JP2000135093A - New amylase for baking and its gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new amylase for baking, containing a specific amino acid sequence, having α-amylase activity, and a specific thermostability suitable for baking, providing a bread having an increased volume and improved softness, effective for preventing bread aging. SOLUTION: This variant α-amylase contains an amino acid sequence in which one or plural amino acids are deleted from, inserted into or substituted for those of an amino acid sequence of the formula, has α-amylase activity, remaining α-amylase activity after treatment at 65 deg.C for 30 minutes lower than 80% before the treatment and thermostability suitable for use in baking, is effective for increasing bread volume, improving softness are preventing aging and is useful for baking. The enzyme is obtained by treating a DNA encoding the enzyme or a cell containing the DNA with a mutagenic substance, transducing the obtained variant DNA into a host cell, screening and selecting an α-amylase having a desirable thermostability.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel mutant α-amylase for baking and a baking method using this enzyme.

[0002]

2. Description of the Related Art α-amylase is used for the production of bread (hereinafter, referred to as bread).
Baking) is used in the fermentation process. α
-The addition of amylase to bread dough has the effect of improving the tenderness of bread and the effect of preventing aging of bread. The former is caused by the fact that the α-amylase acts on the starch granules present as pillars supporting them in the gluten network of the dough and softens the starch granules. The latter is provided by α-amylase preventing recrystallization of amylose in dough.

If the activity of α-amylase added in bread making remains after baking of the dough, the quality of the bread is reduced. The α-amylase currently used in baking is mainly derived from mold (eg, Aspergillus oryzae). This is because mold-derived α-amylase generally has low heat resistance. However, mold-derived α-amylase is expensive because of its cost for production.

[0004] As α-amylase widely used industrially, bacteria belonging to the genus Bacillus (eg, Bacillus amylo) are used.
liquefaciens, Bacillus licheniformis, Bacillus sub
tilis). Bacill
Us bacteria secrete large amounts of enzymes such as α-amylase and protease outside of cells. Therefore, enzymes derived from Bacillus bacteria can be produced at low cost. Α from Bacillus bacteria
Amylase is used, for example, in starch liquefaction. Generally, liquefaction is performed at elevated temperatures. On the other hand, as described above, it is preferable that the enzyme for baking is deactivated during baking. Α-Amylase from Bacillus sp. Is too heat-resistant to be suitable for use in liquefaction but not for use in baking.

[0005]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide an inexpensive α-amylase having heat resistance suitable for use in baking.

[0006]

Means for Solving the Problems The present inventors have intensively studied from the above viewpoint. In the process, Bacillus amy
DN encoding α-amylase from loliquefaciens
A was randomly mutagenized and screened for DNA encoding α-amylase with reduced thermostability. From the various modified α-amylases encoded by the obtained DNA, those having significantly reduced heat resistance compared to the parent strain were selected. The selected enzymes were inactivated during bread baking, so they were effective in improving bread tenderness and preventing aging. The present inventors have isolated the gene encoding this enzyme (mutant α-amylase) and analyzed the nucleotide sequence and the amino acid sequence encoded thereby, thereby completing the present invention.

According to the present invention, the amino acid sequence of SEQ ID NO: 9 includes an amino acid sequence in which one or several amino acids have been deleted, substituted or inserted, has an α-amylase activity, and has There is provided a mutated α-amylase for baking, having a residual α-amylase activity of less than 80% after treatment compared to the activity before treatment.

[0008] Preferably, said deleted, substituted or inserted amino acid sequence is encoded by a nucleotide sequence obtained by treating a plasmid containing the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9 with hydroxylamine, In the treatment, the number of transformants obtained by transforming a host with the plasmid subjected to this treatment is about one-tenth of the number of transformants obtained by transforming the host with a plasmid not subjected to this treatment. This is a kind of processing.

In one embodiment, the mutant amylase for baking comprises an amino acid sequence in which one or two amino acids are substituted in the amino acid sequence of SEQ ID NO: 9.

In one embodiment, the mutated amylase for baking comprises an amino acid sequence in which two amino acids are substituted in the amino acid sequence of SEQ ID NO: 9, and the two substituted amino acids are about 50 amino acids. It exists between the following amino acids.

In one embodiment, the mutated amylase for baking is characterized in that in the amino acid sequence of SEQ ID NO: 9, the alanine residue at position 380 is replaced with a threonine residue.
The phenylalanine residue at position 93 is substituted with a serine residue.

[0012] In one embodiment, the mutated amylase for baking is the amino acid sequence of SEQ ID NO: 9, wherein the serine residue at position 30 is a leucine residue and the aspartic acid residue at position 195 is an asparagine residue. Has been replaced.

In one embodiment, the mutated amylase for bread making has the amino acid sequence of SEQ ID NO: 9 in which the arginine residue at position 154 is substituted with a lysine residue.

[0014] In one embodiment, the mutant amylase for baking is the amino acid sequence of SEQ ID NO: 9, in which the alanine residue at position 192 is replaced with a valine residue and 233.
The second aspartic acid residue has been replaced by asparagine.

[0015] In one embodiment, the above-mentioned mutant α-amylase for baking is characterized in that, in the amino acid sequence of SEQ ID NO: 9, the alanine residue at position 380 is replaced with a threonine residue;
The 393rd phenylalanine residue is substituted with a serine residue.

According to the present invention, the above-described mutated α-
A DNA encoding an amylase is provided.

In one embodiment, the DNA is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.
The nucleotide sequence of

According to the present invention, there is provided an expression vector containing the above DNA.

According to the present invention, there is provided a recombinant host cell containing the above expression vector.

According to the present invention, there is provided a method for producing a mutant α-amylase for baking, which comprises a step of culturing the above-mentioned recombinant host cell and a step of collecting a polypeptide having α-amylase activity from the culture. Is provided.

According to the present invention, there is provided a bread making method comprising the step of adding the above-mentioned α-amylase to bread dough.

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION In the present specification, abbreviations for amino acids, peptides, nucleotides, nucleic acids and the like are expressed as
UPAC, IUB regulations, "Guidelines for preparation of specifications including nucleotide or amino acid sequences"
(Japan Patent Office), and conventional symbols in the art.

Α-Amylase is an enzyme that acts on α-1,4 glucosidic bonds of starch to hydrolyze starch. The α-amylase activity is measured, for example, as follows. The starch solution and the buffer are added to the test tube and this is incubated at a predetermined temperature for a predetermined time. After the incubation, add the enzyme sample to the test tube,
Mix immediately. This mixed solution is incubated at a predetermined temperature for a predetermined time, and the reaction is stopped by adding Fehling B solution. The saccharifying power of each sample is determined by measuring the generated reducing sugars according to the modified Fehling-Lehman-Schole method (JIS K7001 Starch Liquefaction Test (199)).
0); the activity is indicated by JLU (JIS activity unit)). However, since the reaction according to the JIS method is carried out at a high temperature (65 ° C.), it may not be suitable for measuring the activity of α-amylase having reduced thermostability. In an alternative method, the starch gelatinizing power is determined by measuring the reduction of the iodine coloration of the starch associated with the low molecular weight of the linear part of the starch (amylose) when amylase acts on the starch. When the unit is tested under operating conditions,
10 minutes of iodine coloration of potato starch per minute
The amount of enzyme reduced by% is defined as 1 starch paste refining power. Alternatively, α-amylase activity can be conveniently detected on a plate. A predetermined amount of the sample is impregnated on a paper disk, and this is placed on a starch-containing medium. After incubating the plate at the appropriate temperature, it is sprayed with an iodine-potassium iodide solution and the halo formed is observed.

