JPH0870853A - A novel microorganism belonging to the genus Vibrio, a novel collagenolytic enzyme produced by the microorganism, and its use - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To provide a collagen-degrading enzyme useful as a meat tenderizer and a cell dispersant, and a production bacterium belonging to the genus Vibrio that produces the degrading enzyme. [Composition] The production strain is Vibrio sp.
rio sp) 1706B strain. The collagen degrading enzyme has the following physicochemical properties. (1) Action: Remarkable activity for degradation of both soluble and insoluble collagen, and high activity for light chain and heavy chain myosin. Collagenase synthetic substrate (Cbz-Gly-Pro-Leu-G
ly-Pro and Pz-Pro-Leu-Gly-P
It works well on ro-Arg). (2) Optimum pH: 7 to 8 (3) Optimum temperature: 30 ° C to 40 ° C (4) Molecular weight: about 60,000

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel collagen-degrading enzyme-producing bacterium belonging to the genus Vibrio, a novel collagen-degrading enzyme produced by the collagen-degrading enzyme-producing bacterium, and a method of using the collagen-degrading enzyme. The collagen-degrading enzyme is a novel collagen-degrading enzyme that not only acts specifically on collagen but also on myosin and exhibits a weak activity on casein. The collagen degrading enzyme is a useful enzyme that can be used as a meat tenderizer and a cell dispersant.

[0002]

Conventionally, an enzyme such as papain which is a plant protease has been used as a meat tenderizer.
Papain not only requires a reducing agent such as cystine, but also has a relatively weak action, so a stronger enzyme has been desired. It has been reported that it is essential that the degrading enzyme acts on collagen and myosin when the enzyme is used as a meat tenderizer (eg, Masato Ishishita, Kunihiko Samejima, New Food Indust).
ry Vol. 36, p. 53, 1994). However, a collagenolytic enzyme that specifically acts not only on collagen but also on myosin has not been found.

On the other hand, Clostridium histolyticum (Cl) is used as a cell dispersant used in the field of biochemistry.
Collagenase derived from the bacterium Ostrichium histolyticum is known, but its action was insufficient. That is, when the collagenase was used in a small amount, cell dispersion was not successful, and conversely, when used in a large amount, cells were damaged. Therefore, a collagen degrading enzyme which is useful as a cell dispersant without such drawbacks has been desired.

[0004]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a novel collagen-degrading enzyme-producing bacterium that produces a collagen-degrading enzyme without the above-mentioned drawbacks.

A further object of the present invention is to provide a novel collagen degrading enzyme which does not have the above-mentioned drawbacks.

[0006] Another object of the present invention is to provide a meat tenderizer and a cell dispersant containing the novel collagen degrading enzyme.

[0007]

[Means for Solving the Problems] The present inventors isolate a strain belonging to the genus Vibrio that produces a collagen-degrading enzyme free from the above-mentioned drawbacks from the sand soil of Shin-Kiba, Tokyo by the special isolation method described below. This completes the present invention. That is, the isolation of this strain was performed as follows.

The medium having the following composition was used.

Composition of medium (g / l) Insoluble collagen 4.0 Yeast extract 1.0 Potassium phosphate 1 0.5 Dipotassium phosphate 0.5 Magnesium sulfate 0.2 Sodium chloride 5.0 Ferrous sulfate 0 .01 Manganese sulfate 0.01 Calcium chloride 0.6 pH7 In the above medium, ordinary bacteria cannot grow, and only bacteria capable of assimilating collagen can grow. The strain of the present invention is mountain soil,
One of the few strains obtained from a large number of isolates from numerous samples, such as upland soil, wastewater treatment plant sludge, rawhide, coastal sand soil, and ranch soil. More found. The mycological characteristics are as follows.

1) Form: length 2.0 μm, width 1.1 μm
Is a bacillus.

2) Growth condition: Proliferated on ordinary agar medium and gelatin medium, and the colonies were yellow. Aerobic bacteria with no anaerobic growth. The optimum growth temperature is 26 ° C to 35 ° C, and the growthable temperature is 10 ° C to 38 ° C.

