JP2016036327A - Protease-containing cleaning composition and method for stabilizing protease - Google Patents

Abstract

To find a stabilizer that suppresses the inactivation of a novel protease derived from actinomycetes, a novel enzyme composition containing the protease and the stabilizer, a novel protease-containing cleaning composition containing the enzyme composition, and the like A novel washing method using the composition and a method for stabilizing the protease are provided. As a protease stabilizer, skim milk or calcium ion is used. [Selection figure] None

Description

  The present invention relates to a protease-containing cleaning composition and a method for stabilizing protease.

  Conventionally, as a cleaning agent for a medical device, a cleaning composition comprising a nonionic surfactant, a protease, a sequestering agent, benzotriazole, and the like is known (for example, see Patent Document 1). A cleaning composition containing a protease, a nonionic surfactant, and an enzyme stabilizer is also known (see, for example, Patent Document 2).

JP-A-5-279700 JP 2001-31999 A

At present, alkaline detergents are most widely used, but the following problems have been pointed out.
1. Corrosion of aluminum products, and stainless steel products turn black.
2. In the cleaning process, the cleaning device may be damaged, and the piping may be corroded.
3. Dangerous to the human body and the environment.

In order to solve such problems, a novel protease having a strong ability to degrade proteins, and particularly having high detergency against protein stains such as blood, a detergent containing the protease, and a method for producing the same have already been disclosed. A patent application has been filed (PCT / JP2013 / 000724). This protease exhibits activity in a high temperature range (50 to 95 ° C.), and can be used even at 93 ° C. which is a temperature for performing a sterilization / disinfection process such as an ordinary instrument decontamination washer (WD). .
However, protease, which is a protein, is rapidly deactivated at high temperatures, and is also a protease. Therefore, the enzyme itself undergoes self-digestion, and there is a problem that inactivation is further promoted.

  Accordingly, an object of the present invention is to find a stabilizer that suppresses inactivation of the protease, a novel enzyme composition containing the protease and the stabilizer, a novel protease-containing cleaning composition containing the enzyme composition, these And a method for stabilizing the protease.

The present invention
[1] The following polypeptides (a) to (d):
(A) a polypeptide comprising an amino acid sequence at positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2,
(B) a polypeptide comprising an amino acid sequence at positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2 and having protease activity;
(C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence at positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2 and having protease activity; and (d) SEQ ID NO: 2 comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence from position 111 to position 386 of the amino acid sequence represented by 2 and having protease activity At least one protease selected from the group;
An enzyme composition comprising at least one protease stabilizer selected from the group consisting of skim milk and calcium ions;
[2] The protease is
A protease obtained by culturing actinomycetes sp. RD001933 strain (accession number NITE BP-1467),
Proteases obtained by culturing actinomycetes Actinomadura miaoliensis RD000920 strain (Accession No. NITE BP-1468), actinomycetes Actinomadura sp. RD001933 strain and The enzyme composition according to [1], which is at least one protease selected from the group consisting of proteases obtained by mixing and culturing actinomycete Actinomadura miaoliensis RD000920;
[3] The enzyme composition according to [2] or [3], further comprising a surfactant,
[4] A protease-containing cleaning composition comprising the enzyme composition according to any one of [1] to [3],
[5] A cleaning method using the enzyme composition according to any one of [1] to [3] or the protease-containing cleaning composition according to [4],
[6] The following polypeptides (a) to (d):
(A) a polypeptide comprising an amino acid sequence at positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2,
(B) a polypeptide comprising an amino acid sequence at positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2 and having protease activity;
(C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence at positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2 and having protease activity; and (d) SEQ ID NO: 2 comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence from position 111 to position 386 of the amino acid sequence represented by 2 and having protease activity At least one protease selected from the group,
The present invention relates to a method for stabilizing a protease, wherein at least one protease stabilizer selected from the group consisting of skim milk and calcium ions is allowed to coexist.

  According to the present invention, inactivation of a novel protease having a high detergency against protein stains such as blood can be suppressed. More specifically, for example, by maintaining protease activity during storage of the enzyme composition and when stored as a cleaning composition, by inhibiting protease degradation and maintaining protease activity at the reaction temperature (particularly in the high temperature range) during cleaning. , Stabilization of the protease can be achieved.

