elution buffer: 0.1 M Na ₂ SO ₄ , and
0.1 M Tris-HCl buffer (pH 7.5) containing 10 mM CaCl ₂
Flow rate: 0.35 mL / min

  For the fraction in which the absorbance peak was confirmed, the degree of protein purification, the protein concentration and the collagen degradation activity were measured in the same manner as in hydrophobic interaction chromatography.

  The result of the protein elution pattern by the gel filtration chromatography is shown in FIG. As shown in the figure, three peaks (peaks 1 to 3) showing high absorbance were obtained. Among these peaks, no collagen degradation activity was confirmed in peaks 1 and 3, and the same activity was confirmed in peak 2. The confirmation result of the protein purification degree of the peak 2 is shown in FIG. In the figure, lane 1 is the molecular weight marker, and lane 2 is the result of peak 2. As shown in the figure, in lane 2, a single band was confirmed at a molecular weight of about 70 kDa. From this result, it was confirmed that the collagenase derived from the C35 strain was a protein having a molecular weight of about 70 kDa by SDS-PAGE. The enzyme solution of peak 2 had a total protein amount of 0.169 mg and a specific activity of 879 U / mg.

  Table 6 below shows the total amount of protein (mg), collagen degradation activity (U), specific activity (U / mg), and degree of purification (times) for the enzyme samples obtained in each purification step of the C35 strain-derived collagenolytic enzyme. ) And yield (%). The degree of purification (times) was determined as a relative value when the specific activity of the culture supernatant was 1. The yield (%) was determined as a relative ratio (%) when the collagen degradation activity (U) of the culture supernatant was 100%. As shown in the table, the above-described purification process finally yielded a collagenase having a purification degree of 52 times with a yield of 0.064%.

(Example 6)
Regarding the collagen degrading enzyme derived from C35 strain obtained in Example 5, using a fluorescent synthetic substrate, substrate specificity, optimum temperature, optimum pH, temperature stability, influence on collagen degradation activity by various protease inhibitors, enzyme reaction The effect on speed and muscle protein was confirmed.

(1) Measurement of substrate specificity The substrate specificity of the C35 strain-derived collagenolytic enzyme was confirmed using the following two types of synthetic substrates that have been reported to be a substrate for collagenolytic enzyme. First, in order to measure the collagen degradation activity, a measurement reagent containing a synthetic substrate was prepared. Specifically, the measurement reagent is a 0.1 M Tris-HCl containing 50 μL of DMSO in which a synthetic substrate is dissolved to 0.04 mM, 0.1 M NaCl, 10 mM CaCl ₂ and 0.05% Brij-35. It was prepared by mixing 800 μL of buffer (pH 7.5). As the synthetic substrate, the aforementioned MOCAc-Pro-Leu-Gly- Leu-A 2 pr (Dnp) -Ala-Arg-NH 2 ( KK Peptide Institute) and Gly-Pro-pNA.Tos (LTD Peptide Institute) was used. The former is the aforementioned PLGL (D) AR. PLGL (D) AR emits light by MOCAc-Pro-Leu-Gly- Leu-A 2 pr (Dnp) while is degraded. The latter is hereinafter referred to as pNATOs. The latter develops color when Gly-Pro-pNA.Tos is decomposed. Then, the C35 strain-derived collagenolytic enzyme was added to each of the measuring reagents and incubated at 25 ° C. for 30 minutes to carry out an enzyme reaction. After the incubation, 100 μL of 0.1 M EDTA was further added to the reaction solution to stop the reaction, and a measurement solution was obtained. About the said measurement liquid containing the said pNATOs, the light absorbency in 405 nm was measured. Further, with respect to the measurement liquid containing the PLGL (D) AR, the fluorescence intensity was measured using a spectrofluorometer (F-2500, HITACHI) under conditions of an excitation wavelength of 328 nm and an absorption wavelength of 393 nm. In addition, as a control, enzyme reaction and fluorescence intensity or absorbance were measured in the same manner except that the C35 strain-derived collagenolytic enzyme was not added. Then, the true fluorescence intensity or absorbance was calculated by subtracting the corresponding value of the control from the fluorescence intensity or absorbance of the measurement solution (hereinafter the same).

As a result, no color was observed in the measurement solution containing pNATOs, and it was found that the C35 strain-derived collagenolytic enzyme does not act on pNATOs. On the other hand, fluorescence was observed in the measurement solution containing PLGL (D) AR, and it was confirmed that the C35 strain-derived collagenolytic enzyme acts on MOCAc-Pro-Leu-Gly-Leu-A ₂ pr (Dnp). .

