JP2009034063A - Method for improving stability of alkaline protease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for improving the stability of an alkaline protease in a liquid detergent. <P>SOLUTION: The method for stabilizing the alkaline protease composed of a specific amino acid sequence derived from a specified Bacillus sp. to a surfactant is provided. The method for improving the stability to the surfactant (to a sodium straight-chain alkylbenzene sulfonate having particularly strong surface activating actions) comprises the step of substituting an amino acid residue at a specific position with several kinds of specified limited amino acids. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for improving the stability of an alkaline protease to a surfactant.

  Protease use has a long history in the industrial field, ranging from detergents such as garment detergents to fiber modifiers, leather treatment agents, cosmetics, bath agents, food modifiers or pharmaceutical applications. Over. Among them, the most industrially produced mass-produced proteases are detergent proteases such as Alcalase, Sabinase (registered trademark; Novozymes), Maxacar (registered trademark; Genencor), Blapp (registered trademark; Henkel), and KAP ( Kao) is known.

  The purpose of blending protease into the detergent is to break down the soil mainly composed of proteins attached to clothing to lower the molecular weight and promote solubilization with surfactants. There is a composite soil containing a plurality of components in which organic and inorganic substances such as lipids derived from sebum and solid particles are mixed, and a cleaning agent having a high detergency against such composite soil has been desired.

  From this point of view, the present inventor has a molecular weight of about 43,000, which retains sufficient casein degrading activity even in the presence of a high concentration of fatty acid and has excellent detergency against complex soil containing not only proteins but also sebum. Several kinds of alkaline proteases were found and a patent application was filed earlier (see Patent Document 1). Such alkaline protease group is different from subtilisin which is a serine protease derived from Bacillus bacteria conventionally known in that it has a molecular weight, primary structure, enzymological properties, particularly a very strong oxidant resistance, It has been proposed to classify into a new subtilisin subfamily (Non-patent Document 1).

  By the way, cleaning agents can be classified into powder detergents and liquid detergents according to their forms. Liquid detergents are more soluble than powder detergents and have the advantage that the stock solution can be applied directly to the soiled area. Liquid detergents have the above-mentioned advantages not found in powder detergents, but on the other hand, it is widely known that there are technical difficulties not found in powder detergents regarding the stable formulation of enzymes such as proteases. . Originally, storage at room temperature in liquid tends to lead to protein denaturation, and liquid detergents contain surfactants, fatty acids, solvents, etc., pH is also weakly alkaline, and extremely severe conditions for enzymes It has become. In addition, since protease is a proteolytic enzyme, it also has a problem of self-digestion, which makes stable storage in liquid detergent more difficult.

  It is known to add enzyme stabilizers such as carboxylic acids such as calcium ions, borax, boric acid, boron compounds, formic acid, and polyols to such technical problems. In addition, studies have been made to solve the problem of self-digestion by inhibiting the activity of protease, and 4-substituted phenylboronic acid (Patent Document 2) and certain peptide aldehydes and boron compositions (Patent Document). A stabilization method by reversible inhibition of protease according to 3) has been reported. In addition, it has been reported that a protease chemically modified with dextran can improve the stability in an aqueous solution containing a surfactant (Non-patent Document 2).

  However, even with the addition of enzyme stabilizers such as calcium ions and boric acid, the stability of the protease is not sufficient, and the inhibitor also has a problem as a liquid detergent composition considering the inhibitory efficiency depending on the enzyme species and the production cost. It's hard to say how to solve it. Similarly, chemical modification of enzymes has been difficult in terms of production cost. Various measures such as enzyme stabilizers, inhibitors and chemical modifications have been studied, but it is most desirable to improve the stability of the protease itself in a liquid detergent in terms of cost.

  Common liquid detergent compositions include surfactants, alkali agents, anti-staining agents, solvents, fragrances, fluorescent dyes, etc., but the substances that most impair enzyme stability are surfactants, usually anions Often consists of a surfactant and a nonionic surfactant. Nonionic surfactants are generally not very damaging to enzymes, but anionic surfactants stabilize the enzyme in addition to the hydrophobic moiety entering the enzyme and destroying the hydrophobic interaction of the enzyme. In order to supplement divalent metals such as calcium ions, it is considered that the damage to the enzyme is very high (Non-patent Document 3).

