JP2001054392A - MUTANT alpha-AMYLASE - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new mutant α-amylase excellent in resistance to chelating agent and specific activity in alkaline region, used for detergent composition or the like obtained by substituting or deleting a part of amino acid residue of an α-amylase having specific amino acid sequence. SOLUTION: This new mutant α-amylase comprises an α-amylase substituting or deleting one or more amino acid residues corresponding either one of the amino acids of the 11th Tyr, the 16th Glu, the 49th Asn, the 84th Glu, the 144th Ser, the 167th Gln, the 169th Tyr, the 178th Ala, the 188th Glu, the 190th Asn, the 205th His and the 209th Gln of the amino acid sequence expressed by the formula or homology >=70% to the amino acid sequence, and is useful as detergents for an automatic dish washer, detergents for clothes or the like. The mutant enzyme is obtained by applying a site-specific mutagenesis to a liquescent α-amylase gene derived from a microorganism and expressing a product mutant gene.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a mutant liquefied alkali α having excellent heat resistance and particularly useful as an enzyme for detergents.
-Related to amylase and its genes.

[0002]

2. Description of the Related Art Conventionally, when α-amylase [EC.3.2.1.1] is used as a detergent, starch can be decomposed in a highly random manner, is alkaline and stable, and is effective against chelating and oxidative bleaching components. Liquefied alkaline α-amylase, which is stable, is said to be preferred. However, in general, calcium ions are important for maintaining the structure of the liquefied amylase, and its stability is reduced in the presence of a chelating agent. Almost.

[0003] Under such circumstances, the present inventors have proposed an alkaliphilic Bacillus sp KSM-K38 (FERM BP) isolated from soil.
-6946) and KSM-K36 (FERM BP-6945) show no decrease in activity due to the high concentration of the chelating agent which is inactivated with conventional liquefied α-amylase. Having resistance to surfactants and oxidizing agents, and compared to conventional liquefied α-amylase,
It has been found that it has a high activity on the alkali side and is useful for detergents (Japanese Patent Application No. 10-362487).

However, since the enzyme is inactivated at a temperature of 50 ° C. or higher, it is difficult to wash clothes and dishes at 10 to 60 ° C.
Considering that it is generally performed in the vicinity, its heat resistance was somewhat insufficient.

[0005]

DISCLOSURE OF THE INVENTION The present invention relates to a liquefied alkaline α-amylase having high activity on the alkali side, stable against chelating components and oxidative bleaching components, and having excellent heat resistance. It is an object to provide an α-amylase having

[0006]

Means for Solving the Problems The present inventors have obtained various mutant enzymes for liquefied alkaline α-amylase,
As a result of the examination, the mutation of a specific amino acid residue in the amino acid sequence of KSM-K38-derived amylase (SEQ ID NO: 1) causes loss of chelating agent resistance and oxidizing agent resistance and high specific activity in the alkaline region. It has been found that the heat resistance can be improved without any problem and that further heat resistance can be achieved by combining these mutations.

That is, the present invention relates to an amino acid sequence represented by SEQ ID NO: 1 or an α-amylase having 70% or more homology to the amino acid sequence, wherein Tyr at position 11 and Glu at position 16 of the amino acid sequence are used. , 49th A
sn, 84th Glu, 144th Ser, 167
Gln, 169th Tyr, 178th Al
a, 188th Glu, 190th Asn, 205
It is intended to provide a mutant α-amylase obtained by substituting or deleting one or more amino acid residues corresponding to any of the No. His and the No. 209 Gln. The present invention also relates to an amino acid sequence represented by SEQ ID NO: 1 or an α-amino acid having 70% or more homology to the amino acid sequence.
An amylase which provides a mutant α-amylase obtained by replacing a sequence corresponding to 11 to 100 amino acid residues from the amino terminus of the amino acid sequence with an amino acid sequence corresponding to the amino acid sequence of another liquefied α-amylase It is.

Further, the present invention provides a gene encoding the mutant α-amylase, a vector having the gene, a cell transformed with the vector, and a method for culturing the transformed cell. -To provide a method for producing amylase. Further, the present invention provides a detergent composition containing these mutant α-amylases.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The mutant α-amylase of the present invention comprises
It can be obtained by mutating the gene encoding the liquefied alkaline α-amylase having the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 70% or more homology with the sequence. An example in which the heat resistance is improved by substitution has also been performed for a conventional liquefied α-amylase. For example, in the enzyme derived from B. amyloliquefaciens , the 177th Arg to the 178th Gl
Deletion of y residue (J. Biol. Chem., 264, 1893
3, 1989), in which the 133rd His was replaced with Tyr in the enzyme of B. licheniformis (J. Biol. Chem., 2
65, 15481, 1990). However, the liquefied alkaline α-amylase used in the present invention has low amino acid homology with the conventional liquefied α-amylase, and the site corresponding to the 178th Gly residue from the 177th Arg to the 178th Gly residue is already present. The amino acid corresponding to His at position 133 has already been deleted, and the example of the conventional enzyme is not always applicable. That is, the mutation of the amino acid sequence for improving the heat resistance in the present invention is completely different from the examples described above.

Examples of the liquefied alkaline α-amylase include Bacillus sp. Isolated from soil by the present inventors.
An enzyme having the amino acid sequence of SEQ ID NO: 1 derived from KSM-K38 (FERM BP-6946) strain (Japanese Patent Application No. 10-362487).
Or an enzyme derived from the same KSM-K36 (FERMBP-6945) strain and having about 95% homology to the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 4) (Japanese Patent Application No. 10-362487). No. The homology of the amino acid sequence is Lipman-Pea
It is calculated by the rson method (Science, 227, 1435, 1985).

The mutant α-amylase of the present invention is obtained by first cloning a gene encoding the liquefied α-amylase from a microorganism producing liquefied α-amylase. An alternative technique may be used, for example, the method described in JP-A-8-336392 can be used. Examples of the gene include those shown in SEQ ID NO: 3 and SEQ ID NO: 5.

Next, a mutation is given to the obtained gene, and any method can be used as long as it is a commonly used site-specific mutation method. For example, Site-Directed Mutagenesis by Takara Shuzo Co., Ltd. System Mutan-Super Exp
It can be performed using a ress Km kit or the like. In addition, recombinant PCR (polymerase chain reaction) method
By using (PCR protocols, Academic press, New York, 1990), it is possible to replace an arbitrary sequence of a gene with a sequence corresponding to the arbitrary sequence of another gene.

In the present invention, the mutation for improving thermostability is SEQ ID NO: 1.
The amino acid residue corresponding to the 11th Tyr of the amino acid sequence represented by Phe, the amino acid residue corresponding to the 16th Glu to Pro, the amino acid residue corresponding to the 49th Asn to Ser, and the 84th Glu to the Glu The corresponding amino acid residue is Gln, the amino acid residue corresponding to Ser at position 144 is Pro, the amino acid residue corresponding to Gln at position 167 is Glu, the amino acid residue corresponding to Tyr at position 169 is Lys, the amino acid residue corresponding to Tyr at position 178 is The amino acid residue corresponding to Ala is Gln, the amino acid residue corresponding to Glu at position 188 is Asp, the amino acid residue corresponding to Asn at position 190 is Phe, the amino acid residue corresponding to His at position 205 is Arg or 209. A mutation that substitutes the amino acid residue corresponding to the Gln with Val is desirable.

