Highly productive alpha-amylases

Abstract

The invention relates to mutant α-amylases that may be produced at high yield from recombinant microorganisms.

Description

TECHNICAL FIELD

The present invention relates to mutant α-amylases having improved productivity.

BACKGROUND ART

α-Amylases [EC.3.2.1.1.] have been used in a wide range of industrial fields such as starch industry, brewing industry, fiber industry, pharmaceutical industry and food industry. Among them, those capable of degrading starches at high random are suited for detergents. Conventionally known as such are, as well as α-amylases derived from Bacillus licheniformis , liquefying alkaline α-amylases derived from the alkaliphilic strain Bacillus sp. KSM-AP1378 (FERM BP-3048) (WO94/26881) and improved enzymes having improved heat resistance and oxidant resistance (WO98/44126).

The present inventors have recently found liquefying alkaline α-amylases derived from the alkaliphilic strain Bacillus sp. KSM-K38 (FERM BP-6946) and having chelating-agent- and oxidation-resistance (Japanese Patent Application No. Hei 10-362487, Japanese Patent Application No. Hei 10-362488); and improved enzymes having improved heat resistance (Japanese Patent Application No. Hei 11-163569).

In addition to such properties, enzymes for detergents are required to have high productivity in consideration of their industrial production. Although various trials have been made to improve the heat resistance or oxidant resistance of α-amylases for detergent by using protein engineering technique, neither improvement of productivity has been considered sufficiently nor an attempt of production increase by mutation of a structural gene has been reported yet.

An object of the present invention is to provide mutant α-amylases having excellent productivity.

DISCLOSURE OF THE INVENTION

The present inventors introduced, in microorganisms, mutant α-amylase structural gene constructed by site-directed mutagenesis and evaluated productivity of α-amylases. As a result, it has been found that since an α-amylase gene has a site taking part in the improvement of productivity, introduction, into a microorganism, of a recombinant gene having this site mutated makes it possible to produce α-amylases having drastically improved productivity.

In one aspect of the present invention, there is thus provided a mutant α-amylase which is derived from an α-amylase having an amino acid sequence represented by SEQ ED No. 2 or showing at least 60% homology thereto by substitution or deletion of at least one amino acid residue corresponding to any one of Pro 18 , Gln 86 , Glu 130 , Asn 154 , Arg 171 , Ala 186 , Glu 212 , Val 222 , Tyr 243 ) Pro 260 , Lys 269 , Glu 276 , Asn 277 , Arg 310 , Glu 360 , Gln 391 , Trp 439 , Lys 444 , Asn 471 and Gly 476 of the amino acid sequence.

In another aspect of the present invention, there is also provided a mutant α-amylase derived from an α-amylase having an amino acid sequence represented by SEQ ID No. 4 or showing at least 60% homology thereto by substitution or deletion of at least one amino acid residue corresponding to any one of Asp 128 , Gly 140 , Ser 144 , Arg 168 , Asn 181 , Glu 207 , Phe 272 , Ser 375 , Trp 434 and Glu 466 of the amino acid sequence.

In a further aspect of the present invention, there is also provided a gene encoding this mutant α-amylase, a vector containing the gene, a cell transformed with the vector and a production method of a mutant α-amylase which comprises cultivating the transformed cell.

In a still further aspect of the present invention, there is also provided a detergent composition containing this mutant α-amylase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a method of constructing a recombinant plasmid for production of an α-amylase derived from the strain KSM-1378 or KSM-K38.

FIG. 2 is a schematic view illustrating a method of introducing a mutation into an α-amylase gene derived from the strain KSM-1378 or KSM-K38.

BEST MODE FOR CARRYING OUT THE INVENTION

The term “highly productive mutant α-amylase” as used herein means an α-amylase whose yield is increased, upon production of it by cultivating a recombinant microorganism, by at least 5%, preferably at least 10%, more preferably at least 20% compared with that before mutation.

The mutant α-amylase of the present invention is constructed so that out of amino acids constituting the α-amylase, the amino acid residues taking part in the productivity are substituted with another amino acid residues or deleted. Examples of the α-amylase usable here include liquefying α-amylases derived from Bacillus. amyloliquefaciens or Bacillus. licheniformis and liquefying alkaline α-amylases derived from alkaliphilic microorganisms belonging to the Bacillus sp., of which α-amylases having an amino acid sequence represented by SEQ ID No. 2 or SEQ ID No. 4 and α-amylases having at least 60% homology to the above-described amino acid sequence are preferred.

Examples of the α-amylase having the amino acid sequence represented by SEQ ID NO. 2, or α-amylase having at least 60% homology thereto include liquefying alkaline α-amylases derived from the strain Bacillus sp. KSM-AF1378 (FERM BP-3048) (Japanese Patent Application. Laid-Open No. Hei 8-336392) and improved enzymes of the above-described one in heat resistance and oxidant resistance which are constructed by protein engineering technique (WO98/44126).