The mutant α-amylase of the present invention is sufficiently heat-resistant for improving the quality of bread making and is sensitive to heat to the extent that no activity remains after baking of the dough. In one embodiment, the variant α-
Amylase is α-derived from Bacillus amyloliquefaciens.
It has a thermostability between that of amylase and that of α-amylase derived from Aspergillus oryzae. Heat resistance is
For example, it can be determined by performing the incubation in the measurement of the α-amylase activity at an elevated temperature and generating a heat inactivation curve showing a decrease in activity over time. Usually, during the baking of the bread, the internal temperature of the bread does not rise above 90 ° C. Therefore, the mutant α-amylase of the present invention is preferably inactivated at a temperature lower than 90 ° C. (eg, 80 ° C.). The α-amylase from Bacillus amyloliquefaciens has about 100% residual activity compared to the untreated enzyme after 30 minutes incubation at 65 ° C. On the other hand, α-amylase from Aspergillus oryzae (which costs more to produce than α-amylase from Bacillus amyloliquefaciens) has about 10% residual activity compared to untreated enzyme under the same conditions. Having. The mutant α-amylase of the present invention is Bacillus
It has lower thermostability than wild type α-amylase derived from amyloliquefaciens. In one embodiment, the mutant α-amylase of the invention has a activity of less than 80%, preferably 60%, after treatment at 65 ° C. for 30 minutes compared to the activity before the treatment.
%, Most preferably less than 40%, residual α-
Has amylase activity.

The mutant α-amylase mutates DNA encoding α-amylase having relatively high thermostability, and isolates a mutated clone encoding α-amylase having reduced thermostability. By doing so, it can be obtained. As α-amylase having relatively high heat resistance, bacteria belonging to the genus Bacillus (for example, B. amyloliquefaciens,
Α-amylases from B. subtilis, and B. licheniformis) are preferred. Because α derived from Bacillus bacteria
-Amylase is secreted and produced in large amounts outside the cell,
It is easy to purify and can be produced at low cost. In one embodiment, the industrial strain B. amy
loliquefaciens F strain (Seki, T. et al., Int. J. Syst. Bact
eriol. 25: 258-270 (1975)).

The nucleotide sequence of the DNA encoding the α-amylase of B. amyloliquefaciens has been described previously (Takkinen, K. et al., J. Biol. Chem. 258 (2): 1007).
-1013 (1983); GenBank accession no. M12034). Thus, the DNA can be isolated using well-known techniques, such as library screening or the polymerase chain reaction (PCR), using probes or primers designed based on the known nucleotide sequence. Alternatively, the DNA can be isolated by detecting a halo formed around the colony of the clone containing the DNA of interest. Halos can be detected by forming colonies on plates containing starch and then spraying the plates with an iodine-potassium iodide solution.

Mutagenesis of α-amylase-encoding DNA can be performed, for example, in vivo by applying a mutagenic substance to cells containing the DNA (natural cells or recombinant cells), or to the DNA. It can be performed in vitro by random mutagenesis with the action of a mutagen, or by site-directed mutagenesis using an oligonucleotide having the desired sequence. In the in vivo method, the nucleotide sequence other than the DNA encoding α-amylase can be mutated, so that efficient isolation of the clone of interest is difficult. Therefore, the in vitro method is preferred. Also, many mutant α
-Random mutagenesis is preferred, as more and more variants can be obtained, since it is necessary to select the most appropriate one for the baking out of the amylase. Random mutagenesis in vitro can be achieved, for example, by treating the cloned DNA encoding α-amylase with hydroxylamine. Mutagenesis methods using hydroxylamine are described, for example, in Sikorsk.
i, RS and Boeke, JD (Methods Enzymol. 194: 302
-318 (1991)).

A DNA encoding an α-amylase having a reduced thermostability compared to the original α-amylase is screened, for example, as follows. The DNA mutated in vitro as described above is introduced into a suitable host cell. The α-amylase activity produced by the resulting transformant is firstly screened for those whose activity decreases at high temperatures, for example, using the above-described simple detection method on a starch-containing plate. Then, for the selected candidate clones, a secondary screening was performed by creating a heat inactivation curve as described above,
An α-amylase having the desired thermostability is selected.

As the host cell, any host cell used in any recombinant DNA technology can be used. When the DNA to be treated is derived from B. amyloliquefaciens, the same Bacillus bacterium, Bacillus subtilis, is used. Is preferred. Am, the α-amylase gene of B. subtilis
A mutant B. subtilis strain lacking yE is more preferred. amyE
As a defective strain, for example, B. subtilis KY110 (leuC7,
amyE, rm-). Transformation of B. subtilis is performed by a competent cell method or a protoplast method. In the competent cell method, the transformation efficiency is relatively low, but the recombination frequency is high.
-Suitable for producing amylase-deficient strains. on the other hand,
The protoplast method is suitable for screening a large number of clones because of its relatively high transformation efficiency. The protoplast method is described, for example, in Cohen et al. (Molec.
n. Genet. 168: 111-115 (1979)).

The expression vector is prepared based on a vector compatible with the host cell to be used. The vector is
It may be a plasmid vector, a phage vector, a virus vector, or the like. When B. subtilis is used as a host cell, for example, plasmids pUB110, pE194, p
C194 or a derivative thereof is used. Expression vectors may contain, in addition to the α-amylase coding sequence, regulatory sequences required for transcription and translation of this sequence. These regulatory sequences may be sequences that are linked in their natural environment to the α-amylase coding sequence,
Alternatively, the sequence may not be linked to the α-amylase coding sequence in a natural environment. For example, B. amylo
When expressing an α-amylase coding sequence derived from liquefaciens, a promoter, a ribosome binding site, a terminator and the like unique to this sequence can be used.

[0031] The selected mutant α-amylase is produced by culturing a transformant. The culturing is performed under conditions suitable for the growth of the host cell and / or the expression of the gene of interest. If the α-amylase accumulates in the cells, the α-amylase is purified from cell lysates. If α-amylase is secreted extracellularly, α-amylase is purified from the supernatant. Purification is
It is performed by various methods known in the art or a combination thereof. For example, for purification, a mutant α-amylase-producing transformant is cultured in a liquid medium, a supernatant is obtained by centrifuging the culture, the supernatant is concentrated by ultrafiltration, and the concentrate is subjected to alcohol precipitation. And vacuum drying the precipitate.

The utility of the selected mutant α-amylase in bread making is tested by assessing the quality of the bread made using this α-amylase.

The baking is performed, for example, as follows.
Mix the ingredients of the dough (for example, see Table 1). Purified mutant α-amylase is further added thereto. Fermentation and baking of bread dough can be conveniently performed by using a commercially available home bakery.

The hardness of the baked bread is evaluated, for example, as follows. After the bread is baked, it is left at room temperature and stored in a plastic bag for a certain time in order to prevent evaporation of water as much as possible. Then, the hardness of the bread is measured. The hardness measurement can be performed as follows. The bread is sliced, and the center part of the bread is cut into a predetermined size to produce a piece. This piece is compressed and the average value of the maximum stress (g) per unit area (cm ² ) is measured. Furthermore, bread hardness can be evaluated by a sensory test (texture). The aging of the bread is evaluated by sensory tests (texture and / or feel) after storage for a certain period.