Therefore, improving the resistance to the anionic surfactant is a very important factor for improving the stability in the liquid detergent.
International Publication No. 99/18218 Pamphlet Japanese National Patent Publication No. 11-507680 JP 2000-506933 A Saeki et al., Biochem. Biophys. Res. Commun., 279, 313-319, 2000 Cosmetics & Toiletries magazine, 111, p79-88, 1996 Detergent Enzyme: A Challenge! In Handbook of Detergents part A, New York, p639-690, 1999

  The present invention relates to providing a method for improving the stability of alkaline proteases in liquid detergents.

  The present inventor has identified LAS (linear alkylbenzene sulfone) as a representative anionic surfactant by substituting a specific amino acid residue among amino acid residues characteristic of alkaline protease KP43 having a molecular weight of about 43,000. It has been found that the stability of sodium acid) is improved.

That is, the present invention is a method for stabilizing an alkaline protease surfactant comprising the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having 80% or more identity thereto, wherein the amino acid represented by SEQ ID NO: 2 (A) the amino acid residue at position 133 or a position corresponding thereto and / or (b) the amino acid residue at position 195 or a position corresponding thereto is substituted with the following amino acid residue: The present invention relates to a method for improving stability against a surfactant.
(A) position: cysteine, glycine, aspartic acid, glutamic acid or proline residue (b) position: leucine or isoleucine residue

  According to the present invention, the stability of an alkaline protease against an anionic surfactant such as LAS can be improved. Therefore, the alkaline protease obtained by the present invention is active as a liquid detergent blended with a surfactant and has high specific activity, and is useful as an enzyme for blending detergents.

  The alkaline protease that is the subject of the stability improving method of the present invention is an alkaline protease consisting of the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having 80% or more identity thereto. Is sometimes referred to as a parent alkaline protease.

  Examples of the alkaline protease comprising the amino acid sequence represented by SEQ ID NO: 2 include alkaline protease derived from KP43 [Bacillus sp. KSM-KP43 (FERM BP-6532) (WO99 / 18218 pamphlet).

The alkaline protease comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 is different from the amino acid sequence shown in SEQ ID NO: 2, but the amino acid sequence shown in SEQ ID NO: 2, for example, A protein comprising an amino acid sequence having 80% or more or 90% or more identity and having the same function as an alkaline protease comprising the amino acid sequence represented by SEQ ID NO: 2, preferably 95% or more, more preferably 96% or more, A protein having an amino acid sequence having an identity of preferably 97% or more, more particularly preferably 98% or more, most preferably 99% or more and having the same function as an alkaline protease consisting of the amino acid sequence represented by SEQ ID NO: 2 Can be mentioned.
As such an alkaline protease, for example, in the amino acid sequence represented by SEQ ID NO: 2, one or several amino acids are deleted, substituted or added, and the equivalent of the alkaline protease consisting of the amino acid sequence represented by SEQ ID NO: 2 is used. What has a function is included.
Specifically, for example, protease KP9860 [from Bacillus sp. KSM-KP9860 (FERM BP-6534), WO99 / 18218, GenBank accession no. AB046403], protease E-1 [Bacillus no. From D-6 (FERM P-1592), JP-A-49-71191, GenBank accession no. AB044602], protease Ya [FERM BP-1029], JP-A-61-2280268, GenBank accession no. AB 046404], protease SD521 [Bacillus SD521 (FERM P-11162), JP-A-3-191781, GenBank accession no. AB046405], protease A-1 [from NCIB12289, WO88 / 01293, GenBank accession no. AB046406], protease A-2 [derived from NCIB12513, WO 98/56927], protease 9865 [derived from Bacillus SP KSM-9865 (FERM P-18656), GenBank accession no. AB084155], mutant proteases and sequences described in JP-A No. 2002-218189, JP-A No. 2002-306176, JP-A No. 2003-125783, JP-A No. 2004-000122, JP-A No. 2004-057195 Mutant in which amino acid sequence shown by No. 2 is substituted with serine at position 63, mutant in which position 89 is substituted with histidine, mutant in which position 120 is substituted with arginine, and mutant in which positions 63 and 187 are substituted with serine A mutant in which position 226 is substituted with tyrosine, a mutant in which position 296 is replaced with valine, a mutant in which position 304 is replaced with serine (Japanese Patent Laid-Open No. 2004-305175), 15 of the amino acid sequence represented by SEQ ID NO: 2 Mutant with position substituted with histidine, Mutant with position 16 replaced with threonine or glutamine, position 166 A glycine-substituted mutant, a 167-substituted valine-substituted mutant, a 346-substituted arginine-substituted mutant, a 405-position substituted aspartic acid (Japanese Patent Laid-Open No. 2004-305176), and the like Can be mentioned.
Of these, the alkaline protease having the amino acid sequence represented by SEQ ID NO: 2 preferably has any of the following enzymatic properties. 1) It has oxidant resistance, acts on the alkali side (pH 8 or more), and is stable. Here, having an oxidant resistance means that the alkaline protease is left in a 50 mM hydrogen peroxide (containing 5 mM calcium chloride) solution (20 mM Gritton Robinson buffer, pH 10) at 20 ° C. for 20 minutes to remain ( Synthetic substrate method) holds at least 50% or more. 2) Remaining of 80% or more when treated at 50 ° C. and pH 10 for 10 minutes. 3) Inhibited by diisopropylfluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF). 4) The molecular weight by SDS-PAGE is 43,000 ± 2,000.