(2) Measurement of optimum temperature Except that the incubation temperature of the enzyme reaction was set to a predetermined temperature (0, 4, 10, 20, 25, 30, 40, 50 ° C.) and PLGL (D) AR was used as a fluorescent synthesis substrate. In the same manner as (1) measurement of substrate specificity, a reagent for measurement was prepared and fluorescence intensity was calculated. And the relative value (%) of the fluorescence intensity of each measurement liquid was calculated | required by making the highest fluorescence intensity among the calculated fluorescence intensity the relative activity rate 100% (hereinafter, the same).

  The collagen degradation activity of the C35 strain-derived collagenolytic enzyme under various temperature conditions is shown in Table 7 and FIG. As shown in the figure, the C35 strain-derived collagenolytic enzyme exhibited collagenolytic activity in the temperature range of 0 to 50 ° C. The optimum temperature at which the C35 strain-derived collagenolytic enzyme exhibits the highest collagenolytic activity was 30 ° C. For example, the optimum temperature of collagenase N-2 (manufactured by Nitta Gelatin Co., Ltd.), a commercially available collagenolytic enzyme, is 37 ° C. according to the company's report. That is, it was found that the optimum temperature of the C35 strain-derived collagenolytic enzyme was lower than that of the commercially available collagenase N-2. The relative activity rate at 0 ° C. was 6.4%, and the relative activity rate at 4 ° C. was 7.3%. From this result, it was found that the collagen degrading enzyme derived from C35 strain has collagen activity even in a general refrigerated storage temperature range.

(3) Measurement of temperature stability The incubation temperature of the enzyme reaction is set to a predetermined temperature (0, 10, 25, 30, 35, 40, 45, 50, 60 ° C.), and PLGL (D) AR is used as a fluorescent synthesis substrate. Except for the above, the measurement reagent was prepared and the fluorescence intensity was calculated in the same manner as in (1) Measurement of substrate specificity. And the relative value (%) of the fluorescence intensity of each measurement liquid was calculated | required by making the highest fluorescence intensity among the calculated fluorescence intensity the relative activity rate 100% (hereinafter, the same).

  The measurement results of the temperature stability of the C35 strain-derived collagenolytic enzyme are shown in FIG. As shown in the figure, the C35 strain-derived collagenolytic enzyme had high temperature stability in a temperature range of 30 ° C. or lower. The C35 strain-derived collagenolytic enzyme remained active up to 50 ° C. and was completely inactivated at 60 ° C.

(4) Measurement of optimum pH In the preparation of the measurement reagent, each buffer shown below was used instead of 0.1 M Tris-HCl buffer (pH 7.5), and PLGL (d) AR was used as a fluorescent synthesis substrate. In the same manner as in the above (1) measurement of substrate specificity, the measurement reagent was prepared and the fluorescence intensity was calculated. Then, the relative activity was determined from the calculated fluorescence intensity in the same manner as described above.

(buffer)
pH: Type
pH 5: 0.1 M sodium acetate / acetic acid buffer pH 6: 0.1 M sodium acetate / acetic acid buffer pH 6: 0.1 M PIPES-NaOH buffer pH 7: 0.1 M PIPES-NaOH buffer pH 7: 0.1 M Tris-HCl buffer pH 8: 0. 1M Tris-HCl buffer pH 9: 0.1M Tris-HCl buffer

  FIG. 13 shows the collagenolytic activity of the C35 strain-derived collagenolytic enzyme under various pH conditions. As shown in the figure, in the pH range of pH 5-9, the C35 strain-derived collagenolytic enzyme exhibited collagenolytic activity. The optimum pH at which the C35 strain-derived collagenolytic enzyme exhibits the highest collagenolytic activity was pH 7. From this result, it can be said that the C35 strain-derived collagenolytic enzyme is a neutral collagenolytic enzyme.

(5) Effect of protease inhibitor The effect of a protease inhibitor on the collagen degradation activity of C35 strain-derived collagenolytic enzyme was measured. As the protease inhibitor, various inhibitors shown in Table 8 below were used. First, an inhibitor solution was prepared by dissolving the inhibitor in DMSO. This inhibitor solution was added to the measurement reagent containing the fluorescent synthetic substrate PLGL (D) AR so that the final concentration of the inhibitor was as shown in the following table and the final concentration of DMSO was 2.5%. Further, as a control, the above-mentioned reagent for measurement containing no inhibitor and containing DMSO at a final concentration of 2.5% was prepared. Thus, except that the inhibitor solution was added to the measurement reagent, the measurement reagent was prepared and the fluorescence intensity was calculated in the same manner as in (1) Measurement of substrate specificity. Then, the relative activity was determined from the calculated fluorescence intensity in the same manner as described above.