3) Gram stainability: -4) NaCl growth concentration: 0.2 to 7.0% 5) Motility: Yes (due to polar flagella) 6) No spore formation 7) Oxidase: +8) Catalase: + 9) Acid production from glucose: + From the above properties, the novel microorganism of the present invention is recognized as a bacterium belonging to the genus Vibrio and is identified as Vibrio sp. Strain 1706B. On August 26, 1994, this strain was sent to the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, under the contract number FERM P-
Deposited as No. 14493.

As a collagenase-producing bacterium belonging to the genus Vibrio, Vibrio alginoliticum (Vibrio al)
ginoliticum) is known [Reid,
Woods et al. Bacterology
Vol. 142, p. 142 (1980)]. However, it differs from the bacterium of the present invention in the points shown in Table 1.

[0014]

[Table 1] Therefore, the strain of the present invention is a novel strain which is clearly different from Vibrio alginoliticum.

The collagen degrading enzyme of the present invention is the above-mentioned Vi.
brio sp. It is a novel enzyme obtained by culturing strain 1706B. Although the enzyme has a remarkable activity for degrading collagen, it has a weak activity for casein and is characterized by its substrate specificity.

The physicochemical and enzymatic chemical properties of the novel collagen-degrading enzyme of the present invention are as follows.

1) Action: It shows a remarkable degrading activity for collagen (soluble collagen and insoluble collagen). It also acts on gelatin, which is a modified product of collagen, but its activity on casein is weak. That is, it has a titer of about 1/100 as compared with collagen. It also shows high activity on myosin (light and heavy chains). Collagenase synthetic substrate [Cbz-Gly-Pro
-Leu-Gly-Pro (sometimes abbreviated as Z-GPLGP), Pz-Pro-Leu-Gly-Pro-A
rg (sometimes abbreviated as PZ-PLGPR)]. However, in the above formula, Cbz represents a carboxybenzoyl group, and Pz represents a phenylazobenzoxycarbonyl group.

2) Purification method: The collagen-degrading enzyme of the present invention can be purified by ammonium sulfate fraction of the culture supernatant of gelatin medium shown in Table 2 below, Q sepharose column chromatography, and then gel filtration, and SDS polyacrylamide electrophoresis. (Hereinafter, referred to as SDS-PAGE) It is possible to confirm the degree of purification whether or not it has become uniform.

[0019]

[Table 2] 3) Optimum temperature: 30 ° C to 40 ° C (see Fig. 1) 4) Optimum pH: 7 to 8 (see Fig. 2) 5) Thermal stability: 30 ° C or less (see Fig. 3) 6) pH stability: 4.0 to 11.5 (see FIG. 4) 7) Inhibitor: Inhibited by EDTA, pCMB, DE
Not inhibited by P (see Table 3).

[0020]

[Table 3] Here, EDTA is an abbreviation for ethylenediaminetetraacetic acid and is a chelating agent. PCMB is an abbreviation for para-chromel benzoic acid and is an inhibitor of SH enzyme. D
FP is diisopropyl fluorophosphate, an inhibitor of the serine enzyme. APMSF is an abbreviation for aminophenylmethanesulfonyl fluoride, which is an inhibitor of serine enzyme.

8) Molecular weight: 60,000 (SDS-PA
GE) 9) Ultraviolet absorption: absorption maximum (λmax) = 280 nm
(See FIG. 5) A280 / A260 = 1.68 10) Titer measurement method (collagen degradation method): 50 mg Tris-HCl buffer (pH 7.5, containing 4 mM CaCl ₂ ) 0.9 ml Insoluble collagen (Sigma) 10 mg And enzyme solution 0.
After reacting with 1 ml at 30 ° C. for 1 to 3 hours, 1.0 ml of TCA (trichloroacetic acid) reagent is added and centrifuged, and the supernatant is subjected to ninhydrin color development and colorimetric measurement is performed at 570 nm. Titer is 1
One unit is the amount of enzyme that produces 1 μmol Leu per minute.

The titer of the pure collagen-degrading enzyme of the present invention measured by the above-mentioned collagen-degrading method was 20.8 units / mg.

(Casein decomposition method): 50 mM Tris-HCl buffer (pH 7.5, containing 1.1% casein)
4mM CaCl ₂ containing) 0.9m enzyme solution 0.1m
1 is added and reacted at 30 ° C. for 30 minutes. Add 0.9 ml of the reaction stop solution (TCA reagent) and filter. Filtrate 0.
After taking 5 ml, 2.5 ml of 0.4 M Na ₂ CO ₃ solution and 0.5 ml of Folin reagent are added, and the mixture is kept at 30 ° C. for 20 minutes and colorimetrically measured at 660 nm. Titer is 1 minute
The amount of enzyme that produces μmol Tyr is 1 unit.