  The amino acid sequence identity is calculated by the Lippman-Pearson method (Lipman-Pearson method; Science, 227, 1435, (1985)). Specifically, using the homology analysis (Search homology) program of genetic information processing software Genetyx-Win (Ver. 5.1.1; software development), the unit size to compare (ktup) should be set to 2. Is calculated by

The method of the present invention can be carried out by subjecting the parent alkaline protease to amino acid mutation at the target site.
That is, in the amino acid sequence represented by SEQ ID NO: 2, the amino acid residue at position 133 (alanine residue) is substituted with a cysteine, glycine, aspartic acid, glutamic acid or proline residue, and the amino acid residue at position 195 (tyrosine) In the alkaline protease consisting of an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 2 Substituting the amino acid residue at the position corresponding to position 133 with a cysteine, glycine, aspartic acid, glutamic acid or proline residue, substituting the amino acid residue at the position corresponding to position 195 with a leucine or isoleucine residue, It can be carried out.
It should be noted that amino acid substitution at position 133 or a position corresponding thereto, position 195 or a position corresponding thereto may be performed in either one or both simultaneously.

  Here, as a method for specifying the “amino acid residue at the corresponding position”, for example, amino acid sequences are compared using a known algorithm such as the Lippmann-Person method, and conserved amino acids present in the amino acid sequence of each alkaline protease. This can be done by giving maximum homology to the residues. By aligning the amino acid sequences of proteases in this way, it is possible to determine the positions of homologous amino acid residues in the sequences in each protease regardless of insertions or deletions in the amino acid sequences. The homologous position is considered to exist at the same position in the three-dimensional structure, and it can be assumed that the homologous position has a similar effect on the specific function of the target protease.

  That is, the amino acid sequences are aligned by the above method, and (a) the amino acid residue at the position corresponding to the amino acid residue at position 133 (alanine residue) of the amino acid sequence represented by SEQ ID NO: 2 is to use the above method. Thus, for example, in protease Ya, it can be specified as a proline residue at position 132, (b) at a position corresponding to the amino acid residue at position 195 (tyrosine residue) in the amino acid sequence represented by SEQ ID NO: 2. The amino acid residue can be specified as, for example, an isoleucine residue at position 194 in protease E-1.

  Specific examples of (a) position 133, (b) position corresponding to position 195 and amino acid residues of the amino acid sequence of protease KP43, and specific examples of amino acid residues are protease KP9860, protease E-1, protease Ya, protease SD521, and protease A exemplified above. -1, protease A-2 and protease 9865 are shown (Table 1).

  Specifically, the method of the present invention, for example, mutates a cloned gene encoding a parent alkaline protease (SEQ ID NO: 1), transforms a suitable host using the obtained mutated gene, The recombinant host is cultured and collected from the culture. The gene encoding the parent alkaline protease may be cloned by using a general gene recombination technique, for example, according to the methods described in WO99 / 18218 pamphlet and WO98 / 56927 pamphlet.

  As a means for mutating a gene encoding a parent alkaline protease, any of the commonly used methods of partial specific mutation can be employed. More specifically, it can be performed using, for example, the Site-Directed Mutagenesis System Mutan-Super Express Km kit (Takara). In addition, by using a recombinant PCR (polymerase chain reaction) method (PCR protocols, Academic Press, New York, 1990), an arbitrary sequence of a gene is replaced with a sequence corresponding to the arbitrary sequence of another gene. Is possible.