Further, the amino acid sequence corresponding to 11 to 100 amino acid residues from the amino terminus (Asp) of the amino acid sequence of SEQ ID NO: 1 of the present invention, preferably the first Asp
The sequence corresponding to the 19th Gly from other liquefied α
-Heat resistance can also be achieved by substituting an amino acid sequence corresponding to the amino acid sequence of amylase.

Examples of other liquefied α-amylases to be replaced include, for example, an enzyme having the amino acid sequence shown in SEQ ID NO: 2, and the first Asp in the sequence.
The site corresponding to the 19th Gly from is the 1st His
Is the 21st Gly from. The enzyme is Bacillus s
p. KSM-AP1378 (FERM BP-3048) is a liquefied α-amylase derived from the strain, and its gene sequence is described in Japanese Patent Application Laid-Open No. 8-33639.
No. 2.

In the mutant α-amylase of the present invention,
Furthermore, mutations in which two or more kinds of substitutions or deletions selected from the above-mentioned substitutions or deletions of amino acid residues and substitutions of amino acid sequences are also effective, and by combination, a mutant enzyme having improved heat resistance can be obtained. Obtainable. That is, the method of combining mutations is a combination of two or more substitutions or deletions of various amino acid residues, a combination of two or more substitutions of amino acid sequences, and the substitution or deletion of amino acid residues and the substitution of amino acid sequences. A combination of two or more substitutions is preferred, but preferably the 49th A
The amino acid residue corresponding to sn is replaced with Ser, G at position 167.
The amino acid residue corresponding to ln is Glu, the 169th T
The amino acid residue corresponding to yr is Lys,
The amino acid residue corresponding to sn is Phe, the 205th H
A mutation in which the amino acid residue corresponding to is is substituted with Arg or the amino acid residue corresponding to 209th Gln, or a sequence corresponding to the 1st Asp to the 19th Gly is an amino acid represented by SEQ ID NO: 2. Array 1
Any two or more of the mutations that substitute for the amino acid sequence from the first His to the twenty-first Gly may be appropriately combined.

Further, as an example of the optimum combination, 4
The amino acid residue corresponding to the ninth Asn is Ser, 16
The amino acid residue corresponding to the seventh Gln is represented by Glu, 16
The amino acid residue corresponding to the ninth Tyr is Lys, 19
The amino acid residue corresponding to the 0th Asn is Phe, 20
The amino acid residue corresponding to the fifth His is Arg and 2
A combination of mutations replacing the amino acid residue corresponding to Gln at position 09 with Val, or 1 from Asp at position 1
The sequence corresponding to the ninth Gly is represented by the amino acid sequence shown in SEQ ID NO: 2 from the first His to the 21st Gl.
a mutation that substitutes for the amino acid sequence up to y, the amino acid residue corresponding to Gln at position 167 is Glu, the amino acid residue corresponding to Tyr at position 169 is Lys, and the amino acid residue corresponding to Asn at position 190 is Phe; Examples include a combination of mutations in which the amino acid residue corresponding to 209th Gln is substituted with Val.

Further, the above-mentioned mutations may be modified to improve properties other than heat resistance, for example, to further enhance oxidant resistance.
It is also possible to combine a mutation in which the amino acid residue corresponding to the 7th Met is replaced with Leu, and a mutation in which the amino acid residue corresponding to the 188th Glu, which enhances the detergency in clothing detergents, is replaced with Ile. is there.

The thus-obtained mutant α-amylase of the present invention has an excellent property of high chelating agent resistance and high heat stability without losing high specific activity in an alkaline region, and therefore, it is used for automatic dishwashing. It is useful as a machine detergent, a clothing detergent, and a fiber desizing agent. The content of the mutant α-amylase of the present invention in the detergent composition is 0.0
It is preferably from 0.01 to 10% by weight, particularly preferably from 0.01 to 5% by weight.

In addition to the above-mentioned mutant α-amylase, the detergent may further include a branching enzyme (eg, pullulanase, isoamylase, neopurulanase, etc.), α-glucosidase, glucoamylase, protease, cellulase, lipase, pectinase, protopectinase. , Pectate lyase, peroxidase, laccase and catalase.

The detergent may contain 1 to 90% by weight of a surfactant such as an anionic surfactant, an amphoteric surfactant, a nonionic surfactant or a cationic surfactant as a cleaning component. Also, chelating agents, alkaline agents,
Inorganic salts, recontamination inhibitor, chlorine scavenger, reducing agent, bleach,
Fluorescent dye solubilizer, fragrance, anti-caking agent, enzyme activator, antioxidant, preservative, pigment, bluing agent, bleach activator, enzyme stabilizer, regulator, etc. .

The detergent composition of the present invention can be produced according to a conventional method by combining the above-mentioned mutant α-amylase and the above-mentioned known detergent components. The form of the cleaning agent can be selected according to the use, and for example, can be liquid, powder, granules, and the like. Further, the cleaning composition of the present invention,
Clothes detergent, bleach detergent, automatic dishwasher detergent,
Although it can be used as a water pipe cleaning agent, a denture cleaning agent, etc., it can be suitably used as a cleaning agent for clothes, a bleaching agent, and a cleaning agent for automatic dishwashers.

The mutant α-amylase of the present invention can be used as a composition for liquefaction and saccharification of starch. The above enzymes can be blended and act on starch together with the mutant α-amylase.

Further, the mutant α-amylase of the present invention can be used as a desizing agent composition for fibers, and can also contain an enzyme such as pullulanase, isoamylase or neopurulanase.

[0025]

EXAMPLES Measurement of amylase activity and protein content The amylase activity and protein content of each enzyme were determined by the following methods. The amylase activity was measured by the 3,5-dinitrosalicylic acid method (DNS method). After performing a reaction at 50 ° C. for 15 minutes in a reaction solution containing soluble starch in a 50 mM glycine buffer (pH 10), the amount of reducing sugars generated was determined by quantification by a DNS method. The enzyme titer was defined as one unit of the amount of the enzyme producing a reducing sugar corresponding to 1 μmol of glucose per minute. Measurement of protein amount
Using bovine serum albumin as standard, Bio-Rad Protein A
Quantification was performed using a ssay kit.

Reference Example 1 Screening of Alkaline Liquefied Amylase About 0.5 g of soil was suspended in sterilized water and heated at 80 ° C. for 15 minutes. The supernatant of this heat treatment solution was appropriately diluted with sterile water, and applied to an agar medium for separation (medium A). Then
This was cultured at 30 ° C. for 2 days to form a colony. Those that formed a clear zone based on starch dissolution around the colonies were selected and isolated as amylase-producing bacteria. Further, the isolated bacteria were inoculated on the medium B, and aerobically shake-cultured at 30 ° C. for 2 days. After the cultivation, the supernatant of the centrifuged suspension was measured for chelating agent (EDTA) resistance and, further, the optimum pH was measured to screen the alkaline liquefied amylase-producing bacteria of the present invention.