Examples of the α-amylase having the amino acid sequence represented by SEQ ID No. 4 or having at least 60% homology thereto include liquefying alkaline α-amylases derived from the strain Bacillus sp. KSM-K38 (FERM BP-6946) and improved enzymes of the above-described one in heat resistance which are constructed by protein engineering technique (Japanese Patent Application No. Hei 11-163569).

The homology of an amino acid sequence is calculated by Lipman-Pearson method (Science, 227, 1435(1985)).

The mutant α-amylase of the present invention can be obtained first by cloning, from a microorganism producing an α-amylase, a gene encoding the α-amylase. For this purpose, ordinarily employed gene recombinant technique, for example, the method as described in Japanese Patent Application Laid-Open No. Hei 8-336392 may be employed. Examples of the gene usable here include that represented by from the above-described ones and having improved heat resistance and oxidant resistance are also usable.

For mutation of the gene thus obtained by cloning, any site-directed mutagenesis ordinarily employed can be adopted. For example, mutation can be conducted using a “Site-Directed Mutagenesis System Mutan-Super Express Km” kit (product of Takara Shuzo Co., Ltd.).

Mutation for obtaining highly productive α-amylases of the invention can be attained, for example, by substitution or deletion, in an α-amylase having an amino acid sequence represented by SEQ ID No. 2 or having at least 60% homology thereto, of at least one amino acid residue corresponding to any one of Pro 18 , Gln 86 , Glu 130 , Asn 154 , Arg 171 , Ala 186 , Glu 212 , Val 222 , Tyr 243 , Pro 260 , Lys 269 , Glu 276 , Asn 277 , Arg 310 , Glu 360 , Gln 391 , Trp 439 , Lys 444 , Asn 471 and Gly 476 of the amino acid sequence; or by substitution or deletion, in another α-amylase having an amino acid sequence represented by SEQ ID No.4 or having at least 60% homology thereto, of at least one amino acid residue corresponding to any one of Asp 128 , Gly 140 , Ser 144 , Arg 168 , Asn 181 , Glu 207 , Phe 272 , Ser 375 , Trp 434 and Glu 466 of the amino acid sequence. Preferred mutations include, in the amino acid sequence of SEQ ID No. 2 substitution of the amino acid residue corresponding to Pro 18 with Ser or Thr, the amino acid residue corresponding to Gln 86 with Glu, the amino acid residue corresponding to Glu 130 with Val or Gln, the amino acid residue corresponding to Asn 154 with Asp, the amino acid residue corresponding to Arg 171 with Cys or Gln, the amino acid residue corresponding to Ala 186 with Val or Asn, the amino acid residue corresponding to Glu 212 with Asp, the amino acid residue corresponding to Val 222 with Gln, the amino acid residue corresponding to Tyr 243 with Cys or Ser, the amino acid residue corresponding to Pro 260 with Glu, the amino acid residue corresponding to Lys 269 , with Gln, the amino acid residue corresponding to Glu 276 with His, the amino acid residue corresponding to Asn 277 with Ser or Phe, the amino acid residue corresponding to Arg 310 with Ala, the amino acid residue corresponding to Glu 360 with Gln, the amino acid residue corresponding to Gln 391 with Glu, the amino acid residue corresponding to Trp 439 with Arg, the amino acid residue corresponding to Lys 444 with Arg, the amino acid residue corresponding to Asn 471 , with Asp or Glu, or the amino acid residue corresponding to Gly 476 with Asp;

or substitution, in the amino acid sequence of SEQ ID No. 4, of the amino acid residue corresponding to Asp 128 with Val or Gln, the amino acid residue corresponding to Gly 140 with Ser, the amino acid residue corresponding to Ser 144 with Pro, the amino acid residue corresponding to Arg 168 with Gln, the amino acid residue corresponding to Gln 181 , with Val, the amino acid residue corresponding to Glu 270 with Asp, the amino acid residue corresponding to Phe 272 with Ser, the amino acid residue corresponding to Ser 375 with Pro, the amino acid residue corresponding to Trp 434 with Arg or the amino acid residue corresponding to Glu 466 with Asp.

Among the mutations of the amino acid sequence of SEQ ID No. 2, those by substitution of the amino acid residue corresponding to Gln 86 with Glu, the amino acid residue corresponding to Glu 130 with Val or Gln, the amino acid residue corresponding to Ala 186 with Asn, the amino acid residue corresponding to Tyr 243 with Ser, the amino acid residue corresponding to Pro 260 with Glu, the amino acid residue corresponding to Lys 269 with Gln, the amino acid residue corresponding to Asn 277 with Phe and the amino acid residue corresponding to Asn 471 with Asp or Gln can bring about improvement in solubility of the α-amylase in a culture medium or desalted and concentrated solution thereof. More specifically, the above-described mutations make it possible to improve the residual activity of the αamylase in the supernatant after storage at 4° C. for one week in a desalted and concentrated solution by at least 5%, especially 10% compared with the activity before mutation. Accordingly, in the case of the mutant α-amylases of the present invention obtained by such amino acid mutations, a fermented concentrate solution of a high concentration is available at a high yield and enzyme formulation treatment such as ultrafiltration after fermentation production can be conducted efficiently.

