JP2013146227A - Protein solution and detergent composition containing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a protein solution capable of stably maintaining the enzymatic activity of a low temperature-optimum protease for a long period by preventing reduction of the enzymatic activity of the low temperature-optimum protease and improving heat resistance thereof.SOLUTION: There is provided a protein solution (D) containing a low temperature-optimum protease (A), a protease activity inhibitor (B) and a solvent (C), wherein the optimum temperature of the enzymatic reaction of the component (A) is 0-50°C. The protease activity inhibitor (B) is obtained by using one of benzyloxycarbonylphenylalanine nitroanilide, benzoylarginine nitroanilide, furylacryloylglycylleucine amide and succinylalanylalanylprolylphenylalanine nitroanilide as a substrate (E), and has an inhibitory constant Ki of from 1 pM to 10 μM against the component (A) obtained by a Henderson plot under a condition of the optimum temperature, optimum pH and optimum substrate concentration for the activity of the low temperature-optimum protease (A).

Description

  The present invention relates to a protein solution and a detergent composition containing the same.

  Protease is a general term for a group of enzymes that catalyze the hydrolysis of peptide bonds, and is widely known in microorganisms, animals and plants. Its application fields include clothing cleaners, automatic dishwasher cleaners, contact lens cleaners, bath preparations, exfoliating cosmetics, food modifiers (baking, meat softening, marine processing, etc. ), Beer fining agents, leather tanning agents, photographic film gelatin removers, digestion aids and anti-inflammatory agents, etc., and are actively used in many fields.

  As a protease to be added to a cleaning agent, a low-temperature optimal protease that exhibits high activity at the temperature at the time of cleaning has been recently developed. However, the low temperature optimal protease is particularly low in heat resistance among proteases, and is more likely to self-decompose in solution as compared with general proteases (optimal temperature is more than 50 and less than 80 ° C.). There is a problem that it tends to decrease.

On the other hand, a protease activity inhibitor that inhibits the activity of a protease has been studied. For example, Non-Patent Document 1 describes that boronic acid inhibits serine protease and subtilisin.
However, although the above protease activity inhibitor has a certain effect on general proteases, it is not sufficient to stably maintain the enzyme activity of the low temperature optimal protease for a long period of time. Moreover, there is no effect in improving the heat resistance of the low temperature optimal protease.

Molecular & Cellular Biochemistry, 51, 1983, p5-32.

  An object of the present invention is to provide a protein solution that can suppress a decrease in enzyme activity of a low-temperature optimal protease, further improve heat resistance, and can stably maintain the enzyme activity of a low-temperature optimal protease for a long period of time.

As a result of studies to achieve the above object, the present inventors have completed the present invention.
That is, the protein solution of the present invention is a protein solution (D) containing a low-temperature optimum protease (A), the following protease activity inhibitor (B) and a solvent (C), and is suitable for the enzyme reaction of (A). A protein solution having a temperature of 0 ° C to 50 ° C.
Protease activity inhibitor (B): As the substrate (E), any one of benzyloxycarbonylphenylalanine nitroanilide, benzoylarginine nitroanilide, furylacryloylglycylleucinamide and succinylalanylalanylprolylphenylalanine nitroanilide is used. A protease activity inhibitor having an inhibition constant Ki of 1 pM to 10 μM for (A) determined by Henderson plot under the conditions of the optimum temperature, optimum pH and optimum substrate concentration of the low temperature optimum protease (A).

The protein solution of the present invention can suppress a decrease in the enzyme activity of the low temperature optimal protease and can further improve the heat resistance, so that the enzyme activity of the low temperature optimal protease can be stably maintained for a long period of time.
Moreover, the detergent composition of the present invention has a high detergency.
In the present invention, “high cleaning durability” means that the difference between the cleaning performance measured after storage for a certain period of time and the cleaning performance measured immediately before storage is small and exhibits a certain cleaning performance. Means.

FIG. 2 is a Hanes-Woolf plot prepared for obtaining the Michaelis constant Km of a substrate for cannase, where the vertical axis represents the reciprocal ([S] / v) of each substrate concentration [S] according to the initial enzyme reaction velocity v and the horizontal axis represents the substrate. It is the graph plotted as density | concentration [S] of (D). In order to determine the optimum pH of cannase activity, the vertical axis represents the slope coefficient k at pH 4.5 to 9.5, and the horizontal axis represents pH, which is a graph showing the pH dependence of cannase activity. In order to determine the optimum temperature of cannase activity, the vertical axis represents a slope coefficient k at 20 to 70 ° C. and the horizontal axis represents temperature, and is a graph showing the temperature dependence of cannase activity. It is a Henderson plot created in order to obtain | require the inhibition constant with respect to cannase of the protease activity inhibitor (B1) obtained in manufacture example 1, 1 / (alpha) is on a horizontal axis, [Molar concentration of (B1)] / ( It is the graph which plotted 1- (alpha).

The protein solution of the present invention is a protein solution (D) containing a low temperature optimal protease (A), the following protease activity inhibitor (B) and a solvent (C), and the enzyme reaction optimal temperature of (A) is It is a protein solution which is 0-50 degreeC.
Protease activity inhibitor (B): As the substrate (E), any one of benzyloxycarbonylphenylalanine nitroanilide, benzoylarginine nitroanilide, furylacryloylglycylleucinamide and succinylalanylalanylprolylphenylalanine nitroanilide is used. A protease activity inhibitor having an inhibition constant Ki of 1 pM to 10 μM for (A) determined by Henderson plot under the conditions of the optimum temperature, optimum pH and optimum substrate concentration of the low temperature optimum protease (A).

In the present invention, the low temperature optimal protease (A) is a protease having an enzyme reaction optimal temperature of 0 to 50 ° C.
(A) includes known proteases having an optimal enzyme reaction temperature of 0 to 50 ° C., for example, those classified as aspartic proteases {proteases having two aspartic acids in the active site}, cysteine Those classified as proteases {proteases having cysteines that perform nucleophilic attack in the active site}, those classified as metal proteases {proteases having metal ions in the active site} and those classified as serine proteases {catalytic residues Are proteases having serine residues that perform nucleophilic attack, and specifically include cannase (Novozymes), subtilisin S39, subtilisin S41, etc.}.
(A) is preferably one classified as a serine protease from the viewpoint of being widely used in detergent applications.

Specifically, the enzyme reaction optimum temperature of the low temperature optimum protease (A) is determined by the optimum temperature measurement method described later.
In addition, for proteases other than the above, whether or not it is a low-temperature optimal protease is determined by determining the optimal temperature of the enzymatic reaction of the protease by a general protease activity measurement method (for example, an activity measurement method using azocasein) Judgment is made based on whether or not the optimum temperature is 0 to 50 ° C., and an optimum temperature of 0 to 50 ° C. is defined as a low temperature optimum protease.
The low temperature optimal protease is a protease having an optimal temperature of 0 to 50 ° C., and this optimal temperature is preferably 5 to 45 ° C. from the viewpoint of exhibiting high activity at the temperature at the time of use (washing etc.), More preferably, it is 10-40 degreeC.

  The protease activity inhibitor (B) comprises any one of benzyloxycarbonylphenylalanine nitroanilide, benzoylarginine nitroanilide, furylacryloylglycylleucinamide and succinylalanylalanylprolylphenylalanine nitroanilide as a substrate (E). A protease activity inhibitor having an inhibition constant Ki of 1 pM to 10 μM with respect to (A) determined by Henderson plot under conditions of optimum temperature, optimum pH and optimum substrate concentration of low temperature optimum protease (A) activity is there. (B) binds reversibly to (A) at a fixed molar ratio and inhibits the activity of (A).

The benzyloxycarbonylphenylalanine nitroanilide is obtained by binding an oxycarboxybenzyl group to the amino group of phenylalanine and a peptide bond of nitroaniline to the carboxyl group. MP-Biomedical Co., Ltd. (product name: N-cbz-L- (Phenylalanine-p-nitronilide).
The above benzoylarginine nitroanilide is a compound in which carboxybenzyl group is bonded to the amino group of arginine, and nitroaniline is peptide-bonded to the carboxyl group of arginine. Wako Pure Chemical Industries, Ltd. (product name: Nα-benzoyl-DL) -Arginine-p-nitroanilide hydrochloride).
The above-mentioned furylacryloylglycylleucine amide is a peptide in which furylacrylic acid is peptide-bonded to the amino group of glycylleucine amide. Sigma (product name: N- (3- [2-Furyl] acryloyl) -Gly-Leu amide).
The succinylalanylalanylprolylphenylalanine nitroanilide is a succinic anhydride bonded to the terminal amino group of alanylalanylprolylphenylalanine, and nitroaniline is peptide-bonded to the terminal carboxyl group. Product name: Suc-Ala-Ala-Pro-Phe-pNA).

Among the substrates (E), which substrate is used when obtaining Ki is appropriately selected according to the classification of (A) in which the protease activity inhibitor (B) inhibits the activity. Benzyloxycarbonylphenylalanine nitroanilide for the aspartic protease (A-1), benzoylarginine nitroanilide for the cysteine protease (A-2), and furylacryloylglycylleucinamide for the metalloprotease (A-3). In the case of serine protease (A-4), succinylalanylalanylprolylphenylalanine nitroanilide is used.
For proteases not exemplified as (A) above, Reference 1 (Rawlings, ND & Barrett, AJ (1993) Evolutionary families of peptides. Biochem J 290, p205-218), A-1) to (A-4) are classified.
In addition, whether or not a protease not described in Reference 1 is classified as (A-1) to (A-4) is determined in Reference 2 (Woosner & Barrett (1998) Handbook of It can be classified by using a method shown in proteolytic enzymes (Academic Press).

In the present invention, the optimal substrate concentration for the activity of the low temperature optimal protease (A) is 1/3 to 2 times the Michaelis constant Km of the substrate (E) to (A). The Michaelis constant Km is determined by determining the substrate concentration dependency of the initial enzyme reaction rate. Specifically, it is obtained by the following Michaelis constant Km measurement method.
<Michaelis constant Km measurement method>
An enzyme reaction solution (I) prepared to a certain temperature and pH containing a certain amount of substrate (E), low temperature optimum protease (A), pH adjusting agent (M) and water is prepared.
The temperature of the enzyme reaction solution (I) is within the range of 0 to 50 ° C., and the temperature of the enzyme reaction solution (I) is such that the activity of the low temperature optimal protease (A) is not inactivated and the activity can be measured. I) What is necessary is just to be able to keep constant temperature from the time of preparation to the end of measurement.
The pH of enzyme reaction solution (I) should just be in the range of pH 3-12. When the optimum pH described later is known, the optimum pH is preferable.
The pH adjuster (M) used in the enzyme reaction solution (I) is preferably a Good buffer such as a HEPES buffer or MES buffer from the viewpoint of ease of handling and stability. The concentration (molar concentration) of (M) in the enzyme reaction solution (I) is 25 to 500 mM.
The concentration (molar concentration) of the low-temperature optimum protease (A) in the enzyme reaction solution (I) is appropriately selected depending on each protease, and the absorbance change ΔAλ described later is plotted on the vertical axis and the time h is plotted on the horizontal axis. If this is the case, select a concentration that makes the plot a straight line.
The concentration (molar concentration) of the substrate (E) in the enzyme reaction solution (I) may be selected from three or more points between the minimum substrate concentration and the maximum substrate concentration at which the change in absorbance over time can be observed. When the Michaelis constant Km of the similar protease to (A) used for the measurement is known, it is sufficient to select 3 or more points between 1/50 and 10 times the Km of the similar protease.
For the enzyme reaction solution (I), using a spectrophotometer, the absorbance Aλ at a wavelength at which the product of the enzyme reaction has a maximum absorption is measured over time within a wavelength range of 300 to 450 nm. The temperature at the time of measurement is the same temperature as when the enzyme reaction solution (I) was prepared. The measurement time varies depending on the activity of the enzyme, but it is sufficient that the absorbance changes by 0.1 or more within a range not exceeding 0.8. The absorbance immediately after mixing the substrate (E) and the low temperature optimal protease (A) is Aλ ₀ , the absorbance after h hours is Aλ _h, and the change in absorbance ΔAλ (ΔAλ = Aλ _h −Aλ ₀ ) and measurement of the product The initial rate of enzyme reaction v (M / s) is calculated from the following mathematical formula (1) using the molar extinction constant ε at the wavelength.
v = ΔAλ / (ε × h × 3600) (1)
Further, using the enzyme reaction solution (I) having a different concentration of the substrate (E), the same measurement is performed to calculate the enzyme reaction initial velocity v.
The Michaelis constant Km is obtained by a Hanes-Woolf plot derived from the following Michaelis-Menten equation (Equation (2)) using the calculated initial reaction velocity v.
v = Vmax [S] / (Km + [S]) (2)
In the above formula (2), v is the initial enzyme reaction rate (M / s), Vmax is the maximum velocity (M / s), and [S] is the substrate concentration (M) in the enzyme reaction solution.
The Hanes-Woolf plot shows the concentration [S] of each substrate (E) on the horizontal axis (x-axis) and the reciprocal ([S ] / V) is plotted, and the intersection of the approximate straight line of the plot and the x-axis is −Km.