  Protease using the obtained mutant gene can be obtained, for example, by ligating the mutant gene to a DNA vector capable of stably amplifying and transforming the host strain, or on the chromosomal DNA of the host strain capable of stably maintaining the mutant gene. It is possible to adopt a method such as introducing them. Examples of hosts satisfying this condition include Bacillus bacteria, Escherichia coli, mold, yeast, actinomycetes, etc., and using these strains, inoculating a medium containing an assimilable carbon source, nitrogen source and other essential nutrients, What is necessary is just to culture | cultivate according to a conventional method.

  The alkaline protease thus obtained has resistance to oxidizing agents, is not inhibited by casein degradation activity by a high concentration of fatty acid, has a molecular weight of 43,000 ± 2,000 recognized by SDS-PAGE, and is active in the alkaline region. In addition, it has a new property that it has activity and high specific activity even in liquid detergents containing an anionic surfactant and the like compared to a parent alkaline protease. Therefore, the alkaline protease is useful as an enzyme for blending various detergent compositions.

Hereinafter, the present invention will be described in more detail based on examples.
Example 1
Bacillus sp. KSM-KP43 strain-derived alkaline protease structural gene from about 2.0 kb (SEQ ID NO: 1) to the mature enzyme region (1) position 195, (2) site-specific mutation at position 133 Each primer to be introduced was designed.
(1) In order to introduce leucine at position 195, primer 1 (SEQ ID NO: 3), primer 3 (SEQ ID NO: 5), primer 4 (SEQ ID NO: 6) and primer 2 (SEQ ID NO: 4)
In order to introduce isoleucine at position 195, primer 1 (SEQ ID NO: 3), primer 3 (SEQ ID NO: 5), primer 5 (SEQ ID NO: 7) and primer 2 (SEQ ID NO: 4)
(2) In order to introduce cysteine at position 133, primer 1 (SEQ ID NO: 3), primer 6 (SEQ ID NO: 8), primer 7 (SEQ ID NO: 9) and primer 2 (SEQ ID NO: 4)
To introduce glycine at position 133, primer 1 (SEQ ID NO: 3), primer 6 (SEQ ID NO: 8), primer 8 (SEQ ID NO: 10) and primer 2 (SEQ ID NO: 4)
In order to introduce aspartic acid at position 133, primer 1 (SEQ ID NO: 3), primer 6 (SEQ ID NO: 8), primer 9 (SEQ ID NO: 11) and primer 2 (SEQ ID NO: 4)
In order to introduce glutamic acid at position 133, primer 1 (SEQ ID NO: 3), primer 6 (SEQ ID NO: 8), primer 10 (SEQ ID NO: 12) and primer 2 (SEQ ID NO: 4)
In order to introduce proline at position 133, primer 1 (SEQ ID NO: 3), primer 6 (SEQ ID NO: 8), primer 11 (SEQ ID NO: 13) and primer 2 (SEQ ID NO: 4)
PCR was performed using each.

Primer 1 is provided with a Bam HI linker on the 5 ′ end side of the sense strand, Primer 2 is provided with an Xba I linker on the 5 ′ end side of the antisense strand, Primer 3, Primer 4 and Primer 5, and Primer 6 Primer 7, primer 8, primer 9, primer 10 and primer 11 were designed to complement each other with a length of 10 to 15 bp from their 5 ′ ends. Pyrobest (Takara) is used as the DNA polymerase for PCR. PCR conditions are as follows: Denaturation of template DNA at 94 ° C for 2 minutes, then 1 cycle at 94 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 1 minute And 30 cycles were reacted. After purifying the amplified DNA fragments with a PCR product purification kit (Roche), the corresponding amplified fragments alone were used to denature the DNA at 94 ° C for 2 minutes, then at 94 ° C for 1 minute, at 55 ° C for 1 minute, 72 Recombinant PCR was performed by reacting 30 cycles of 1 minute at 1 ° C. PCR was performed on the obtained amplified fragment using primer 1 and primer 4. The template DNA was denatured at 94 ° C. for 2 minutes, and then reacted for 30 cycles of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes to obtain a full-length gene into which the mutation was introduced. After purification of the amplified fragment, the restriction enzyme linker attached to the end was cleaved with Bam HI and Xba I (Roche). Amplified DNA fragment previously Bam HI, Xba I and treated plasmid PHA64 (Japanese Patent No. 349293: Bam downstream of the promoter 64 HI, having Xba I cleavage site) was mixed with the Ligation High (Toyobo), ligase reaction Went. The plasmid recovered from the reaction solution by ethanol precipitation was used to transform the host strain Bacillus sp. KSM9865 (FERM P-18566).

