JP2019080493A - Protease, detergent composition, washing method, and microorganism - Google Patents

Abstract

To provide a protease having an optimum pH near neutrality, inactivated by heat treatment at a temperature of about 50°C, and sufficiently maintaining protease activity in the presence of a chelating agent, and to provide a detergent composition containing the protease.SOLUTION: In order to solve the problem, the present invention provides a protease having an optimum pH of 4.0 to 8.5, having a residual activity of 50% or more with respect to citric acid of 10 mM, and having the residual activity after treatment for 30 minutes at 50°C of 5% or less, and a detergent composition containing the protease.SELECTED DRAWING: None

Description

  The present invention relates to a protease having high residual activity in the presence of a chelating agent and a microorganism producing this protease. The present invention also relates to a detergent composition containing a protease having high residual activity in the presence of a chelating agent, and a cleaning method using the detergent composition.

  In food factories, hygiene is maintained by washing daily so that microorganisms such as bacteria do not grow. When protein adheres to equipment and equipment for food production, it is difficult to wash off with a neutral detergent. Therefore, the protein is decomposed and washed off with a high concentration alkaline detergent or the like.

  In addition, detergents to which a thermophilic protease having protein degrading activity is added are known. For example, Patent Document 1 discloses the invention of a thermophilic protease which has an optimum pH of 7.5 and is inactivated when treated at 50 ° C. for about 15 minutes, and as an application example thereof, a detergent composition for clothing It is stated to be added to

JP-A-8-214878

  There is a problem that deterioration of a resin such as polyurethane is likely to proceed, for example, when a protein attached to a device or facility for food production is washed with a high concentration alkaline detergent. Therefore, it was investigated to lower the pH of the detergent composition and to decompose the protein by protease.

  In the case of adding a protease to a detergent composition used to clean food manufacturing equipment and equipment, it is necessary to be careful that the protease does not remain in the equipment after cleaning. If the protease remains in the apparatus after washing, the protease is mixed in the processed food etc., and the protein of the food is decomposed during the storage period, and there is a problem that the texture and taste change. Therefore, as a property of the protease, the property of being completely inactivated by heat treatment at a temperature of about 50 ° C. is required.

  In addition, the cleaning composition contains a chelating agent such as citric acid or EDTA for the purpose of disinfecting, antibacterial or enhancing the cleaning effect of the surfactant. Conventional proteases have a significant decrease in activity in the presence of a chelating agent, and therefore, when added to detergent compositions, there is a problem that sufficient protease activity can not be obtained.

  Therefore, the subject of the present invention is characterized in that it has an optimum pH near neutrality, is inactivated by heat treatment at a temperature of about 50 ° C., and sufficiently maintains protease activity in the presence of a chelating agent. The present invention provides a protease and a detergent composition containing the protease.

  Another object of the present invention is to provide a washing method in which no protease activity remains after washing.

As a result of intensive studies to solve the above problems, the present inventors can inactivate at 50 ° C. out of proteases produced by various microorganisms and can sufficiently maintain the protease activity in the presence of a chelating agent. By selecting one, it was found that the protease produced by the Flavobacterium microorganism can be inactivated at 50 ° C. and sufficiently maintain the protease activity in the presence of the chelating agent, thus completing the present invention.
That is, the present invention is the following protease, detergent composition, washing method, and microorganism.

The protease of the present invention for solving the above problems is a protease having an optimum pH of 4.0 to 8.5, and has 50% or more of residual activity against 10 mM citric acid, and it is maintained at 50 ° C. for 30 minutes. It is characterized in that the residual activity after treatment is 5% or less.
According to this protease, since sufficient protease activity can be maintained even in the presence of citric acid as a chelating agent, it can be added to a detergent composition containing a chelating agent. In addition, since the reaction is inactivated by treatment at 50 ° C. for 30 minutes, the action of the protease can be stopped by performing a simple heat treatment.

Furthermore, according to one embodiment of the protease of the present invention, the protease is characterized in that the optimum temperature is 20-40 ° C.
According to this feature, since the protease activity can be sufficiently exhibited at 20 to 40 ° C., an excellent cleaning action is observed even at normal temperature. In addition, when the protease of the present invention is used to modify a food product, modification processing can be performed at a low temperature, so that deterioration or the like of the food product during modification processing can be suppressed.