A combination of two or more substitutions or deletions of various amino acid residues is also effective for such amino acid mutations. It is also possible to use the above-exemplified mutation in combination with a mutation for improving enzymatic properties, for example, in an α-amylase having an amino acid sequence represented SEQ ID No. 2 or having at least 60% homology thereto, a mutation for improving heat resistance by deleting amino acid residues corresponding to Arg 181 and Gly 182 , a mutation for improving oxidant resistance by substituting the amino acid residue corresponding to Met 222 with Thr or a mutation for improving solubility by substituting the amino acid residue corresponding Lys 484 with Gln; or in an α-amylase having an amino acid sequence represented by SEQ ID No. 4 or having at least 60% homology thereto, a mutation for further reinforcing oxidant resistance by substituting the amino acid residue corresponding to Met 107 with Leu or a mutation for heightening detergency of a laundry detergent by substituting the amino acid residue corresponding Glu 188 with Ile.

A mutant α-amylase is available at a high yield by appropriately combining a mutant α-amylase structural gene with a control gene and a proper plasmid vector, thereby constructing a plasmid for the production of the α-amylase, introducing the resulting plasmid into a microorganism such as Bacillus subtilis or Escherichia coli , preferably, Bacillus subtilis and cultivating the resulting recombinant microorganism.

The mutant α-amylase thus obtained has improved productivity by about 10 to 500% as shown later in Examples while maintaining biochemical properties as an enzyme, thus having excellent properties. By the above-described mutation of the amino acid residues of liquefying alkaline α-amylases having heat resistance, chelating agent resistance, oxidant resistance and high solubility, it is therefore possible to create useful enzymes having drastically improved productivity in a recombinant microorganism without losing the above-described original properties.

The detergent compositions of the present invention may contain, in addition to the α-amylase of the invention, one or more than one enzymes selected from debranching enzymes (such as pullulanase, isoamylase and neopullulanase), α-glucosidase, glucoamylase, protease, cellulase, lypase, pectinase, protopectinase, pectate lyase, peroxidase, laccase and catalase.

The detergent composition may contain, in addition, components ordinarily added to a detergent, for example, surfactants such as anionic surfactants, amphoteric surfactants, nonionic surfactants and cationic surfactants, chelating agents, alkali agents, inorganic salts, anti-redeposition agents, chlorine scavengers, reducing agents, bleaching agents, fluorescent dye solubilizing agents, perfumes, anti-caking agents, enzyme activating agents, antioxidants, antiseptics, blueing agents, bleach activating agents, enzyme stabilizing agents and regulator.

The detergent compositions of the invention can be produced in a manner known per se in the art from a combination of the highly productive α-amylase available by the above-described method and the above-described known detergent components. The form of the detergent can be selected according to the using purpose and examples include liquid, powder and granule. The detergent compositions of the present invention are suited as laundry detergents, bleaching detergents, detergents for automatic dish washer, pipe cleaners, and artificial tooth cleaners, of which they are especially suited as laundry detergents, bleaching detergents and detergents for automatic dish washer.

The highly productive mutant α-amylases of the invention are also usable as starch liquefying saccharifying compositions. Moreover, these mutant α-amylases, after addition thereto of one or more than one enzymes selected from glucoamylase, maltase, pullulanase, isoamylase and neopullulanase, can be allowed to act on starches.

Furthermore, the mutant α-amylases of the present invention are usable as a desizing composition of fibers and an enzyme such as pullulanase, isoamylase or neopullulanase can be incorporated in the composition.

EXAMPLES

Measurement of Amylase Activity and Protein Content

Amylase activity and protein content of the enzymes each produced from recombinant Bacillus subtilis were measured in accordance with the below-described methods.

Amylase activity was measured by the 3,5-dinitrosalicylic acid method (DNS method). After reaction at 50° C. for 15 minutes in a reaction mixture of a 40 mM glycine-sodium hydroxide buffer (pH 10) containing soluble starch, the reducing sugar thus formed was quantitatively analyzed by the DNS method. As the titer of the enzyme, the amount of the enzyme which formed reducing sugar equivalent to 1 μmol of glucose in one minute was defined as one unit.

The protein content was determined by “Protein Assay Kit” (product of Bio-Rad Laboratories) using bovine serum albumin as standard.