The optimum pH for the activity of the low temperature optimum protease (A) can be determined by measuring the absorbance of the enzyme reaction solution at various pHs. Specifically, it is the pH determined by the following optimum pH measurement method.
<Optimum pH measurement method>
An enzyme reaction solution (II) having a pH adjusted to 3 to 12 containing a certain amount of substrate (E), low temperature optimal protease (A), pH adjusting agent (M) and water is prepared. The pH interval of each solution (II) is about 1 (for example, pH 3.0, 4.0, 5.0, etc.).
The temperature of the enzyme reaction solution (II) is within a range of 0 to 50 ° C., and the temperature of the enzyme reaction solution ( II) It should be possible to maintain a constant temperature from the time of creation to the end of measurement.
The pH adjuster (M) used in the enzyme reaction solution (II) is preferably a Good buffer such as a HEPES buffer or a MES buffer from the viewpoint of ease of handling and stability. The concentration (molar concentration) of the pH adjuster (M) in the enzyme reaction solution (II) is 25 to 500 mM.
The concentration (molar concentration) of the substrate (E) in the enzyme reaction solution (II) is 4 to 6 times the Michaelis constant Km with respect to the substrate (E) of the low temperature optimum protease (A).
The concentration (molar concentration) of the low temperature optimal protease (A) in the enzyme reaction solution (II) is appropriately selected depending on each protease, and the absorbance change ΔAλ described later is plotted on the vertical axis and the time h is plotted on the horizontal axis. Select the concentration at which the plot is a straight line.
In the enzyme reaction solution (II), using a spectrophotometer, the absorbance Aλ at a wavelength at which the product of the enzyme reaction has a maximum absorption is measured over time within a wavelength range of 300 to 450 nm. The temperature at the time of measurement is the same temperature as when the enzyme reaction solution (II) was prepared. Although the measurement time h varies depending on the activity of the enzyme, it may be changed by 0.1 or more as long as the absorbance does not exceed 0.8. The absorbance immediately after mixing the substrate (E) and the low temperature optimal protease (A) is Aλ ₀ , the absorbance after h hours is Aλ _h, and the change in absorbance ΔAλ (ΔAλ = Aλ _h −Aλ ₀ ) is plotted on the vertical axis, The time h is plotted on the horizontal axis, and the slope of the straight line (coefficient k) is obtained. Furthermore, using the coefficient k measured using the enzyme reaction solution (II) of each pH (3 to 12), the vertical axis represents the coefficient k, the horizontal axis represents the pH, and the pH at which the coefficient k becomes the maximum value is plotted. Is the optimum pH.
When adjusting the pH, when the buffer type is changed, the protease activity differs depending on the buffer, and thus it is necessary to correct this appropriately. Examples of the correction method include a method in which the activity is measured with both buffers at the pH of the boundary at which the buffer type is changed, and the activity is corrected based on the value measured with one buffer.

The optimum temperature for the activity of the low temperature optimum protease (A) is determined by measuring the absorbance of the enzyme reaction solution in the range of 0 to 50 ° C. Specifically, it is the temperature obtained by the following optimum temperature measurement method.
<Optimum temperature measurement method>
An enzyme reaction solution (III) adjusted to an optimum pH containing a certain amount of substrate (E), low temperature optimum protease (A), pH adjusting agent (M) and water is prepared.
The enzyme reaction solution (III) is prepared at temperatures of 0 ° C, 5 ° C, 10 ° C, 20 ° C, 30 ° C, 40 ° C, 50 ° C, 60 ° C, 70 ° C and 80 ° C, respectively. When measuring at 0 ° C., it is prepared by dissolving sodium chloride to 1M.
The pH adjuster (M) used in the enzyme reaction solution (III) is preferably a Good buffer such as a HEPES buffer or a MES buffer from the viewpoint of ease of handling and stability. The concentration (molar concentration) of (M) in the enzyme reaction solution (III) is 25 to 500 mM.
The concentration (molar concentration) of the substrate (E) in the enzyme reaction solution (III) is 4 to 6 times the Michaelis constant Km with respect to the substrate (E) of the low-temperature optimal protease (A) described later.
The concentration (molar concentration) of the low temperature optimal protease (A) in the enzyme reaction solution (III) is appropriately selected depending on each protease, and the absorbance change ΔAλ described later is plotted on the vertical axis and the time h is plotted on the horizontal axis. Select the concentration at which the plot is a straight line.
For the enzyme reaction solution (III), using a spectrophotometer, the absorbance Aλ at a wavelength at which the product of the enzyme reaction has a maximum absorption is measured over time within a wavelength range of 300 to 450 nm. The temperature at the time of measurement is the same temperature as when the enzyme reaction solution (III) was prepared. Although the measurement time h varies depending on the activity of the enzyme, it may be changed by 0.1 or more as long as the absorbance does not exceed 0.8. The absorbance immediately after mixing the substrate (E) and the low temperature optimal protease (A) is Aλ ₀ , the absorbance after h hours is Aλ _h, and the change in absorbance ΔAλ (ΔAλ = Aλ _h −Aλ ₀ ) is plotted on the vertical axis, The time h is plotted on the horizontal axis to determine the slope of the straight line (coefficient k). Using the coefficient k measured in the enzyme reaction solution (III) prepared at each temperature of 0 to 50 ° C., the vertical axis represents the coefficient k and the horizontal axis represents the temperature. The temperature at which the coefficient k reaches the maximum value is the optimum temperature for the activity of the protease (A).

<Measurement method of inhibition constant Ki>
Low temperature optimal protease / protease activity inhibitor mixed solution containing a certain amount of low temperature optimal protease (A), protease activity inhibitor (B), pH adjuster (M) and water adjusted to the optimal temperature and optimal pH Create (i). Also, several types of (i) having different concentrations of (B) are created. The prepared (i) is allowed to stand for several tens of minutes to several hours. Substrate (E) is added to each (i) after standing to prepare an enzyme reaction solution (IV).
The pH adjuster (M) used in (i) and (IV) is preferably a Good buffer such as a HEPES buffer or MES buffer from the viewpoint of ease of handling and stability. The concentration (molar concentration) of (M) in (i) and (IV) is 25 to 500 mM, as long as it can be adjusted to the optimum pH.
The concentration (molar concentration) of the low temperature optimal protease (A) in (i) is close to the concentration of (A) when the above-mentioned Km was determined, and the substrate (D) to be added to (i) later The molar concentration is 1/1000 to 1/10. The molar concentration of (A) in (IV) is 1/1000 to 1/10 of the molar concentration of substrate (E) in (IV).
The concentration (molar concentration) of the protease activity inhibitor (B) in (i) and (IV) is 1 to 1000 times the concentration (molar concentration) of the low-temperature optimal protease (A), and four or more types. Create one that does not include (B).
The molar concentration of the substrate (E) in the enzyme reaction solution (IV) is the optimum substrate concentration (1 to 2 times the third of the Michaelis constant Km).
The standing time of (i) is the time until (A) and (B) are sufficiently combined and the activity of the low-temperature optimal protease (A) becomes constant, and is determined in advance through experiments.
The time until the activity of the low temperature optimal protease (A) becomes constant can be determined by an activity measurement method using absorbance described later. Specifically, after preparing the protease / protease activity inhibitor mixed solution (i), the slope of the straight line (coefficient k) is determined at regular intervals by an activity measurement method using absorbance described later. The time until the coefficient k becomes constant is the time until the activity of (A) becomes constant.
After preparing the enzyme reaction solution (IV) under the conditions of optimal temperature and optimal pH for protease activity, using a spectrophotometer, the absorbance at a wavelength where the product has a maximum absorption within a wavelength range of 300 to 450 nm Aλ is measured over time. Although the measurement time h varies depending on the activity of the enzyme, it may be changed by 0.1 or more within a range where the absorbance does not exceed 0.8. The initial enzyme reaction rate v is calculated from the absorbance change ΔAλ per unit time (ΔAλ = Aλ _h −Aλ ₀ ) and the molar absorption constant ε at the measurement wavelength of the product using the above equation (1). Further, the enzyme reaction solution (IV) prepared using (i) having different concentrations of the protease activity inhibitor (B) is also measured in the same manner, and the initial reaction rate vi is calculated. The initial enzyme reaction rate when (B) is not included is vo, and the relative activity α (x = vi / vo) at each concentration (B) is calculated.
Plot 1 / α on the horizontal axis and [Molar concentration of (B)] / (1-α) on the vertical axis to create a Henderson plot. The slope Ki (app) when drawing an approximate straight line on this plot, the substrate concentration [S] at the time of measurement, and the Michaelis constant Km are applied to the equation Ki = Ki (app) / (1+ [S] / Km) to obtain Ki. calculate.

  The Henderson plot is specifically described in Biochem. J. et al. 127, 1972, p21-333.

  In the present invention, the inhibitory constant Ki of the protease activity inhibitor (B) is 1 pM to 10 μM, and suppresses and recovers the low temperature optimal protease activity within the appropriate low temperature optimal protease concentration range, and the heat resistance of the low temperature optimal protease. From the viewpoint that it can be improved, 1 nM to 1 μM is preferable.

When the inhibition constant Ki is less than 1 pM, the low-temperature optimum protease (A) and the protease activity inhibitor (B) are strongly bound. Therefore, in order to recover the activity of (A), it must be diluted in a large amount. For example, when (B) having a Ki of 0.1 pM is used in an equimolar amount with (A) and the activity of (A) is 20% or less, (A) in the protein solution is about 1.0 × 10 ⁻ The activity of (A) cannot be recovered to 50% or more unless it is diluted to a concentration of ¹⁰ % by weight. However, when the concentration of (A) is 1.0 × 10 ⁻¹⁰ wt% or less, when a low-temperature optimum protease is used as a cleaning agent for clothing, a useful effect due to addition cannot be obtained.
On the other hand, when the inhibition constant Ki of (B) is larger than 10 μM, a large amount of (B) is required to suppress the activity of (A). Moreover, when (B) is added in a large amount, the activity of (A) cannot be recovered unless it is diluted in a large amount.

  In the present invention, (B) includes known protease activity inhibitors that satisfy the above conditions. (B) includes proteinaceous protease activity inhibitors (B1) and non-protein protease activity inhibitors (B2).