According to the method described above, Bacillus sp. KSM-K38
(FERM BP-6946) strain and Bacillus sp. KSM-K36 (FERM B
P-6945) We were able to acquire the strain.

[0028] Table 1 shows the bacteriological properties of the KSM-K38 strain and the KSM-K36 strain.

[0029]

[Table 1]

Reference Example 2 KSM-K38 strain and KSM-
Culture of strain K36 In the liquid medium B of Reference Example 1, strain KSM-K38 or strain K
The SM-K36 strain was inoculated and cultured at 30 ° C. aerobically with shaking for 2 days. Amylase activity (pH
As a result of measurement of 8.5), 1 L of the culture solution had 557 U and 1177 U of activity, respectively.

Reference Example 3 Purification of Liquefied Alkaline Amylase The culture supernatant of the KSM-K38 strain obtained in Reference Example 2
After adding ammonium sulfate to a 0% saturation concentration and stirring, the formed precipitate was collected and dissolved in a 10 mM Tris-HCl buffer (pH 7.5) containing ₂ mM CaCl ₂ ,
The buffer was dialyzed overnight. The resulting dialysis solution was applied to a DEAE-Toyopearl 650 M column equilibrated with the same buffer, and the protein was eluted with the same buffer by a concentration gradient of 0-1 M sodium chloride. The active fraction was dialyzed against the same buffer, and the active fraction obtained by gel filtration column chromatography was dialyzed against the above buffer to obtain polyacrylamide gel electrophoresis (gel concentration: 10%) and sodium dodecyl sulfate (SDS). ) A purified enzyme giving a single band by electrophoresis could be obtained. In addition, KSM-K
Purified enzymes could be obtained from culture supernatants of 36 strains in the same manner.

Reference Example 4 Enzyme characteristics The characteristics of both purified enzymes are as follows. (1) Action Both of them decompose α-1,4 glucoside bonds of starch, amylose, amylopectin and their partially decomposed products,
Glucose (G1) and maltose (G
2) produce maltotriose (G3), maltotetraose (G4), maltopentaose (G5), maltohexaose (G6) and maltoheptaose (G7). However, it does not act on pullulan. (2) pH stability (Britton-Robinson buffer) In all cases, pH 6.5 at 40 ° C. for 30 minutes.
It shows a residual activity of 70% or more in the range of ~ 11.0. (3) Working temperature range and optimum working temperature Both work in a wide range of 20 to 80 ° C, and the optimum working temperature is 50 to 60 ° C. (4) Temperature stability The temperature was changed in a 50 mM sodium glycine hydroxide buffer solution (pH 10), and treatment was performed at each temperature for 30 minutes to examine the inactivation conditions. It showed residual activity, and showed about 60% residual activity even at 45 ° C. (5) Molecular weight In each case, the molecular weight measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 55,000 ±.
5,000. (6) Isoelectric point In each case, the isoelectric point measured by the isoelectric focusing method is around 4.2.

(7) Influence of a surfactant Sodium linear alkyl benzene sulfonate, alkyl
Sulfuric acid ester sodium salt, polyoxyethylene alkyl
Sodium sulfate, α-olefin sulfone
Sodium, α-sulfonated fatty acid ester sodium
, Sodium alkyl sulfonate, SDS, soap and
In a 0.1% solution of various surfactants such as sophthanol, p
H10, 30 ° C for 30 min.
It does not suffer from sex inhibition (activity residual rate 90% or more). (8) Influence of metal salts Coexist with various metal salts at pH 10, 30 ° C for 30 minutes
Processing and examining the effects. K38 is 1 mM Mn²⁺
(Inhibition rate about 75%), 1 mM Sr ²⁺Passing
And Cd²⁺(Inhibition rate about 30%). K
36 is 1 mM Mn²⁺(Inhibition rate of about 95
%) 1 mM Hg²⁺, Be²⁺And Cd²⁺Slightly inhibited by
(Inhibition rate 30-40%).

Example 1 Liquefied α-Amylase Gene
Cloning From the cells of KSM-K38 strain, the method of Saito and Miura (Bioc
him. Biophys. Acta, 72, 619, 1961) and using the chromosomal DNA extracted as a template, primer K38U
Using S (SEQ ID NO: 19) and K38DH (SEQ ID NO: 20), a gene fragment encoding a liquefied alkaline α-amylase (hereinafter referred to as K38AMY) having the amino acid sequence shown in SEQ ID NO: 1 by PCR reaction (about 1) .
5 kb) was amplified. After digestion with the restriction enzyme SalI , the expression vector pHSP64 (JP-A-6-217) is used.
781) into the SalI -SmaI site to obtain Bacillus sp. KSM-64 (F
A recombinant plasmid pHSP-K38 having a structural gene of K38AMY bound thereto was constructed downstream of a strong promoter derived from the alkaline cellulase gene of ERM P-10482) strain (FIG. 1). Similarly, Bacillus sp. KSM-AP1378 (F
PCR using primers LAUS (SEQ ID NO: 21) and LADH (SEQ ID NO: 22) with chromosomal DNA extracted from ERM BP-3048) strain (Japanese Unexamined Patent Publication No. 9-336392) as a template.
Liquefying α- amylase having the amino acid sequence shown in SEQ ID NO: 2 was amplified by the reaction (hereinafter, described as LAMY) gene fragment encoding a (about 1.5 kb), in the same manner as described above for the expression vector pHSP64 Sal I- Sma
I site, the recombinant plasmid pH
SP-LAMY was constructed (FIG. 1).

Example 2 Preparation of Mutant K38AMY Gene
-1 Site-Directed Mutagene by Takara Shuzo Co., Ltd.
The sis System Mutan-Super Express Km kit was used.
First, the recombinant plasmid pHSP- obtained in Example 1 was used.
Using K38 as a template, primer CLUBG (SEQ ID NO: 2)
3) and a PCR reaction using K38DH (SEQ ID NO: 20) to amplify an approximately 2.1 kb fragment from the upstream of the strong promoter derived from the KSM-64 strain to the downstream of the liquefied alkaline α-amylase gene. And the plasmid vector pKF19k attached to the above kit.
Inserted into the Sma I site, the recombinant plasmid p
KF19-K38 was constructed (FIG. 2).

Next, various oligonucleotides for site-directed mutagenesis shown in SEQ ID NOs: 6 to 15 were
After 5 ′ phosphorylation by 4DNA kinase, a mutagenesis reaction was carried out using this and pKF19-K38 according to the method of the kit, and Escherichia coli MV was determined by the reaction product.
Transformation of 1184 strain (competent cell MV1184, manufactured by Takara Shuzo) was performed. A recombinant plasmid was extracted from the resulting transformant, and the nucleotide sequence was analyzed to confirm the mutation.