Referential Example 1

Screening of Liquefying Alkaline Amylase

About 0.5 g of soil was suspended in sterilized water and the resulting suspension was heat treated at 80° C. for 15 minutes. The supernatant of the heat treated mixture was diluted with an adequate amount of sterilized water, followed by applying to an isolating agar medium (Medium A). The medium was then cultured at 30° C. for 2 days to grow colonies. The colonies which formed transparent zones in their peripheries due to starch dissolution were selected and isolated as amylase producing strains. The resulting isolated strains were inoculated in Medium B, followed by aerobic shaken culture at 30° C. for 2 days. After cultivation, the chelating agent (EDTA) resisting capacity of the supernatant obtained by centrifugation was measured and in addition, the optimum working pH was measured. Thus, strain Bacillus sp. KSM-K38 (FERM BP-6946) was obtained.

	Medium A:	Tryptone	1.5%
		Soytone	0.5%
		Sodium chloride	0.5%
		Colored starch	0.5%
		Agar	1.5%
		Na 2 Co 3	0.5%
		(pH 10)
	Medium B:	Tryptone	1.5%
		Soytone	0.5%
		Sodium chloride	0.5%
		Soluble starch	1.0%
		Na 2 CO 3	0.5%
		(pH 10)

The mycological properties of strain KSM-K38 are shown in Table 1.

                                 TABLE 1
                                 Strain KSM-K38
(a) Observation under microscope Cells are rods of a size of 1.0 to 1.2 μm × 2.4 to 5.4 μm in the
                                 strain K36 and 1.0 to 1.2 μm × 1.8 to 3.8 μm in the strain
                                 K38, and form an elliptical endospore (1.0 to 1.2 μm × 1.2 to
                                 1.4 μm) at their subterminals or center. They have flagella
                                 and are motile. Gram's staining is positive. Acid fastness:
                                 negative.
(b) Growth in various culture mediums.
The strains are alikaliphilic so that 0.5%
sodium carbonate was added to the
culture medium in the tests described
hereinafter.
Nutrient agar plate culture      Growth of cells is good. Colony has a circular shape, with its
                                 surface being smooth and its peripheral end being smooth.
                                 The color of the colony is yellowish brown.
Nutrient agar slant culture      Cells can grow.
Nutrient broth                   Cells can grow.
Stab culture in nutrient-broth gelatin Growth of cells is good. Liquefaction of gelatin is not
                                 observed.
Litmus milk medium               No change in growth.
(c) Physiological properties
Nitrate reduction and denitrification Nitrate reduction: positive Denitrification: negative
MR test                          Indeterminable because the medium is an alkaline medium.
V-P test                         Negative
Production of indole             Negative
Production of hydrogen sulfide   Negative
Hydrolysis of starch             Positive
Utilization of citric acid       Positive in Christensen's medium but negative in Koser's
                                 medium and Simmon's medium.
Utilization of inorganic nitrogen sources Nitrate is utilized but ammonium salts are not.
Production of colorants          Negative
Urease                           Negative
Oxidase                          Negative
Catalase                         Positive
Growth range                     Growth temperature range: 15 to 40° C., optimum growth
                                 temperature: 30° C., growth pH range: pH 9.0 to 11.0,
                                 optimum growth pH range: same
Behavior on oxygen               Aerobic
O-F test                         Cells do not grow
Sugar utilization                L-galactose, D-xylose, L-arabinose, lactose, glycerin,
                                 melibiose, ribose, D-glucose, D-mannose, maltose, sucrose,
                                 trehalose, D-mannitol, starch, raffinose and D-fructose are
                                 utilized.
Growth in a salt-containing medium Cells can grow when salt concentration is 12%, but not when
                                 salt concentration is 15%.

Referential Example 2

Cultivation of Strain KSM-K38

In the liquid medium B of Referential Example 1, the strain KSM-K38 was inoculated, followed by aerobic shaken culture at 30° C. for 2 days. The amylase activity (at pH 8.5) of each of the supernatants isolated by centrifugation was measured. As a result, the activity in 1 L of the culture medium was found to be 1177 U.

Referential Example 3

Purification of Liquefying Alkaline Amylase

Ammonium sulfate was added to the supernatant of the culture medium of the strain KSM-K38 obtained in Referential Example 2 to give 80% saturation, followed by stirring. The precipitate thus formed was collected and dissolved in a 10 mM tris-HCl buffer (pH 7.5) containing 2 mM CaCl 2 to dialyze the resulting solution against the buffer overnight. The dialysate was loaded on a DEAE-Toyopearl 650M column equilibrated with the same buffer and protein was eluted in a linear gradient of 0 to 1 M of NaCl in the same buffer. The active fraction obtained by gel filtration column chromatography after dialysis against the same buffer was dialyzed against the buffer, whereby purified enzyme exhibited a single band on polyacrylamide gel electrophoresis (gel concentration: 10%) and sodium dodecylsulfate (SDS) electrophoresis was obtained.