  The proteinaceous protease activity inhibitor (B1) is a protein extracted from a natural product or expressed by incorporating a gene into a host cell, and has a property of inhibiting the activity of a low-temperature optimal protease. Examples of (B1) include proteinaceous protease activity inhibitors (B1-1) and proteinaceous protease activity inhibitors (B1-2) belonging to the potato inhibitor 1 family.

  As a proteinaceous protease activity inhibitor (B1-1) belonging to the potato inhibitor 1 family, a proteinaceous protease activity inhibitor having a sequence (Y) in which 1 to 8 amino acid sequences of the amino acid sequence of SEQ ID NO: 1 are substituted ( B1-1-1), a proteinaceous protease activity inhibitor (B1-1-2) having 90% or more homology with the amino acid sequence of (B1-1-1), having the amino acid sequences of SEQ ID NOs: 26 to 28 Proteinaceous protease activity inhibitors (B1-1-3) and other proteinaceous protease activity inhibitors (B1-1-4) are included.

A protease activity inhibitor (B1-1-1) is a protein having a sequence (Y) in which 1 to 8 amino acids of the amino acid sequence in the amino acid sequence of SEQ ID NO: 1 are replaced with an amino acid different from the amino acid before the change It is a protein protease activity inhibitor, Comprising: It is a protein protease activity inhibitor which satisfy | fills at least 1 condition among following (1)-(8).
(1) The amino acid at position 12 of (Y) is the following amino acid (X1) (2) The amino acid at position 38 of (Y) is the following amino acid (X2) (3) The amino acid at position 48 of (Y) is The following amino acid (X3) (4) The amino acid at position 50 of (Y) is the following amino acid (X4) (5) The amino acid at position 51 of (Y) is the following amino acid (X5) (6) (Y The amino acid at position 52 in () is the following amino acid (X6) (7) The amino acid at position 53 in (Y) is the following amino acid (X7) (8) The amino acid at position 70 in (Y) is the following amino acid (X8) (X1): amino acid other than Glu among amino acids (X0): amino acid other than Val among amino acids (X0) (X3): amino acids other than Met among amino acids (X0) (X4): amino acids ( X0) except for Tyr Noic acid (X5): Amino acid other than Arg among amino acids (X0) (X6): Amino acid other than Ile among amino acids (X0) (X7): Amino acid other than Asp among amino acids (X0) (X8): Amino acids ( Amino acid other than Arg (X0): Ala, Arg, Asn, Asp, Cys, Gly, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val

In the condition (1) above, (X1) is an amino acid other than Glu among the amino acids (X0), but moderate suppression of low-temperature optimal protease activity, improvement in heat resistance of low-temperature optimal protease, and low-temperature optimality during dilution. From the viewpoint of easy recovery of suitable protease activity, Asp, Ala, Asn, Gln, Leu, Ser, Thr and Val are preferable, more preferably Asp, Ala, Asn and Gln, and particularly preferably Ala and Asp. is there.
In the above condition (2), (X2) is an amino acid other than Val among the amino acids (X0), but moderate suppression of low-temperature optimal protease activity, improvement in heat resistance of low-temperature optimal protease, and low-temperature optimality during dilution. From the viewpoint of easy recovery of suitable protease activity, Ala, Gly, Ile, Leu, Phe, Ser, Thr and Trp are preferred, Ala, Leu and Ile are more preferred, and Ala and Ile are particularly preferred.
In the condition (3) above, (X3) is an amino acid other than Met among the amino acids (X0), but moderate suppression of low-temperature optimal protease activity, improvement of heat resistance of low-temperature optimal protease, and low-temperature optimality during dilution. From the viewpoint of easy recovery of suitable protease activity, Ala, Gly, Ile, Leu, Ser, Thr and Val are preferred, more preferably Ala, Gly, Leu, Ser and Val, particularly preferably Ala and Gly. is there.
In the above condition (4), (X4) is an amino acid other than Tyr among the amino acids (X0), but moderate suppression of low temperature optimal protease activity, improvement of heat resistance of low temperature optimal protease, and low temperature optimality during dilution. From the viewpoint of easy recovery of suitable protease activity, Ala, Phe, Gly, Ile, Leu, Ser, Thr and Val are preferable, more preferably Ala, Phe and Leu, and particularly preferably Ala, Phe and Leu. is there.
In the condition (5) above, (X5) is an amino acid other than Arg among the amino acids (X0), but moderate suppression of low-temperature optimal protease activity, improvement in heat resistance of low-temperature optimal protease, and low-temperature optimality during dilution. From the viewpoint of easy recovery of suitable protease activity, Ala, Lys, His, Ile, Leu, Ser, Thr and Val are preferable, and Ala, Lys and His are more preferable.
In the condition (6) above, (X6) is an amino acid other than Ile among the amino acids (X0), but moderate suppression of low temperature optimal protease activity, improvement of heat resistance of low temperature optimal protease, and low temperature optimality during dilution. From the viewpoint of easy recovery of suitable protease activity, Glu, Ala, Asn, Gln, Leu, Ser, Thr and Val are preferable, and Ala, Val and Gln are more preferable.
In the condition (7) above, (X7) is an amino acid other than Asp among the amino acids (X0), but moderate suppression of low-temperature optimal protease activity, improvement of heat resistance of low-temperature optimal protease, and low-temperature optimality during dilution. From the viewpoint of easy recovery of suitable protease activity, Glu, Ala, Asn, Gln, Ile, Leu, Ser, Thr and Val are preferable, and Glu, Ala, Asn and Gln are more preferable.
In the above condition (8), (X8) is an amino acid other than Arg among the amino acids (X0), but moderate suppression of low temperature optimal protease activity, improvement of heat resistance of low temperature optimal protease, and low temperature optimality during dilution. From the viewpoint of easy recovery of suitable protease activity, Ala, Lys, His, Ile, Gly, Leu, Ser, Thr and Val are preferred, more preferably Ala, Lys, Gly, Ile and Leu, particularly preferably. Ala, Gly and Lys.

  (B1-1-1) is a proteinaceous protease inhibitor having a sequence (Y) in which 1 to 8 amino acids of the amino acid sequence of SEQ ID NO: 1 are substituted with amino acids different from the amino acids before change, The number of amino acid substitutions in (Y) is 1 to 5 from the viewpoints of moderate suppression of low temperature optimal protease activity, improvement of heat resistance of low temperature optimal protease, and ease of recovery of low temperature optimal protease activity upon dilution. The number is preferable, and more preferably 1 to 3.

  (B1-1-1) only needs to have at least one sequence (Y), which is suitable for moderate suppression of low temperature optimal protease activity, improvement of heat resistance of low temperature optimal protease, and low temperature optimal protease activity during dilution. From the viewpoint of easy recovery, 1 to 4 is preferable.

In the present invention, (B1-1-1) is a proteinaceous protease inhibitor having the sequence (Y), which may consist of only the sequence (Y), and is arranged at the C-terminus and / or N-terminus of the sequence (Y). It may have one or a plurality of (Z), and may be an arrangement in which these are repeated.
The sequence (Z) is a peptide sequence in which one amino acid or two or more amino acids are bound.
The number of amino acids constituting the sequence (Z) is from the viewpoint of appropriate suppression of low temperature optimal protease activity, improvement of heat resistance of low temperature optimal protease, and ease of recovery of low temperature optimal protease activity upon dilution. 100 is preferable, and more preferably 1 to 50.
In the case where (B1-1-1) has the sequence (Z), the number of the sequences (Z) is appropriate suppression of low temperature optimal protease activity, improvement of heat resistance of the low temperature optimal protease, and low temperature optimal protease during dilution. From the viewpoint of easy recovery of activity, 1 to 100 is preferable.

  In addition, the number of amino acids other than the sequence (Y) in the amino acid sequence constituting (B1-1-1) includes moderate suppression of low-temperature optimal protease activity, improvement in heat resistance of low-temperature optimal protease, and low temperature during dilution. From the viewpoint of easy recovery of optimal protease activity, 1 to 100 is preferable, and 1 to 50 is more preferable.

(B1-1-1) is a proteinaceous protease inhibitor having the sequence (Y), and specific examples include proteinaceous protease inhibitors of SEQ ID NOs: 2 to 25.
As (B1-1-1), from the viewpoints of moderate suppression of low temperature optimal protease activity, improvement of heat resistance of low temperature optimal protease and ease of recovery of low temperature optimal protease activity upon dilution, SEQ ID NOs: 2 to 25 Is preferred. Moreover, 4, 12, 18, 19, 22, 23, 24 and 25 are preferable from the viewpoint of the persistence of the protease.

The protease activity inhibitor (B1-1-2) is a proteinaceous protease activity inhibitor having 90% or more homology with the amino acid sequence of (B1-1-1), and includes the following (9) to (16): ) Is a proteinaceous protease activity inhibitor (B1-1-2) that satisfies at least one of the conditions.
(9) In (B1-1-1), the amino acid at the position corresponding to position 12 of the sequence (Y) is the following amino acid (X1) (10) In (B1-1-1), In (11) (B1-1-1), the amino acid at the position corresponding to position 38 is the following amino acid (X2), and the amino acid at the position corresponding to position 48 in the sequence (Y) is the following amino acid (X3) ( 12) In (B1-1-1), the amino acid at the position corresponding to position 50 of the sequence (Y) is the following amino acid (X4): (13) In (B1-1-1), 51 of the sequence (Y) In (14) (B1-1-1), the amino acid at the position corresponding to the position is the following amino acid (X5), and the amino acid at the position corresponding to position 52 in the sequence (Y) is the following amino acid (X6) (15 ) (B1-1-1), the sequence (Y) In amino acid position corresponding to position 53-position is the following amino acids (X7) (16) (B1-1-1), amino acid at the position corresponding to position 70 of SEQ (Y) is the following amino acid (X8)

In the present invention, the amino acid sequence of (B1-1-2) is a proteinaceous protease activity inhibitor having 90% or more homology with the amino acid sequence of (B1-1-1). From the viewpoints of moderate suppression of heat resistance, improvement in heat resistance of low temperature optimal protease and ease of recovery of low temperature optimal protease activity upon dilution, it is preferable to have 95% or more homology, more preferably 97% or more It is to have homology.
The homology of amino acid sequences is analyzed using the blastp algorithm of the homology analysis program “BLAST” provided by National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi). .

  The proteinaceous protease activity inhibitor (B1-1-3) having the amino acid sequence of SEQ ID NOs: 26 to 28 may be any one having the amino acid sequence of SEQ ID NOs: 26 to 28, and specifically, SEQ ID NO: 26 Those having an amino acid sequence of ˜28 are included.

  Examples of other proteinaceous protease activity inhibitors (B1-1-4) include Eglin C and Wheat Subtilisin / Chymotrypsin inhibitor.

  The proteinaceous protease activity inhibitor (B1-2) includes known proteinase protease activity inhibitors that satisfy the above conditions other than the proteinaceous protease activity inhibitor (B1-1) belonging to the potato inhibitor 1 family, Specific examples include Fungal Protease Inhibitor F, Streptomyces Subtilisin Inhibitor, and Human LEKTI.

  The non-protein protease activity inhibitor (B2) is a compound having a molecular weight of 50 to 1000 other than the above (B1), forms a reversible bond with the low temperature optimal protease (A), and the activity of the low temperature optimal protease It inhibits. Specific examples of (B2) include 2,4-dichlorophenylboronic acid.

Among the above (B), from the viewpoints of availability, suppression of low-temperature optimal protease activity and improvement of heat resistance, proteinaceous protease activity inhibitor (B1) is preferred, and protein properties belonging to the potato inhibitor 1 family are more preferred. Protease activity inhibitor (B1-1), particularly preferably Eglin C and Wheat Subtilisin / Chymotrypsin inhibitor.
Of the above (B), a protein protease activity inhibitor (B1-1) belonging to the potato inhibitor 1 family is preferable from the viewpoints of suppression of low temperature optimal protease activity, improvement of heat resistance and stability of other proteins. More preferred are a protease activity inhibitor (B1-1-1) and a protease activity inhibitor (B1-1-2).