Further, according to one embodiment of the protease of the present invention, the protease may be Flavobacterium frigidarium FF-No. It is characterized in that it is produced by the microorganism of 26 strains or its mutant.
According to this feature, by culturing a microorganism, a protease having the characteristics of the present invention can be easily obtained.

The detergent composition of the present invention for solving the above-mentioned problems is characterized by containing the above-mentioned protease of the present invention.
According to this detergent composition, it is possible to provide a detergent composition excellent in the protein degrading action. Moreover, since the protease can be inactivated by treating at 50 ° C. for 30 minutes, the action of the protease can be stopped by a simple heat treatment.

Furthermore, according to one embodiment of the detergent composition of the present invention, the detergent composition is characterized by having a pH of 5.0 to 9.0.
According to this feature, since the pH of the detergent composition is near neutral, deterioration of the resin product can be suppressed in the washing of the resin product such as polyurethane. In addition, since the pH of the detergent composition overlaps with the optimum pH of the protease of the present invention, the protease activity is more exerted.

The cleaning method of the present invention for solving the above-mentioned problems is characterized in that an object to be cleaned is cleaned using the cleaning composition of the present invention.
According to this washing method, since the protein is degraded by the detergent composition, it is possible to wash out the protein attached to the object to be washed.

The cleaning method of the present invention for solving the above problems comprises cleaning the object to be cleaned using a detergent composition containing a protease having a residual activity of 5% or less after treatment at 50 ° C. for 30 minutes. It is characterized by
According to this washing method, since the protein is degraded by the detergent composition, it is possible to wash out the protein attached to the object to be washed.

Furthermore, according to one embodiment of the cleaning method of the present invention, after cleaning the object to be cleaned, the object to be cleaned is heat-treated to 50 ° C. or higher.
According to this feature, since the cleaning object is heated to 50 ° C. or higher after cleaning the cleaning object, the activity of the protease remaining in the cleaning object can be reduced.

Furthermore, according to one embodiment of the cleaning method of the present invention, the object to be cleaned is characterized as being an apparatus or equipment for food production.
When proteases are mixed in foods and the like, there is a problem that the quality is reduced during storage of the foods (for example, changes in taste and texture due to unintended softening of meat and the like). The detergent composition of the present invention is reduced in protease activity by heat treatment at 50 ° C. Therefore, even if protease remains in the object to be cleaned, simple heat treatment is applied to the object to be cleaned The activity of the protease remaining in the object to be washed can be reduced. Therefore, even when applied to an apparatus or the like for manufacturing food, the influence of protease on food or the like can be suppressed. In addition, even if the food contains proteases, the action of the protease can be stopped by subjecting the food to a simple heat treatment. From such a point of view, the cleaning method of the present invention can be suitably used for cleaning an apparatus or facility for food production.

Furthermore, according to one embodiment of the cleaning method of the present invention, the object to be cleaned is characterized as being a resin product.
The detergent composition of the present invention can be suitably used for washing a resin product which is easily degraded by an alkali or the like because the protein is degraded by the protease even in the vicinity of neutrality.

The microorganism of the present invention for solving the above-mentioned problems is characterized by producing the protease of the present invention.
According to this microorganism, the protease of the present invention can be easily produced by culturing the microorganism.

The microorganism of the present invention for solving the above-mentioned problems is a microorganism capable of producing a protease, such as Flavobacterium frigidarium FF-No. 26 strains or variants thereof.
According to this microorganism, the protease of the present invention can be easily produced by culturing the microorganism.

  According to the present invention, a protease having an optimum pH near neutrality, being inactivated when heat-treated at a temperature of about 50 ° C., and sufficiently maintaining protease activity in the presence of a chelating agent And a detergent composition containing the protease can be provided.

FIG. 1 is a graph showing the optimum temperature of the protease of the example of the present invention. FIG. 2 is a graph showing the residual activity against heat treatment of the protease of the example of the present invention. FIG. 3 is a graph showing the optimum pH of the protease of the example of the present invention. FIG. 4 is a graph showing the results of the washing test with the protease sample of the example of the present invention. FIG. 5 is a graph showing the results of the washing test with the detergent composition containing the protease sample of the example of the present invention.