Referential Example 4

Enzymological Properties

The properties of the purified enzyme are as follows:

(1) Action

It acts on starch, amylose, amylopectin and α-1,4-glycoside bond which is a partially degraded product thereof to degrade them and produce, from amylose, glucose (G1), maltose (G2), maltotriose (G3), maltotetraose (G4), maltopentaose (G5), maltohexaose (G6) and maltoheptaose (G7). But it does not act on pullulan.

(2) pH Stability (Britton-Robinson Buffer)

It exhibits residual activity of 70% or more within a range of pH 6.5 to 11.0 under treating conditions at 40° C. for 30 minutes.

(3) Working Temperature Range and Optimum Working Temperature

It acts in a wide temperature range of from 20 to 80° C., with the optimum working temperature being 50 to 60° C.

(4) Temperature Stability

The temperature at which the enzyme loses its activity was examined by causing a temperature change in a 50 mM glycine-sodium hydroxide buffer (pH 10) and then, treating at each temperature for 30 minutes. The residual activity of the enzyme is 80% or more at 40° C. and about 60% even at 45° C.

(5) Molecular Weight

The molecular weight as measured by sodium-dodecylsulfate polyacrylamide gel electrophoresis is 55,000 ±5,000.

(6) Isoelectric Point

Its isoelectric point as measured by isoelectric focusing electrophoresis is about 4.2.

(7) Effects of Surfactants

It is almost free from activity inhibition (activity remaining ratio: 90% or more) even when treated at pH 10 and 30° C. for 30 minutes in a 0.1% solution of a surfactant such as sodium linear alkylbenzene sulfonate, alkyl sulfate ester sodium salt, polyoxyethylene alkylsulfate ester sodium salt, sodium α-olefin sulfonate, sodium α-sulfonated fatty acid ester, sodium alkylsulfonate, SDS, soap and softanol.

(8) Effects of Metal Salts

It was treated at pH 10 and 30° C. for 30 minutes in each of the reaction systems containing various metal salts and their effects were studied. Its activity is inhibited by 1 mM of Mn 2+ (inhibition ratio: about 75%) and slightly inhibited by 1 mM of Sr 2+ and Cd 2+ (inhibition ratio: about 30%).

Example 1

Preparation of Various Recombinant Plasmids Having an α-Amylase Gene Ligated Thereto

In accordance with the method as described in WO98/44126, genes encoding a mutant α-amylase (which will hereinafter be described as “ΔRG”) having improved heat resistance and a mutant α-amylase (“ΔRG-M202T”) having improved oxidant resistance as well as improved heat resistance were constructed, respectively, by deleting Arg 181 and Gly 182 of the α-amylase (“LAMY”) which was derived from the strain Bacillus sp. KSM-AP1378 (FERM BP-3048) and had the amino acid sequence represented by SEQ ID No. 2; and by, in addition to this mutation by deletion, substituting Thr for Met 202 of the amino acid sequence represented by SEQ ID No. 2. With the genes as a template, gene fragments (about 1.5 kb) encoding these mutant α-amylases were amplified by the PCR reaction using primers LAUS (SEQ ID No. 5) and LADH (SEQ ID No. 6). After cutting of them with a restriction enzyme SalI, each of the fragments was inserted into the SalI-SmaI site of an expression vector pHSP64 (Japanese Patent Application Laid-Open No. Hei 6-217781), whereby a recombinant plasmid having a structural gene of each of the mutant α-amylases bonded thereto was constructed downstream of a strong promoter derived from an alkaline cellulase gene of the strain Bacillus sp. KSM-64 (FERM P-10482).

In the meantime, with a chromosomal DNA, which had been extracted from the cells of the strain Bacillus sp. KSM-K38 (FERM BP-6946) by the method of Saito and Miura (Biochim. Biophys. Acta, 72, 619 (1961)), as a template, PCR reaction was effected using primers K38US (SEQ ID No. 7) and K38DH (SEQ ID No. 8) shown in Table 2, whereby a structural gene fragment (about 1.5 kb) encoding an α-amylase (which will hereinafter be described as “K38AMY”) having an amino acid sequence of SEQ ID No.4 was amplified. After cutting of it with a restriction enzyme SalI, the resulting fragment was inserted into the SalI-SmaI site of an expression vector pHSP64 to construct, downstream of a strong promoter derived from an alkaline cellulase gene of the strain Bacillus sp. KSM-64 (FERM P-10482) contained in pHSP64, a recombinant plasmid having a structural gene of K38AMY bonded thereto (FIG. 1 ). With this recombinant plasmid as a template, PCR reaction was effected using the primers CLUBG (SEQ ID. No. 9) and K38DH (SEQ. ID. No.8) to amplify a gene fragment (about 2.1 kb) having the strong promoter and K38AMY bonded thereto.