Further, gene introduced mutation, in the same manner as described above, by inserting the Sma I site of pKF19k expression promoter region and a mutant K38AMY gene portion again becomes a template plasmid upon introducing different mutations, the Still another mutation was introduced in the same manner as described above.

Using each of the obtained mutant recombinant plasmids as a template, primers CLUBG (SEQ ID NO: 23) and K38D
H (SEQ ID NO: 20) to perform a PCR reaction to amplify each mutant K38AMY gene fragment, cut it with SalI , and then use the expression vector pHSP
Sal I- Sma I 64 (JP-A-6-217881).
A plasmid for producing mutant K38AMY was constructed by insertion into the site (FIG. 1).

Example 3 Preparation of Mutant K38AMY Gene
-2 (chimera with LAMY gene) Recombinant PCR was used for the mutation that replaces the N-terminal region of the K38AMY gene with the corresponding region of the LAMY gene (Fig. 3). First, using the recombinant plasmid pHSP-K38 obtained in Example 1 as a template, primer K38
DH (SEQ ID NO: 20) and LA-K38 (SEQ ID NO: 1)
By performing a PCR reaction using 7), Gln20 of the amino acid sequence of K38AMY shown in SEQ ID NO: 1
The fragment encoding the sequence from to the C-terminal downstream was amplified.
On the other hand, using the recombinant plasmid pHSP-LAMY as a template, primer CLUBG (SEQ ID NO: 23) and LA-K
By PCR reaction using 38R (SEQ ID NO: 18),
A gene fragment encoding from the upstream of the strong promoter to Gly at position 21 of the amino acid sequence of LAMY of SEQ ID NO: 2 was amplified. Next, by performing a second PCR reaction using both the obtained DNA fragments, the primer CLUBG (SEQ ID NO: 23) and K38DH (SEQ ID NO: 20), the primer LA-K38 (SEQ ID NO: 1) was added to the end.
7) and both fragments having complementary sequences derived from LA-K38R (SEQ ID NO: 18) bind to each other, and downstream of the strong promoter, from the 1st His to 21st Gly of LAMY.
Followed by Gln2 of K38AMY
A gene fragment (about 2.1 kb) encoding a substitution mutant enzyme (abbreviated as LA-K38AMY) to which a region encoding from 0 to the C-terminal was bound was amplified. This was cut with Sal I, and inserted into the Sal I- Sma I site of the expression vector pHSP64 (Japanese Patent Laid-Open 6-217781), were constructed mutant K38AMY for production plasmid (Figure 1).

Example 4 Mutually liquefied alkali α-amilla
Production of various K38AMY mutant plasmids obtained in Examples 2 and 3 was carried out by the protoplast method (Mol. Gen. Genet., 16).
8, 111, 1979) by Bacillus subtilis ISW1214 strain (leu A me
t B5 hsd M1), and the resulting recombinant Bacillus subtilis was transformed into a liquid medium (corn steep liquor, 8%; meat extract, 1%;
Potassium phosphate, 0.02%; maltose, 5%; calcium chloride, 5 mM; tetracycline, 15 μg /
(ml) at 30 ° C for 3 days. The obtained culture supernatant was dialyzed against Tris-HCl buffer (pH 7.0),
DEAE-Toyopearl 650M equilibrated with the same buffer
The column was adsorbed and eluted with a concentration gradient of sodium chloride. This eluate was added to a 10 mM glycine buffer (pH 1).
0.0), each mutant K38AMY
Was obtained.

Example 5 Assay for heat resistance- 1 According to the method described in Examples 1, 2, and 4, an enzyme in which Tyr at position 11 in SEQ ID NO: 1 was substituted with Phe (Y11
F), an enzyme in which the 49th Asn is substituted with Ser (abbreviated as N49S), an enzyme in which the 84th Glu is substituted with Gln (abbreviated as E84Q), and the 144th Se.
an enzyme in which r is replaced with Pro (abbreviated as S144P), 1
Enzyme in which Gln at position 67 was replaced with Glu (Q167E
169th Tyr was replaced with Lys (abbreviated as Y169K), 178th Ala was replaced with Gl
n-substituted enzyme (abbreviated as A178Q), 188th Glu was substituted with Asp (abbreviated as E188D), and 190th Asn was substituted with Phe (N
190F), and 209th Gln is Val
A purified sample of the enzyme (abbreviated as Q209V) was obtained and its heat resistance was assayed by the following method. Wild-type K38AMY was used as a control.

A 50 mM glycine buffer (pH
10.0) at 50 ° C.
After adding the enzyme to about 1.2 U / mL, sampling was performed 30 minutes later, and the remaining amylase activity was measured by the method described in the above example. The relative activity was determined by defining the activity at the start as 100%, and defined as the amylase residual activity. The results are shown in Table 2, where the residual activity of the wild-type K38AMY after 30 minutes was reduced to 15%, whereas all mutant enzymes showed higher residual activities than the wild-type.

[0043]

[Table 2]

Example 6 Test of heat resistance-2 Among the mutations shown in Example 5, Q167E and Y169
Mutant enzymes in which K, N190F and Q209V were combined as follows were prepared by the methods described in Examples 1, 2 and 4. Q167E / Y169K (using the primer of SEQ ID NO: 16, abbreviated as QEYK) N190F / Q209V (abbreviated as NFQV) Q167E / Y169K / N190F / Q209V (Q
EYK / NFQV)

The heat resistance of each of them was tested in the same manner as in Example 5. However, the temperature of the heat treatment was 55 ° C., and Q167E, Y169K, N190F
And Q209V. As a result, as shown in Table 3, all the mutations showed an improvement in heat resistance by the combination, and the QEYK / NF obtained by combining the four types of mutations
QV showed 85% residual activity after 30 minutes even at 55 ° C.

[0046]

[Table 3]

Example 7 Assay of thermostability-3 Mutant enzyme obtained by combining the mutation shown in Example 6 with the mutation shown in Example 5 and S144P in addition to the mutant NFQV shown in Example 6,
Mutant enzymes combined with a mutation (abbreviated as E16P) that substitutes Glu at position 6 for Pro were produced by the methods described in Examples 1, 2, and 4. S144P / NFQV (abbreviated as SP / NFQV) E16P / S144P / NFQV (EPSP / NFQV
These were tested for heat resistance in the same manner as in Example 5 (50 ° C.). As a result, as shown in Table 4, SP
An improvement in heat resistance was observed by combining E16P with / NFQV.