[Protease]
The protease of the present invention is a protease having an optimum pH of 4.0 to 8.5, and has a residual activity of 50% or more against 10 mM citric acid, and the residual activity after treatment at 50 ° C. for 30 minutes is It is characterized by being 5% or less.

(Measurement of protease activity)
In addition, unless otherwise indicated, the measuring method of the activity of the protease in this invention was performed according to the following azo casein method.
<Azocasein Method: Degradation of Protein by Azocasein>
30 μL of the sample enzyme solution was added to 150 μL of 0.05 M Tris-HCl buffer (pH 7.0) containing 1% (W / V) azocasein, and the reaction was allowed to stand at 30 ° C. for 2 hours. After the reaction was stopped by adding 120 μL of 15% trichloroacetic acid to the reaction solution, centrifugation was carried out at 1,000 × g and 4 ° C. for 10 minutes. The supernatant was measured for absorbance at 440 nm with a spectrophotometer. The relative value between each sample was determined from the absorbance value to evaluate the degradation activity of the protein.

(Optimal pH)
The protease of the present invention has an optimum pH of 4.0 to 8.5. The lower limit is preferably 5.0 or more, more preferably 5.5 or more, still more preferably 6.0 or more, and particularly preferably 6.5 or more. The upper limit is preferably 8.0 or less, more preferably 7.8 or less, and particularly preferably 7.5 or less. Thereby, the protein can be degraded without using an alkaline agent.
In addition, the method of determining the optimum pH is 100 mg each of acetic acid-sodium acetate buffer (pH 4.0-5.0) and phosphoric acid-sodium buffer (pH 6.0-) as the composition of the buffer of the reaction liquid. 8.0), Tris-HCl buffer (pH 9.0), carbonic acid-bicarbonate buffer (pH 10.0), phosphoric acid-sodium hydroxide buffer (pH 11.0-12.0) The pH of the solution is adjusted, and after addition of the protease, the activity of the protease is measured. Then, the protease activity was measured for various pH conditions, and the pH condition with the highest protease activity was taken as the optimum pH.

(Stability to chelating agents)
The protease of the present invention has a residual activity of 50% or more against 10 mM citric acid. The residual activity against 10 mM citric acid is preferably 55% or more, more preferably 60% or more, and particularly preferably 65% or more. Thus, even when added to a detergent composition containing citric acid, a processed food, etc., the protease activity can be sufficiently maintained.
In addition, the measuring method of the residual activity with respect to 10 mM citric acid is added so that the final concentration of citric acid may be 10 mM to the solution of protease, and after maintaining for 60 minutes at 20 ° C., the activity of the remaining protease is measured. The residual activity (%) is the percentage of the ratio of the protease activity with citric acid to the protease activity without citric acid.

Moreover, it is preferable that the protease of the present invention is also excellent in stability to other chelating agents. For example, the residual activity against 10 mM EDTA is preferably 15% or more, more preferably 20% or more.
In addition, the measuring method of the residual activity with respect to EDTA substitutes said citric acid to EDTA.

(Stability to heat)
The protease of the present invention has a residual activity of 5% or less after treatment at 50 ° C. for 30 minutes. When the residual activity is 5% or less, it is recognized that the protease is inactivated. The residual activity after treatment at 50 ° C. for 30 minutes is preferably 3% or less, more preferably 1% or less, particularly preferably 0. In the case of 5% or less. Thereby, the action of the protease can be stopped by a simple heat treatment.
Further, from the viewpoint of inactivating the protease by a simpler heat treatment, the heat treatment time at 50 ° C. is preferably 20 minutes, and particularly preferably 15 minutes.
In addition, the measuring method of the residual activity after heat processing at 50 degreeC hold | maintained the solution of the protease at 50 degreeC for predetermined time, and measured the activity of the remaining protease. The residual activity (%) is the percentage of the ratio of protease activity after heat treatment to protease activity before heat treatment.