By the recombinant PCR method as described below, a gene encoding chimeric α-amylase between K38AMY and LAMY was constructed. Described specifically, with a chromosomal DNA of the strain K.SM-K38 (FERM BP6946) as a template, PCR reaction was conducted using primers K38DH (SEQ ID No. 8) and LA-K38 (SEQ ID No. 10) shown in Table 2, whereby a fragment encoding the sequence from Gln 20 downstream to the C-terminal of the amino acid sequence of K38AMY represented by SEQ ID No. 4 was amplified. With the above-described recombinant plasmid containing the LAMY gene and strong promoter as a template, PCR reaction was conducted using primers CLUBG (SEQ ID No. 9) and LA-K38R (SEQ ID No. 11) shown in Table 2, whereby a gene fragment encoding from the upstream strong promoter to Gly 21 of the amino acid sequence of LAMY of SEQ ID No. 2 was amplified. By the second PCR reaction using the resulting two DNA fragments and primers CLUBG (SEQ ID No. 9) and K38DH (SEQ ID No. 8) shown in Table 2, the resulting two fragments having, at the end thereof, complementary sequences derived from primers LA-K38 (SEQ ID No. 10) and LA-K38R (SEQ ID No. 11) respectively were combined, whereby a gene fragment (about 2.1 kb) encoding a chimeric α-amylase (which will hereinafter be described as “LA-K38AMY”) which has, successively bonded thereto, a region encoding from His 1 to Gly 21 of the LAMY downstream of the strong promoter and a region encoding from Gln 20 to the C-terminal of the K38AMY was amplified.

By using a “Site-Directed Mutagenesis System MutanSuper Express Km” kit (product of Takara Shuzo Co., Ltd.), the below-described mutations were introduced to the K38AMY and LA-K38AMY. First, the K38AMY and LA-K38AMY gene fragments (about 2.1 kb) were inserted into the site SmaI of a plasmid vector pKF19k attached to the kit to construct a mutagenic recombinant plasmid (FIG. 2 ). A site-directed mutagenic oligonucleotide primer N190F (SEQ ID No. 50) shown in Table 2 was 5′-phosphorylated with T4 DNA kinase. Using this and the above-described mutagenic recombinant plasmid, mutagenesis was effected in accordance with the method of the kit and by using the reaction product, the strain Escherichia coli MV1184 (“Competent cell MV1184”, product of Takara Shuzo Co., Ltd.) was transformed. From the transformant thus obtained, a recombinant plasmid was extracted, followed by analysis of a basic sequence, whereby mutation by substitution of Phe for Asn 190 was confirmed. By repeated introduction of mutagenic reactions into the mutated gene by successively using primers A209V (SEQ ID No. 51) and QEYK (SEQ ID No. 49) in a similar manner as above, thereby substituting Asn 190 and Gln 209 , each of the amino acid sequence of the K38AMY represented by SEQ ID No. 4 with Phe and Val, respectively, and the sequence from Asp 1 to Gly 19 of the amino acid sequence of the K38AMY represented by SEQ ID No. 4 with the sequence from His 1 to Gly 21 of the amino acid sequence of the LAMY represented by SEQ ID No. 2; by substituting Gln 167 , Tyr 169 , Asn 190 and Gln 209 , each of the amino acid sequence of the K38AMY, with Glu, Lys, Phe and Val, respectively and the sequence from Asp 1 to Gly 19 of the amino acid sequence of the K38AMY with the sequence from His 1 to Gly 2 , of the amino acid sequence of the LAMY; and substituting Gln 167 and Tyr 169 , Asn 190 and Gln 209 , each of the amino acid sequence of the K38AMY, with Glu, Lys, Phe and Val, respectively, genes encoding a mutant α-amylase (which will hereinafter be described as “LA-K38AMY/NFQV”) having improved heat resistance, a mutant α-amylase (“LA-K38AMY/QEYKINFQV”) having drastically improved heat resistance, and a mutant α-amylase (“QEYK/NFQV”) having improved heat resistance were constructed, respectively.

With these genes as a template, PCR reaction was conducted using primers K38US (SEQ ID No. 7) and K38DH (SEQ ID No. 8) to amplify structural gene fragments (about 1.5 kb) encoding the mutant α-amylases were amplified. They were then inserted into the SalI-SmaI site of an expression vector pHSP64 in a similar manner as above, whereby a recombinant plasmid having structural genes of these mutant α-amylases bonded each other was constructed (FIG. 1 ).