[0048]

[Table 4]

Example 8 Assay of heat resistance-4 Among the mutations shown in Example 4, QEYK / NFQV had a mutation in which Met at position 107 in SEQ ID NO: 1 was replaced with Leu (abbreviated as M107L), and a mutation at position 205 The following mutant enzymes combining N49S among the mutations in which His was replaced with Arg (abbreviated as H205R) and the mutations shown in Example 3 were produced by the methods described in Examples 1, 2, and 4. M107L / QEYK / NFQV (ML / QEYK / N
N49S / M107L / QEYK / NFQV (NSML)
/ QEYK / NFQV) N49S / M107L / H205R / QEYK / NFQ
V (abbreviated as NSMLHR / QEYK / NFQV) The heat resistance of these was tested in the same manner as in Example 5. However, the temperature of the heat treatment was 60 ° C. As a result, ML / QEYK / NFQV has N49S and H2
By combining 05R, the heat resistance was additionally improved, and NSMLHR / QEYK / NFQV showed 75% residual activity after 30 minutes even at 60 ° C (Table 5).

[0050]

[Table 5]

Example 9 Test for heat resistance-5 According to the method described in Examples 1, 3, and 4, K38AMY was used.
Sequence from Asp1 to Gly at position 19 is LAMY
The mutant enzyme LA-K38AMY was obtained by substituting the sequence from the 1st His to the 21st Gly. As a result of testing the heat resistance of this enzyme by the method of Example 5, as shown in Table 6, improvement of the heat resistance by substitution was observed.

[0052]

[Table 6]

Example 10 Assay of thermostability -6 The sequence of the mutant enzyme QEYK / NFQV shown in Example 6 from the first Asp to the 19th Gly by the same method as in Examples 1 and 3. Introduced a mutation that replaces the sequence from the first His to the twenty-first Gly of LAMY. Mutant enzyme LA-K38AMY / QEY obtained by the method of Example 4 using this gene
The heat resistance of K / NFQV was tested in the same manner as in Example 8 (heat treatment was 60 ° C.). As a result, the heat resistance is additively improved by the combination, and LA-K38AM
Y / QEYK / NFQV showed 63% residual activity after 30 minutes even at 60 ° C (Table 7).

[0054]

[Table 7]

Example 11 Composition of detergent for automatic dishwasher
A detergent composition for an automatic dishwasher was manufactured with the formulation shown in Table 8, and a washing test was conducted by blending each mutant enzyme with the detergent. As a result, when the enzyme having the same activity value was added, the mutant enzyme showed an excellent washing effect as compared with the wild-type enzyme.

[0056]

[Table 8]

[0057]

The mutant α-amylase of the present invention has excellent properties of high chelating agent resistance, high specific activity in an alkaline region, and also has excellent heat stability. Therefore, detergents for automatic dishwashers, clothing detergents,
It is useful as a starch liquefaction, saccharification composition, and fiber desizing agent.

[0058]

[Sequence List] SEQUENCE LISTING <110> KAO CORPORATION <120> New mutant alpha-amylase <130> P02591206 <150> JP P1999-163569 <151> 1999-06-10 <160> 23 <210> 1 <211> 480 <212> PRT <213> Bacillus sp.KSM-K38 <400> 1 Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr Glu Trp His Leu Glu 5 10 15 Asn Asp Gly Gln His Trp Asn Arg Leu His Asp Asp Ala Ala Ala Leu 20 25 30 Ser Asp Ala Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45 Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60 Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80 Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys Ser Asn Asp Ile Asn 85 90 95 Val Tyr Gly Asp Val Val Met Asn His Lys Met Gly Ala Asp Phe Thr 100 105 110 Glu Ala Val Gln Ala Val Gln Val Asn Pro Thr Asn Arg Trp Gln Asp 115 120 125 Ile Ser Gly Ala Tyr Thr Ile Asp Ala Trp Thr Gly Phe Asp Phe Ser 130 135 140 Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp Arg Trp Phe His Phe 145 150 155 160 Asn Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu Asn Hi s Ile Phe Arg 165 170 175 Phe Ala Asn Thr Asn Trp Asn Trp Arg Val Asp Glu Glu Asn Gly Asn 180 185 190 Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe Ser His Pro Glu Val 195 200 205 Gln Asp Glu Leu Lys Asp Trp Gly Ser Trp Phe Thr Asp Glu Leu Asp 210 215 220 Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His Ile Pro Phe Trp Tyr 225 230 235 240 Thr Ser Asp Trp Val Arg His Gln Arg Asn Glu Ala Asp Gln Asp Leu 245 250 255 Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val Gly Ala Leu Glu Phe 260 265 270 Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu Phe Asp Val Pro Leu 275 280 285 Asn Tyr Asn Phe Tyr Arg Ala Ser Gln Gln Gly Gly Ser Tyr Asp Met 290 295 300 300 Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala His Pro Met His Ala 305 310 315 320 Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Glu Ser Leu Glu 325 330 335 Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala Tyr Ala Thr Ile Leu 340 345 350 Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr Gly Asp Tyr Tyr Gly 355 360 365 Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp Met Ile As p Glu Leu 370 375 380 Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe 385 390 395 400 400 Asp His Trp Asp Val Val Gly Trp Thr Arg Glu Gly Ser Ser Ser Arg 405 410 415 Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn Gly Pro Gly Gly Ser 420 425 430 Lys Trp Met Tyr Val Gly Arg Gln Asn Ala Gly Gln Thr Trp Thr Asp 435 440 445 Leu Thr Gly Asn Asn Gly Ala Ser Val Thr Ile Asn Gly Asp Gly Trp 450 455 460 Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser Val Tyr Val Asn Gln 465 470 475 480

<210> 2 <211> 485 <212> PRT <213> Bacillus sp.KSM-AP1378 <400> 2 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30 Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Ser Gln Leu Gln Gly Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Gly Thr Glu Met Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn 115 120 125 Gln Glu Ile Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys 165 170 175 Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190 Ile Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200 205 Asp His Pro Glu Val Ile Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Tyr Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr 245 250 255 Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Ala Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300 Gly Tyr Phe Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys 305 310 315 320 His Pro Ile His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Gly Glu Ala Leu Glu Ser Phe Val Gln Ser Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ser Met Lys Ser 370 375 380 Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Tyr Ala Tyr Gly Thr 385 390 395 400 400 Gln His Asp Tyr PheAsp His His Asp Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asp Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys His Lys Ala Gly 435 440 445 Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Ser Gly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Thr Val Asn Gly Gly Ala Val Ser 465 470 475 480 480 Val Trp Val Lys Gln 485