Bread is produced by mixing various ingredients, fermenting the prepared dough, and then baking it. Materials for producing bread include flour, yeast, yeast food, and fats and oils (shortening, lard, margarine, butter, liquid oil, water-in-oil emulsion composition, oil-in-water emulsion composition, etc.) ), Water (ninis), dairy products, salt, sugars and the like. If necessary, hydrophilic emulsifiers, seasonings (glutamic acids, nucleic acids), preservatives, enhancers such as vitamins and calcium, proteins, chemical swelling agents, flavors and the like are added.

[0036] Filled breads, such as fillings, are also intended to be included in the breads according to the invention. That is, the bread according to the present invention includes bread, special bread, cooked bread, sweet bread, steamed bread and the like. For example, examples of the bread include white bread, black bread, French bread, varative red, and rolls (table rolls, buns, butter rolls, etc.). Special breads include grissini, muffins and rusks. Hot docks, hamburgers,
Pizza pie and the like. Sweet breads include jam bread, anpan bread, cream bread, raisin bread, melon bread, sweet rolls, and rich goods (croissants, brioches, danish pastries) and the like. Examples of the steamed bread include meat and anman.

Hereinafter, the present invention will be described in detail with reference to examples. However, the invention is not limited by these examples.

[0038]

Examples (Example 1: Examination of treatment conditions with hydroxylamine) Bacillus subtilis strain KY110 (l
euC7, amyE, rm-) was used. Bacillus subtilis KY
110 strains were isolated as follows. B.su, a Marburg strain
btilis MI114 (leuC7, trpC2, rm-) was replaced with B. subtilis
Transformation was performed with chromosomal DNA extracted from 118 strains (aroI, amyE). Among the non-tryptophan non-auxotrophs, a strain having no α-amylase activity was converted to a starch medium (0.6% sugarcane starch, 0.1 M phosphate buffer (pH 7.0), 0.1%).
05% NaN _3, 1% agar) on iodine - were selected as strains that do not form a halo when spraying potassium iodide solution. 0.5% of tryptophan non-auxotrophs were α-amylase deficient strains. One strain was named B. subtilis KY110.

B. subtilis protoplasts were transformed according to the method of Cohen et al. (Molec. Gen. Genet. 168: 111-115 (1979)). The host cells were pre-cultured overnight in 5 ml of BHI liquid medium. Add 200 ml of this culture to Pena
ssay broth (Antibiotical bro
th III (Difco), 17.5 g / ml), and cultured with shaking at 37 ° C. for about 2 hours. The cells pelleted by centrifugation (8,000 rpm, 10 minutes) are mixed with 20 ml of SMMP (0.5 M sucrose, 0.002 M maleic acid (pH 6.5), 0.0
2 M MgCl ₂ , antibiotic broth
III). 40 mg of lysozyme was added to this suspension, and the mixture was gently shaken at 37 ° C. for 1 to 2 hours. The obtained protoplasts were centrifuged (4,000 rpm,
10 minutes) and washed once with SMMP. 0.5 ml of the cells suspended in 5 ml of SMMP were placed in a small test tube, and DN was added thereto at a concentration of 1 μg / ml in SMM.
100 μl of A was added. Then add 1.5 m to this mixture
1 of 40% PEG (MW 6,000) was added, mixed gently and left on ice for 2 minutes. Then
Dilute the PEG by adding 5 ml of SMMP,
Then, the mixture was centrifuged (4,000 rpm, 10 minutes).
The pelleted cells were suspended in 1 ml of SMMP and gently shaken at 30 ° C. for about 2 hours. Then, the appropriately diluted cell suspension was added to DM3 regeneration medium (4% agar,
1M sodium succinate, 5% casamino acid, 10% yeast extract, 3.5% K ₂ HPO ₄ , 1.5% KH ₂ PO ₄ , 5
% Glucose, 2% BSA, kanamycin 100 μg /
and kanamycin resistant transformants were selected by culturing at 37 ° C. for 2 days.

The DNA encoding α-amylase was isolated as follows. B. amylo prepared by a conventional method
The chromosomal DNA of liquefaciens F strain was digested with restriction enzyme BglII and ligated to pUB110 digested with restriction enzyme BamHI. Bacillus subtilis strain KY110 (leuC7, amyE,
rm-) was transformed with this ligation mixture to obtain a kanamycin resistant strain. The α-amylase positive strain was selected as a halo-forming colony upon spraying iodine-potassium iodide on a starch medium containing kanamycin (5 μg / ml). Plasmid DNA was extracted from the selected strain and named pAMY110. The structure of pAMY110 is shown in FIG. The nucleotide sequence of the insert in pAMY110 was as previously described for B. amyloliquefacie.
ns nucleotide sequence of the α-amylase coding sequence (Tak
Kinen, K. et al., J. Biol. Chem. 258 (2): 1007-1013 (198
3)). The deduced amino acid sequence encoded by this nucleotide sequence is shown in SEQ ID NO: 9. This plasmid was used in the following experiments. Next, Siko
rski, RS and Boeke, JD (Methods Enzymol. 194:
302-318 (1991)). 10 μl of a solution of plasmid pAMY110 (1 μg / ml) and 1 M hydroxylamine solution (0.
1 M NaCl, 50 mM sodium phosphate (pH 7.
0), 2 mM EDTA, 1 M hydroxylamine hydrochloride)
500 μl and incubated at 70 ° C. for 0, 30, 60, or 120 minutes, and then the reaction was stopped by cooling on ice. Hydroxylamine was removed from the DNA by isopropanol precipitation. This plasmid DNA
Was used to transform Bucillus subtilis KY110 strain. The number of transformants decreased as the treatment time with hydroxylamine increased, and the number of transformants treated with 60 or 120 minutes was about 10 minutes less than the number of transformants without treatment. It became 1. Preliminary screening for changes in α-amylase thermostability (see Example 2) was performed and a treatment time of 60 minutes was selected as a treatment condition that resulted in efficient mutagenesis.

(Example 2: Primary screening for clones encoding α-amylase with reduced thermostability) Mutagenesis was performed according to the method described in Example 1. 100 colonies forming a halo on a starch medium were selected from the transformants into which pAMY110 had been treated with hydroxylamine for 60 minutes, and 5 ml of BHI was selected.
And incubated at 37 ° C. overnight. The culture solution was centrifuged, and the supernatant or the supernatant that had been heat-treated at 80 ° C. for 30 minutes was 5 μl a paper disc (ToyoRosh).
i company, 8 mm thickness), and starch medium (0.
6% sugarcane starch, 0.1 M phosphate buffer (pH 7.
0), 0.05% NaN ₃ , 1% agar). 3
After overnight incubation at 0 ° C., the iodine-potassium iodide solution was sprayed and the size of the halo formed was compared. FIG. 2 shows an example of the result. In FIG. 2, 1
Has pAMY110 containing the wild-type α-amylase gene
Shows a halo formed by B. subtilis KY110 strain,
2-9 show halos formed by transformants with plasmids treated with hydroxylamine. FIG.
As shown in the figure, the halo when heat-treated was significantly smaller than the control without heat-treatment, that is, α
-Several clones were found which seemed to have reduced thermostability of amylase. The primary screen using this starch medium could be reproducibly repeated.