  The measurement results of the relative activity rate of the C35 strain-derived collagenolytic enzyme in the presence of each protease inhibitor are shown in Table 9 below. As shown in the table, by applying 10 mM EDTA, EGTA, and 1,10-phenanthroline monohydrate, the relative activity rates of the C35 strain-derived collagenolytic enzymes were 4.6%, 7.7% and It decreased to 23%. Thus, since the C35 strain-derived collagenolytic enzyme was inhibited by the metalloprotease inhibitors EDTA, EGTA and 1,10-phenanthroline monohydrate, the C35 strain-derived collagenolytic enzyme was It is suggested to be a protease.

(6) Analysis of enzyme reaction rate The PLGL (D) AR is used as a substrate, and a predetermined concentration (0.32, 0.16, 0.08, 0.04, 0.02, 0.01,. The reagent for the measurement was prepared in the same manner as in the above (1) measurement of substrate specificity except that it was adjusted to 005, 0.0025 mM). Fluorescence intensity of the product MOCAc-Pro-Leu-Gly by the enzymatic reaction in the same manner as in the above (1) measurement of substrate specificity, except that the final concentration of the C35 strain-derived collagenolytic enzyme was 1.336 μg / mL Was measured. And the concentration of the product produced | generated by the enzyme reaction was computed from the above-mentioned measured value using the analytical curve which shows the relationship between the density | concentration of MOCAc-Pro-Leu-Gly and fluorescence intensity which were produced beforehand. In addition, as a reference substance of the MOCAc-Pro-Leu-Gly, (7-methoxycoumarin-4-yl) acetyl-prolyl-leucyl-glycine (MOCAc-Pro-Leu-Gly, manufactured by Peptide Institute, Inc.) is used. Using. Then, using the following formula (3), the reaction rate (mM / mg protein / min) at each substrate concentration was calculated, and a Lineweaver-Burk plot was created from the obtained reaction rate. From the slope and intercept of the obtained linear equation, K _m and V _max were calculated, and k _cat and k _cat / K _m were calculated.

A graph showing the enzyme reaction rate of the collagen degrading enzyme derived from the C35 strain when the PLGL (D) AR is used as a substrate is shown in FIG. In the figure, the horizontal axis represents the substrate concentration (mM), and the vertical axis represents the reaction rate (mM / mg protein · min). Further, a Lineweaver-Burk plot is shown in FIG. Furthermore, Table 10 below shows the comparison results of K _m , k _cat , and k _cat / K _m of the C35 strain-derived collagenolytic enzyme and other enzymes. Examples of the other enzymes include almerysin, vimelysin and thermolysin. The armericin and the vimelicin are metalloproteinases having activity at low temperatures, and the thermolysin is a thermostable proteolytic enzyme. These other enzymes have already been reported for the same temperature conditions. The data for armericin are cited from Shibata et al. (Bioscience, Biotechnology, and Biochemistry, 1997, Vol. 61, No. 4, p. 710-715), and the data for vimelisin and thermolysin are reported by Takahashi et al. (FEBS Letters, 1992, Vol. 294, p.263-266). As shown in the following table, k _cat / K _m of the collagen degrading enzyme derived from the C35 strain was slightly larger than that of vimelicin and thermolysin, and was considerably larger than that of armelicin. From this result, it was found that the catalytic efficiency of the C35 strain-derived collagenolytic enzyme with respect to the substrate was higher than that of other enzymes.

(7) Action on muscle protein (collagen) The C35 strain-derived collagenolytic enzyme and a commercially available proteolytic enzyme were reacted with collagen, and the degradation activity of each enzyme on collagen was measured. The following three types of commercially available proteolytic enzymes were used.

(Commercial proteolytic enzyme)
Collagenase N-2 (derived from Streptomyces pabras, manufactured by Nitta Gelatin Co., Ltd.)
Collagenase GRADEII (from Clostridium historicum, manufactured by Wako Pure Chemical Industries, Ltd.)
Papain (manufactured by Sigma)

  10 μL of each 0.01 mg / mL enzyme solution was added to 30 μL of the same collagen substrate solution as in Example 4, and reacted at 25 ° C. for 1 hour or at 4 ° C. for 24 hours. As papain, 0.01 mg of papain powder was previously mixed with 1 mL of a 2 mM EDTA solution containing 5 mM L-cysteine, and incubated at 37 ° C. for 15 minutes, and used as the enzyme solution. Then, 8 μL of the SDS solution described in Table 5 and 2 μL of mercaptoethanol were added to each reaction solution obtained, and heated at 100 ° C. for 2 minutes to prepare an SDS-PAGE sample. Each SDS-PAGE sample was subjected to SDS-PAGE in the same manner as in Example 4, and CBB staining was performed.