<210> 3 <211> 1753 <212> DNA <213> Bacillus sp.KSM-K38 <220> <221> sig # peptide <222> (162) .. (224) <220> <221> mat # peptide <222> (225) .. (1664) <220> <221> CDS <222> (162) .. (1664) <400> 3 gtatgcgaaa cgatgcgcaa aactgcgcaa ctactagcac tcttcaggga ctaaaccacc 60 ttttttccaa aaatgaatcat atataaacaaatgtgt ttatgatata tgtaagcgtt atcattaaaa ggaggtattt g atg aga aga tgg gta 176 gta gca atg ttg gca gtg tta ttt tta ttt cct tcg gta gta gtt gca 224 gat gga ttg aac ggt acg atg atg cag tat tat ggg cat tat tt gag gat cat tat gg g cat tat tt gg g cat tat tat gg tg cat g Thr Met Met Gln Tyr Tyr Glu Trp His Leu Glu 1 5 10 15 aac gac ggg cag cat tgg aat cgg ttg cac gat gat gcc gca gct ttg 320 Asn Asp Gly Gln His Trp Asn Arg Leu His Asp Asp Ala Ala Ala Leu 20 25 30 agt gat gct ggt att aca gct att tgg att ccg cca gcc tac aaa ggt 368 Ser Asp Ala Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45 aat agt cag gcg gat gtt ggg tac ggt gca tac gat ctt tat gat tta 416 Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60 gga gag ttc aat caa aag ggt act gtt cga acg aaa tac gga act aag 464 Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80 gca cag ctt gaa cga gct att ggg tcc ctt aaa tct aat gat atc aat 512 Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys Ser Asn Asp Ile Asn 85 90 95 gta tac gga gat gtc gtg atg aat cat aaa atg gga gct gat ttt acg 560 Val Tyr Gly Asp Val Val Met Asn His Lys Met Gly Ala Asp Phe Thr 100 105 110 gag gca gtg caa gct gtt caa gta aat cca acg aat cgt tgg cag gat 608 Glu Ala Val Gln Ala Val Gln Val Asn Pro Thr Asn Arg Trp Gln Asp 115 120 125 att tca ggt gcc tac acg att gat gcg tgg acg ggt ttc gac ttt tca 656 Ile Ser Gly Ala Tyr Thr Ile Asp Ala Trp Thr Gly Phe Asp Phe Ser 130 135 140 ggg cgt aac aac gcc tat tca gat ttt aag tgg aga tgg ttc cat ttt 704 Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp Arg Trp Phe His Phe 145 150 155 160 aat ggt gtt gac tgg gat cag cgc tat caa gaa aat cat att ttc cgc 752 Asn Gly Val Asp Trp Asp Gln Ar g Tyr Gln Glu Asn His Ile Phe Arg 165 170 175 ttt gca aat acg aac tgg aac tgg cga gtg gat gaa gag aac ggt aat 800 Phe Ala Asn Thr Asn Trp Asn Trp Arg Val Asp Glu Glu Asn Gly Asn 180 185 190 tat gat tac ctg tta gga tcg aat atc gac ttt agt cat cca gaa gta 848 Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe Ser His Pro Glu Val 195 200 205 caa gat gag ttg aag gat tgg ggt agc tgg ttt acc gat gag tta gat 896 Gln Asp Glu Leu Lys Asp Trp Gly Ser Trp Phe Thr Asp Glu Leu Asp 210 215 220 ttg gat ggt tat cgt tta gat gct att aaa cat att cca ttc tgg tat 944 Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His Ile Pro Phe Trp Tyr 225 230 235 240 aca tct gat tgg gtt cgg cat cag cgc aac gaa gca gat caa gat tta 992 Thr Ser Asp Trp Val Arg His Gln Arg Asn Glu Ala Asp Gln Asp Leu 245 250 255 ttt gtc gta ggg gaa tat tgg aag gat gac gta ggt gct ctc gaa ttt 1040 Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val Gly Ala Leu Glu Phe 260 265 270 270 tat tta gat gaa atg aat tgg gag atg tct cta ttc gat gtt cca ctt 1088 Glu Leu AsuMet Asn Trp Glu Met Ser Leu Phe Asp Val Pro Leu 275 280 285 aat tat aat ttt tac cgg gct tca caa caa ggt gga ag tat gat atg 1136 Asn Tyr Asn Phe Tyr Arg Ala Ser Gln Gln Gly Gly Ser Tyr Asp Met 290 295 300 cgt aat att tta cga gga tct tta gta gaa gcg cat ccg atg cat gca 1184 Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala His Pro Met His Ala 305 310 315 320 gtt acg ttt gtt gat aat cat gat act cag cca ggg gag tca tta gag 1232 Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Glu Ser Leu Glu 325 330 335 tca tgg gtt gct gat tgg ttt aag cca ctt gct tat gcg aca att ttg 1280 Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala Tyr Ala Thr Ile Leu 340 345 350 acg cgt gaa ggt ggt tat cca aat gta ttt tac ggt gat tac tat ggg 1328 Thr Arg Glu Gly Gly Gly Tly Pro Asn Val Phe Tyr Gly Asp Tyr Tyr Gly 355 360 365 att cct aac gat aac att tca gct aaa aaa gat atg att gat gag ctg 1376 Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp Met Ile Asp Glu Leu 370 375 380 ctt gat gca cgt caa aat tac gca tat ggc acg cag cat gac tat ttt 24 Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe 385 390 395 400 gat cat tgg gat gtt gta gga tgg act agg gaa gga tct tcc tcc aga 1472 Asp His Trp Asp Val Val Gly Trp Thr Arg Glu Gly Ser Ser Ser Arg 405 410 415 cct aat tca ggc ctt gcg act att atg tcg aat gga cct ggt ggt tcc 1520 Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn Gly Pro Gly Gly Ser 420 425 430 aag tgg atg tat gta gga cgt cag aat gca gga caa aca tgg aca gat 1568 Lys Trp Met Tyr Val Gly Arg Gln Asn Ala Gly Gln Thr Trp Thr Asp 435 440 445 tta act ggt aat aac gga gcg tcc gtt aca att aat ggc gat gga tgg 1616 Leu Thr Gly Asn Asn Gly Ala Ser Val Thr Ile Asn Gly Asp Gly Trp 450 455 460 ggc gaa ttc ttt acg aat gga gga tct gta tcc gtg tac gtg aac caa 1664 Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser Val Tyr Val Asn Gln 465 470 475 480 taacaaaaag ccttgagaag ggattcctcc ctaactcaag gctttcttta tgtcgcttag 1724 cttaacgctt ctacgacttt gaagcttta 1753

<210> 4 <211> 480 <212> PRT <213> Bacillus sp.KSM-K36 <400> 4 Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr Glu Trp His Leu Glu 5 10 15 Asn Asp Gly Gln His Trp Asn Arg Leu His Asp Asp Ala Glu Ala Leu 20 25 30 Ser Asn Ala Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45 Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60 Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80 Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys Ser Asn Asp Ile Asn 85 90 95 Val Tyr Gly Asp Val Val Met Asn His Lys Leu Gly Ala Asp Phe Thr 100 105 110 Glu Ala Val Gln Ala Val Gln Val Asn Pro Ser Asn Arg Trp Gln Asp 115 120 125 Ile Ser Gly Val Tyr Thr Ile Asp Ala Trp Thr Gly Phe Asp Phe Pro 130 135 140 Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp Arg Trp Phe His Phe 145 150 155 160 Asn Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu Asn His Leu Phe Arg 165 170 175 Phe Ala Asn Thr Asn Trp Asn Trp Arg Val Asp Glu Glu Asn Gly Asn 180 185 190 Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe Ser His Pro Glu Val 195 200 205 Gln Glu Glu Leu Lys Asp Trp Gly Ser Trp Phe Thr Asp Glu Leu Asp 210 215 220 Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His Ile Pro Phe Trp Tyr 225 230 235 240 Thr Ser Asp Trp Val Arg His Gln Arg Ser Glu Ala Asp Gln Asp Leu 245 250 255 Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val Gly Ala Leu Glu Phe 260 265 270 Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu Phe Asp Val Pro Leu 275 280 285 Asn Tyr Asn Phe Tyr Arg Ala Ser Lys Gln Gly Gly Ser Tyr Asp Met 290 295 300 Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala His Pro Ile His Ala 305 310 315 320 Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Glu Ser Leu Glu 325 330 335 Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala Tyr Ala Thr Ile Leu 340 345 350 Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr Gly Asp Tyr Tyr Gly 355 360 365 Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp Met Ile Asp Glu Leu 370 375 380 Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe 385 390 395 400 Asp His Trp Asp Ile Val Gly Trp Thr Arg Glu Gly Thr Ser Ser Arg 405 410 415 Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn Gly Pro Gly Gly Ser 420 425 430 Lys Trp Met Tyr Val Gly Gln Gln His Ala Gly Gln Thr Trp Thr Asp 435 440 445 Leu Thr Gly Asn His Ala Ala Ser Val Thr Ile Asn Gly Asp Gly Trp 450 455 460 Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser Val Tyr Val Asn Gln 465 470 475 480