Example 3 Secondary Screening by Heat Resistance Test No. 2 selected in the primary screening described in Example 2 21, no. 22, no. 24, and no. For α-amylase produced by 25 4 clones, 55 ° C, 60 ° C, 65 ° C, 70 ° C, 75 ° C
A heat deactivation curve at 80 ° C, 85 ° C, 85 ° C, or 90 ° C was created.

The amylase activity was measured as the starch gelatinizing power. One starch gelatinizing power unit is defined as the amount of enzyme that reduces iodine coloration of potato starch by 10% per minute when tested under the conditions of the following procedure. The measurement procedure is as follows. Substrate solution 10m
After heating at 37 ± 0.5 ° C. for 10 minutes, the sample solution 1
Mix with ml. This solution is incubated at 37 ± 0.5 ° C. for 10 minutes. 1 ml of this solution is mixed with 10 ml of a 0.1N hydrochloric acid solution. Add 0.5 ml of this solution to 0.0004
Mix with 10 ml of N iodine solution. 660nm of this solution
The absorbance at is measured with water symmetric (A _T ). Absorbance obtained by using 1 ml of water instead of the sample solution is expressed as A _B
And The starch refining power is calculated according to the following formula. Starch Norisei of force (unit _{/ g) = (A B -A} T) / A
_B x 1 / W. Here, the sample solution is adjusted to a concentration of 0.2 to 0.5 starch paste refining unit / ml. The substrate solution was prepared by adding 20 ml of water and a sodium hydroxide solution (2 →
25) Add 5 ml, warm, add 25 ml of water, add 2N
Neutralize with hydrochloric acid solution and prepare by adding 10 ml of 1 M acetic acid / sodium acetate buffer (pH 6.0).

The saccharifying power at each time point at each temperature was shown as a relative value with the saccharifying power at 0 minute as 100. An α-amylase (BAA) derived from the original B. amyloliquefaciens F strain and an α-amylase derived from Aspergillus oryzae (Sumiteam L, manufactured by Shin Nippon Chemical Co., Ltd.) were used as controls.

FIG. 3 shows the results. Of the four clones, No. Twenty-one enzymes had the lowest heat resistance. Although the original α-amylase (LA) derived from the B. amyloliquefaciens F strain was hardly inactivated by treatment at 65 ° C. for 30 minutes, No. The 21 mutant α-amylases had little residual activity under the same conditions.

(Example 4: Purification of mutant α-amylase) The four mutant α-amylase-producing strains selected in Example 3 were mixed with 500 ml of BHIS liquid medium in 500 ml.
It was inoculated into a Nosakaguchi flask and cultured with shaking at 37 ° C. overnight.
0.5 ml of the culture solution was inoculated into each of 30 culture media having the same composition and volume, and cultured at 37 ° C. for 72 hours with shaking. The obtained culture was centrifuged to obtain 1,500 ml of the supernatant. This supernatant was concentrated about 10 times by ultrafiltration. Next, a three-fold amount of alcohol was added to the concentrated solution to form a precipitate, and the precipitate was dried under vacuum to obtain a crude enzyme powder.

Example 5 Bread Making Using Mutant α-Amylase Bread dough ingredients for bread and French bread were mixed according to Table 1.

[0048]

[Table 1]

To this, the four crude enzyme powders prepared in Example 4 were further added. Controls include B. a
α-amylase from myloliquefaciens (BAA) or α-amylase from Aspergiluus oryzae (Sumizyme L) was added or no enzyme was added.
The amount of the enzyme added was 40 units of the enzyme other than Sumizyme L or 104 units of Sumiteam L in the case of bread, and 28 units of the enzyme other than Sumizyme L or 73 units of Sumiteam L in the case of French bread. The dough was baked using a National Automated Home Bakery (SD-BT6). The baked bread was evaluated as follows.

After baking, leave the bread at room temperature for 1.5 hours and place it in a plastic bag at 20 ° C to minimize evaporation of water.
For 24 or 72 hours, and then the bread hardness was measured. The hardness was measured as follows. Slice the bread to a thickness of 2 cm, and measure the center of this bread to 5 cm x
The pieces were cut into 5 cm pieces. This fragment was repeatedly compressed to 1 cm twice at a compression speed of 1 mm / sec using a rheometer (COMPAC-100) manufactured by Sun Chemical. Maximum stress (g) per unit area (cm ² )
Was measured. Four measurements were performed for one sample, and the average value was determined.

The softness and texture were evaluated as follows: It was best when 21 enzymes were used (data not shown). As shown in Table 2, The bread to which the enzyme No. 21 was added had significantly improved quality compared to the bread to which no enzyme was added. The addition of α-amylase from Aspergillus oryzae to French bread is described in It was not as effective as the addition of 21 enzymes. This is due to Aspergil
It can be considered that the heat resistance of lus oryzae-derived α-amylase is low, and that the mode of action on the starch in bread is not appropriate as compared with the α-amylase derived from B. amyloliquefaciens.

[0052]

[Table 2]

Example 6: Clone No. 21, No.
22, no. 24 and No. 25) The clone No. selected in Example 5 21, no. 22,
No. 24 and No. The nucleotide sequence of the insert of the 25 plasmids was determined. The sequencing reaction was performed by the dideoxy method (Proc. Natl. Acad. Sci. USA, 74: 5463).
(1997)). The reaction was carried out using an autoread sequencing kit (Pharmacia) using a fluorescently labeled M13 universal primer and T7 DNA polymerase. The reaction product is L. F. The nucleotide sequence was determined using a DNA sequencer (Pharmacia).

SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 21, no. 22, N
o. 24 and No. Each of the 25 nucleotide sequences is shown. SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7 have clone No. 21, no. 22,
No. 24 and No. Each of the 25 deduced amino acid sequences is shown.

Clone No. The nucleotide sequence of No. 21 was different from the nucleotide of the α-amylase coding region of the original B. amyloliquefaciens strain F in two places (A in nucleotide position 1138 and C in nucleotide position 1178 in SEQ ID NO: 2 were respectively different from each other). G and T in the strains with. As a result, it was found that the codon GCC for alanine at position 380 in the amino acid sequence of SEQ ID NO: 9 was changed to codon ACC for threonine, and the codon TTC for phenylalanine at position 393 was changed to TCC for serine.

Clone No. The nucleotide sequence of SEQ ID NO: 22 also differed from the nucleotide of the α-amylase coding region of the original B. amyloliquefaciensF strain in two places (the T at nucleotide position 89 and the A at nucleotide position 583 of SEQ ID NO: 4 were each different). C and G in the strains with. As a result, in the amino acid sequence of SEQ ID NO: 9, the codon TCA for serine at position 30 is the codon TTA for leucine, and the codon GAT for aspartic acid at position 195 is the AAT for asparagine.
It was found that each changed.

Clone No. The nucleotide sequence of 24 differed from the nucleotides of the α-amylase coding region of the original B. amyloliquefaciens F strain in three places (A at nucleotide position 461, T at nucleotide position 702 and T at nucleotide position 1086 of SEQ ID NO: 6). A of
G, C and G in the original strains, respectively). As a result, it was found that the codon AGA for arginine at position 154 of the amino acid sequence of SEQ ID NO: 9 was changed to codon AAA for lysine.