  In the present invention, examples of the solvent (C) include water, organic solvents, and mixtures thereof.

  The water is not particularly limited, and examples thereof include tap water, ion exchange water, distilled water, and reverse osmosis water. Moreover, the buffer aqueous solution etc. which contain the pH adjuster (M) mentioned later in water are mentioned.

  Examples of the organic solvent include alcohols (alcohols having 1 to 18 carbon atoms, specifically methanol, ethanol, butanol, ethylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), ethers (tetrahydrofuran). , Diethyl ether, ethylene glycol monoalkyl ether, propylene glycol monoalkyl ether, cyclic ether, etc.), sulfoxide (dimethyl sulfoxide, etc.), aliphatic or alicyclic hydrocarbons (n-hexane, n-heptane, mineral spirit, cyclohexane, etc.) ) And fluorine-containing compounds (tetrafluoroethylene, etc.). Of these organic solvents, alcohol is preferred from the viewpoint of protein stability.

  As the solvent (C), alcohol and water are preferable from the viewpoint of stability of the low temperature optimal protease, more preferably water, and particularly preferably a buffer aqueous solution containing a pH adjuster (M).

The protein solution (D) in the present invention contains the aforementioned low-temperature optimal protease (A), protease activity inhibitor (B) and solvent (C).
The content (% by weight) of (A) in the protein solution (D) is 0.000001 to 50 based on the total weight of (A), (B), and (C) from the viewpoint of exerting a proteolytic effect. % By weight is preferable, more preferably 0.00001 to 40% by weight, and particularly preferably 0.0001 to 30% by weight.
The content (% by weight) of (B) in the protein solution (D) is 0 on the basis of the total weight of (A), (B) and (C) from the viewpoint of the activity-inhibiting effect of the low-temperature optimal protease. 0.000001 to 50% by weight is preferable, 0.00001 to 50% by weight is more preferable, and 0.0001 to 50% by weight is particularly preferable.
The content (% by weight) of (C) in the protein solution (D) is 0. 0% based on the total weight of (A), (B) and (C) from the viewpoint of the stability of the low temperature optimal protease. It is preferably from 00000001 to 99.999999% by weight, more preferably from 10 to 99.99998% by weight, and particularly preferably from 20 to 99.99998%.

  The molar ratio of the protease activity inhibitor (B) and the low temperature optimal protease (A) in the protein solution (D) (number of moles of (B) / number of moles of (A)) is effective for the low temperature optimal protease. From the viewpoint of inhibition, 1 to 100,000 is preferable, more preferably 1 to 1000, and particularly preferably 1 to 500.

  In the protein solution (D) in the present invention, in addition to the above-mentioned low temperature optimal protease (A), protease activity inhibitor (B) and solvent (C), inorganic salt (G), sugar (H), amino acid (I ), Low molecular organic compound (J), fatty acid (K), fat (L), pH adjuster (M), protein (N) other than (A) and (B), and surfactant (O) be able to.

  Examples of the inorganic salt (G) include sodium chloride, sodium borate, calcium chloride, magnesium chloride, sodium formate, magnesium sulfate, and ammonium sulfate.

  Examples of the sugar (H) include trehalose, sucrose, dextrin, cyclodextrin, maltose, fructose, hyaluronic acid and chondroitin sulfate.

  As amino acid (I), glycine, alanine, aspartic acid, asparagine, phenylalanine, tryptophan, tyrosine, leucine, lysine, histidine, cysteine, glutamine, glutamic acid, isoleucine, methionine, proline, serine, threonine, valine and their salts Can be mentioned.

  Examples of the fatty acid (K) include oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid, and eicosapentaenoic acid.

  Examples of the fat (L) include mono-, di-, and triglycerides of the above fatty acid (K).

  As the pH adjuster (M), a conventional pH adjuster can be used, and examples thereof include borate buffer, phosphate buffer, acetate buffer, Tris buffer, HEPES buffer, and citric acid.

  The protein (N) other than (A) and (B) is not particularly limited, but examples include enzymes other than low-temperature optimal proteases, recombinant proteins, antibodies, peptides, and the like. Specifically, serum albumin , Collagen, casein, gelatin, silk peptide and the like.

  As the surfactant (O), a surfactant (O) described later is included, and preferable ones are also the same.

  Examples of the low molecular weight organic compound (J) include low molecular weight organic compounds having a molecular weight of 1000 or less other than those described above, specifically, benzyl acetate, methyl salicylic acid, benzyl salicylic acid, hydroxybenzoic acid, cinnamic acid, caffeic acid, Examples include catechins, ascorbic acid, and carotenoids.

The content (% by weight) of the inorganic salt (G) contained in the protein solution (D) of the present invention is preferably 0 to 10%, more preferably from the weight of the protein solution (D) from the viewpoint of protein stability. Is 0 to 5%, particularly preferably 0 to 3%.
The content (% by weight) of the saccharide (H) contained in the protein solution (D) of the present invention is preferably 0 to 50%, more preferably from the weight of the protein solution (D), from the viewpoint of protein stability. 0 to 30%, particularly preferably 0 to 15%.
The content (% by weight) of amino acid (I) contained in the protein solution (D) of the present invention is preferably from 0 to 50%, more preferably from the weight of the protein solution (D) from the viewpoint of protein stability. 0 to 30%, particularly preferably 0 to 15%.
The content (% by weight) of the low molecular weight organic compound (J) contained in the protein solution (D) of the present invention is preferably 0 to 50% with respect to the weight of the protein solution (D) from the viewpoint of protein stability. More preferably, it is 0 to 30%, and particularly preferably 0 to 15%.
The content (% by weight) of the fatty acid (K) contained in the protein solution (D) of the present invention is preferably 0 to 50%, more preferably the weight of the protein solution (D) from the viewpoint of protein stability. 0 to 30%, particularly preferably 0 to 15%.
The content (% by weight) of the fat (L) contained in the protein solution (D) of the present invention is preferably 0 to 50%, more preferably from the weight of the protein solution (D) from the viewpoint of protein stability. 0 to 30%, particularly preferably 0 to 15%.
The content (% by weight) of the pH adjusting agent (M) contained in the protein solution (D) of the present invention is preferably 0 to 50% with respect to the weight of the protein solution (D) from the viewpoint of protein stability. Preferably it is 0 to 30%, particularly preferably 0 to 15%.
The content (% by weight) of the protein (N) other than the low-temperature optimal protease contained in the protein solution (D) of the present invention is 0 to 50% based on the weight of the protein solution (D) from the viewpoint of protein stability. Is more preferable, 0 to 30% is more preferable, and 0 to 15% is particularly preferable.
The content (% by weight) of the surfactant (O) contained in the protein solution (D) of the present invention is preferably 0 to 50% based on the weight of the protein solution (D) from the viewpoint of protein stability. Preferably it is 0 to 25%, particularly preferably 0 to 10%.

The protein solution (D) in this invention is obtained by mixing each component, and a manufacturing method is not specifically limited. An example is shown below.
(1) A pH adjuster (M) and a protease activity inhibitor (B) are added to the solvent (C), and the mixture is stirred at 20 to 40 ° C. until uniform.
(2) Add low temperature optimal protease (A) and stir until uniform at 20 ° C. to 40 ° C. (from the viewpoint of the binding rate between low temperature optimal protease (A) and protease activity inhibitor (B) 30 minutes or more).
(3) Finally, (G) to (O) are added and stirred at 25 ° C. until uniform to produce a protein solution (D).

The pH of the protein solution (D) in the present invention is preferably pH 3 to 10, more preferably pH 5 to 9, from the viewpoint of protein stability.
The pH of (D) can be appropriately adjusted with a pH adjuster (M).

  The protein solution (D) in the present invention is a protein solution (S) from the viewpoint that the protease activity Xi by the activity measurement method using absorbance prevents degradation of the protease itself and / or other proteins due to the remaining protease activity. Is preferably 20% or less, more preferably 0.0001 to 10%, and particularly preferably 0.0001 to 3%.

  The protein solution (S) is a protein solution obtained by replacing (B) with (C) having the same weight as (B) in the protein solution (D). For example, the content (% by weight) of (A), (B) and (C) in (D) is 2% by weight based on the total weight of (A), (B) and (C). %, (B) is 18 wt%, and (C) is 80 wt%, (A) in (S) is 2 wt% and (C) is 98 wt%.

In the present invention, the activity measurement method using absorbance is an enzyme reaction using a substrate (D) under conditions of a constant temperature, a constant pH, and an optimal substrate concentration for the activity of the above-mentioned low temperature optimal protease (A). Is a method of measuring the absorbance Aλ at a wavelength at which the product has a maximum absorption over time.
Which substrate is used as the substrate (E) is appropriately selected according to the classification of the low-temperature optimum protease (A) in the protein solution (D), as with the substrate used when obtaining Ki.
Specifically, the activity measurement method using absorbance is determined by the following measurement.

<Activity measurement method using absorbance>
○ Protease activity Xi
An enzyme reaction solution (V) is prepared by adding a certain amount of protein solution (D) and a certain amount of substrate (E).
The temperature of the enzyme reaction solution (V) is within the range of 0 to 50 ° C., the temperature of the low temperature optimal protease (A) is not inactivated, the activity is active, and the absorbance can be measured. I hope I can keep it up.
The molar concentration of the substrate (E) in the enzyme reaction solution (V) is 1 to 2 times the above Michaelis constant Km at the optimal substrate concentration, and the low temperature optimal protease (A) in (V) The concentration may be 5 to 100,000 times the molar concentration.
For the enzyme reaction solution (V), using a spectrophotometer, the absorbance Aλ at a wavelength at which the product of the enzyme reaction has a maximum absorption is measured over time within a wavelength range of 300 to 450 nm. The temperature at the time of measurement is the same temperature as when the enzyme reaction solution (V) was prepared. The absorbance Aλ ₀ immediately after preparation of the enzyme reaction solution (V) and the absorbance Aλ _h after the preparation of the enzyme reaction solution (V) are measured to determine the change in absorbance ΔAλ (ΔAλ = Aλ _h −Aλ ₀ ). The measurement time varies depending on the activity of the enzyme, but it is sufficient that the absorbance changes by 0.1 or more within a range not exceeding 0.8. The absorbance change ΔAλ is plotted on the vertical axis and the time h is plotted on the horizontal axis to determine the slope of the straight line (coefficient k _x ).

The absorbance is measured in the same manner except that the protein solution (S) is used instead of the protein solution (D), and the slope of the straight line (coefficient k _S ) is obtained.

  The protein solutions (D) and (S) are used from 30 minutes to 6 hours after preparation.

The protease activity Xi of (D) is determined by the following mathematical formula (3) from the slope coefficients k _X and k _S of the straight line using the protein solution (D) and the protein solution (S).
Protease activity Xi (%) = {k _X / k _S } × 100 (3)

  The method of using the protein solution (D) of the present invention is a conventional method.

  The detergent composition of the present invention is a detergent composition containing the protein solution (D) and the surfactant (O).

  In the present invention, the surfactant (O) is a nonionic surfactant (O-1), an anionic surfactant (O-2), a cationic surfactant (O-3), or an amphoteric surfactant ( O-4).