(Optimum temperature)
The optimum temperature of the protease of the present invention is not particularly limited, but is preferably 20 to 40 ° C. The lower limit is more preferably 25 ° C. or more, and the upper limit is more preferably 35 ° C. or less. Thereby, since protease activity can fully be exhibited at 20-40 degreeC, the outstanding cleaning effect is recognized also at normal temperature. In addition, when the protease of the present invention is used to modify a food product, modification processing can be performed at a low temperature, and therefore it is possible to suppress the decay of food during modification processing.
In addition, the determination method of the optimal temperature adjusts the temperature of a substrate solution, and after adding a protease to this, the activity of a protease is measured. And protease activity was measured about various temperature conditions, and the temperature with the highest protease activity was made optimal temperature.

[Method for producing protease]
The method for producing the protease of the present invention is not particularly limited, and includes, for example, a method of producing it by a natural microorganism or animal, an artificially mutated microorganism or animal, etc., a method of synthesizing and producing an amino acid, etc. . From the viewpoint of being easily obtained in large quantities, a method of producing with a natural microorganism or an artificially mutated microorganism is preferred. More preferably, it is a method of production by a microorganism of the genus Flavobacterium, particularly preferably Flavobacterium frigidarium FF-No. It is a method of producing with the microorganism of 26 strains or its mutant.

(Microbe)
The microorganism in the present invention forms a clear zone around colonies by the decomposition of skimmed milk after cultivation (5 days at 10 ° C.) on a skimmed milk-based agar medium (skimmed milk added standard agar medium). Furthermore, after heating the supernatant of the culture broth in which the microorganism is cultured at 50 ° C. for 15 minutes, no protease activity is detected, and the microorganism is a protease having activity in the presence of 10 mM EDTA. Here, the fact that no protease activity is detected after heat treatment at 50 ° C. for 15 minutes means that it is determined as “−” in “(1) Heat treatment test” in the following examples. In addition, having protease activity in the presence of 10 mM EDTA means that it is determined as "+-" or "+" in "(2) Chelation resistance test" in the following examples, and preferably It is a microorganism judged as "+".

  Furthermore, from the viewpoint of excellent protease degradation, a microorganism that is determined to be “+” in “(3) protease activity test” in the following examples is preferable, and a microorganism that is determined to be “++” is particularly preferable.

(Flavobacterium frigidarium FF-No. 26 strain or a mutant thereof)
The microorganism in the present invention is preferably a microorganism of the genus Flavobacterium, particularly preferably Flavobacterium frigidarium FF-No. 26 strains of microorganisms. This Flavobacterium frigidarium FF-No. The 26 strains have an optimum pH of 4.0 to 8.5, a residual activity of 50% or more against 10 mM citric acid, and a residual activity of 5% or less after treatment at 50 ° C. for 30 minutes. A protease having an optimum temperature of 20 to 40 ° C. can be produced. In addition, Flavobacterium frigidarium FF-No. Twenty-six strains have been deposited at the Patent Organism Depositary Center of the National Institute of Technology and Evaluation (NITE) (Accession No .: NITE P-02555).

  In addition, Flavobacterium frigidarium FF-No. The microorganisms of the 26 mutant strains include Flavobacterium frigidarium FF-No. These are microorganisms in which mutations have been introduced into 26 strains, and can be produced, for example, by spontaneous mutation or known artificial mutation.

  As a method of causing a natural microorganism or an artificially mutated microorganism (hereinafter simply referred to as "the microorganism") to produce, a culture solution in which the microorganism is cultured can be prepared as a protease solution. Preferably, it is a culture solution from which microorganisms have been removed by centrifugation, filtration or the like, and particularly preferably a culture solution from which microorganisms have been removed by filtration. From the viewpoint of reliably removing microorganisms, the upper limit of the filtration opening is preferably 1 μm or less, more preferably 0.5 μm or less, and particularly preferably 0.3 μm or less. From the viewpoint of increasing the filtration amount, the lower limit of the filtration opening is preferably 0.01 μm or more, and particularly preferably 0.1 μm or more. Examples of the filtration method include a total amount filtration method of filtering the whole amount of the culture solution, and a cross flow method of filtering while flowing the culture solution parallel to the membrane surface.