Clone No. The nucleotide sequence of 25 was different from the nucleotide of the α-amylase coding region of the original B. amyloliquefaciens strain F in two places (T at nucleotide position 575 and A at nucleotide position 697 in SEQ ID NO: 8 were each different). And C
And G). As a result, the codon GCG for alanine at position 192 and the codon GAC for aspartic acid at position 233 in the amino acid sequence of SEQ ID NO: 9 correspond to ATG for asparagine.
AC was found to be altered in each case. Therefore, it was suggested that these amino acid substitutions are responsible for the decrease in heat resistance. Clone No. The positions of amino acid substitutions in 21 mutant α-amylases are schematically shown in FIG.

No. The Bacillus subtilis KY110 (pAMY21) strain, which is a 21-mutant α-amylase-producing strain, was deposited with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology on July 23, 1998, and has a deposit number of FERM P-169.
No. 05.

The amino acid residue of α-amylase derived from B. amyloliquefaciens was changed to B. licheniformi at the corresponding position.
Studies have been reported to increase the heat resistance of α-amylase derived from B. amyloliquefaciens by substituting the amino acid residue of α-amylase derived from s (see JP-A-63-680).
84; Suzuki, Y. et al., J. Biol. Chem. 264 (32): 18933-189.
38 (1989)). Suzuki et al. Identified two regions located at the center of the amino acid sequence of α-amylase as being able to participate in thermostability, but identified at positions 30 and 154,
192, 195, 233, 380 and 393
Positions are not included in these regions. That is, the mutations found in the present application cannot be obtained from site-directed mutagenesis based on the description in this document. This means
It shows that the method for screening for a mutant α-amylase by random mutation of the present invention is useful for isolating α-amylase having reduced thermostability suitable for baking.

[0061]

According to the present invention, an α-amylase having heat resistance suitable for use in bread making is provided.

[0062]