Example 2

Introduction of a Mutation for Improving α-Amylase Productivity

A “Site-Directed Mutagenesis System Mutan-Super Express Km” kit of Takara Shuzo Co., Ltd. was used for site-directed mutagenesis for improving amylase productivity of recombinant Bacillus subtilis. With various recombinant plasmids obtained in Example 1 as a template, PCR reactions were effected using primers CLUBG (SEQ ID No. 9) and LADH (SEQ ID No. 6) for ARG and ΔRG/M202T, while using primers CLUBG (SEQ ID No. 9) and K38DH (SEQ ID No. 8) for K38AMY, LA-K38AMY/NFQV, LA-K38AMY/QEYK/NFQV and QEYK/NFQV, whereby fragments of about 2.1 kb from the upstream strong promoter derived from the strain KSM-64 to the downstream α-amylase gene were amplified. These amplified fragments were inserted into the SmaI site of a plasmid vector pKF19k attached to the above-described kit, whereby various mutagenetic recombinant plasmids were constructed (FIG. 2 ).

Various oligonucleotide primers for site-directed mutagenesis shown in Table 2 (SEQ ID Nos. 12 to 51) were 5′-phosphorylated with T4DNA kinase, and by using the resultant products and the above mutagenetic recombinant plasmids, mutagenesis was conducted in accordance with the method as described in the kit. With the reaction products, the strain Escherichia coli MV1184(“Competent Cell MV1184” product of Takara Shuzo Co., Ltd.) was transformed. From the resulting transformants, a recombinant plasmid was extracted, followed by analysis of a base sequence to confirm mutation.

TABLE 2
SeQ
ID			Using
No.	Primer	Base sequence (5′-3′)	purpose
5	LAUS	GAGTCGACCAGCACAAGCCCATCATAATGG	PCR for
6	LADH	TAAAGCTTCAATTTATATTGG	recombi-
7	K38US	GGGTCGACCAGCACAAGCCGATGGATTGAACGGTACGATG	nation
8	K38DH	TAAAGCTTTTGTTATTGGTTCACGTACAC
9	CLUBG	CCAGATCTACTTACCATTTTAGAGTCA
10	LA-K38	ATTTGCCAAATGACGGGCAGCATTGGAATCGGTT
11	LA-K38R	AACCGATTCCAATGCTGCCCGTCATTTGGCAAAT
12	P18S	TTTGAATGGCATTTGTCAAATGACGGGGAACCAC	Site-directed
13	Q86E	ACAAGGAGTCAGTTGGAAGGTGCCGTGACATCT	mutagenesis
14	E130V	CGAAACCAAGTAATATCAGGT	(ΔRG)
15	N154D	AATACCCATTCCGATTTTAAATGGCGC
16	R171C	GATTGGGATCAGTCATGYCAGCTTCAGAACAAA
17	A186V	AAATTCACCGGAAAGGTATGGGACTGGGAAGTA
18	E212D	TCATCCAGATGTAATCAATG
19	V222E	CTTAGAAATTGGGGAGAATGGTATACAAATACA
20	Y243C	GTGAAACATATTAAATGCAGCTATACGAGAGAT
21	P260E	AACACCACAGGTAAAGAAATGTTTGCAGTTGCA
22	K269Q	AGAATTTTGGCAAAATGACCT
23	E276H	TTGCTGCAATCCATAACTATTTAAAT
24	N277S	CTTGCTGCAATCGAAAGYTATTTAAATAAAACA
25	R310A	GGCTATTTTGATATGGCAAATATTTTAAATGGT
26	E360Q	TCTGACAAGGCAGCAAGGTTA
27	Q391E	GATCCACTTCTGGAAGCACGTCAAACG
28	W439R	GGGGGTAATAAAAGAATGTATGTCGGG
29	K444R	ATGTATGTCGGGCGACATAAAGCTGG
30	N471D	GATGGTTGGGGGGATTTCACTGTAA
31	G476D	TTCACTGTAAACGATGGGGCAGTTTCG
32	K484Q	GGTTTGGGTGCAGCAATAAAT
33	P18X	TTTGAATGGCATTTGNNNAATGACGGGAACCAC	Site-directed
34	A186X	AAATTCACCGGAAAGNNNTGGGACTGGGAAGTA	mutagenesis
35	Y243X	GTGAAACATATTAAANNNAGCTATACGAGAGAT	(for ΔRG/
36	N277X	CTTGCTGCAATCGAANNNTATTTAAATAAAACA	M2027)
37	N471E	GATGGTTGGGGGGAATTCACTGTAA
38	D128V	CCAACGAATCGTTGGCAGGTAATTTCAGGTGCCTACACG	Site-directed
39	G140S	ATTGATGCGTGGACGAGTTTCGACTTTTCAGGG	mutagenesis
40	S144P	TTTCGACTTTCCAGGGCGTAA	for
41	R168Q	GGTGTTGACTGGGATCAGCAATATCAAGAAAATCATATTTTCC	K38AMY)
42	N181V	CATATTTTCCGCTTTGCAAATACGGTNTGGAACAGGCGAGTG
43	E207D	AATATCGACTTTAGTCATCCAGATGTACAAGATGAGTTGAAGGA
44	F272S	GACGTAGGTGCTCTCGAATCTTATTTAGATGAAATGAATTGGG
45	S375P	CGATAACATTCCAGCTAAAAA
46	W434R	GACCTGGTGGTTCCAAGAGAATGTATGTAGGACGTCAG
47	E466D	AATGGCGATGGATGGGGCGATTTCTTTACGAATGGAGGATCT
48	D128X	CCAACGAATCGTTGGCAGNNNATTTCAGGTGCCTACACG
49	QEYK	GTTGACTGGGATGAGCGCAAACAAGAAAATCAT
50	N190F	TGGATGAAGAGTTCGGTAATTATGA
51	Q209	AGTCATCCAGAGGTCGTAGATGAGTTGAAGGAT
The “N” in the base sequence means a mixed base of A, T, G and C, while “Y” means a mixed base of T and C.