  As nonionic surfactant (O-1), aliphatic alcohol (carbon number 8-24) alkylene oxide (carbon number 2-8) adduct (degree of polymerization = 1-100) [oleyl alcohol ethylene oxide 11 mol addition Products], (poly) oxyalkylene (carbon number 2-8, polymerization degree = 1-100) glycol higher fatty acid (carbon number 8-24) ester [polyethylene glycol monostearate (polymerization degree = 20) and polyethylene distearate Glycol (degree of polymerization = 30), etc., polyvalent (divalent to 10-valent or higher) alcohol fatty acid (carbon number 8-24) ester [glyceryl monostearate, ethylene glycol monostearate, sorbitan monolaurate, etc.], Multivalent (divalent to 10-valent or higher) alcohol higher fatty acid (8 to 24 carbon atoms) (Poly) alkylene oxide adduct (alkylene group having 2 to 8 carbon atoms, polymerization degree = 1 to 100) [sorbitan monolaurate ethylene oxide (polymerization degree = 10) adduct and methyl glucose dioleate ethylene oxide (polymerization) Degree = 50) adducts, etc.], fatty acid N-hydroxyalkylamide [1: 1 type coconut oil fatty acid diethanolamide, 1: 1 type lauric acid diethanolamide, etc.], alkyl (C1-22) (poly) oxyalkylene (C2-8, degree of polymerization = 1-100) phenyl ether, alkyl (C8-8) (poly) oxyalkylene (C2-8, degree of polymerization = 1-100) -aminoalkyl (carbon number) 8-24) -ethers and alkyls (8-24 carbon atoms) dialkyl (1-6 carbon atoms) amine oxides [lauryl Methylamine oxide, and the like] and the like.

  Examples of the anionic surfactant (O-2) include an alkyl ether carboxylic acid having 8 to 24 carbon atoms or a salt thereof and an alkyl (poly) oxyethylene ether carboxylic acid having 8 to 24 carbon atoms or a salt thereof [(poly) oxy Ethylene (degree of polymerization = 1 to 100) sodium lauryl ether acetate and (poly) oxyethylene (degree of polymerization = 1 to 100) disodium lauryl sulfosuccinate, etc.], alkyl sulfate salts having 8 to 24 carbon atoms and carbon numbers 8 to 8 24 alkyl (poly) oxyethylene sulfate esters [sodium lauryl sulfate, lauryl (poly) oxyethylene (degree of polymerization = 1-100) sodium sulfate and lauryl (poly) oxyethylene (degree of polymerization = 1-100) sulfate-tri Ethanolamine salts, etc.], palm oil fatty acid monoethanolamide sulfate sulfonate Lithium, alkyl phenyl sulfonates having 8 to 24 carbon atoms [sodium dodecylbenzene sulfonate, etc.], alkyl phosphate salts having 8 to 24 carbon atoms and alkyl (poly) oxyethylene phosphate salts having 8 to 24 carbon atoms [Sodium lauryl phosphate and (poly) oxyethylene (degree of polymerization = 1-100) sodium lauryl ether phosphate, etc.], fatty acid salt [sodium laurate, triethanolamine laurate, etc.], acylated amino acid salt [coconut oil fatty acid Methyl taurine sodium, coconut oil fatty acid sarcosine sodium, coconut oil fatty acid sarcosine triethanolamine, N-coconut oil fatty acid acyl-L-glutamic acid triethanolamine, N-coconut oil fatty acid acyl-L-glutamic acid sodium and lauroylmethyl-β -Alanine Sodium etc.].

  As the cationic surfactant (O-3), quaternary ammonium salt type [stearyl trimethyl ammonium chloride, behenyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride, ethyl lanolin sulfate fatty acid aminopropylethyl dimethyl ammonium, etc.] and amine salts Type [diethylaminoethylamide stearate lactate, dilaurylamine hydrochloride, oleylamine lactate, etc.] and the like.

  As the amphoteric surfactant (O-4), a betaine-type amphoteric surfactant [coconut oil fatty acid amidopropyldimethylaminoacetic acid betaine, lauryldimethylaminoacetic acid betaine, 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazoli Nium betaine, laurylhydroxysulfobetaine, lauroylamidoethylhydroxyethylcarboxymethylbetaine hydroxypropyl sodium phosphate, etc.] and amino acid type amphoteric surfactants [sodium β-laurylaminopropionate, etc.].

  As surfactant (O), 1 type (s) or 2 or more types can be used. When two or more kinds of surfactants are used, combinations thereof include nonionic surfactants and anionic surfactants, nonionic surfactants and cationic surfactants, and nonionic surfactants and amphoteric surfactants. Examples include combinations of agents.

As the surfactant (O), from the viewpoint of detergency, it is preferable to use a nonionic surfactant alone or a combination of a nonionic surfactant and an anionic surfactant.
The nonionic surfactant is preferably an aliphatic alcohol (8 to 24 carbon atoms) ethylene oxide adduct (degree of polymerization = 1 to 100), more preferably an aliphatic alcohol (12 to 12 carbon atoms) from the viewpoint of detergency. 18) Ethylene oxide adduct (degree of polymerization 4-20), then more preferably aliphatic alcohol (carbon number 12-15) ethylene oxide adduct (degree of polymerization = 8-12), particularly preferably oleyl alcohol ethylene oxide 11 Mole adduct.
As an anionic surfactant, from a viewpoint of detergency, an alkylphenyl sulfonate having 8 to 24 carbon atoms, a fatty acid salt, an alkyl sulfate ester having 8 to 24 carbon atoms, and an alkyl (poly) having 8 to 24 carbon atoms. Preferred are oxyethylene sulfate salts, more preferably alkyl phenyl sulfonates having 12 to 16 carbon atoms and fatty acid salts having 8 to 16 carbon atoms, and more preferably dodecylbenzenesulfonic acid monoethanolamine salts and lauric acid. Sodium.

The content (% by weight) of the protein solution (D) in the detergent composition is preferably 0.1 to 99% by weight, based on the total weight of (D) and (O), from the viewpoint of detergency. Preferably it is 0.5 to 80 weight%, Most preferably, it is 1 to 70 weight%.
The content (% by weight) of the surfactant (O) in the detergent composition is preferably 1 to 99.9% by weight based on the total weight of (D) and (O) from the viewpoint of detergency. More preferably, it is 20-99.5 weight%, Most preferably, it is 30-99 weight%.

The content (% by weight) of the protein solution (D) in the detergent composition is preferably 0.1 to 99% by weight, more preferably 0.5 based on the weight of the detergent composition from the viewpoint of detergency. -60% by weight, particularly preferably 1-45% by weight.
From the viewpoint of detergency, the content (% by weight) of the surfactant (O) in the detergent composition is preferably 1 to 99.99999% by weight, more preferably 10 to 10%, based on the weight of the detergent composition. It is 89.998% by weight, particularly preferably 30 to 78.998% by weight.
The content (% by weight) of the low temperature optimal protease (A) in the detergent composition is preferably 0.000001 to 5% by weight, more preferably 0, based on the weight of the detergent composition from the viewpoint of detergency. 0.0001 to 4% by weight, particularly preferably 0.001 to 3% by weight.
The content (% by weight) of the protease activity inhibitor (B) in the detergent composition is preferably 0.000001 to 50% by weight, based on the weight of the detergent composition, from the viewpoint of durability of detergency. Preferably it is 0.0001 to 40 weight%, Most preferably, it is 0.001 to 30 weight%.
The content (% by weight) of the solvent (C) in the detergent composition is 1 based on the weight of the detergent composition from the viewpoint of the stability of the low temperature optimum protease (A) and the protease activity inhibitor (B). It is preferably -90% by weight, more preferably 10-80% by weight, particularly preferably 20-70% by weight.

  The pH of the detergent composition is preferably pH 5.0 to 9.0, more preferably pH 6.5 to 8.5, from the viewpoint of detergency.

  In addition to the protein solution (D) and the surfactant (O), the detergent composition of the present invention includes a builder (P), a chelating agent (Q), a dye (R), a fragrance (S), and a fluorescent whitening agent (T ), An antifoaming agent (U), an enzyme (W) other than the low temperature optimal protease (A), and the like.

  The builder (P) is not particularly limited, and a builder used in a conventional detergent composition can be used. For example, polycarboxylates (such as acrylate homopolymer and maleate homopolymer), Examples include polyvalent carboxylates (such as citric acid and malic acid), and alkali builders (such as caustic soda, soda ash, ammonia, triethanolamine, sodium tripolyphosphate, and sodium silicate).

  The chelating agent (Q) is not particularly limited, and chelating agents used in conventional detergent compositions can be used. For example, nitrilotriacetic acid, iminodiacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, glycol Aminopolyacetic acid such as etherdiaminetetraacetic acid, hydroxyethyliminodiacetic acid, triethylenetetraaminehexaacetic acid and diencoric acid or salts thereof, diglycolic acid, oxydisuccinic acid, carboxymethyloxysuccinic acid, citric acid, lactic acid, tartaric acid, sulphate Organic acids such as acids, malic acid, oxydisuccinic acid, gluconic acid, carboxymethyl succinic acid and carboxymethyl tartaric acid or salts thereof, aminotri (methylenephosphonic acid), 1-hydroxyethylidene-1,1-diphosphonic acid, ethylenediaminetetra ( Chirenhosuhon acid), diethylenetriamine penta (methylene phosphonic acid) or the like of the alkali metal or lower amine salts.

  The content (% by weight) of the builder (P) and the chelating agent (Q) in the detergent composition is preferably 0 to 10% by weight, more preferably based on the weight of the detergent composition, from the viewpoint of detergency. 0.1 to 5% by weight.

  The dye (R) is not particularly limited, and dyes used in conventional detergent compositions can be used, and examples thereof include bromophenol blue.

  The fragrance (S) is not particularly limited, and fragrances used in conventional detergent compositions can be used, and examples thereof include phenylethyl alcohol.

  The fluorescent brightening agent (T) is not particularly limited, and the fluorescent brightening agent used in conventional detergent compositions can be used, and examples thereof include sodium biphenyl sulfonate.

  The antifoaming agent (U) is not particularly limited, and antifoaming agents used in conventional detergent compositions can be used, such as silicone-based antifoaming agents, polyoxyalkylene-based antifoaming agents and minerals. Examples thereof include oil-based antifoaming agents.

  The content (% by weight) of the dye (R), fragrance (S), fluorescent whitening agent (T) and antifoaming agent (U) in the detergent composition is the weight of the detergent composition from the viewpoint of detergency. Based on 0 to 10% by weight, more preferably 0 to 5% by weight.

  Enzymes (W) other than the low temperature optimal protease (A) include cellulase (W-1), amylase (W-2), lipase (W-3) and oxidoreductase (W-4).

Cellulases (W-1) include those of animal, plant or microbial origin, and those of microbial origin are preferred from the viewpoint of availability. Cellulases also include chemically or genetically modified variants.
Specific examples of the cellulase include Cellozyme ^™ and Carezyme ^™ manufactured by Novozymes.

Amylase (W-2) includes those of bacterial or fungal origin. Amylases also include chemically or genetically modified variants. Examples of the amylase include B.I. described in detail in British Patent No. 1,296,839. And α-amylase obtained from a special strain of B. licheniformis.
Commercially available amylases, Novozymes of Duramyl ^TM, Termamyl ^TM, etc. Fungamyl ^TM and BAN ^TM and Gist-Brocades Inc., Rapidase ^TM and Maxamyl P ^TM, and the like.

  Lipases (W-3) include those of bacterial or fungal origin. Lipases also include chemically or genetically modified variants. Examples of lipases include Humicola langinosa lipase (EP 258 068 and EP 305 216), Rhizomucor miehei lipase and Candida (Candida) Patent No. 238 023), C.I. C. ntartica lipase A and B, Pseudomonas lipase (European Patent No. 247661), P. P. pseudoalcaligenes and P. p. P. alcaligenes lipase (European Patent No. 218272), P. a. P. cepacia lipase (European Patent No. 331376); P. stutzeri lipase, P. p. P. fluorescens lipase and Bacillus lipase (British Patent No. 1,372,034); B. subtilis lipase (Dartois et al. (1993), Biochemica et Biophysica Acta 1131, 253-260), B. stearothermophilus lipase (Japanese Patent Publication No. 64-744992) and B. stearothermophilus lipase. And B. pumilus lipase (International Publication No. 91/16422).