[Sequence List] SEQUENCE LISTING <110> Daiwa Kasei KK <120> Novel amylase for making bread and gene therefor <130> J199120165 <140> <141> <150> 10-237839 <151> 1998-08-24 <160> 9 <170> PatentIn Ver. 2.0 <210> 1 <211> 514 <212> PRT <213> Bacillus amyloliquefaciens, clone No. 21 <400> 1 Met Ile Gln Lys Arg Lys Arg Thr Val Ser Phe Arg Leu Val Leu Met 1 5 10 15 Cys Thr Leu Leu Phe Val Ser Leu Pro Ile Thr Lys Thr Ser Ala Val 20 25 30 Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp Gly 35 40 45 Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp Ile 50 55 60 Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser Gln 65 70 75 80 Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe 85 90 95 Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu Leu 100 105 110 Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr Gly 115 120 125 Asp Val Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp Val 130 135 140 Thr Ala Val Glu Val Asn Pro Ala Asn Arg As n Gln Glu Thr Ser Glu 145 150 155 160 Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg Gly 165 170 175 Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Ala 180 185 190 Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg Gly 195 200 205 Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr 210 215 220 Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val 225 230 235 240 Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser Leu 245 250 255 Asp Gly Phe Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser Phe Leu 260 265 270 270 Arg Asp Trp Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu Met Phe 275 280 285 Thr Val Ala Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn Tyr 290 295 300 Leu Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu His 305 310 315 320 Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met Arg 325 330 335 Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala Val 340 345 350 Thr Thr Phe Val Glu Asn His Asp Thr Gln Pro G ly Gln Ser Leu Glu Ser 355 360 365 Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Thr Phe Ile Leu Thr 370 375 380 Arg Glu Ser Gly Tyr Pro Gln Val Ser Tyr Gly Asp Met Tyr Gly Thr 385 390 395 400 Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile Glu 405 410 415 Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His Asp 420 425 430 Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp Ser 435 440 445 Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly 450 455 460 Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr Trp 465 470 470 475 480 Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser Asp 485 490 495 Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr Val 500 505 510 Gln Lys 514 <210> 2 <211> 1545 <212> DNA <213> Bacillus amyloliquefaciens, clone No. 21 <220> <221> CDS <222> (1) .. (1545) <220> <221> sig_peptide <222> (1) .. (93) <220> <221> mat_peptide <222> (94) .. (1542) <400> 2 ATGATTCAAA AACGAAAGCG GACAGTTTCG TTCAGACTTG TGCTTATGTG CACGCTGTTA 60 TTTGTCAGTT TGCCGATTAC AAAAACATCA GCCGTAAATG GCACGCTGAT GCAGTATTTT 120 GAATGGTATA CGCCGAACGA CGGCCAGCAT TGGAAACGAT TGCAGAATGA TGCGGAACAT 180 TTATCGGATA TCGGAATCAC TGCCGTCTGG ATTCCTCCCG CATACAAAGG ATTGAGCCAA 240 TCCGATAACG GATACGGACC TTATGATTTG TATGATTTAG GAGAATTCCA GCAAAAAGGG 300 ACGGTCAGAA CGAAATACGG CACAAAATCA GAGCTTCAAG ATGCGATCGG CTCACTGCAT 360 TCCCGGAACG TCCAAGTATA CGGAGATGTG GTTTTGAATC ATAAGGCTGG TGCTGATGCA 420 ACAGAAGATG TAACTGCCGT CGAAGTCAAT CCGGCCAATA GAAATCAGGA AACTTCGGAG 480 GAATATCAAA TCAAAGCGTG GACGGATTTT CGTTTTCCGG GCCGTGGAAA CACGTACAGT 540 GATTTTAAAT GGCATTGGTA TCATTTCGAC GGAGCGGACT GGGATGAATC CCGGAAGATC 600 AGCCGCATCT TTAAGTTTCG TGGGGAAGGA AAAGCGTGGG ATTGGGAAGT ATCAAGTGAA 660 AACGGCAACT ATGACTATTT AATGTATGCT GATGTTGACT ACGACCACCC TGATGTCGTG 720 GCAGAGACAA AAAAATGGGG TATCTGGTAT GCGAATGAAC TGTCATTAGA CGGCTTCCGT 780 ATTGATGCCG CCAAACATAT TAAATTTTCA TTTCTGCGTG ATTGGGTTCA GGCGGTCAGA 840 CAGGCGACGG GAAAAGAAAT GTTTACGGTT GCGGAGTATT GGCAGAATAA TGCCGGGAAA 900 CTCGAA AACT ACTTGAATAA AACAAGCTTT AATCAATCCG TGTTTGATGT TCCGCTTCAT 960 TTCAATTTAC AGGCGGCTTC CTCACAAGGA GGCGGATATG ATATGAGGCG TTTGCTGGAC 1020 GGTACCGTTG TGTCCAGGCA TCCGGAAAAG GCGGTTACAT TTGTTGAAAA TCATGACACA 1080 CAGCCGGGAC AGTCATTGGA ATCGACAGTC CAAACTTGGT TTAAACCGCT TGCATACACC 1140 TTTATTTTGA CAAGAGAATC CGGTTATCCT CAGGTGTCCT ATGGGGATAT GTACGGGACA 1200 AAAGGGACAT CGCCAAAGGA AATTCCCTCA CTGAAAGATA ATATAGAGCC GATTTTAAAA 1260 GCGCGTAAGG AGTACGCATA CGGGCCCCAG CACGATTATA TTGACCACCC GGATGTGATC 1320 GGATGGACGA GGGAAGGTGA CAGCTCCGCC GCCAAATCAG GTTTGGCCGC TTTAATCACG 1380 GACGGACCCG GCGGATCAAA GCGGATGTAT GCCGGCCTGA AAAATGCCGG CGAGACATGG 1440 TATGACATAA CGGGCAACCG TTCAGATACT GTAAAAATCG GATCTGACGG CTGGGGAGAG 1500 TTTCATGTAA ACGATGGGTC CGTCTCCATT TATGTTCAGA AATAA 1545 <210> 3 <211> 514 <212> PRT <213> Bacillus amyloliquefaciens, clone No. 22 <400 > 3 Met Ile Gln Lys Arg Lys Arg Thr Val Ser Phe Arg Leu Val Leu Met 1 5 10 15 Cys Thr Leu Leu Phe Val Ser Leu Pro Ile Thr Lys Thr Leu Ala Val 20 25 30 Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp Gly 35 40 45 Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp Ile 50 55 60 Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser Gln 65 70 75 80 Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe 85 90 95 Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu Leu 100 105 110 Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr Gly 115 120 125 Asp Val Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp Val 130 135 140 Thr Ala Val Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser Glu 145 150 155 160 Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg Gly 165 170 175 Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Ala 180 185 190 Asp Trp Asn Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg Gly 195 200 205 Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr 210 215 220 Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val 225 230 235 240 Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser Leu 245 250 255 Asp Gly Phe Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser Phe Leu 260 265 270 270 Arg Asp Trp Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu Met Phe 275 280 285 Thr Val Ala Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn Tyr 290 295 300 Leu Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu His 305 310 315 320 Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met Arg 325 330 335 Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala Val 340 345 350 Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser 355 360 365 Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr 370 375 380 Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr 385 390 395 400 400 Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile Glu 405 410 415 Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His Asp 420 425 430 Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp Ser 435 440 445 445 Ser Ala Ala Lys Ser Gly LeuAla Ala Leu Ile Thr Asp Gly Pro Gly 450 455 460 Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr Trp 465 470 475 480 Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser Asp 485 490 495 Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr Val 500 505 510 Gln Lys 514 <210> 4 <211> 1545 <212> DNA <213> Bacillus amyloliquefaciens, No. 22 <220> <221> CDS <222> (1) .. (1545) <220> <221> sig_peptide <222> (1) .. (93) <220> <221> mat_peptide <222> (94) .. (1542) <400 > 4 ATGATTCAAA AACGAAAGCG GACAGTTTCG TTCAGACTTG TGCTTATGTG CACGCTGTTA 60 TTTGTCAGTT TGCCGATTAC AAAAACATTA GCCGTAAATG GCACGCTGAT GCAGTATTTT 120 GAATGGTATA CGCCGAACGA CGGCCAGCAT TGGAAACGAT TGCAGAATGA TGCGGAACAT 180 TTATCGGATA TCGGAATCAC TGCCGTCTGG ATTCCTCCCG CATACAAAGG ATTGAGCCAA 240 TCCGATAACG GATACGGACC TTATGATTTG TATGATTTAG GAGAATTCCA GCAAAAAGGG 300 ACGGTCAGAA CGAAATACGG CACAAAATCA GAGCTTCAAG ATGCGATCGG CTCACTGCAT 360 TCCCGGAACG TCCAAGTATA CGGAGATGTG GTTTTGAATC ATAAGGCTGG TGCTGATGCA 420 ACAGAAGATG TAACTGCCG T CGAAGTCAAT CCGGCCAATA GAAATCAGGA AACTTCGGAG 480 GAATATCAAA TCAAAGCGTG GACGGATTTT CGTTTTCCGG GCCGTGGAAA CACGTACAGT 540 GATTTTAAAT GGCATTGGTA TCATTTCGAC GGAGCGGACT GGAATGAATC CCGGAAGATC 600 AGCCGCATCT TTAAGTTTCG TGGGGAAGGA AAAGCGTGGG ATTGGGAAGT ATCAAGTGAA 660 AACGGCAACT ATGACTATTT AATGTATGCT GATGTTGACT ACGACCACCC TGATGTCGTG 720 GCAGAGACAA AAAAATGGGG TATCTGGTAT GCGAATGAAC TGTCATTAGA CGGCTTCCGT 780 ATTGATGCCG CCAAACATAT TAAATTTTCA TTTCTGCGTG ATTGGGTTCA GGCGGTCAGA 840 CAGGCGACGG GAAAAGAAAT GTTTACGGTT GCGGAGTATT GGCAGAATAA TGCCGGGAAA 900 CTCGAAAACT ACTTGAATAA AACAAGCTTT AATCAATCCG TGTTTGATGT TCCGCTTCAT 960 TTCAATTTAC AGGCGGCTTC CTCACAAGGA GGCGGATATG ATATGAGGCG TTTGCTGGAC 1020 GGTACCGTTG TGTCCAGGCA TCCGGAAAAG GCGGTTACAT TTGTTGAAAA TCATGACACA 1080 CAGCCGGGAC AGTCATTGGA ATCGACAGTC CAAACTTGGT TTAAACCGCT TGCATACGCC 1140 TTTATTTTGA CAAGAGAATC CGGTTATCCT CAGGTGTTCT ATGGGGATAT GTACGGGACA 1200 AAAGGGACAT CGCCAAAGGA AATTCCCTCA CTGAAAGATA ATATAGAGCC GATTTTAAAA 1260 GCGCGTAAGG AGTACGCATA CGGGCCCCAG CA CGATTATA TTGACCACCC GGATGTGATC 1320 GGATGGACGA GGGAAGGTGA CAGCTCCGCC GCCAAATCAG GTTTGGCCGC TTTAATCACG 1380 GACGGACCCG GCGGATCAAA GCGGATGTAT GCCGGCCTGA AAAATGCCGG CGAGACATGG 1440 TATGACATAA CGGGCAACCG TTCAGATACT GTAAAAATCG GATCTGACGG CTGGGGAGAG 1500 TTTCATGTAA ACGATGGGTC CGTCTCCATT TATGTTCAGA AATAA 1545 <210> 5 <211> 514 <212> PRT <213> Bacillus amyloliquefaciens, No 24 <400> 5 Met Ile Gln Lys Arg Lys Arg Thr Val Ser Phe Arg Leu Val Leu Met 1 5 10 15 Cys Thr Leu Leu Phe Val Ser Leu Pro Ile Thr Lys Thr Ser Ala Val 20 25 30 Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp Gly 35 40 45 Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp Ile 50 55 60 Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser Gln 65 70 75 80 Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe 85 90 95 Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu Leu 100 105 110 Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr Gly 115 120 125 Asp Val Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp Val 130 135 140 Thr Ala Val Glu Val Asn Pro Ala Asn Lys Asn Gln Glu Thr Ser Glu 145 150 155 160 Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg Gly 165 170 175 Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Ala 180 185 190 Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg Gly 195 200 205 Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr 210 215 220 Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val 225 230 235 240 Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser Leu 245 250 255 Asp Gly Phe Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser Phe Leu 260 265 270 Arg Asp Trp Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu Met Phe 275 280 285 Thr Val Ala Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn Tyr 290 295 300 Leu Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu His 305 310 315 320 Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tly Asp Met Arg 325 330 335 Arg Leu Leu Asp Gly ThrVal Val Ser Arg His Pro Glu Lys Ala Val 340 345 350 Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser 355 360 365 Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr 370 375 380 Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr 385 390 395 400 Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile Glu 405 410 415 Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His Asp 420 425 430 Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp Ser 435 440 445 Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly 450 455 460 Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr Trp 465 470 475 480 480 Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser Asp 485 490 495 Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr Val 500 505 510 Gln Lys 514 <210> 6 <211> 1545 <212> DNA <213> Bacillus amyloliquefaciens, No. 24 <220> <221> CDS <222> (1) .. (1545) <220> <221> sig_peptide <222> (1) .. (93) <220> <221> mat_pep tide <222> (94) .. (1542) <400> 6 ATGATTCAAA AACGAAAGCG GACAGTTTCG TTCAGACTTG TGCTTATGTG CACGCTGTTA 60 TTTGTCAGTT TGCCGATTAC AAAAACATCA GCCGTAAATG GCACGCTGAT GCAGTATTTT 120 GAATGGTATA CGCCGAACGA CGGCCAGCAT TGGAAACGAT TGCAGAATGA TGCGGAACAT 180 TTATCGGATA TCGGAATCAC TGCCGTCTGG ATTCCTCCCG CATACAAAGG ATTGAGCCAA 240 TCCGATAACG GATACGGACC TTATGATTTG TATGATTTAG GAGAATTCCA GCAAAAAGGG 300 ACGGTCAGAA CGAAATACGG CACAAAATCA GAGCTTCAAG ATGCGATCGG CTCACTGCAT 360 TCCCGGAACG TCCAAGTATA CGGAGATGTG GTTTTGAATC ATAAGGCTGG TGCTGATGCA 420 ACAGAAGATG TAACTGCCGT CGAAGTCAAT CCGGCCAATA AAAATCAGGA AACTTCGGAG 480 GAATATCAAA TCAAAGCGTG GACGGATTTT CGTTTTCCGG GCCGTGGAAA CACGTACAGT 540 GATTTTAAAT GGCATTGGTA TCATTTCGAC GGAGCGGACT GGGATGAATC CCGGAAGATC 600 AGCCGCATCT TTAAGTTTCG TGGGGAAGGA AAAGCGTGGG ATTGGGAAGT ATCAAGTGAA 660 AACGGCAACT ATGACTATTT AATGTATGCT GATGTTGACT ATGACCACCC TGATGTCGTG 720 GCAGAGACAA AAAAATGGGG TATCTGGTAT GCGAATGAAC TGTCATTAGA CGGCTTCCGT 780 ATTGATGCCG CCAAACATAT TAAATTTTCA TTTCTGCGTG ATTGGGTTCA GGCG GTCAGA 840 CAGGCGACGG GAAAAGAAAT GTTTACGGTT GCGGAGTATT GGCAGAATAA TGCCGGGAAA 900 CTCGAAAACT ACTTGAATAA AACAAGCTTT AATCAATCCG TGTTTGATGT TCCGCTTCAT 960 TTCAATTTAC AGGCGGCTTC CTCACAAGGA GGCGGATATG ATATGAGGCG TTTGCTGGAC 1020 GGTACCGTTG TGTCCAGGCA TCCGGAAAAG GCGGTTACAT TTGTTGAAAA TCATGACACA 1080 CAGCCAGGAC AGTCATTGGA ATCGACAGTC CAAACTTGGT TTAAACCGCT TGCATACGCC 1140 TTTATTTTGA CAAGAGAATC CGGTTATCCT CAGGTGTTCT ATGGGGATAT GTACGGGACA 1200 AAAGGGACAT CGCCAAAGGA AATTCCCTCA CTGAAAGATA ATATAGAGCC GATTTTAAAA 1260 GCGCGTAAGG AGTACGCATA CGGGCCCCAG CACGATTATA TTGACCACCC GGATGTGATC 1320 GGATGGACGA GGGAAGGTGA CAGCTCCGCC GCCAAATCAG GTTTGGCCGC TTTAATCACG 1380 GACGGACCCG GCGGATCAAA GCGGATGTAT GCCGGCCTGA AAAATGCCGG CGAGACATGG 1440 TATGACATAA CGGGCAACCG TTCAGATACT GTAAAAATCG GATCTGACGG CTGGGGAGAG 1500 TTTCATGTAA ACGATGGGTC CGTCTCCATT TATGTTCAGA AATAA 1545 <210> 7 <211> 514 <212> PRT <213> Bacillus amyloliquefaciens, No. 25 <400> 7 Met Ile Gln Lys Arg Lys Arg Thr Val Ser Phe Arg Leu Val Leu Met 1 5 10 15 Cys Thr Leu Leu Phe Val Ser Leu Pro Ile Thr Lys Thr Ser Ala Val 20 25 30 Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp Gly 35 40 45 Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp Ile 50 55 60 Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser Gln 65 70 75 80 Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe 85 90 95 Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu Leu 100 105 110 Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr Gly 115 120 125 Asp Val Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp Val 130 135 140 Thr Ala Val Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser Glu 145 150 155 160 Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg Gly 165 170 175 Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Val 180 185 190 Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg Gly 195 200 205 Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr 210 215 220 Asp Tyr Leu Met Tyr Ala Asp Val Asn Tyr Asp His Pro Asp Val Val 225 230 235 240 Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser Leu 245 250 255 Asp Gly Phe Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser Phe Leu 260 265 270 Arg Asp Trp Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu Met Phe 275 280 285 Thr Val Ala Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn Tyr 290 295 300 Leu Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu His 305 310 315 320 Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met Arg 325 330 335 Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala Val 340 345 350 Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser 355 360 365 Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr 370 375 380 Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr 385 390 395 400 Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile Glu 405 410 415 Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His Asp 420 425 430 Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp Ser 435 440 445 445 Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly 450 455 460 Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr Trp 465 470 475 480 Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser Asp 485 490 495 Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr Val 500 505 510 Gln Lys 514 <210> 8 <211> 1545 <212> DNA <213> Bacillus amyloliquefaciens, No. 25 <220> <221> CDS <222> (1) .. (1545) <220> <221> sig_peptide <222> (1) .. (93) <220> <221> mat_peptide <222> (94) .. (1542) <400> 8 ATGATTCAAA AACGAAAGCG GACAGTTTCG TTCAGACTTG TGCTTATGTG CACGCTGTTA 60 TTTGTCAGTT TGCCGATTAC AAAAACATCA GCCGTAAATG GCACGCTGAT GCAGTATTTT 120 GAATGGTATA CGCCGAACGA CGGCCAGCAT TGGAAACGAT TGCAGAATGA TGCGGAACAT 180 TTATCGGATA TCGGAATCAC TGCCGTCTGG ATTCCTCCCG CATACAAAGG ATTGAGCCAA 240 TCCGATAACG GATACGGACC TTATGATTTG TATGATTTAG GAGAATTCCA GCAAAAAGGG 300 ACGGTCAGAA CGAAATACGG CACAAAATCA GAGCTTCAAG ATGCGATCGG CTCACTGCAT 360 TCCCGGAAC G TCCAAGTATA CGGAGATGTG GTTTTGAATC ATAAGGCTGG TGCTGATGCA 420 ACAGAAGATG TAACTGCCGT CGAAGTCAAT CCGGCCAATA GAAATCAGGA AACTTCGGAG 480 GAATATCAAA TCAAAGCGTG GACGGATTTT CGTTTTCCGG GCCGTGGAAA CACGTACAGT 540 GATTTTAAAT GGCATTGGTA TCATTTCGAC GGAGTGGACT GGGATGAATC CCGGAAGATC 600 AGCCGCATCT TTAAGTTTCG TGGGGAAGGA AAAGCGTGGG ATTGGGAAGT ATCAAGTGAA 660 AACGGCAACT ATGACTATTT AATGTATGCT GATGTTAACT ACGACCACCC TGATGTCGTG 720 GCAGAGACAA AAAAATGGGG TATCTGGTAT GCGAATGAAC TGTCATTAGA CGGCTTCCGT 780 ATTGATGCCG CCAAACATAT TAAATTTTCA TTTCTGCGTG ATTGGGTTCA GGCGGTCAGA 840 CAGGCGACGG GAAAAGAAAT GTTTACGGTT GCGGAGTATT GGCAGAATAA TGCCGGGAAA 900 CTCGAAAACT ACTTGAATAA AACAAGCTTT AATCAATCCG TGTTTGATGT TCCGCTTCAT 960 TTCAATTTAC AGGCGGCTTC CTCACAAGGA GGCGGATATG ATATGAGGCG TTTGCTGGAC 1020 GGTACCGTTG TGTCCAGGCA TCCGGAAAAG GCGGTTACAT TTGTTGAAAA TCATGACACA 1080 CAGCCGGGAC AGTCATTGGA ATCGACAGTC CAAACTTGGT TTAAACCGCT TGCATACGCC 1140 TTTATTTTGA CAAGAGAATC CGGTTATCCT CAGGTGTTCT ATGGGGATAT GTACGGGACA 1200 AAAGGGACAT CGCCAAAGGA AAT TCCCTCA CTGAAAGATA ATATAGAGCC GATTTTAAAA 1260 GCGCGTAAGG AGTACGCATA CGGGCCCCAG CACGATTATA TTGACCACCC GGATGTGATC 1320 GGATGGACGA GGGAAGGTGA CAGCTCCGCC GCCAAATCAG GTTTGGCCGC TTTAATCACG 1380 GACGGACCCG GCGGATCAAA GCGGATGTAT GCCGGCCTGA AAAATGCCGG CGAGACATGG 1440 TATGACATAA CGGGCAACCG TTCAGATACT GTAAAAATCG GATCTGACGG CTGGGGAGAG 1500 TTTCATGTAA ACGATGGGTC CGTCTCCATT TATGTTCAGA AATAA 1545 <210> 9 <211> 514 <212> PRT <213> Bacillus amyloliquefaciens <400> 9 Met Ile Gln Lys Arg Lys Arg Thr Val Ser Phe Arg Leu Val Leu Met 1 5 10 15 Cys Thr Leu Leu Phe Val Ser Leu Pro Ile Thr Lys Thr Ser Ala Val 20 25 30 Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp Gly 35 40 45 Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp Ile 50 55 60 Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser Gln 65 70 75 80 Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe 85 90 95 Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu Leu 100 105 110 Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr Gly 115 120 125 Asp Val Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp Val 130 135 140 Thr Ala Val Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser Glu 145 150 155 160 Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg Gly 165 170 175 Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Ala 180 185 190 Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg Gly 195 200 205 Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr 210 215 220 Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val 225 230 235 240 Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser Leu 245 250 255 Asp Gly Phe Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser Phe Leu 260 265 270 Arg Asp Trp Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu Met Phe 275 280 285 Thr Val Ala Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn Tyr 290 295 300 Leu Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu His 305 310 315 320 Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met Arg 325 330 335 Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala Val 340 345 350 Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser 355 360 365 Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr 370 375 380 Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr 385 390 395 400 Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile Glu 405 410 415 Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His Asp 420 425 430 Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp Ser 435 440 445 Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly 450 455 460 Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr Trp 465 470 475 475 480 Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser Asp 485 490 495 Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr Val 500 505 510 Gln Lys 514