The titer of the pure collagen-degrading enzyme of the present invention as determined by the casein decomposition method was 0.21 unit / mg.

11) SDS in the purification step of the collagen-degrading enzyme of the present invention (ammonium sulfate salting-out → Q sepharose → gel filtration)
-PAGE is as shown in Figure 6. Here, a gel concentration of 10% was used.

The collagen degrading enzyme of the present invention can be used as it is or as a meat softener by diluting it with a suitable solvent such as Tris buffer, physiological saline, water and the like. The collagen-degrading enzyme of the present invention is a low-temperature type and has an activity of 60% at the optimum temperature (35 ° C) even at 5 ° C (see Fig. 1).
It has the advantage that the meat can be softened even during low temperature storage. Therefore, for example, it is possible to soften the hard meat by dipping or spraying the hung meat during low-temperature storage after slaughter in an enzyme solution. Meat can be softened by other methods known to those skilled in the art.

Since the collagen-degrading enzyme of the present invention acts specifically on collagen and myofibrils, it can be used as a cell dispersant in the field of biochemistry. The cell dispersant is the collagen degrading enzyme of the present invention as it is or in a suitable solvent, for example,
It can be prepared by diluting with Tris buffer, physiological saline, water or the like. The cell dispersant of the present invention can be used by methods known to those skilled in the art.

[0028]

Examples of the present invention will be described below. (Example 1) The collagen degrading enzyme of the present invention was reacted with insoluble collagen at 30 ° C, and the time course of the decomposition of insoluble collagen was observed. The test method is 50 mM Tris HC
1 buffer solution (containing pH 7.5, ₄ mM CaCl ₂ ) 0.9
After reacting 10 mg of insoluble collagen (manufactured by Sigma) in 0.1 ml with 0.1 ml of enzyme solution containing 38 μg of collagen-degrading enzyme (pure product) at 30 ° C. for 1, 2, 4, 8, 12, 16, and 24 hours Then, 1.0 ml of TCA (trichloroacetic acid) reagent is added to each reaction product, and the mixture is centrifuged. The supernatant is subjected to ninhydrin color development and colorimetric measurement is performed at 570 nm. Trypsin [100 μg; titer = 18,3 as control
A blank test (without enzyme addition) was also performed using 00 units / mg (BAEE method); manufactured by Sigma. Figure 7
Shown in As shown in FIG. 7, when the collagen-degrading enzyme of the present invention was used, insoluble collagen was completely degraded in 24 hours. Visual observation revealed that the insoluble matter in the reaction solution disappeared in 8 hours. The control showed a near constant value with a low level of degradation over time. The ninhydrin level of the blank did not decompose even after 24 hours. (Example 2) The time course of the decomposition of the soluble collagen when the collagen-degrading enzyme of the present invention was allowed to act on the soluble collagen (substrate) was observed.

50 mM Tris HCl buffer (pH 7.
5,4 mM CaCl _{2 is included} ), the weight ratio of the collagen degrading enzyme of the present invention to the soluble collagen is 1: 2500.
And allowed to react under ice cooling. 1, 2, 5, 30 and 6
After reacting for 0 minutes, it was boiled immediately to inactivate the enzyme and stop the reaction. SDS- for each sample after reaction
PAGE was performed. The results are shown in Fig. 8. In the figure, SCL is the electrophoretic pattern of soluble collagen before the reaction,
β, α1, and α2 are the main bands of soluble collagen. The numbers 1, ..., 60 represent the action time (minutes).
M (H) is a molecular weight marker. The gel concentration is 4%. As can be seen from FIG. 8, the main components of soluble collagen (β, α1, α2) were observed as thick bands before the reaction. A band similar to that of the soluble collagen before the reaction was observed up to the action time of 1 to 2 minutes, but it was found that at the action time of 5 minutes or longer, the molecular weight was lowered and the soluble collagen was finely cut.

In the enzyme reaction, the enzyme-substrate is usually 1: 100 and is allowed to act at room temperature. Therefore, it is possible to act under the above conditions (enzyme-substrate = 1: 2500, ice-cooled (0 ° C.)). It can be seen that the collagen degrading enzyme of the present invention has a very strong action.