It is a graph which shows the temperature dependence of the specific activity about protease (purified enzyme) derived from RD001933 strain and protease (purified enzyme) derived from RD000920 strain. It is a graph which shows the pH dependence of the specific activity about the protease (purified enzyme) derived from RD001933 strain and the protease (purified enzyme) derived from RD000920 strain. It is a graph which shows the temperature dependence of the relative activity about the crude enzyme obtained from the mixed culture solution of RD001933 strain and RD000920 strain. It is a graph which shows the pH dependence of the relative activity about the crude enzyme obtained from the mixed culture solution of RD001933 strain and RD000920 strain. It is a graph which shows the stabilization effect of skim milk at the time of storing as a solution about the crude enzyme obtained from the mixed culture solution of RD001933 strain and RD000920 strain. It is a graph which shows the stabilization effect of skim milk at the time of incubating at 80 degreeC about the crude enzyme obtained from the mixed culture solution of RD001933 strain and RD000920 strain. It is a graph which shows the stabilization effect of the calcium ion at the time of incubating at 80 degreeC about the crude enzyme obtained from the mixed culture solution of RD001933 strain and RD000920 strain. It is a graph which shows the stabilization effect of a calcium ion at the time of changing the calcium ion concentration and incubating at 80 degreeC about the crude enzyme obtained from the mixed culture solution of RD001933 strain and RD000920 strain. It is a graph which shows the stabilization effect of the calcium ion at the time of incubating in the high temperature range about the crude enzyme obtained from the mixed culture solution of RD001933 strain and RD000920 strain.

In the present invention, as the protease, for example,
(A) a polypeptide comprising an amino acid sequence at positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2,
(B) a polypeptide comprising an amino acid sequence at positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2 and having protease activity;
(C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence at positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2 and having protease activity (hereinafter referred to as homologous polypeptide and )
(D) an amino acid sequence at positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2 comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added, and having protease activity Polypeptide (hereinafter referred to as functional equivalent variant)
Can be used.

  The amino acid sequence represented by SEQ ID NO: 2 is, for example, a sequence encoded by the base sequence (1161 bp) represented by SEQ ID NO: 1, and encodes a protease consisting of 386 amino acids. In the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence at positions 1 to 26 is a signal sequence, the amino acid sequence at positions 27 to 110 is a pro sequence, and the amino acid sequence at positions 111 to 386 is a mature type. (Active form) sequence.

  Examples of the polypeptide comprising the amino acid sequence at positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2 and having protease activity include a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 2 A polypeptide consisting of the amino acid sequence of positions 27 to 386 of the amino acid sequence represented by the formula: N-terminal of the amino acid sequence of positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2 and And / or a polypeptide to which a protease activity is not reduced, for example, a signal sequence, a detection marker, and a purification tag sequence (polypeptides such as histidine and GST consisting of 6 residues) are listed. be able to.

Protease activity refers to enzyme activity that degrades proteins. The protease activity can be confirmed, for example, by the following azocasein method or casein method.
In the azocasein method, first, 50 μL of 3% azocasein (final concentration 0.6%) is mixed with 198 μL of Tris-HCl buffer (pH 7.5, final concentration 80 mM). And 2 microliters of samples which confirm enzyme activity are added, and it is made to react at 65 degreeC for 5 minutes or 10 minutes. After the enzymatic reaction, 50 μL of 20% trichloroacetic acid is added to stop the reaction. After stopping the reaction, the reaction solution is centrifuged, and the absorbance of the supernatant is measured at 340 nm. An enzyme amount of 1 U (unit) is an amount that generates 1 μmol of an azo dye equivalent per minute.

  In the casein method, 5 μL of a sample for confirming enzyme activity and 50 μL of a 2% casein solution are added to 45 μL of Tris-HCl buffer. The reaction is carried out at 65 ° C. for 5 minutes. After the enzyme reaction, 25 μL of 35% trichloroacetic acid is added to stop the reaction. After stopping the reaction, the reaction solution is centrifuged, 20 μL of the supernatant is taken, 180 μL of 0.1N NaOH is added, and the absorbance at 280 nm is measured.

  “Homologous polypeptide” includes “an amino acid sequence having 80% or more identity with the amino acid sequence at positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2 and having protease activity” However, a polypeptide comprising an amino acid sequence whose identity is preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more is preferable.

In the present specification, the “identity” means a value Identity obtained by using a parameter prepared as a default by a NEEDLE program (J Mol Biol 1970; 48: 443-453) search. The parameters are as follows:
Gap penalty = 10
Extended penalty = 0.5
Matrix = EBLOSUM62

  The number of amino acids that can be substituted, deleted, and / or inserted in the “functionally equivalent variant” is 1 to several, preferably 1 to 10, more preferably 1 to 7, and most preferably 1. ~ 5.