[Brief description of the drawings]

FIG. 1 shows the structure of plasmid pAMY110.

FIG. 2 shows the results of primary screening on a plate.

FIG. 3. Mutant α obtained in secondary screening
FIG. 2 is a view showing a heat inactivation curve of amylase.

FIG. 21, no. 22, no. 2
4 and No. FIG. 25 is a view showing the position of amino acid substitution in 25.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:07) (C12N 1/21 C12R 1: 125) (C12N 9/28 C12R 1: 125) (72 Inventor Satoko Takahashi 5 Wakatake-cho, Kosai-cho, Koga-gun, Shiga Prefecture (72) Inventor Yumiko Hidaki 253-4, Saiji-cho, Ishibe-cho, Koga-gun, Shiga Prefecture (72) Inventor Tetsu Hashimoto 5 Wakatake-cho, Kosai-cho, Koka-gun, Shiga Prefecture

Claims (14)

[Claims] 1. An amino acid sequence of SEQ ID NO: 9 comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or inserted, has an α-amylase activity, and is treated at 65 ° C. for 30 minutes before treatment. 8 compared to the activity of
A variant α-amylase for baking having a residual α-amylase activity of less than 0%. 2. The deletion, substitution or insertion of the amino acid sequence is encoded by a nucleotide sequence obtained by treating a plasmid containing the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9 with hydroxylamine. However, the number of transformants obtained by transforming a host with the plasmid subjected to the treatment is about 1/10 of the number of transformants obtained by transforming the host with a plasmid not subjected to the treatment. The mutated α-amylase for baking according to claim 1, which is subjected to such treatment. 3. The mutated α-amylase for baking according to claim 1, comprising an amino acid sequence in which one or two amino acids have been substituted in the amino acid sequence of SEQ ID NO: 9. 4. The method according to claim 1, comprising an amino acid sequence in which two amino acids have been substituted in the amino acid sequence of SEQ ID NO: 9, wherein the substituted two amino acids are present across about 50 amino acids or less. The mutated α-amylase for baking according to the above. 5. The amino acid sequence of SEQ ID NO: 9
The mutant α-amylase for baking according to claim 1, wherein the 80th alanine residue is substituted with a threonine residue, and the 393rd phenylalanine residue is substituted with a serine residue. 6. The amino acid sequence of SEQ ID NO: 9
The mutant α-amylase for baking according to claim 1, wherein the serine residue at position 0 is substituted with a leucine residue and the aspartic acid residue at position 195 is substituted with an asparagine residue. 7. The amino acid sequence of SEQ ID NO: 9
The mutant α-amylase for baking according to claim 1, wherein the 54th arginine residue is substituted with a lysine residue. 8. The amino acid sequence of SEQ ID NO: 9
The alanine residue at position 92 is substituted with a valine residue and the aspartic acid residue at position 233 is substituted with asparagine;
The mutated α-amylase for baking according to claim 1. 9. A DNA encoding the mutated α-amylase for baking according to any one of claims 1 to 8. 10. The DNA of claim 9, comprising the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. An expression vector comprising the DNA according to claim 9 or 10. A recombinant host cell comprising the expression vector according to claim 11. 13. A method for producing a mutated α-amylase for baking, comprising a step of culturing the recombinant host cell according to claim 12 and a step of collecting a polypeptide having α-amylase activity from the culture. . 14. The α according to claim 1, wherein
-A baking method, comprising the step of adding amylase to dough.