Separately from the above test, when the collagen-degrading enzyme of the present invention: soluble collagen = 1: 2500 was allowed to act at 25 ° C., the band pattern of soluble collagen disappeared immediately after the reaction (within 1 minute). It was observed. Example 3 The action of the collagen-degrading enzyme of the present invention on myosin (light chain and heavy chain) was observed.

1) Action on myosin light chain (fragment 1; manufactured by Sigma)

50 mM Tris HCl buffer (pH 7.
5,4 mM CaCl ₂ contained), the weight ratio of the collagen degrading enzyme of the present invention to the myosin light chain was set to 1: 200,
The reaction was carried out at 5 ° C. The reaction time is 0.5, 1, 2, 5,
At 8, 16 and 24 hours, the reaction was stopped by boiling for 1 minute as in Example 2, and SDS-PAGE was performed.

The results are shown in FIG. Left side in the figure (vertical direction)
The numbers given in are the values of the molecular weight marker kDa.
E (bottom direction) described is the collagen degrading enzyme of the present invention, M (H) is a molecular weight marker, St is an unacted light chain myosin, and 0.5, ... 24 are acted. It is time. The gel concentration was 7.5%. As can be seen from FIG. 9, the collagen degrading enzyme of the present invention is 6
A band was shown at 0 kD. It was observed that the myosin light chain was decomposed and disappeared with the progress of the reaction, and instead a single band having a low molecular weight was produced. From this, it is understood that the degrading enzyme of the present invention acts on the specific cleavage point for myosin light chain.

2) Action on myosin heavy chain (manufactured by Sigma).

The myosin heavy chain was reacted under the same conditions as the myosin light chain, and treated in the same manner as in SDS-PAG.
E was done.

The results are shown in FIG. The display in the figure is shown in FIG.
Is the same as. As can be seen from FIG. 10, unlike the myosin light chain, it is found that it is subdivided and is considerably finely cut by the action for 2 hours.

From the above results, it can be seen that the degrading enzyme of the present invention is useful as a meat tenderizer. (Example 4) The degrading enzyme of the present invention was allowed to act on meat with a large amount of muscle (beef fillet of beef), and the observation was performed. The enzyme used an ammonium sulfate salting-out product, and the titer was 1 / 6.3 of that of the pure product. For comparison, papain [titer = 3,000 units / mg (casein decomposition method); Wako Pure Chemical Industries], pancreatin [titer =
10.7 units / mg (casein decomposition method); Mikuni Chemical Industry] was also used.

50 mM Tris HCl buffer (pH 7.
5,4 mM CaCl _{2 (} containing 0.2 ml), 30 mg of the above-mentioned hard meat was added, and each enzyme 3.0, 1.5 and 0.75
After adding mg, the mixture was kept at 5 ° C. and treated for a predetermined number of days. Papain was performed in the presence of 0.1 M cystine.

The results are shown in Table 4.

[0041]

[Table 4] As can be seen from Table 4, in the case of the degrading enzyme of the present invention, 3.
The product treated with 0 mg and 1.5 mg for 4 days completely decomposed. Even when 0.75 mg was added, the product treated for 4 days decomposed about 3/4. In the case of papain, the one treated with 3.0 mg for 4 days decomposed only about 3/4.
In the case of pancreatin, even if it was treated with any addition amount for 4 days, the shape of the hard meat was slightly destroyed and it hardly acted.

In the above test, the enzyme / substrate ratio was made to act at a high value in order to obtain a clear result, but if a milder result is desired, the enzyme / substrate ratio may be lowered or the treatment temperature may be lowered. Or the conditions such as the use of an appropriate inhibitor can be changed to make the adjustment according to the purpose. (Example 5) The degrading enzyme of the present invention was further allowed to act on beef top meat and observed.

50 mM Tris HCl buffer (pH 7.
5,4 mM CaCl _{2 (} containing 0.2 ml), 30 mg of the above beef meat was added, and the same ammonium sulfate salting-out enzyme (titer as 1 / 6.3 of pure product) used in Example 4 was added to 3.0 ml. , 1.5 and 0.75 mg were added, the mixture was kept at 5 ° C., treated for 24 hours, and then observed.