In the present invention, the amino acid substituted for maintaining the function of the peptide is preferably an amino acid having properties similar to the amino acid before substitution. For example, amino acids belonging to each group as shown below are amino acids having properties similar to each other within the group. Substituting these amino acids with other amino acids in the group often does not compromise the essential function of the protein. Such amino acid substitution is called conservative substitution and is known as a technique for converting an amino acid sequence while retaining the function of a polypeptide.
Nonpolar amino acids: Gly, Ala, Val, Leu, Ile, Pro, Met, Phe, and Trp
Neutral amino acids: Ser, Thr, Cys, Tyr, Asn, and Gln
Acidic amino acids: Asp and Glu
Basic amino acids: Lys, Arg, and His

  The polypeptide consisting of the amino acid sequence of positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2 that can be used in the present invention is, for example, Actinomydra sp. RD001933 strain ( (Accession No. NITE BP-1467), Actinomadura miaoliensis RD000920 strain (Accession No. NITE BP-1468) can be prepared. Each of these microorganisms can be cultivated alone or in a mixed culture.

  In addition, a polypeptide that can be used in the present invention, for example, a polypeptide comprising an amino acid sequence at positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2, or 111 of the amino acid sequence represented by SEQ ID NO: 2 Polypeptides containing the amino acid sequence at positions 386 can also be prepared using genetic recombination techniques. For example, a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 is incorporated into an appropriate expression vector, an appropriate host is transformed with the vector of the present invention, and the resulting transformant is cultured to obtain the desired The polypeptide can be prepared.

  In the present invention, the culture supernatant obtained by removing the cells from the culture solution obtained in these cultures can be used as it is as a protease-containing fraction, or various proteins can be used using the culture supernatant as a starting material. A crudely purified fraction or purified enzyme obtained by the purification method can also be used as a protease.

In the present invention, skim milk or calcium ions (Ca ²⁺ ) is used as a protease stabilizer. One of these protease stabilizers can be used alone, or both can be used in combination.
In the present invention, by adding the stabilizer, for example, maintenance of protease activity when storing the enzyme composition and when stored as a cleaning composition, inhibition of protease degradation at the reaction temperature (particularly in the high temperature range) during cleaning. And maintenance of protease activity can be achieved.

  When skim milk is used as a protease stabilizer, the final concentration in the reaction system is, for example, 0.04 to 4% (W / V), preferably 0.04 to 1.2% (W / V), Preferably, it can be added at 0.5 to 1.2% (W / V). The enzyme composition or cleaning composition is, for example, 0.04 to 5% (W / V), preferably 0.04 to 4% (W / V), more preferably 0.5 to 3% (W / V).

  When calcium ions are used as a protease stabilizer, the final concentration in the reaction system is, for example, 0.01-5 mM, preferably 0.05-1 mM, more preferably 0.1-0.3 mM. Can do. Moreover, as an enzyme composition or a washing | cleaning composition, it can contain in the density | concentration of 0.01-200 mM, Preferably it is 0.05-50 mM, More preferably, it is 0.1-30 mM, for example.

  In this invention, in addition to the said stabilizer, the arbitrary additives which can be added to a normal cleaning composition can be contained. Examples of the additive include surfactants, buffers, preservatives, rust inhibitors, dyes, chelating agents, foam modifiers, fragrances, dechlorinating agents, antibacterial agents, and viscosity modifiers.

  As the surfactant, for example, a nonionic surfactant, an anionic surfactant, a cationic surfactant, or an amphoteric surfactant can be used.

  Specific examples of the nonionic surfactant include polyoxyethylene alkyl ether [eg, polyethylene glycol mono-para-iso-octylphenyl ester (Triton X-100)], polyoxyethylene alkyl allyl ether, polyoxy Ethylene derivatives, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters [for example, polyoxyethylene monolaurate (Tween 20)], polyoxyethylene sorbitol fatty acid esters, glycerin fatty acid esters, polyoxyethylene alkylamines, alkyl alkanolamides, etc. Can be mentioned.

  Specific examples of the anionic surfactant include fatty acid salts, alkyl sulfate esters [for example, sodium lauryl sulfate (SDS)], alkyl benzene sulfonates, alkyl naphthalene sulfonates, and alkyl sulfosuccinates. Can be mentioned.

  Specific examples of the cationic surfactant include alkylamine salts or quaternary ammonium salts [for example, cetyltrimethylammonium bromide (CTAB)].

  Specific examples of amphoteric surfactants include alkyl betaines, amine oxides, and dimethylammoniopropane sulfonic acid.