  An electrophoretic photograph showing the degradation activity of each enzyme against collagen is shown in FIG. In the figure, each lane is as shown in Table 11 below. As shown in the figure, when the reaction was carried out at 4 ° C. for 24 hours, the C35 strain-derived collagen degrading enzyme strongly degraded the collagen. In contrast, commercially available papain, collagenase N-2 and collagenase GRADEII had low degradation activity. In addition, when the reaction was performed at 25 ° C. for 1 hour, the results were the same as when the reaction was performed at 4 ° C. for 24 hours.

(Table 11)
Lane number Reaction temperature Reaction time Enzymes used in the reaction
14 ℃ 24 hours Control (no enzyme)
2 24 ° C. for 24 hours Papain 3 4 ° C. for 24 hours Collagenase N-2
4 4 ℃ 24 hours Collagenase GRADEII
5 4 ° C. 24 hours Collagenase derived from C35 strain 6 25 ° C. 1 hour Papain 7 25 ° C. 1 hour Collagenase N-2
8 25 ℃ 1 hour Collagenase GRADEII
9 25 ° C for 1 hour Collagenase derived from C35 strain

(8) Action on Muscle Protein (Actomyosin) The C35 strain-derived collagenolytic enzyme was reacted with actomyosin, and the degradation activity of each enzyme on actomyosin was measured. Specifically, the measurement was performed in the same manner as the measurement of the action on (7) muscle protein (collagen) except that the substrate solution was prepared using actomyosin instead of collagen. In addition, the actomyosin used what was made in-house based on the reference (Journal of Food Science, 1986, Vol.51, No.4, p.946-950).

  FIG. 17 shows an electrophoresis photograph showing the degradation activity of each of the four types of enzymes on actomyosin. In the figure, each lane is the same as in Table 11 above. As shown in the figure, when the reaction was carried out at 4 ° C. for 24 hours, almost no degradation products could be confirmed for all the enzymes. On the other hand, when the reaction was carried out at 25 ° C. for 1 hour, papain decomposed actomyosin and particularly strongly decomposed myosin heavy chain. On the other hand, collagen C-derived collagen degrading enzyme, collagenase N-2 and collagenase GRADEII could hardly be confirmed even at 25 ° C.

  As shown in the results of (7) and (8) above, the C35 strain-derived collagenolytic enzyme acts strongly on collagen among muscle proteins even at low temperatures, and on actomyosin, temperature Despite this, it hardly worked. From this, it was found that the collagen-degrading enzyme derived from the C35 strain preferentially degrades collagen related to meat hardening among muscle proteins, and has low activity on actomyosin constituting muscle fibers. Therefore, it can be said that if the C35 strain-derived collagenolytic enzyme is used, the texture of the meat is not impaired and the meat can be softened at a low temperature.

  If the psychrophilic bacterium of the present invention is used, the collagenolytic enzyme of the present invention can be easily produced by culturing the bacterium. If the collagenolytic enzyme of the present invention is used, the meat can be softened under low temperature conditions without damaging the texture of the meat. Furthermore, since the manufacturing method of the softened meat using the collagenolytic enzyme of this invention can be implemented on low temperature conditions, it can suppress the propagation of microorganisms and is excellent in food safety, for example. The use of the collagenolytic enzyme of the present invention can be used for, for example, animal processing, fish and shellfish processing such as peeling squid and fish eggs, leather product processing, and production of collagen peptides. It can be used in a wide range of chemical products such as chemicals.