Commercially available lipases, Genencor M1 Lipase ^TM, Luma fast ^TM and Lipomax ^TM, Novozymes of Lipolase ^TM and Lipolase Ultra ^TM and Amano Enzyme Inc. of Lipase P "Amano" ^TM, and the like.

Oxidoreductase (W-4) includes peroxidase and oxidase (for example, laccase).
Peroxidases include those of plant, bacterial or fungal origin. Peroxidases also include chemically or genetically modified variants. Peroxidases include Coprinus (e.g., derived from C. cinereus or C. macrolithus strains), Bacillus (from B. pumilus strains). And the peroxidase described in WO 91/05858 are preferable, and the peroxidase described in WO 91/05858 is particularly preferable.
Laccases include those of bacterial or fungal origin. Laccases include Trametes (eg, derived from strains of T. villosa or T. versicolol), Coprinus (eg, strains of C. cinereus). Derived from] and Myceliophthora [for example, derived from a strain of M. thermophila] and the like.

  From the viewpoint of detergency, the content (% by weight) of the enzyme (W) other than (A) in the detergent composition is preferably 0 to 10% by weight, more preferably 0, based on the weight of the detergent composition. 0.001 to 8% by weight, particularly preferably 0.01 to 5% by weight.

The detergent composition of the present invention is obtained by mixing each component, and the production method is not particularly limited. An example is shown below.
(1) A predetermined amount of the protein solution (D) is added to the surfactant (O) and stirred until it becomes uniform at 25 ° C.
(2) Add a predetermined amount of other components and stir until uniform at 25 ° C.

The method of using the detergent composition of the present invention may be the same as the method of using the conventional detergent composition, and is not particularly limited. An example of usage as a laundry detergent is shown below.
(1) The washing machine containing the laundry is filled with tap water, the detergent composition is added at 25 ° C., and lightly stirred to dissolve.
(2) Wash the laundry with a washing machine.
(3) Drain the liquid from the washing machine and rinse with tap water once or twice.
(4) Appropriate dehydration.

  The present invention will be further described with reference to the following examples and comparative examples, but the present invention is not limited thereto.

<Production Example 1>
The gene encoding the amino acid sequence of SEQ ID NO: 1 (restriction enzyme NcoI added to the 5 ′ end, restriction enzyme BamHI recognition sequence added to the 3 ′ end, and artificial synthesis was requested from Hokkaido System Science) restriction enzyme NcoI Then, the plasmid was treated with BamHI to bind to the NcoI restriction enzyme site and the BamHI restriction enzyme site of the pET-22b plasmid (Novagen) to prepare a plasmid (P1) that expresses the protein of SEQ ID NO: 1. Using this plasmid and mutation introduction primer SEQ ID NOs: 29 and 30 for replacing glutamic acid at position 12 with aspartic acid, mutation was introduced into the plasmid (P1) under the following conditions. Specifically, 0.5 μL (10 ng) of plasmid (P1), 0.75 μL (7.5 pg) of each 10 μM primer for mutagenesis, 2.5 μL of 10 times concentration buffer for PCR (manufactured by Takara Bio Inc.), 2 mM deoxynucleotide After mixing 2 μL of 3-phosphate (dNTP) mixture (Takara Bio), DNA polymerase ExTaq 0.25 μL (Takara Bio) and 17 μL of deionized water, PCR was performed using TaKaRa Thermal Cycler Dice (Takara Bio). The reaction conditions were heat denaturation at 94 ° C. for 2 minutes, followed by 30 cycles of 98 ° C. for 10 seconds, 50 ° C. for 10 seconds, and 68 ° C. for 6.5 minutes. After purifying the obtained PCR product with QIAquick Gel Extraction Kit (manufactured by Qiagen) (50 μL), 6 μL of 10 times concentrated DpnI buffer solution and 3 μL of restriction enzyme DpnI (manufactured by Takara Bio) were added for 1 hour at 37 ° C. The template was disassembled. The obtained restriction enzyme-treated PCR product (5 μL) was subjected to transformation into E. coli DH5α. That is, 5 μL of restriction enzyme-treated PCR product was converted to 100 μL of E. coli. It was added to Coli DH5α competent cell (manufactured by TOYOBO), stored on ice for 30 minutes, and then heated at 42 ° C. for 90 seconds. Here, 900 μL of SOC medium (manufactured by TOYOBO) was added, followed by static culture at 37 ° C. for 1 hour. 100 μL of the culture solution was applied to an LB / ampicillin plate and cultured at 37 ° C. overnight. The appearing colonies were cultured in 1 mL of LB medium for 12 hours and then subjected to Quantumprep Miniprep Kit (manufactured by Bio-Rad) to purify the plasmid (P2) expressing the protein of SEQ ID NO: 2 (100 μL). The obtained plasmid was analyzed by a DNA sequence analysis service (Macrogen Japan), and it was confirmed that the mutation was introduced.
The resulting plasmid (P2) was transformed into E. coli E. coli. Coli BL21 (DE3) was transformed in the same manner. The obtained protease activity inhibitor-expressing strain was inoculated into 1 mL of LB culture solution (containing 100 mg / L of ampicillin) and cultured at 30 ° C. for 12 hours to prepare an overnight culture solution. 0.5 ml of LB culture solution ( Inoculated into 5 ml of ampicillin (containing 100 mg / L), and cultured with shaking at 30 ° C. for 3 hours to prepare 7 types of precultures. Each of the seven types of pre-culture solution is 50 mL of culture solution {the content of each component in 50 mL of water is 1.2 g of yeast extract (manufactured by Nippon Pharmaceutical Co., Ltd.), 0.6 g of polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.), phosphoric acid 0.47 g of dipotassium, 0.11 g of monopotassium phosphate, 0.35 g of ammonium sulfate, 0.66 g of disodium phosphate dodecahydrate, 0.02 g of sodium citrate dihydrate, 0.2 g of glycerol, hydrolyzed lactalbumin 1.5 g, antifoam (made by Shin-Etsu Silicone, “KM-70”) 0.3 g, 1 mM magnesium sulfate, trace metal solution (calcium chloride 18.9 μg, iron (III) chloride 500 μg, zinc sulfate heptahydrate 9.0 μg, copper sulfate 5.1 μg, manganese chloride tetrahydrate 6.7 μg, cobalt chloride 4.9 μg, ethylenediaminetetraacetic acid tetrasodium 200 μg), 00mg / L ampicillin} inoculated microorganisms culture apparatus in the culture was performed while maintaining the pH6.8,30 ℃ by using the (Able Co., Ltd., product name "BioJr.8"). After the start of culture, 0.15 mL of 1M IPTG solution was added. From 14 hours after the start of the culture, glycerol / protein solution (50% glycerol, 50 g / L lactalbumin hydrolyzate, 33 g / L antifoam (manufactured by Shin-Etsu Silicone, “KM-70”), 100 mg / L ampicillin) was added dropwise. Started. The culture solution (K-1) was collected 48 hours after the start of the culture.
The obtained culture solution (K-1) was separated with a carrier for purification of His-tag (Ni Sepharose 6 Fast Flow manufactured by GE Healthcare), and the glutamic acid at position 12 in SEQ ID NO: 1 was replaced with aspartic acid. A protease activity inhibitor (B1) solution (L-1) having the amino acid sequence was obtained. When the amount of the protease activity inhibitor (B1) in (L-1) was measured by SDS-PAGE, it was 1 g / L.

<Production Example 2>
Instead of “Primer for mutagenesis for substituting glutamic acid at position 12 for aspartic acid (SEQ ID NOs: 29, 30)” in Production Example 1, “Primer for mutagenesis for substituting tyrosine at position 50 for leucine” (SEQ ID NO: 31, 32) ”was used in the same manner except that the protease activity inhibitor (B2) solution (L-2) of the amino acid sequence of SEQ ID NO: 10 in which tyrosine at position 50 of SEQ ID NO: 1 was substituted with leucine was used. Obtained. When the amount of the protease activity inhibitor (B2) in (L-2) was measured by SDS-PAGE, it was 1 g / L.

<Production Example 3>
Instead of “Primer for mutagenesis for substituting glutamic acid at position 12 for aspartic acid (SEQ ID NO: 29, 30)” in Production Example 1, “Mutation for substituting valine at position 38 of SEQ ID NO: 1 for alanine” Plasmid (P3) in which valine at position 38 was substituted with alanine was obtained using the primers (SEQ ID NOs: 31 and 32).
Also, in Production Example 1, the arginine at position 51 and the isoleucine at position 52 were each replaced with alanine instead of “primer for mutagenesis to replace glutamic acid at position 12 with aspartic acid (SEQ ID NOs: 29, 30)”. The valine at position 38 in SEQ ID NO: 1 was substituted with alanine in the same manner except that the plasmid P3 was used instead of the plasmid P1 using the mutation-introducing primers (SEQ ID NOs: 33 and 34) ”. A protease activity inhibitor (B3) solution (L-3) having the amino acid sequence of SEQ ID NO: 22 in which arginine and isoleucine at position 52 were each substituted with alanine was obtained. When the amount of the protease activity inhibitor (B3) in (L-3) was measured by SDS-PAGE, it was 1 g / L.

<Measurement of optimum pH of cannase protease activity>
10 mg of cannase (Novozymes) was dissolved in 1 L of buffer 1 [50 mM Na phosphate aqueous solution (pH 8.5, 25 ° C.) aqueous solution] to obtain a cannase solution (A1) (0.37 μM).
1570 μL of (A1) was placed in a spectrophotometer cell (bottom area 1 cm × 1 cm). The sample chamber of the spectrophotometer was kept at 25 ° C., and the cell was allowed to stand in the sample chamber for 5 minutes to control the temperature. 400 μL of a substrate solution (1) adjusted to 25 ° C. (Succinyl-Ala-Ala-Pro-Phe-nitronelide dissolved in buffer 1 to a concentration of 5 mg / mL (0.119 mM)) was added thereto. An enzyme reaction solution (II-1) was obtained. Absorbance at 405 nm was measured immediately after preparation of (II-1) and every 5 seconds for 2 minutes.
Similarly, instead of buffer 1, buffer 2 [50 mM Na acetate buffer (pH 5.5, 25 ° C. aqueous solution)], buffer 3 [50 mM Na acetate buffer (pH 6.5, 25 ° C. aqueous solution)], buffer 4 [50 mM phosphoric acid Na buffer (pH 6.5, 25 ° C. aqueous solution)], buffer 5 [50 mM aqueous sodium phosphate buffer (pH 7.5, 25 ° C.) aqueous solution], buffer 7 [50 mM TAPS-NaOH buffer (pH 8.5, 25 ° C. aqueous solution)] And cannase solutions (A2) to (A8) and substrate solutions (2) to (8) were prepared using Buffer 8 [50 mM TAPS-NaOH buffer (pH 9.5, 25 ° C. aqueous solution)], respectively.
In the same manner except that (A2) to (A8) are changed to (A1) and the substrate solutions (2) to (8) are used instead of the substrate solution (1), the enzyme reaction solutions (II-2) to (II) are used. -8) was prepared, and the absorbance at 405 nm was measured immediately after the preparation of (II-2) to (II-8) and every 5 seconds for 2 minutes.
For the enzyme reaction solutions (II-1) to (II-8), the absorbance immediately after preparing (II-1) to (II-8) was Aλ ₀ , the absorbance after 2 minutes was Aλ _h , and the absorbance The change ΔAλ (ΔAλ = Aλ _h −Aλ ₀ ) is plotted on the vertical axis and the time h is plotted on the horizontal axis to obtain the slope coefficient k of the straight line. Plotting with the vertical axis representing the coefficient k and the horizontal axis representing the pH (FIG. 2), the pH at which the coefficient k is a maximum was determined, and the optimum pH was 7.5.
The activity was measured with both buffers at the boundary pH (pH 6.5 and 8.5) at which the buffer type was changed, and the activity was corrected based on the value measured with one buffer. At pH 6.5 (buffers 3 and 4), the value obtained using buffer 4 is the result at pH 6.5, and the coefficient k obtained using buffer 2 is the ratio of buffers 3 and 4 (with buffer 4). The coefficient k / the coefficient k in buffer 3 was corrected. Similarly, at pH 8.5 (buffers 1 and 7), the value in buffer 1 is the result of pH 8.5, and the coefficient k obtained using buffer 8 is the ratio of buffers 1 and 7 (coefficient k in buffer 1). / Correction by multiplying by coefficient k) in buffer 7. The optimum pH for cannase activity was 8.5.