When a surfactant is added as the additive, for example, Triton X-100, Tween 20, SDS, or CTAB is preferably used. It has been confirmed that these surfactants have not only the original cleaning effect but also the effect of increasing the enzyme activity of the protease used in the present invention.
Moreover, as a chelating agent, for example, ethylenediaminetetraacetic acid (EDTA) can be used, and EDTA has been confirmed to have an effect of increasing the enzyme activity of the protease used in the present invention.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

<< Example 1: Purification of protease (RD001933 strain), evaluation of enzymological properties, and sequencing >>
(1) Single culture of microorganism (RD001933 strain) RD001933 strain (accession number: NITE BP-1467) belonging to the genus Actinodura sp.
First, 490 mL of ISP2 medium (yeast extract 0.6%, malt extract 1.4%, glucose 0.6%) was prepared, and 70 mL was dispensed into a 500 mL Erlenmeyer flask. One spring coil was put in this, and steam sterilization was performed at 121 ° C. for 20 minutes. Furthermore, 30 mL of 2.5% skimmed milk that was sterilized separately was added to a final concentration of 0.75%.
Then, 50 μL of glycerol stock cells were inoculated into a φ18 test tube (18 × 180 mm) containing 5 mL of ISP2 medium, and cultured with shaking at 45 ° C. until good growth was obtained. 1 mL of this culture solution was inoculated into 100 mL of the previously sterilized medium, and cultured with shaking at 45 ° C. for about 96 hours. The supernatant was recovered from this culture using a centrifuge.

(2) Purification of protease (a) Acetone precipitation and ammonium sulfate fraction Acetone is added to the culture supernatant recovered in (1) above so as to be 60% (v / v) or more, and the resulting precipitate is centrifuged. (10,000 rpm, 10 minutes, 4 ° C.). This precipitate was dissolved in 15 mL of 20 mM Tris-HCl buffer (pH 8.0) containing ammonium sulfate having a final concentration of 1 M to obtain a crude enzyme solution.

(B) Toyopearl Phenyl-650M column chromatography The Toyopearl Phenyl-650M column (inner diameter) obtained by previously equilibrating the crude enzyme solution obtained in (a) above with 20 mM Tris-HCl buffer (pH 8.0) containing 1 M ammonium sulfate. 26 mm, height 38 mm, manufactured by Tosoh Corporation). After washing the column with the same buffer, the protein was eluted with a linear gradient of ammonium sulfate (from 1 M to 0 M).

(C) HiTrap Q HP column chromatography The active fractions obtained in (b) above were collected and desalted by dialysis using 20 mM Tris-HCl buffer (pH 9.0). After applying the column to a HiTrap Q HP (5 mL) column (manufactured by GE Healthcare Biosciences) previously equilibrated with 20 mM Tris-HCl buffer (pH 9.0), the column was washed with the same buffer, and then sodium chloride ( The protein was eluted with a linear gradient from 0M to 1M.

(D) HiTrap SP HP column chromatography The active fractions obtained in (c) above were collected and desalted by dialysis using 20 mM MES-sodium hydroxide buffer (pH 5.5). This was applied to a HiTrap SP (1 mL) column (manufactured by GE Healthcare Biosciences) pre-equilibrated with 20 mM MES-sodium hydroxide buffer (pH 5.5), and the column was washed with the same buffer. The protein was eluted with a linear gradient of sodium chloride (0M to 1M).

As described above, a purified enzyme was obtained from the RD001933 strain belonging to the genus Actinodura sp.
The enzyme activity of the protease was measured as follows. First, the reaction liquid shown in Table 1 was allowed to react at 65 ° C. and pH 7.5 for 5 minutes, and then 50 μL of 20% trichloroacetic acid was added to stop the reaction. Thereafter, the reaction solution was centrifuged, and the absorbance at 340 nm of the supernatant was measured. The amount of enzyme 1U (unit) was set to an amount capable of producing 1 μmol of an azo dye corresponding to 1 minute. Hereinafter, activity measurement using azocasein may be referred to as azocasein method.

  The active fractions eluted in (d) above were collected and analyzed for molecular weight by SDS-PAGE (12% (w / v) polyacrylamide gel). A single band was observed in the active fraction, and the molecular weight of the purified enzyme (polypeptide) was about 31 kDa.

(3) Examination of Enzymatic Properties of Purified Protease The enzymatic properties of protease (purified enzyme) derived from RD001933 strain were examined.

(A) Working temperature After reacting the reaction liquid shown in Table 1 at each temperature and pH 7.5 for 5 minutes to react, 50 μL of 20% trichloroacetic acid was added to stop the reaction. Thereafter, the reaction solution was centrifuged, and the absorbance at 340 nm of the supernatant was measured to determine the enzyme activity.
FIG. 1 is a graph showing specific activities of enzyme activities at various reaction temperatures. As shown in the graph of FIG. 1, the RD001933-derived protease (purified enzyme) exhibits activity at 50 to 90 ° C., and the optimum temperature for the reaction is in the range of 60 to 80 ° C., preferably 70 It was around -75 ° C.