FIG. 1 is a photograph of an image obtained by observing the Gram-stained psychrophilic bacterium C35 strain of the present invention using an inverted microscope. FIG. 2 is a photograph of a gel obtained by agarose gel electrophoresis of 16S rRNA gene DNA of the psychrophilic bacterium C35 strain in the present invention. FIG. 3 is a phylogenetic tree showing the results of phylogenetic analysis of the psychrophilic bacteria C35 strain in the present invention. FIG. 4 is a graph showing the temperature range in which the psychrophilic bacteria C35 strain can grow in the present invention. FIG. 5 is a graph showing the growth rate when the psychrophilic bacterium C35 strain of the present invention is cultured using different media. FIG. 6 is a photograph showing the collagenolytic activity when the psychrophilic bacterium C35 strain in the present invention is cultured using different media. FIG. 7 is a protein elution chart when the pyrolytic bacterial C35-derived collagen degrading enzyme in the present invention is purified using hydrophobic interaction chromatography. FIG. 8 is an electrophoretogram showing the degree of protein purification of the collagenolytic enzyme derived from the psychrophilic bacterium C35 strain of the present invention, which was purified using hydrophobic interaction chromatography. FIG. 9 is a protein elution chart when the pyrolytic bacterium C35 collagen-degrading enzyme in the present invention is purified by gel filtration chromatography. FIG. 10 is an electrophoretogram showing the degree of protein purification of the collagenolytic enzyme derived from the low-temperature bacterium C35 strain according to the present invention, which was purified using gel filtration chromatography. FIG. 11 is a graph showing the collagen degrading activity of the psychrotrophic bacterial C35 strain-derived collagen degrading enzyme in the present invention under different temperature conditions. FIG. 12 is a graph showing the temperature stability of the collagenolytic enzyme derived from the psychrophilic bacteria C35 strain in the present invention. FIG. 13 is a graph showing the collagenolytic activity of the psychrophilic bacteria C35 strain-derived collagenolytic enzyme in the present invention under different pH conditions. FIG. 14 is a graph showing the enzyme reaction rate of the psychrophilic bacterial C35 strain-derived collagenolytic enzyme in the present invention. FIG. 15 is a graph showing a Lineweaver-Burk plot of a collagenolytic enzyme derived from a psychrophilic bacterium C35 strain in the present invention. FIG. 16 is an electrophoretogram showing the action of the low-temperature bacterial C35 strain-derived collagenase in the present invention on collagen. FIG. 17 is an electrophoretogram showing the action of the psychrophilic bacterium C35 strain-derived collagen degrading enzyme in the present invention on actomyosin.

Claims (6)

Having the traits of the following (1) to (8), producing the collagenolytic enzyme shown in the following (9),
The bacteria name is Shewanella sp. Strain C35,
A psychrophilic bacterium with NITE P-520 as the accession number of the Patent Microorganism Depositary.
(1) Motile bacilli (2) Size is 3μm × 1μm
(3) Gram-negative (4) Cell color at the time of collection is red (5) Range of viable temperature is 4 to 37 ° C
(6) The optimal growth temperature range is 20 to 33 ° C.
(7) under aerobic conditions, does not produce hydrogen sulfide (8) nucleotide sequence of the 16S rRNA gene, nucleotide sequence according to the base sequence (a) SEQ ID NO: 1 described in the following (a)
( 9) The molecular weight by SDS-PAGE method is 60-72 kDa,
It has collagen degradation activity in the temperature range of 0-50 ° C, the optimal temperature is in the range of 20-40 ° C,
It has collagen degradation activity in the pH range of pH 5-9, the optimum pH is in the range of pH 6-8,
Collagen degradation activity is inhibited by EDTA, EGTA and 1,10-phenanthroline monohydrate,
Collagen degradation to degrade (7-methoxycoumarin-4-yl) acetyl-prolyl-leucyl-glycyl-leucyl- [N ^β- (2,4-dinitrophenyl) -2,3-diaminopropionyl] -alanyl-argininamide enzyme The enzyme derived from a psychrotrophic bacterium according to claim 1,
The molecular weight by SDS-PAGE method is 60-72 kDa,
It has collagen degradation activity in the temperature range of 0-50 ° C, the optimal temperature is in the range of 20-40 ° C,
It has collagen degradation activity in the pH range of pH 5-9, the optimum pH is in the range of pH 6-8,
Collagen degradation activity is inhibited by EDTA, EGTA and 1,10-phenanthroline monohydrate,
(7-methoxycoumarin-4-yl) acetyl-prolyl-leucyl-glycyl-leucyl- [N ^β- (2,4-dinitrophenyl) -2,3-diaminopropionyl] -alanyl-argininamide,
A collagenolytic enzyme that does not degrade myosin heavy chain. A method for producing a collagenolytic enzyme according to claim 2,
A method for producing a collagenolytic enzyme, comprising culturing the psychrophilic bacteria according to claim 1 to produce the collagenolytic enzyme. Furthermore, the manufacturing method of the collagenolytic enzyme of Claim 3 which has the collection | recovery process which collect | recovers the supernatant containing the said collagenolytic enzyme from the culture solution of the said low temperature bacteria. A method for producing softened meat that softens meat by enzymatic treatment,
The manufacturing method of the softened meat which uses the collagenolytic enzyme of Claim 2 as an enzyme. The method for producing softened meat according to claim 5, wherein the enzyme treatment is performed under a temperature condition of 0 to 37 ° C.