As a result, the meat to which 3 mg was added was broken into pieces by only shaking. The samples to which 1.5 mg and 0.75 mg were added did not disintegrate only by shaking, but when they were placed on a glass plate and crushed with a spatula, they easily disintegrated.

FIG. 11 is a photomicrograph when the above 3.0 mg was added and treated (magnification: 250 times). 12
Is a similar micrograph when 0.75 mg was added and treated. In each case, it was observed that it was cut into roughly square thin plate-like pieces. The side of this cut small square was approximately 0.1 mm. For comparison, FIG. 13 shows a micrograph of a blank test which was similarly treated without the addition of the enzyme.

Separately, papain [potency = 3,000 units /
mg (casein decomposition method); Wako Pure Chemical Industries; reaction in the presence of 0.1 M cystine] and pancreatin [titer = 10.
7 units / mg (casein decomposition method); Mikuni Chemical Industry] was also operated under the same conditions, but the effect was weak and the results were almost similar to the results of the blank test.

In the above test, as in Example 4, a high enzyme / substrate ratio was used in order to obtain a clear result.
If a milder result is desired, conditions such as lowering the enzyme / substrate ratio, lowering the processing temperature, or using an appropriate inhibitor can be changed to make the adjustment depending on the purpose.

From the results of Examples 4 and 5 described above, it can be seen that the collagen degrading enzyme of the present invention can be expected to be used as a cell dispersant.

[Brief description of drawings]

FIG. 1 is a table showing the optimum temperature of the collagen-degrading enzyme of the present invention. As a substrate, a synthetic substrate Z-GPL
GP was used.

FIG. 2 shows the optimum p of the collagen degrading enzyme of the present invention.
It is a chart showing H. a) shows the optimum pH for the synthetic substrate Z-GPLGP, and b) shows the optimum pH for insoluble collagen.

FIG. 3 is a chart showing the temperature stability of the collagen-degrading enzyme of the present invention. Synthetic substrate Z-GPL as substrate
GP was used.

FIG. 4 is a chart showing the pH stability of the collagen-degrading enzyme of the present invention. As a substrate, a synthetic substrate Z-GP
LGP was used.

FIG. 5 is a chart showing an ultraviolet absorption curve of the collagenolytic enzyme of the present invention.

FIG. 6 is a photograph showing SDS-PAGE at the purification step of the collagenolytic enzyme of the present invention. In the figure, H and L are molecular weight markers. 1 is an ammonium sulfate salting out precipitate,
2 is purified by Q-Sepharose chromatography,
3 shows what was refined by Superose 6 (gel filtration) chromatography.

FIG. 7 is a chart showing the ability of the collagen-degrading enzyme and trypsin of the present invention to degrade insoluble collagen.

FIG. 8 is a photograph of SDS-PAGE showing the ability of the collagen-degrading enzyme of the present invention to degrade soluble collagen.

FIG. 9 is a photograph of SDS-PAGE showing the ability of the collagen-degrading enzyme of the present invention to degrade myosin light chain.

FIG. 10 is a SDS-PAGE photograph showing the ability of the collagen-degrading enzyme of the present invention to degrade myosin heavy chains.

FIG. 11 is a micrograph showing the cell-dispersing ability of the collagen degrading enzyme of the present invention in bovine upper meat.

FIG. 12 is a micrograph showing the cell dispersal ability of the collagen degrading enzyme of the present invention in bovine upper meat.

FIG. 13 is a micrograph showing the cell-dispersing ability of beef upper meat without the addition of the collagenolytic enzyme of the present invention.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location (C12N 9/52 C12R 1:63)

Claims (4)

[Claims] 1. A strain of Vibrio sp 1706B that produces a collagen-degrading enzyme. 2. The method according to claim 1, which has the following physicochemical properties.
Collagen-degrading enzyme produced by the described strain. (1) Action: Remarkable activity for the decomposition of both soluble and insoluble collagen and high activity for myosin. Collagenase synthetic substrate (Cbz-Gly-Pro-Leu-Gly-Pro and Pz-Pro-Leu-Gly-Pro-Arg)
Works well on. (2) Optimum pH: 7 to 8 (3) Optimum temperature: 30 ° C to 40 ° C (4) Molecular weight: about 60,000 3. A meat tenderizer containing the collagenolytic enzyme according to claim 2. 4. A cell dispersant containing the collagenolytic enzyme according to claim 2.