  The protease solution obtained as a culture solution is preferably concentrated. The method for concentration is not particularly limited as long as the method is not heating to 50 ° C. or higher, and examples thereof include methods such as ammonium sulfate precipitation and vacuum concentration. From the viewpoint of being able to concentrate by a simple operation, the concentration method by ammonium sulfate precipitation is preferred. Alternatively, it may be dried to form a solid.

[Detergent composition]
The detergent composition of the present invention is a protease having an optimum pH of 4.0 to 8.5, which has a residual activity of 50% or more against 10 mM citric acid, and after treatment at 50 ° C. for 30 minutes It is characterized in that it contains a protease having a residual activity of 5% or less.

  The pH of the detergent composition of the present invention is not particularly limited, but is preferably 5.0 to 9.0. The lower limit is more preferably 5.5 or more, still more preferably 6.0 or more, and particularly preferably 6.5 or more. The upper limit is more preferably 8.5 or less, still more preferably 8.0 or less, and particularly preferably 7.5 or less. Thereby, since the pH of the detergent composition overlaps with the optimum pH of the protease of the present invention, the protease activity is more exerted. Moreover, in washing | cleaning of resin products, such as a polyurethane, deterioration of resin products can be suppressed.

  The form of the detergent composition is not particularly limited, and examples thereof include liquid, gel, solid, and powder. Moreover, it is good also as foam form with foaming apparatuses, such as a pump former.

[Washing method]
The cleaning method of the present invention is characterized in that the object to be cleaned is cleaned using the above-mentioned detergent composition.
Further, the cleaning method of the present invention is characterized in that the cleaning object is cleaned using a detergent composition containing a protease having a residual activity of 5% or less after treatment at 50 ° C. for 30 minutes. It is.

  The object to be cleaned is not particularly limited, and examples thereof include resin products, metal products, glass products, pottery products, textile products and the like. The cleaning method of the present invention can be suitably used for cleaning a resin product which is likely to be deteriorated with respect to an alkaline cleaning composition, since it has protein decomposability near neutral pH. The material of the resin product is not particularly limited, and examples thereof include polyurethane, polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polycarbonate, natural rubber, silicone rubber, vinyl chloride, nylon and the like. The cleaning method of the present invention is suitable for cleaning a resin product made of polyurethane from the viewpoint of being easily deteriorated with respect to an alkaline cleaning composition.

  Moreover, since the detergent composition of the present invention can inactivate the protease by a simple heat treatment, it can be suitably used for cleaning an apparatus or equipment for food production. As an apparatus for food production, for example, there are a feed tank, a mixing apparatus, a filtration apparatus and the like. In the filtration filter of the filtration apparatus, since the protease tends to remain in fine pores, the cleaning method of the present invention in which the protease is inactivated by simple heating can be suitably used. Moreover, as an installation for food manufacture, a pallet, a conveyor belt, etc. are mentioned.

The cleaning method of the present invention is, for example, a cleaning method comprising the following steps.
Step 1: A step of washing an object to be washed with the detergent composition of the present invention.
Step 2: heat treating the object to be cleaned at 50 ° C. or higher.
Step 3: Rinsing the object to be cleaned and washing out the detergent composition.

  The above step 1 is not particularly limited as long as it is an operation to wash the cleaning object with the cleaning composition of the present invention, but for example, the operation to dip the cleaning object into the cleaning composition or its diluted solution Washing), an operation of scrubbing with a washing tool (brush, sponge etc.) adhering to the washing composition, and the like.

  The pickling time in pickling washing is not particularly limited, and is, for example, 1 minute to 24 hours. In consideration of the decomposition action of the protease, the lower limit is preferably 5 minutes or more, and particularly preferably 10 minutes or more. On the other hand, in consideration of the working time etc., the upper limit value is preferably 1 hour or less, and particularly preferably 30 minutes or less. The temperature is not particularly limited as long as it is 50 ° C. or less, but in view of the optimum temperature of the protease, it is preferably 20 to 40 ° C.