(B) Working pH
The reaction liquid shown in Table 2 was allowed to react at each pH and 75 ° C. for 5 minutes, and then the reaction was stopped by adding 50 μL of 20% trichloroacetic acid. Thereafter, the reaction solution was centrifuged, and the absorbance at 340 nm of the supernatant was measured to determine the enzyme activity.
The buffer used was as follows.
MES-sodium hydroxide buffer: pH 5.5, pH 6
Bis-Tris buffer: pH 6, pH 6.5, pH 7.2
Tris-HCl buffer: pH 7.2, pH 8, pH 8.8
Glycine-NaOH buffer solution: pH 9, pH 9.5

  FIG. 2 is a graph showing the enzyme activity at various reaction pHs as specific activity. As can be seen from the graph of FIG. 2, the protease (purified enzyme) derived from the RD001933 strain exhibits activity in a wide range of pH 5.5 to 9.0, and the optimum pH of the reaction is around 7.2 (for example, pH 6 0.0 to 8.8).

(4) Determination of the base sequence of the protease gene For the protease (purified enzyme) derived from the above RD001933 strain, N-terminal amino acid sequence analysis and internal amino acid sequence analysis of the peptide sample obtained by trypsin digestion were performed. The gene was cloned by the PCR method and the base sequence was determined. As a result, the base sequence (1161 bp) represented by SEQ ID NO: 1 was obtained. The amino acid sequence represented by SEQ ID NO: 2 is an amino acid sequence corresponding to the codon of this sequence (SEQ ID NO: 1).
As a result of sequence analysis, it was revealed that the gene encoding protease derived from RD001933 strain consists of 1161 bp nucleotides and encodes an amino acid of 386 residues. In the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence at positions 1 to 26 is a signal sequence, the amino acid sequence at positions 27 to 110 is a pro sequence, and the amino acid sequence at positions 111 to 386 is a mature type. (Active form) sequence.

<< Example 2: Purification of protease (RD000920 strain), evaluation of enzymatic properties, and sequencing >>
(1) Microbial culture and protease purification Example 1 (1) except that RD000920 strain (accession number: NITE BP-1468) belonging to the genus Actinomadura miaoliensis of actinomycetes was used as the microbial cell. ) And Example 1 (2) were repeated to obtain RD000920 strain-derived protease (purified enzyme).

When the molecular weight was analyzed by SDS-PAGE (12% (w / v) polyacrylamide gel), a single band was observed in the active fraction, and the molecular weight of the purified enzyme (polypeptide) was about 31 kDa.
Further, when the base sequence was determined by the same method as in Example 1 (4), it almost coincided with the base sequence represented by SEQ ID NO: 1 and the amino acid sequence represented by SEQ ID NO: 2 100% agreement.

(2) Examination of Enzymatic Properties of Purified Protease The enzymatic properties of RD000920 strain-derived protease (purified enzyme) were examined by the procedure described in Example 1 (3).
The results regarding the working temperature and working pH are shown in FIG. 1 and FIG.

(A) Action temperature Protease (purified enzyme) derived from strain RD000920 exhibits activity at 50 to 90 ° C, and the optimum temperature for the reaction is in the range of 60 to 80 ° C, preferably around 70 to 75 ° C. there were.

(B) Working pH
RD000920 strain-derived protease (purified enzyme) exhibits activity in a wide range of pH 5.5 to 9.0, and the optimum pH of the reaction is around 7.2 (for example, pH 6.0 to 8.8). It was.

<< Example 3: Purification of protease (mixed culture of two strains) and evaluation of enzymological properties >>
(1) Mixed culture of microorganisms (RD001933 strain and RD000920 strain) As cells, RD001933 strain (accession number: NITE BP-1467) belonging to the genus Actinomadura sp. And actinomydra of actinomycetes RD000920 strain (accession number: NITE BP-1468) belonging to the genus (Actinomadura miaoliensis) was used.
First, 4.2 L of ISP2 medium (yeast extract 0.6%, malt extract 1.4%, glucose 0.6%) was prepared and placed in a 10 L desktop culture device (manufactured by Maruhishi Bioengineer), 121 ° C. For 20 minutes. Furthermore, 1.8 L of 2.5% skimmed milk that was sterilized separately was added to a final concentration of 0.75%.
Then, 500 μL of two types of glycerol stock cells were inoculated into 500 mL Erlenmeyer flasks containing 50 mL of ISP2 medium, and cultured with shaking at 45 ° C. until good growth was obtained. 30 mL of these two types of culture solutions were inoculated into 6 L of the previously sterilized medium and cultured at 45 ° C., 500 rpm, 1 vvm for 1 to 7 days. The ratio of the two types of culture solutions at the start of the culture is preferably 1: 1, but there is no special provision. The culture period is preferably 1 to 7 days, and more preferably 3 to 5 days. The supernatant was recovered from this culture using a centrifuge.