<210> 5 <211> 1625 <212> DNA <213> Bacillus sp.KSM-K36 <220> <221> sig # peptide <222> (40) .. (102) <220> <221> mat # peptide <222> (103) .. (1542) <220> <221> CDS <222> (40) .. (1542) <400> 5 atgatatatg taagcgttat cattaaaagg aggtatttg atg aaa aga tgg gta 54 gta gca atg ctg gca gtg tta ttt tta ttt cct tcg gta gta gtt gca 102 gat ggc ttg aat gga acg atg atg cag tat tat gag tgg cat cta gag 150 Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr Glu Trp His Leu Glu 1 5 10 15 aat gat ggg caa cac tgg aat cgg ttg cat gat gat gcc gaa gct tta 198 Asn Asp Gly Gln His Trp Asn Arg Leu His Asp Asp Ala Glu Ala Leu 20 25 30 agt aat gcg ggt att aca gct att tgg ata ccc cca gcc tac aaa gga 246 Ser Asn Ala Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45 aat agt cag gct gat gtt ggg tat ggt gca tac gac ctt tat gat tta 294 Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60 ggg gag ttt aat caa aaa ggt acc gtt cga acg aaa tac ggg aca aag 342 Gly Glu Phe Asn Gln Lys Gly Thr Val Arg T hr Lys Tyr Gly Thr Lys 65 70 75 80 gct cag ctt gag cga gct ata ggg tcc cta aag tcg aat gat atc aat 390 Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys Ser Asn Asp Ile Asn 85 90 95 gtt tat ggg gat gtc gta atg aat cat aaa tta gga gct gat ttc acg 438 Val Tyr Gly Asp Val Val Met Asn His Lys Leu Gly Ala Asp Phe Thr 100 105 110 gag gca gtg caa gct gtt caa gta aat cct tcg aac cgt tgg cag gat 486 Glu Ala Val Gln Ala Val Gln Val Asn Pro Ser Asn Arg Trp Gln Asp 115 120 125 att tca ggt gtc tac acg att gat gca tgg acg gga ttt gac ttt cca 534 Ile Ser Gly Val Tyr Thr Ile Asp Ala Trp Thr Gly Phe Asp Phe Pro 130 135 140 ggg cgc aac aat gcc tat tcc gat ttt aaa tgg aga tgg ttc cat ttt 582 Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp Arg Trp Phe His Phe 145 150 155 160 aat ggc gtt gac tgg gat caa cgc tat caa gaa aac cat ctt ttt cgc 630 Asn Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu Asn His Leu Phe Arg 165 170 175 ttt gca aat acg aac tgg aac tgg cga gtg gat gaa gag aat ggt aat 678 Phe Tla Asn Thrn Asn Trp Arg Val Asp Glu Glu Asn Gly Asn 180 185 190 tat gac tat tta tta gga tcg aac att gac ttt agc cac cca gag gtt 726 Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe Ser His Pro Glu Val 195 200 205 caa gag gaa tta aag gat tgg ggg agc tgg ttt acg gat gag cta gat 774 Gln Glu Glu Leu Lys Asp Trp Gly Ser Trp Phe Thr Asp Glu Leu Asp 210 215 220 tta gat ggg tat cga ttg gat gct att aag cat att cca ttc tgg 822 Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His Ile Pro Phe Trp Tyr 225 230 235 240 acg tca gat tgg gtt agg cat cag cga agt gaa gca gac caa gat tta 870 Thr Ser Asp Trp Val Arg His Gln Arg Ser Glu Ala Asp Gln Asp Leu 245 250 255 ttt gtc gta ggg gag tat tgg aag gat gac gta ggt gct ctc gaa ttt 918 Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val Gly Ala Leu Glu Phe 260 265 270 tat tta gat gaa atg aat tgg atg tct cta ttc gat gtt ccg ctc 966 Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu Phe Asp Val Pro Leu 275 280 285 aat tat aat ttt tac cgg gct tca aag caa ggc gga agc tat gat atg 1014 Asn Tyr As Arg Ala Ser Lys Gln Gly Gly Ser Tyr Asp Met 290 295 300 cgt aat att tta cga gga tct tta gta gaa gca cat ccg att cat gca 1062 Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala His Pro Ile His Ala 305 310 315 320 gtt acg ttt gtt gat aat cat gat act cag cca gga gag tca tta gaa 1110 Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Glu Ser Leu Glu 325 330 335 tca tgg gtc gct gat tgg ttt aag cca ctt gct tat gcg aca atc ttg 1158 Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala Tyr Ala Thr Ile Leu 340 345 350 acg cgt gaa ggt ggt tat cca aat gta ttt tac ggt gac tac tat ggg 1206 Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr Gly Asp Tyr Tyr Gly 355 360 365 att cct aac gat aac att tca gct aag aag gat atg att gat gag ttg 1254 Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp Met Ile Asp Glu Leu 370 375 380 ctt gat gca cgta caca aat tac gca tat ggc aca caa cat gac tat ttt 1302 Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe 385 390 395 400 gat cat tgg gat atc gtt gga tgg aca aga gaa ggt aca tcc tca cgt 1 350 Asp His Trp Asp Ile Val Gly Trp Thr Arg Glu Gly Thr Ser Ser Arg 405 410 415 cct aat tcg ggt ctt gct act att atg tcc aat ggt cct gga gga tca 1398 Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn Gly Pro Gly Gly Ser 420 425 430 aaa tgg atg tac gta gga cag caa cat gca gga caa acg tgg aca gat 1446 Lys Trp Met Tyr Val Gly Gln Gln His Ala Gly Gln Thr Trp Thr Asp 435 440 445 445 tta act ggc aat cac gcg gcg tcg gtt acg att aat ggt gat ggc tgg 1494 Leu Thr Gly Asn His Ala Ala Ser Val Thr Ile Asn Gly Asp Gly Trp 450 455 460 ggc gaa ttc ttt aca aat gga gga tct gta tcc gtg tat gt gtg aac caa 1542 Gly Glu Phe Phe Phe Asn Gly Gly Ser Val Ser Val Tyr Val Asn Gln 465 470 475 480 taataaaaag ccttgagaag ggattcctcc ctaactcaag gctttcttta tgtcgtttag 1602 ctcaacgctt ctacgaagct tta 1625