  The process 2 is not particularly limited as long as it is an operation of subjecting the object to be cleaned to a heat treatment at a temperature of 50 ° C. or higher. The heat treatment operation is, for example, an operation of immersing the cleaning object in warm water of 50 ° C. or more, an operation of pouring the warm water of 50 ° C. or more over the cleaning object, an operation of spraying steam of 100 ° C. or more on the cleaning object Can be mentioned. In the operation of immersing the object to be cleaned in warm water of 50 ° C. or more, the immersion time is not particularly limited, but is preferably 5 minutes or more, more preferably 10 minutes or more from the viewpoint of inactivating the protease. And particularly preferably 15 minutes or more.

[Screen strain]
Each sample collected from flatfish, sea squirt, burdock, aquatic organisms such as shrimp, dirt in the refrigerator, minced meat, water in the canal, etc. was diluted fourfold with sterile physiological saline and crushed for 30 seconds with a stomacher. Each sample solution was streaked on agar medium (skim milk added standard agar medium) using skimmed milk as a substrate, and cultured at 10 ° C. for 5 days. After culture, among the appeared colonies, 103 strains of bacteria that showed clear zones around the colonies due to the decomposition of skimmed milk were isolated. The method for producing a standard agar medium containing skimmed milk is shown below. Table 1 shows the isolated strains.

(Skim milk added standard agar medium)
The standard agar medium (Nissui Pharmaceutical Co., Ltd.) was adjusted to a normal double concentration and sterilized at 121 ° C. for 15 minutes. Further, the skimmed milk was added to distilled water so as to be 2.0%, and sterilization was performed at 121 ° C. for 15 minutes. An equal volume of a double concentration standard agar medium and a 2.0% skimmed milk solution were added, stirred, and dispensed in a sterile petri dish to be solidified.

  Next, the isolates shown in Table 1 were inoculated into Tryptosia bouillon (Nichisui Pharmaceutical Co., Ltd.) and cultured at 10 ° C. for 15 days. After cultivation, centrifugation is carried out (9,000 × g, 4 ° C., 10 minutes), and the collected supernatant is filter-sterilized with a 0.22 μm syringe filter (Merck Millipore Co., Ltd.), and the culture supernatant and did. The obtained culture supernatant was screened in stages by the following (1) heat treatment test, (2) chelating agent test, and (3) protease activity test. In (1) heat treatment test, (2) chelating agent test, and (3) protease activity test, protease activity was evaluated by the following plate assay method.

<Plate assay method: protein degradation activity by skimmed milk standard agar medium>
A well with a diameter of 6.5 mm was aseptically opened in a standard agar medium containing skimmed milk, and 30 μL of the culture supernatant was injected into each well and allowed to stand at 48 ° C for 48 hours. After the reaction, the diameter of the clear zone formed around the well was measured, the relative value between each sample was determined, and the protease activity was evaluated.

(1) Heat treatment test The supernatant of the obtained culture broth was heat treated at 50 ° C. for 15 minutes, and then the protease activity was measured by the plate assay method described above.
Those with a clear zone size of less than 6.5 mm were evaluated as "-", and those with a clear zone size of 6.5 mm or more were evaluated as "+". From the results of the heat treatment test, five strains (strains marked with “*” in Table 1) whose protease activity was reduced by heat treatment at 50 ° C. for 15 minutes were selected, and the following (2) chelating agent resistance test , (3) were subjected to protease activity test.

(2) Chelate resistance test EDTA was added to the supernatant of the culture solution of five selected strains to a final concentration of 10 mM, and after 60 minutes of storage at 20 ° C., protease activity was determined by the above plate assay. It was measured.
If the size of the clear zone is less than 6.5 mm "-", if the size of the clear zone is more than 6.5 mm and less than 10 mm "+-", if the size of the clear zone is 10 mm or more " It evaluated by ", and it showed in Table 2.

(3) Protease Activity Test The supernatant of the culture solution of the selected 5 strains was subjected to the above plate assay to measure the protease activity.
Clear zone sizes of less than 6.5 mm are evaluated as "-", 6.5 mm or more and less than 10 mm as "+-", 10 mm or more and less than 13 mm as "+", and 13 mm or more as "++". The

  (2) No. 26 strain (No. 26 strain, strain marked with "*" in Table 2) showing good results in any of the tests from the results of the chelating agent test and (3) protease activity test I selected.