(2) Purification of protease (a) Acetone precipitation Acetone at −20 ° C. so that it becomes 40%, 50%, 60%, 70%, 80% (v / v) in the culture supernatant recovered in (1) above. And the precipitate produced at each concentration was collected by centrifugation (10,000 rpm, 10 minutes, 4 ° C.). This precipitate was dissolved in 50 mL of 20 mM Tris-HCl buffer (pH 8.0) to obtain a crude enzyme solution. Table 3 shows enzyme recovery.

(B) Ethanol precipitation Ethanol was added to the culture supernatant collected in the above (1) so as to be 40%, 50%, 60%, 70%, and 80% (v / v), and each concentration was generated. The precipitate was collected by centrifugation (10,000 rpm, 10 minutes, 4 ° C.). This precipitate was dissolved in 50 mL of 20 mM Tris-HCl buffer (pH 8.0) to obtain a crude enzyme solution. Table 3 shows enzyme recovery.

(C) Ammonium sulfate precipitation To the culture supernatant collected in (1) above, ammonium sulfate powder was added so that the saturation amount was 40%, 50%, 60%, 70%, and 80%. It was recovered by centrifugation (10,000 rpm, 10 minutes, 4 ° C.). This precipitate was dissolved in 50 mL of 20 mM Tris-HCl buffer (pH 8.0) to obtain a crude enzyme solution. Table 3 shows enzyme recovery.

(2) Examination of Enzymatic Properties of Crude Enzyme Solution Enzymatic properties of protease (crude enzyme) derived from RD001933 strain and RD000920 strain were examined.

(A) Working temperature After reacting the reaction solution shown in Table 2 (enzyme solution obtained by 80% saturated ammonium sulfate precipitation) at each temperature and pH 7.5 for 10 minutes, 50 μL of 20% trichloroacetic acid was reacted. In addition, the reaction was stopped. Thereafter, the reaction solution was centrifuged, and the absorbance at 340 nm of the supernatant was measured to determine the enzyme activity.

  FIG. 3 is a graph showing enzyme activities at various reaction temperatures as relative activities based on the activity (100%) when the reaction temperature is 70 ° C. As shown in the graph of FIG. 3, the RD001933 strain and the RD000920 strain-derived protease (crude enzyme) exhibit activity at 55 to 85 ° C., and the optimum temperature for the reaction is in the range of 65 to 80 ° C. Preferably, it was around 70 ° C.

(B) Working pH
The reaction solution shown in Table 3 (enzyme solution obtained by 80% saturated ammonium sulfate precipitation) was allowed to react for 10 minutes at each pH and 65 ° C., and then the reaction was stopped by adding 50 μL of 20% trichloroacetic acid. It was. Thereafter, the reaction solution was centrifuged, and the absorbance at 340 nm of the supernatant was measured to determine the enzyme activity.
The buffer used was as follows.
Acetic acid-Na acetate buffer: pH 5
Bis-Tris buffer: pH 6
Tris-HCl buffer: pH 7.2, pH 8
Glycine-NaOH buffer: pH 9
FIG. 4 is a graph showing enzyme activities at various reaction pHs as relative activities based on the enzyme activity when the reaction pH is 8.0 (100%). As can be seen from the graph of FIG. 4, the RD001933 strain and the RD000920 strain-derived protease (crude enzyme) exhibit activity in a wide range of pH 5.0 to 9.0, and the optimum pH of the reaction is around 7.5. (For example, pH 7.0 to 8.0).

<< Example 4: Cleaning test >>
(1) Dropping test As a protease-containing detergent, RD001933-derived protease (purified enzyme), RD000920 strain-derived protease (purified enzyme), RD001933 strain and RD000920 strain-derived protease (crude enzyme) were each contaminated with simulated blood. The solution was dropped on a piece (cleaning evaluation indicator “TOSI-Gold”, Nithion Co., Ltd.), and the pseudo blood was wiped off every predetermined time for washing. This washing was performed at 65 ° C. by adjusting the amount of each enzyme to 5 μL (concentration: about 5 U / mL). The cleaning result of the test piece was determined visually by the following criteria. The results are shown in Table 4.
“◎”: completely cleaned “○”: almost cleaned “△”: very little residue (pseudo-blood stain) “×”: residue (pseudo-blood stain) remains State

(2) Flask test As detergents, RD001933 strain and RD000920 strain-derived protease (crude enzyme), commercially available enzyme nattokinase (Wako Pure Chemical Industries, Ltd. “147-08801”), commercially available streptokinase (Wako Pure Chemical Industries, Ltd.) "593-20581"), commercially available enzyme urokinase (Mitsubishi Tanabe Pharma Corporation "873954"), commercially available laundry detergent (Kao Corporation "Attack Neo"), commercially available enzyme thermolysin (Sigma-Aldrich "P1512-1G") Using.
30 mL of each of the above cleaning agents was put into a 200 mL flask, and further a test piece (N cleaning evaluation indicator “TOSI-Gold”, Nithion Co., Ltd.) was added and immersed. And the inside of said flask was stirred with the stirring bar on the conditions of temperature and time shown in Table 5. Then, after taking out said test piece and lightly washing with water, the washing | cleaning result was determined on the same basis as the above. The results are shown in Table 5.