<Measurement of optimum temperature for cannase protease activity>
1570 μL of cannase solution (A1) (0.37 μM) obtained by dissolving 10 mg of cannase (Novozymes) in 1 L of buffer 1 [50 mM Na phosphate buffer (pH 8.5, 25 ° C.) aqueous solution], a spectrophotometer cell ( The bottom area was 1 cm × 1 cm. The sample chamber of the spectrophotometer was kept at 20 ° C., and the cell was allowed to stand in the sample chamber for 5 minutes.
Next, 400 μL of the substrate solution (1) (0.119 mM, pH 8.5) adjusted to 20 ° C. was added to the cell to obtain an enzyme reaction solution (III-1). Absorbance at 405 nm was measured immediately after preparation of (III-1) and every 5 seconds for 2 minutes.
Set the spectrophotometer's sample chamber to 0 ° C, 5 ° C, 10, 20, 30 ° C, 40 ° C, 50 ° C, 60 ° C, 70 ° C, or 80 ° C and adjust the temperature, and the substrate solution (7) is also a sample. Enzyme reaction solutions (III-2) to (III-6) were prepared in the same manner except that the temperature was controlled at the same temperature as the chamber and then added to the cell. Absorbance at 405 nm was measured immediately after preparation of (III-2) to (III-6) and every 5 seconds for 2 minutes.
For the enzyme reaction solutions (III-1) to (III-6), the absorbance immediately after preparing (III-1) to (III-6) was Aλ ₀ , the absorbance after 2 minutes was Aλ _h , and the absorbance The change ΔAλ (ΔAλ = Aλ _h −Aλ ₀ ) is plotted on the vertical axis and the time h is plotted on the horizontal axis to obtain the slope coefficient k of the straight line. When the vertical axis is plotted with the coefficient k and the horizontal axis is plotted with temperature (FIG. 3), the temperature at which the coefficient k reaches the maximum value was determined, and the optimum temperature was 40 ° C.

<Measurement of Michaelis constant Km of substrate for cannase>
1570 μL of cannase solution (A1) (0.37 μM) obtained by dissolving 10 mg of cannase (Novozymes) in 1 L of buffer 1 [50 mM Na phosphate buffer (pH 8.5, 25 ° C.) aqueous solution], a spectrophotometer cell ( The bottom area was 1 cm × 1 cm. The sample chamber of the spectrophotometer was kept at 20 ° C., and the cell was allowed to stand in the sample chamber for 5 minutes. 400 μL of a substrate solution (1) adjusted to 40 ° C. (Succinyl-Ala-Ala-Pro-Phe-nitronelide dissolved in buffer 7 to a concentration of 5 mg / mL (0.119 mM)) was added thereto. An enzyme reaction solution (I-1) was obtained. Absorbance at 405 nm was measured immediately after preparation of (I-1) and every 5 seconds for 2 minutes.
In the substrate solution (7), the molar concentrations of Succinyl-Ala-Ala-Pro-Phe-nitronelide in buffer 1 were 0.024 mM (substrate solution (8), 1 mg / mL), 0.237 mM (substrate solution (9)). 10 mg / mL), 0.474 mM (substrate solution (10), 20 mg / mL) and 1.19 mmM (substrate solution (11), 50 mg / mL) were prepared. Enzyme reaction solutions (I-2) to (I-5) were prepared in the same manner except that the substrate solutions (8) to (11) were used instead of the substrate solution (5). Absorbance at 405 nm was measured immediately after preparation of (I-2) to (I-5) and every 5 seconds for 2 minutes.
The absorbance immediately after preparation of (I-1) to (I-5) is Aλ ₀ , the absorbance after h hours is Aλ _h, and the change in absorbance ΔAλ (ΔAλ = Aλ _h −Aλ ₀ ) and the molar absorption constant at 405 nm, respectively. The initial enzyme reaction velocity v (M / s) was calculated from the following formula (1) using ε.
v = ΔAλ / (ε × h × 3600) (1)
Using the calculated enzyme reaction initial velocity v, the horizontal axis (x-axis) represents the respective substrate concentration [S], and the vertical axis (y-axis) represents the reciprocal [S of the respective substrate concentration [S] by the enzyme reaction initial velocity v [S ] / V was plotted to create a Hanes-Woolf plot (FIG. 1). From the intersection (−Km) between the approximate straight line of the plot and the x-axis, the Michaelis constant Km was 0.04 mM.

<Measurement of Inhibition Constant Ki for Cannase of Protease Activity Inhibitor>
1 mL of protease activity inhibitor (B1) solution (L-1) (1 g / L) obtained in Production Example 1 was diluted with 49 mL of buffer 1 (pH 8.5), and protease activity inhibitor solution (B1-1) ( 2.7 μM). (B1-1)-(B1-5) were prepared by diluting (B1-1) with buffer 1 to 1/50, 1/30, 1/20, and 1/10 times the molar concentration.
Buffer 1 (1530 μL) was placed in a spectrophotometer cell (bottom area 1 cm × 1 cm), 40 μL of cannase solution (A1) and 40 μL of (B1-1) were added, and protease / protease activity inhibitor mixed solution (i-1 )created.
The sample chamber of the spectrophotometer was kept at 40 ° C., and the cell was left in the sample chamber for 30 minutes. 400 μL of the substrate solution (12) adjusted to 40 ° C. {Succinyl-Ala-Ala-Pro-Phe-nitronelide dissolved in buffer 7 to a concentration of 10 mg / mL} is added here. Then, an enzyme reaction solution (IV-1) was prepared. The absorbance at 405 nm for 2 minutes was recorded immediately after preparation of (IV-1) and every 5 seconds.
A cannase / protease activity inhibitor (B1) mixed solution (i-2) to (i-6) except that (B1-2) to (B1-5) are used instead of (B1-1). The enzyme reaction solutions (IV-2) to (IV-6) were prepared using the substrate solution (12), and the absorbance was recorded in the same manner.
The absorbance immediately after preparation of (IV-1) to (IV-6) is Aλ ₀ , the absorbance after h hours is Aλ _h, and the change in absorbance ΔAλ (ΔAλ = Aλ _h −Aλ ₀ ) and the molar absorption constant at 405 nm, respectively. The initial enzyme reaction velocity v (M / s) was calculated from the following formula (1) using ε.
v = ΔAλ / (ε × h × 3600) (1)
The initial reaction rate vi of (IV-1) to (IV-5) is vi, the initial reaction rate of (IV-6) is vo, and the relative activity α (x = vi / vo) at each (B1) concentration is Calculated.
1 / α is plotted on the horizontal axis, and [Molar concentration of (B1)] / (1-α) is plotted on the vertical axis, thereby creating a Henderson plot (FIG. 4). The slope Ki (app) (FIG. 4 to 3.06) of the approximate line of this plot, the substrate concentration [S] at the time of measurement, and the Michaelis constant Km are expressed as Ki = Ki (app) / (1+ [S] / Km). As a result of calculation, Ki was 1.4 nM.

  In the above, the protease activity inhibitor (B2 obtained in Production Example 2 and Production Example 3) was changed to “Protease activity inhibitor (B1) solution (L-1) (1 g / L) 1 mL obtained in Production Example 1]”. ) And (B3) (B2-1) to (B2-5) and (B3-1) in the same manner except that 1 mL each of the solutions (L-2) and (L-3) (1 g / L) is used. To (B3-5) were prepared, and the inhibition constant Ki was measured. The Ki of (B2) was 30 nM, and the Ki of (B3) was 90 nM.

<Measurement of inhibition constant Ki for carboxyphenylboronic acid cannase>
50 mg of 4-carboxyphenylboronic acid (Tokyo Chemical Industry Co., Ltd.) was dissolved in 50 mL of buffer 1 (pH 8.5) to obtain a phenylboronic acid solution (B′1) (8.2 mM). Dilutions of (B′1) with buffer 1 to molar concentrations of 1/10, 1/4, 1/2, and 3/4 were designated as (B′2) to (B′5).
In the above “measurement of inhibition constant Ki for protease activity inhibitor against cannase”, (B′1) to (B′5) are used instead of (B1-1) to (B1-5), and phenylboronic acid is used. The inhibition constant Ki for cannase was determined to be 19 μM.

<Example 1>
40 μL of the aforementioned cannase solution (A1) and 40 μL of 20 mg / L protease activity inhibitor solution (B1-1) were added to 1530 μL of buffer 1 to prepare a protein solution (D1).

<Example 2>
A protein solution (D2) was prepared in the same manner as in Example 1 except that the “protease inhibitor solution (B2-1)” was used instead of the “protease activity inhibitor solution (B1-1)”.

<Example 3>
In Example 1, a protein solution (D3) was prepared in the same manner except that the “protease inhibitor solution (B3-1)” was used instead of the “protease activity inhibitor solution (B1-1)”.

<Comparative Example 1>
40 μL of the aforementioned cannase solution (A1) and 40 μL of the aforementioned 4-carboxyphenylboronic acid solution (B′1) were added to 1530 μL of the buffer 1 to prepare a protein solution (D′ 1).

<Comparative example 2>
80 μL of the aforementioned cannase solution (7) was added to 1530 μL of buffer 1 to prepare a protein solution (D′ 2).

<Protease activity immediately after compounding>
For the protein solutions (D1) to (D3) and (D′ 1) to (D′ 2), 16 μL of the protein solution immediately after compounding and 1584 μL of buffer 1 were mixed in the cell of the spectrophotometer, Protein solutions (Y1) to (Y5) were prepared. The protease activities (Y1) to (Y5) were measured by an activity measurement method using the following absorbance. The results are shown in Table 1.

<Protease activity when stored at 30 ° C for 3 months>
In “Protease activity immediately after compounding”, protease activity was calculated in the same manner except that “protein solution stored at 30 ° C. for 3 months” was used instead of “protein solution immediately after compounding”.

<Long-term persistence of protease activity>
The ratio between the protease activity immediately after compounding and the protease activity after storage at 30 ° C. for 3 months was calculated as the long-term persistence of the protease activity by the following formula.
Long-term persistence of protease activity (%) = (protease activity after 3 months storage at 30 ° C.) / (Protease activity immediately after preparation) × 100

<Protease activity when stored for 1 week at 30 ° C. or 40 ° C.>
In “Protease activity immediately after blending”, protease activity was calculated in the same manner except that “protein solution stored at 30 ° C. or 40 ° C. for 1 week” was used instead of “protein solution immediately after blending”.