<210> 6 <211> 30 <212> DNA <213> Artificial Sequence <400> 6 atgatgcagt attttgagtg gcatttggaa 30

<210> 7 <211> 33 <212> DNA <213> Artificial Sequence <400> 7 tatgagtggc atttgccaaa cgacgggcag cat 33

<210> 8 <211> 33 <212> DNA <213> Artificial Sequence <400> 8 ccagcctaca aaggtagtag tcaggcggat gtt 33

<210> 9 <211> 21 <212> DNA <213> Artificial Sequence <400> 9 gcacagcttc aacgagctat t 21

<210> 10 <211> 21 <212> DNA <213> Artificial Sequence <400> 10 tttcgacttt ccagggcgta a 21

<210> 11 <211> 33 <212> DNA <213> Artificial Sequence <400> 11 catattttcc gctttcaaaa tacgaactgg aac 33

<210> 12 <211> 33 <212> DNA <213> Artificial Sequence <400> 12 aactggcgag tggatgatga gaacggtaat tat 33

<210> 13 <211> 25 <212> DNA <213> Artificial Sequence <400> 13 tggatgaaga gttcggtaat tatga 25

<210> 14 <211> 33 <212> DNA <213> Artificial Sequence <400> 14 aatatcgact ttagtcgtcc agaagtacaa gat 33

<210> 15 <211> 33 <212> DNA <213> Artificial Sequence <400> 15 agtcatccag aggtcgtaga tgagttgaag gat 33

<210> 16 <211> 33 <212> DNA <213> Artificial Sequence <400> 16 gttgactggg atgagcgcaa acaagaaaat cat

<210> 17 <211> 34 <212> DNA <213> Artificial Sequence <400> 17 atttgccaaa tgacgggcag cattggaatc ggtt 34

<210> 18 <211> 34 <212> DNA <213> Artificial Sequence <400> 18 aaccgattcc aatgctgccc gtcatttggc aaat 34

<210> 19 <211> 40 <212> DNA <213> Artificial Sequence <400> 19 gggtcgacca gcacaagccg atggattgaa cggtacgatg 40

<210> 20 <211> 29 <212> DNA <213> Artificial Sequence <400> 20 taaagctttt gttattggtt cacgtacac 29

<210> 21 <211> 30 <212> DNA <213> Artificial Sequence <400> 21 gagtcgacca gcacaagccc atcataatgg 30

<210> 22 <211> 21 <212> DNA <213> Artificial Sequence <400> 22 taaagcttca atttatattg g 21

<210> 23 <211> 27 <212> DNA <213> Artificial Sequence <400> 23 ccagatctac ttaccatttt agagtca 27

[Brief description of the drawings]

FIG. 1. KSM-K38 strain and KSM-AP1378
FIG. 2 is a diagram showing a method for constructing a recombinant plasmid for producing α-amylase derived from a strain.

FIG. 2 is a schematic diagram showing a method for introducing a mutation into an α-amylase gene derived from KSM-K38 strain.

FIG. 3 is a diagram showing a method of substituting the N-terminal sequence of α-amylase gene derived from KSM-K38 strain with the N-terminal region of α-amylase gene derived from KSM-AP1378 strain.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12N 9/28 9/28 5/00 A (72) Inventor Yasuhiro Hayashi Shellfish in Haga-gun, Tochigi Prefecture 2606 Kao Corporation, Research Laboratories in Kao Corporation (72) Inventor Hiroshi Hagiwara 2606 Kabanecho, Kaigacho, Haga-gun, Tochigi Prefecture Kao Corporation Research Laboratory (72) Inventor Katsuya Ozaki 2606 Akabane, Kaigamachi, Haga-gun, Tochigi Research by Kao Corporation Inside

Claims (9)

[Claims] 1. An amino acid sequence represented by SEQ ID NO: 1 or α having at least 70% homology to the amino acid sequence.
-In amylase, the 11th T of the amino acid sequence
yr, 16th Glu, 49th Asn, 84th Glu, 144th Ser, 167th Gln,
Substitution or deletion of one or more amino acid residues corresponding to any of Tyr at position 169, Ala at position 178, Glu at position 188, Asn at position 190, His at position 205 and Gln at position 209 Mutation α
Amylase. 2. An amino acid sequence represented by SEQ ID NO: 1 or α having 70% or more homology to the amino acid sequence.
A mutant α-amylase obtained by replacing a sequence corresponding to 11 to 100 amino acid residues from the amino terminus of the amino acid sequence with an amino acid sequence corresponding to the amino acid sequence of another liquefied α-amylase. 3. The first A of the amino acid sequence of SEQ ID NO: 1.
The mutant α- according to claim 2, wherein the sequence corresponding to sp to the 19th Gly is replaced with an amino acid sequence corresponding to the amino acid sequence of another liquefied α-amylase.
amylase. (4) another liquefied α-amylase has the sequence of SEQ ID NO: 2;
The mutant α-amylase according to claim 2 or 3, which has an amino acid sequence represented by the formula: 5. An amino acid sequence represented by SEQ ID NO: 1 or α having at least 70% homology to the amino acid sequence.
-Amylase is mutated by combining two or more substitutions or deletions selected from the substitution or deletion of the amino acid residue according to claim 1 and the substitution of the amino acid sequence according to any one of claims 2 to 4. Mutated α-amylase. 6. The substitution of an amino acid residue in the amino acid sequence of SEQ ID NO: 1 corresponds to the amino acid residue corresponding to Tyr at position 11 and the amino acid residue corresponding to Glu at position 16 corresponds to Phe.
ro, the amino acid residue corresponding to the 49th Asn
r, the amino acid residue corresponding to Gln at position 167 is represented by Gl
u, the amino acid residue corresponding to the 169th Tyr is Ly
s, the amino acid residue corresponding to Asn at position 190 is Ph
e, the amino acid residue corresponding to His at position 205 is Ar
g or the amino acid residue corresponding to Gln at position 209 is represented by V
al, and the amino acid sequence is replaced with the amino acid sequence from Asp at position 1 to Gly at position 19 in SEQ ID NO: 1 from His at position 1 in SEQ ID NO: 2.
The mutant α-amylase according to claim 5, which is obtained by substituting the amino acid sequence up to the first Gly. 7. A gene encoding the mutant α-amylase according to claim 1 or a vector containing the gene. 8. A cell transformed with the vector according to claim 7. 9. A method for producing a mutant α-amylase, which comprises culturing the transformed cell according to claim 8. 10. A detergent composition comprising the mutant α-amylase according to any one of claims 1 to 6.