  As is apparent from Table 5, it was considered that proteases (crude enzymes) derived from RD001933 and RD000920 strains had the highest cleaning effect, and then the commercially available enzyme thermolysin had the highest cleaning effect.

<< Example 5: Evaluation of stabilizing effect of skim milk >>
(1) Storage stability when stored as a crude enzyme solution The effect of adding skim milk when protease was stored as a solution was evaluated.
Ammonium sulfate was added to the supernatant collected from the culture medium in Example 3 (1) so that the saturation amount was 60%, and the resulting precipitate was collected by centrifugation (10,000 rpm, 10 minutes, 4 ° C.). This precipitate was dissolved in 20 mM Tris-HCl buffer (pH 8.0) so as to be concentrated 90 times to obtain a crude enzyme solution. Skimmed milk was added to the resulting crude enzyme solution to a concentration of 0.04% (W / V). Storage was performed at 40 ° C., and storage stability with and without skim milk was evaluated. In addition, the said temperature is a temperature used in the acceleration test in the storage stability test of an enzyme, and is an index of about 6 times acceleration. Moreover, about the thing which does not add skim milk, the storage at 4 degreeC which is a general refrigeration temperature was also performed.

After a predetermined number of days, the enzyme activity of each crude enzyme was measured as follows. First, the reaction solution shown in Table 1 (the crude enzyme solution was diluted 200-fold with tap water) was allowed to react at 65 ° C. for 10 minutes, and then 50 μL of 20% trichloroacetic acid was added to stop the reaction. . Thereafter, the reaction solution was centrifuged, and the absorbance at 340 nm of the supernatant was measured. The activity on the first day was taken as the standard (100%).
The results are shown in FIG. The horizontal axis is the number of days elapsed from the start of the storage stability test, and the vertical axis is the relative activity based on the protease activity (according to the azocasein method) at the start of the test (100%).
It was confirmed that the storage stability in the solution was greatly improved by adding skim milk.

(2) Stability at the reaction temperature of the enzyme reaction The protease used in the present invention has an optimum reaction temperature in a high temperature range and can be washed at a high temperature. The effect of adding skim milk at the reaction temperature of the enzyme reaction (temperature: 80 ° C.) was evaluated.
A crude enzyme solution was obtained as a protease solution in the same manner as in Example 5 (2), diluted with 20 mM Tris-HCl buffer (pH 7.5) to 0.2 mg / mL, and skim milk was 0.04%. It added so that it might become the density | concentration of (W / V). After heating the crude enzyme solution (50 μL) diluted to a predetermined concentration to 80 ° C., the protease activity was measured by the following procedure after a predetermined time (3 minutes, 5 minutes, 13 minutes, 20 minutes) had elapsed. Each sample (50 μL) was incubated at 65 ° C. for 3 minutes, after which 50 μL of 2% casein solution was added and incubated at 65 ° C. for 5 minutes. After the enzymatic reaction, 25 μL of 35% trichloroacetic acid was added to stop the reaction, the reaction solution was centrifuged, 20 μL of the supernatant was added, 180 μL of 0.1N NaOH was added, and the absorbance at 280 nm was measured (hereinafter, Sometimes called casein method).

The results are shown in FIG. The horizontal axis represents the standing time at 80 ° C., and the vertical axis represents the protease activity (residual activity) indicated by the absorbance at a wavelength of 280 nm.
By adding skim milk, it was confirmed that the activity as an enzyme solution was improved and enzyme stability at high temperature was improved as compared with the case where skim milk was not added.

Example 6: Evaluation of calcium ion stabilization effect
(1) Stability at the reaction temperature of the enzyme reaction The procedure of Example 5 (2), except that calcium ions are used as an additive, and that the standing at 80 ° C. is 5 minutes, 10 minutes, 20 minutes, and 30 minutes. Was repeated. The addition concentration of calcium ions was 2 mM.
The results are shown in FIG. The horizontal axis is the standing time at 80 ° C., and the vertical axis is the relative activity based on the protease activity (by the casein method) at the start of the test (100%).
It was confirmed that by adding calcium ions, the activity as an enzyme solution was improved as compared with the case where calcium ions were not added, and the enzyme stability at high temperature was improved.