[Identification of strain]
No. Morphological and physiological property investigations were performed on 26 strains according to a conventional method. Further, as a result of discrimination of the species based on the homology of the nucleotide sequence of 16S rDNA, it was identified as Flavobacterium frigidarium. The results are shown in Table 3. The nucleotide sequence of 16S rDNA is described in the sequence listing (SEQ ID NO: 1). No. Twenty-six shares were collected from the Patent Organism Depositary Center of the National Institute of Technology and Evaluation (NITE) (Corporation 2-5th, No. 5-122, Kazusa, Kisarazu City, Chiba Prefecture, Japan). It was deposited as 26 strains (Accession No .: NITE P-02555).

[Flavobacterium frigidarium FF-No. Characterization of 26 Proteases]
Flavobacterium frigidarium FF-No. The proteases produced by the 26 strains were evaluated for their properties on residual activity against citric acid and EDTA, optimum temperature, residual activity against heat treatment, and optimum pH.
(Preparation of Protease Sample)
Flavobacterium frigidarium FF-No. The 26 strains were cultured at 10 ° C. for 15 days. After culture, centrifugation is performed (9,000 × g, 4 ° C., 10 minutes), and the collected culture supernatant is sterilized by filtration using a 0.22 μm syringe filter (Merck Millipore Co., Ltd.), and then the culture solution is collected. The supernatant of the culture solution and an equal volume of a saturated ammonium sulfate solution were added to the suspension while stirring, and the mixture was allowed to stand at room temperature for 30 minutes and centrifuged (9,000 × g, 4 ° C., 20 minutes). Thereafter, the supernatant was removed and the pellet was dissolved with 0.05 M Tris-HCl buffer (pH 7.0) to an arbitrary concentration.

(Characteristics evaluation 1: Residual activity to a chelating agent)
To the above protease sample, add citric acid to a final concentration of 10 mM as a chelating agent, or EDTA to a final concentration of 1 mM or 10 mM, store it at 20 ° C for 60 minutes, and then use the azocasein method for protease activity. It was measured. The relative activity was calculated with the case where the chelating agent was not added as 100%. The results are shown in Table 4.

Looking at Table 4, Flavobacterium frigidarium FF-No. The protease produced by the 26 strains has a residual activity of 70% against 10 mM citric acid and is found to have sufficient protease activity even when used in combination with citric acid.
In addition, Flavobacterium frigidarium FF-No. The protease produced by the 26 strains is found to have a residual activity of 32% for 1 mM EDTA and a residual activity of 20% for 10 mM EDTA, and has sufficient protease activity even when used in combination with EDTA.

(Characteristics evaluation 2: optimum temperature)
The above protease samples were subjected to the azocasein method to measure protease activity at each temperature (10 to 50 ° C.). The results are shown in FIG. Referring to FIG. 1, Flavobacterium frigidarium FF-No. The protease produced by the 26 strains is found to have an optimum temperature of 30 ° C.

(Characteristics evaluation 3: Residual activity to heat treatment)
The above-mentioned protease sample was heat-treated at each temperature (40 ° C., 50 ° C., 60 ° C.) for a fixed time (15 minutes, 30 minutes, 60 minutes), and then the protease activity was measured by the azocasein method. The results are shown in FIG. 2 (open triangles in FIG. 2 are results at 60 ° C., open squares are results at 50 ° C., and open circles are results at 40 ° C.). Referring to FIG. 2, the Flavobacterium frigidarium FF-No. The protease produced by the 26 strains is found to be completely inactivated when heat-treated at 50 ° C. or more for 15 minutes or more. Also, it can be seen that the residual activity decreases to about 20% when heat treatment is performed at 40 ° C. for 30 minutes.

(Characteristics evaluation 4: optimum pH)
The above protease samples were subjected to the azocasein method, and protease activity at each pH (4 to 10) was measured. The results are shown in FIG. Referring to FIG. 3, Flavobacterium frigidarium no. The protease produced by the 26 strains is found to have an optimum pH of 7.