<Thermal resistance of protease>
The ratio of the protease activity after 1 week storage at 30 ° C. and the protease activity after 1 week storage at 40 ° C. was calculated as the heat resistance of the protease by the following formula.
Heat resistance of protease (%) = (protease activity after 1 week storage at 40 ° C.) / (Protease activity after 1 week storage at 30 ° C.) × 100

<Activity measurement method using absorbance>
1570 μL of each of the protein solutions (Y1) to (Y4) was placed in a spectrophotometer cell (bottom area 1 cm × 1 cm), the spectrophotometer sample chamber was kept at 40 ° C., and the cell was allowed to stand in the sample chamber for 30 minutes. . Here, a substrate solution (27) whose temperature was adjusted to 40 ° C. (Succinyl-Ala-Ala-Pro-Phe-nitronelide dissolved in buffer 1 to a concentration of 0.5 mg / mL (0.012 mM)) 400 μL was added to prepare an enzyme reaction solution (V-1). The absorbance at 405 nm for 2 minutes was recorded immediately after preparation of (V-1) and every 5 seconds.
The absorbance immediately after creating (V-1) is Aλ ₀ , the absorbance after h seconds is Aλ _h , the change in absorbance ΔAλ (ΔAλ = Aλ _h −Aλ ₀ ) is plotted on the vertical axis, and the time h is plotted on the horizontal axis. Then, the slope coefficient k _{X of the} straight line was obtained.
As a blank, 16 μL of protein solution (D0) containing 1570 μL of buffer 7 and 40 μL of cannase solution (A7) and 1584 μL of buffer 1 were mixed in a spectrophotometer cell to prepare a protein solution (Y0). did. Similarly, protease activity was measured, and a linear slope coefficient k _S was obtained.
Using the coefficients k _X and the coefficient k _S, it was calculated protease activity Xi proteins from the following equation (3) solution (Y1) and (Y2).
Protease activity Xi (%) = {k _X / k _S } × 100 (3)

  From the evaluation results in Table 1, compared to the protein solutions of Comparative Examples 1 and 2, the protein solutions of the present invention of Examples 1 to 3 have a high protease activity persistence and can maintain the protease activity in the long term. I understand that I can do it. It can also be seen that the protease activity after storage at 30 ° C. for 1 week and the protease activity after storage at 40 ° C. for 1 week are both high, and the heat resistance of the protease can be improved. Furthermore, the optimum temperature for the activity of cannase is 40 ° C. Usually, it tends to self-decompose when stored at 40 ° C, but the protease activity after storage at 30 ° C for 1 week and protease after storage at 40 ° C for 1 week From the comparison of the activities, it can be seen that even when stored at 40 ° C., the decrease in the activity is small and the self-decomposition can be efficiently suppressed.

<Examples 4 to 6>
The detergent composition of the present invention was obtained by blending at a rate of Table 2 at 40 ° C.

<Comparative Examples 3-5>
The ratio of Table 2 was mix | blended at 40 degreeC, and the detergent composition for a comparison was obtained.

なお、表２中、各成分の割合は、重量部で示した。

In Table 2, the ratio of each component is shown in parts by weight.

The following detergency tests were conducted using the detergent compositions prepared in Examples 4 to 6 and Comparative Examples 3 to 5.
<Detergency test>
<Washing removal rate immediately after blending>
For the detergent compositions obtained in Examples 4 to 6 and Comparative Examples 3 to 5, 0.8 g of the detergent composition was dissolved in 999.2 g of water immediately after the preparation of the detergent composition to obtain a solution. Five wet artificially contaminated cloths (4 cm × 4 cm) were put into this solution, washed and rinsed under the following conditions using a targotometer (manufactured by Daiei Chemical Co., Ltd.), then the cloths were taken out, and a gear oven (TABAI) The product was dried at 70 ° C. for 60 minutes using GPS-222) to obtain a test cloth. Next, using a multi-light source spectrocolorimeter (manufactured by Suga Test Instruments), the reflectance of the test cloth at 540 nm was adjusted to 4 places, 2 on each side of the test cloth, for a total of 4 places (a total of 20 places with 5 pieces of test cloth). ), The average value was obtained, and the cleaning removal rate (%) was calculated by the following formula. The results are shown in Table 2.
(Cleaning conditions)
Time: 10 minutes, temperature: 25 ° C., rotation speed: 120 rpm
(Rinsing conditions)
Time: 1 minute, temperature: 25 ° C., rotation speed: 120 rpm
(Washing removal rate)
Cleaning removal rate (%) = {(R _W −R _S ) / (R _I −R _S )} × 100
R _I represents the reflectance of the cleaning cloth, R _W represents the reflectance of the cleaning cloth, and R _S represents the reflectance of the contaminated cloth.
Moreover, the used wet artificial contamination cloth is a wet artificial contamination cloth (reflectance at 540 nm of 40 ± 5%) manufactured by the Japan Laundry Science Association having the dirt composition shown in Table 3.

<Washing removal rate after storage at 30 ° C for 3 months>
For the detergent compositions obtained in Examples 4 to 6 and Comparative Examples 3 to 5, in the “cleaning removal rate immediately after compounding”, the “detergent composition was immediately after creation” was changed to “after creation of the detergent composition”. A detergency test was conducted in the same manner except that the detergent composition “after storage at 30 ° C. for 3 months” was used, and the washing removal rate was calculated. The results are shown in Table 2.

<Washing removal rate after storage for 1 week at 30 ° C and 40 ° C>
For the detergent compositions obtained in Examples 4 to 6 and Comparative Examples 3 to 5, in the “cleaning removal rate immediately after compounding”, the “detergent composition was immediately after creation” was changed to “after creation of the detergent composition”. A detergency test was conducted in the same manner except that the detergent composition “after storage at 30 ° C. or 40 ° C. for 1 week” was used, and the cleaning removal rate was calculated. The results are shown in Table 2.

<Durability of detergency>
The ratio of the washing removal rate immediately after compounding and the washing removal rate after storage at 30 ° C. and 40 ° C. for 3 months was calculated as the sustainability of the washing property by the following formula.
Persistence of detergency (%) = (washing removal rate after storage at 30 ° C. for 3 months−washing removal rate of Comparative Example 4) / (washing removal rate immediately after blending—washing removal rate of Comparative Example 4) × 100

<Cleaning heat resistance>
The ratio of the washing removal rate after storage at 30 ° C. or 40 ° C. for 1 week was calculated as the heat resistance of the washing property by the following formula.
Detergency heat resistance (%) = (Wash removal rate after storage at 40 ° C. for 1 week−Wash removal rate of Comparative Example 4) / (Wash removal rate after storage for 1 week at 30 ° C.−Wash removal of Comparative Example 4) Rate) x 100

  As shown in Table 2, the detergent compositions of Comparative Examples 3 and 4 have a high washing removal rate immediately after compounding, but the washing removal rate after storage at 30 ° C. for 3 months is the detergent of Comparative Example 5 that does not contain cannase. It was lowered to the same level as the composition, and the durability of the cleaning property was as low as 5 to 11%. On the other hand, the detergent composition of the present invention has a cleaning removal rate of 10% or more higher than that of the detergent composition of Comparative Example 5 which does not contain cannase even after storage at 30 ° C. for 3 months, and has a long cleaning performance. It was very high at 53-79%. Therefore, it turns out that the detergent composition of the present invention has a high detergency. Furthermore, both the washability after storage at 30 ° C. for 1 week and the washability after storage at 40 ° C. for 1 week are both high, the heat resistance of the washability is 61-68%, and the heat resistance of the detergent composition is also It can be seen that it can be improved.

  The protein solution of the present invention comprises a detergent for clothing, a detergent for automatic dishwashers, a detergent for contact lenses, a bath preparation, a cosmetic for removing keratin, a food modifier (baking, softening meat, marine processing Etc.), beer fining agent, leather tanning agent, photographic film gelatin remover, digestion aid and flame retardant.

Claims (6)

A protein solution (D) containing a low temperature optimal protease (A), the following protease activity inhibitor (B) and a solvent (C), wherein the optimal temperature of the enzyme reaction of (A) is 0 ° C. to 50 ° C. Protein solution.
Protease activity inhibitor (B): As the substrate (E), any one of benzyloxycarbonylphenylalanine nitroanilide, benzoylarginine nitroanilide, furylacryloylglycylleucinamide and succinylalanylalanylprolylphenylalanine nitroanilide is used. A protease activity inhibitor having an inhibition constant Ki of 1 pM to 10 μM for (A) determined by Henderson plot under the conditions of the optimum temperature, optimum pH and optimum substrate concentration of the low temperature optimum protease (A). The protein solution according to claim 1, wherein the substrate (E) is succinylalanylalanylprolylphenylalanine nitroanilide. The protein solution according to claim 1 or 2, wherein the protease activity inhibitor (B) is a proteinaceous protease activity inhibitor (B1). The protein solution according to any one of claims 1 to 3, wherein the protease activity inhibitor (B) is a proteinaceous protease activity inhibitor (B1-1) belonging to the potato inhibitor 1 family. The protein solution according to any one of claims 1 to 4, wherein the protease activity inhibitor (B) is the following protease activity inhibitor (B1-1-1) or protease activity inhibitor (B1-1-2).
Protease activity inhibitor (B1-1-1): A sequence obtained by substituting 1 to 8 amino acids of the amino acid sequence of (F) with an amino acid different from the amino acid before change in the amino acid sequence (F) of SEQ ID NO: 1. A proteinaceous protease activity inhibitor having (Y), which satisfies at least one of the following conditions (1) to (8):
Protease activity inhibitor (B1-1-2): a proteinaceous protease activity inhibitor having 90% or more homology with the amino acid sequence of (B1-1-1), comprising the following (9) to (16): A proteinaceous protease activity inhibitor which is a proteinaceous protease activity inhibitor (B1-1-2) satisfying at least one of the conditions.
(1) The amino acid at position 12 of (Y) is the following amino acid (X1) (2) The amino acid at position 38 of (Y) is the following amino acid (X2) (3) The amino acid at position 48 of (Y) is The following amino acid (X3) (4) The amino acid at position 50 of (Y) is the following amino acid (X4) (5) The amino acid at position 51 of (Y) is the following amino acid (X5) (6) (Y The amino acid at position 52 in () is the following amino acid (X6) (7) The amino acid at position 53 in (Y) is the following amino acid (X7) (8) The amino acid at position 70 in (Y) is the following amino acid (X8) (9) in (B1-1-1), the amino acid at the position corresponding to position 12 of the sequence (Y) is the following amino acid (X1) (10) in (B1-1-1), the sequence (Y ) Of the position corresponding to position 38 is the following amino acid (X2) (11) (B1-1-1), the amino acid at the position corresponding to position 48 of the sequence (Y) is the following amino acid (X3) (12) (B1-1-1), the sequence (Y) In (13) (B1-1-1), the amino acid at the position corresponding to position 51 in the sequence (Y) is the following amino acid (X5). (14) In (B1-1-1), the amino acid at the position corresponding to position 52 of the sequence (Y) is the following amino acid (X6). (15) In (B1-1-1), the amino acid of the sequence (Y) In (16) (B1-1-1), the amino acid at the position corresponding to position 53 is the following amino acid (X7), and the amino acid at the position corresponding to position 70 in the sequence (Y) is the following amino acid (X8) ( X1): Glu of amino acids (X0) Amino acid (X2): Amino acid other than Val among amino acids (X0) (X3): Amino acid other than Met among amino acids (X0) (X4): Amino acid other than Tyr among amino acids (X0) (X5): Amino acids Amino acid other than Arg among (X0): Amino acid other than Ile among amino acids (X0) (X7): Amino acid other than Asp among amino acids (X0) (X8): Other than Arg among amino acids (X0) Amino acid (X0): Ala, Arg, Asn, Asp, Cys, Gly, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val A detergent composition comprising the protein solution (D) according to any one of claims 1 to 5 and a surfactant (O).

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