(2) Stability of enzyme reaction at reaction temperature (effect of calcium ion concentration)
To examine the effect of calcium ion concentration on the enzyme stabilization effect, the procedure of Example 6 (1) was repeated with the standing time at 80 ° C. being 15 minutes and changing the calcium ion concentration. It was.
The results are shown in FIG. The horizontal axis represents the calcium ion concentration, and the vertical axis represents the protease activity (by the casein method) indicated by the absorbance at a wavelength of 280 nm.
It was confirmed that calcium ions showed a high stabilizing effect even at a low concentration.

(3) Stability of enzyme reaction at reaction temperature (effect at high temperature)
Regarding the enzyme stabilization effect, in order to examine the effect in a high temperature range, the addition concentration of calcium ion is 2 mM, and the standing time at a predetermined temperature (70 ° C., 80 ° C., 85 ° C., 90 ° C., 100 ° C.) is 5 The procedure of Example 6 (1) was repeated for 15 minutes or 15 minutes.
The results are shown in FIG. The horizontal axis is the incubation temperature at which the reaction is allowed to stand for 5 minutes or 15 minutes, and the vertical axis is the relative activity based on the protease activity (by the casein method) before being left at the predetermined temperature (100%).
As shown in FIG. 9, when calcium ions were not added, the activity almost disappeared even at 80 ° C. for 15 minutes, and the activity almost disappeared after 5 minutes at 90 ° C. On the other hand, when calcium ions were added, it was confirmed that almost 100% of the activity was maintained at 90 ° C. for 5 minutes and disappeared to about 35% at 90 ° C. for 15 minutes. In particular, even at 100 ° C., it was confirmed that 30% of activity was still retained for 5 minutes.
Usually, sterilization and disinfection processes such as a washer disinfector (WD) are often performed at 93 ° C. for 10 minutes, but by adding calcium ions to the protease used in the present invention, washing and sterilization are performed.・ Can be disinfected at the same time.

  The present invention can be used as a cleaning agent, particularly as a cleaning agent for medical devices.

Actinomadura sp. RD001933 strain was established on November 22, 2012 by the National Institute of Technology and Evaluation of Microorganisms (NPMD) (Kazusa-Kazusa, Kisarazu City, Chiba Prefecture 292-0818). 2-5-8), deposited under the accession number NITE P-1467, and transferred to the international deposit (accession number NITE BP-1467) on April 19, 2013.
Actinomadura miaoliensis RD000920 strain was established on November 22, 2012 by the National Institute of Technology and Evaluation of Microorganisms (NPMD), Kisarazu City, Chiba Prefecture 292-0818. 2-5-8) was deposited as accession number NITE P-1468, and was transferred to the international deposit (accession number NITE BP-1468) on April 19, 2013.

Claims (6)

The following polypeptides (a) to (d):
(A) a polypeptide comprising an amino acid sequence at positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2,
(B) a polypeptide comprising an amino acid sequence at positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2 and having protease activity;
(C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence at positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2 and having protease activity; and (d) SEQ ID NO: 2 comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence from position 111 to position 386 of the amino acid sequence represented by 2 and having protease activity At least one protease selected from the group;
An enzyme composition comprising at least one protease stabilizer selected from the group consisting of skim milk and calcium ions. The protease is
A protease obtained by culturing actinomycetes sp. RD001933 strain (accession number NITE BP-1467),
Proteases obtained by culturing actinomycetes Actinomadura miaoliensis RD000920 strain (Accession No. NITE BP-1468), actinomycetes Actinomadura sp. RD001933 strain and The enzyme composition according to claim 1, wherein the enzyme composition is at least one protease selected from the group consisting of proteases obtained by mixing and culturing actinomycetes, Actinomadura miaoliensis RD000920.   The enzyme composition according to claim 1 or 2, further comprising a surfactant.   A protease-containing cleaning composition comprising the enzyme composition according to any one of claims 1 to 3.   A cleaning method using the enzyme composition according to any one of claims 1 to 3 or the protease-containing cleaning composition according to claim 4. The following polypeptides (a) to (d):
(A) a polypeptide comprising an amino acid sequence at positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2,
(B) a polypeptide comprising an amino acid sequence at positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2 and having protease activity;
(C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence at positions 111 to 386 of the amino acid sequence represented by SEQ ID NO: 2 and having protease activity; and (d) SEQ ID NO: 2 comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence from position 111 to position 386 of the amino acid sequence represented by 2 and having protease activity At least one protease selected from the group,
A method for stabilizing a protease, wherein at least one protease stabilizer selected from the group consisting of skim milk and calcium ions is allowed to coexist.