[Washing test]
A wash test was performed using the protease sample and a detergent composition comprising the protease sample. As objects to be cleaned, a rubber plate with a thickness of about 5 mm is placed on the four sides of a urethane-impregnated conveyor belt (Folvo-Selling Japan Ltd.), and the sample liquid placed on the conveyor belt does not leak out After that, 1.5 g of salted sausage meat was placed on the center of the conveyor belt, spread evenly, and allowed to stand at 6 ° C. for 10 minutes.
Moreover, the residual activity with respect to heat processing was measured using the washing | cleaning liquid after the washing test by the said protease sample.

(Washing test 1: Washing with protease sample)
Add 2 mL each of the above protease sample, a commercially available protease (Protease P "Amano" 3 SD, Amano Enzyme Co., Ltd.) and distilled water to each of the objects to be washed and allow to stand for 5 hours at 20 ° C. The dry weight of the cured meat was measured. The results are shown in FIG. The graph of FIG. 4 shows the relative ratio when the dry weight of the cured meat left after washing with distilled water is 100%.
Referring to FIG. 4, the protease of the present invention was found to have a better cleaning effect than the commercially available protease.

(Washing test 2: Washing with a detergent composition containing a protease sample)
The neutral detergent and the neutral detergent containing 2 mL each of the above-mentioned protease sample were respectively added to the above-mentioned to-be-cleaned matter, rinsed after being left standing at 20 ° C. for 5 hours, and the dry weight of the remaining salted meat was measured. The results are shown in FIG. The graph of FIG. 5 shows the relative ratio when the dry weight of the cured meat left after washing with a neutral detergent is 100%.
Referring to FIG. 5, the neutral detergent containing the protease of the present invention was found to have a better cleaning effect than the neutral detergent.

(Washing test 3: Residual activity for heat treatment of washing liquid after washing test)
The protease activity after heat treatment at 50 ° C. for 30 minutes was measured for each of the washings after washing the above-mentioned protease sample and a commercially available protease with a washing target. Protease activity was evaluated by the method of the above-mentioned "(3) Protease activity test". The results are shown in Table 5.

  As shown in Table 5, while the washing solution for the protease of the present invention is completely inactivated by heat treatment at 50 ° C. for 30 minutes, the commercially available washing solution for protease is heat treated at 50 ° C. for 30 minutes. Also, it can be seen that the protease activity does not decrease.

  The protease of the present invention is a neutral region, and can be suitably used for processing to degrade proteins under conditions including a chelating agent. In addition, since the protease activity can be almost completely inactivated by a simple heat treatment, it can be suitably used in the case where problems such as quality deterioration occur when the protease remains, such as food. Specifically, it can be suitably used as an additive for food production such as softening treatment of meat, or as a detergent composition of an apparatus or facility for food production.

  Also, the protease of the present invention can impart protein degradation to neutral detergents. Therefore, it can be suitably used for the detergent composition for resin products which is easily deteriorated when it is washed with an alkaline detergent.

Claims (12)

A protease having an optimum pH of 4.0 to 8.5,
50% or more of residual activity against 10 mM citric acid,
A protease characterized by having a residual activity of 5% or less after treatment at 50 ° C. for 30 minutes.   The protease according to claim 1, wherein the protease has an optimum temperature of 20 to 40C.   The above-mentioned proteases are exemplified by Flavobacterium frigidarium FF-No. The protease according to claim 1 or 2, which is produced by a microorganism of 26 strains or a mutant thereof.   A detergent composition comprising the protease according to any one of claims 1 to 3.   The detergent composition according to claim 4, wherein the detergent composition has a pH of 5.0 to 9.0.   A cleaning method comprising cleaning the object to be cleaned using the cleaning composition according to claim 4 or 5.   What is claimed is: 1. A washing method comprising: washing a to-be-cleaned object using a detergent composition containing a protease having a residual activity of 5% or less after treatment at 50 ° C. for 30 minutes.   The cleaning method according to claim 6, wherein the cleaning object is heat-treated to 50 ° C. or higher after the cleaning object is cleaned.   The cleaning method according to any one of claims 6 to 8, wherein the object to be cleaned is an apparatus or facility for manufacturing food.   The cleaning method according to any one of claims 6 to 9, wherein the object to be cleaned is a resin product.   A microorganism, characterized in that it produces the protease according to claim 1 or 2.   Flavobacterium frigidarium FF-No. A microorganism which is 26 strains or a mutant thereof.

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