Transformants of 9865 strain were added to skim milk-containing alkaline agar medium [Skim milk (Difco) 1% (w / v), Bactotripton (Difco) 1%, Yeast extract (Difco) 0.5%, Sodium chloride 1%, Agar 1.5% , Sodium carbonate 0.05%, tetracycline 15 ppm], and the presence or absence of mutated protease gene introduction was determined according to the state of halo formation. The transformant was 5 ml of seed medium [6.0% (w / v) polypeptone S, 0.05% yeast extract, 1.0% maltose, 0.02% magnesium sulfate heptahydrate, 0.1% potassium dihydrogen phosphate, 0.25% carbonate. Sodium, 30 ppm tetracycline] was inoculated, and cultured with shaking at 30 ° C. for 16 hours. Then seed culture in 30 ml main medium [8% Polypeptone S, 0.3% yeast extract, 10% maltose, 0.04% magnesium sulfate heptahydrate, 0.2% potassium dihydrogen phosphate, 1.5% anhydrous sodium carbonate, 30ppm tetracycline] The solution was inoculated with 1% (v / v) and cultured with shaking at 30 ° C. for 3 days.
The obtained culture solution is centrifuged to obtain a culture supernatant, which is then applied to a DEAE-Toyopearl (Tosoh) column equilibrated with 10 mM Tris-HCl buffer (pH 7.5) containing 2 mM calcium chloride. Was recovered to obtain almost uniform protease. The amount of protein was measured using a protein assay kit (Wako Pure Chemical Industries).

A 500 μg protein amount of the obtained protease was added to an aqueous solution containing a final concentration of 50 mM Tris-HCl buffer (pH 8), a final concentration of 5% LAS, and a final concentration of 0.05% calcium chloride to make a total of 1 ml, which was used as a sample for stability evaluation. The prepared stability evaluation sample was immediately diluted as appropriate, and then the protease activity was measured as the initial activity.
After maintaining the stability evaluation sample at 30 ° C., the protease activity was measured over time, and the ratio of the activity after storage to the initial activity was defined as the residual activity (%).
The LAS resistance of the mutant protease was evaluated by comparing with the LAS resistance value of the culture supernatant when the transformant having the wild type enzyme gene was cultured under the same conditions (Tables 2 and 3).

  The above-mentioned alkaline protease mutant of the present invention has the characteristics of the parent alkaline protease except that it improves LAS resistance, that is, has resistance to oxidizing agents, is not inhibited by casein degradation activity by a high concentration of fatty acid, and is recognized by SDS-PAGE. The molecular weight was 43,000 ± 2,000, and it was confirmed that it retains the property of having activity in the alkaline region.

<Protease activity measurement method>
100 mM AAPL (Protein Laboratory; dissolved in Glt-Ala-Ala-Pro-Leu-pNA dimethyl sulfoxide: used as final concentration 3 mM) and 200 mM borate buffer (pH 10.5: used as final concentration 50 mM) and diluted as appropriate Add 50 μl of stability evaluation sample to 100 μl, measure the absorbance at 414 nm over time using a microplate reader (Versa Max: manufactured by Molecular Devices) with shaking at 30 ° C. for 15 minutes. The change in absorbance (OD414 / min) was determined. The value obtained by multiplying the obtained slope by the enzyme dilution rate was used as the protease titer.

Claims (2)

A method for stabilizing an alkaline protease consisting of an amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having 80% or more identity thereto, which comprises (a) 133 of the amino acid sequence represented by SEQ ID NO: 2 Or the position corresponding thereto and / or (b) the amino acid residue at position 195 or the position corresponding thereto is substituted with the following amino acid residue: How to improve.
(A) position: cysteine, glycine, aspartic acid, glutamic acid or proline residue (b) position: leucine or isoleucine residue   2. The method of claim 1, wherein the surfactant is sodium linear alkylbenzene sulfonate.