By inserting an expression promoter region and the mutant α-amylase gene portion into the SmaI site of pKF19k again in a similar manner as the above, the mutation-introduced gene became a template plasmid upon introduction of another mutation. Another mutation was thus introduced in a similar manner to the above-described method.

With these mutated recombinant plasmids thus obtained as a template, PCR reaction was conducted using primers CLUBG (SEQ ID No. 9) and LADH (SEQ ID No. 6) or primers CLUBS (SEQ ID No. 9) and K38DH (SEQ ID No. 8) to amplify the mutated gene fragments. After they were cut with SalI, they were inserted into the site of SalI-SmaI site of an expression vector pHSP64, whereby various plasmids for producing mutant α-amylases were constructed (FIG. 1 ).

Example 3

Production of Mutant α-Amylases

The various plasmids for producing mutant α-amylases obtained in Example 2 were each introduced into the strain Bacillus subtilis ISW1214 (leuA metB5 hsdM1) in accordance with the protoplast method. The recombinant Bacillus subtilis thus obtained was cultivated at 30° C. for 4 days in a liquid medium (corn steep liquor, 4%; tryptose, 1%; meet extract, 1%, monopotassium phosphate, 0.1%, magnesium sulfate, 0.01%, maltose, 2%, calcium chloride, 0.1%, tetracycline, 15 μg/mL). The activity of each of the various mutant α-amylases was measured using the supernatant of the culture medium.

Example 4

Evaluation of Amylase Productivity-1

Each of an enzyme having Pro 18 of ΔRG substituted with Ser (which will hereinafter be abbreviated as “P18S/ΔRG”), an enzyme having Gln 86 substituted with Glu (“Q86E/ΔRG”), an enzyme having Glu 130 substituted with Val (“E130V/ΔRG”), an enzyme having Asn 154 substituted with Asp (“N154D/ΔRG”), an enzyme having Arg 171 substituted with Cys (“R171C/ΔRG”), an enzyme having Ala 186 substituted with Val (“A186V/ΔRG”), an enzyme having Glu 212 substituted with Asp (“E212D/ΔRG”), an enzyme having Val 222 substituted with Glu (“V222E/ΔRG”), an enzyme having Tyr 243 substituted with Cys (“Y243C/ΔRG”), an enzyme having Pro 260 substituted with Glu (“P260E/ΔRG”), an enzyme having Lys 269 substituted with Gln (“K269E/ΔRG”), an enzyme having Glu 276 substituted with His (“E276H/ΔRG”), an enzyme having Asn 277 substituted with Ser (“N277S/ΔRG”), an enzyme having Arg 310 substituted with Ala (“R310A/ΔRG”), an enzyme having Glu 360 substituted with Gln (“E360Q/ΔRG”), an enzyme having Gln 391 substituted with Glu (“Q391E/ΔRG”), an enzyme having Trp 439 substituted with Arg (“W439R/ΔRG”), an enzyme having Lys 444 substituted with Arg (“K444R/ΔRG”), an enzyme having Asn 471 substituted with Asp (“N471D/ΔRG”), and an enzyme having Gly 476 substituted with Asp (“G476D/ΔRG”) was assayed for amylase productivity. As a control, ΔRG was employed. A relative value (%) of amylase productivity was determined from the amylase productivity of ΔRG set at 100%. The results are shown in Table 3.

TABLE 3
          Relative amylase
Enzyme    productivity (%)
ΔRG       100
P18S/ΔRG  277
Q86E/ΔRG  119
E130V/ΔRG 362
N154D/ΔRG 146
R171C/ΔRG 235
A186V/ΔRG 485
E212D/ΔRG 327
V222E/ΔRG 135
Y243C/ΔRG 350
P260E/ΔRG 142
K269Q/ΔRG 142
E276H/ΔRG 231
N277S/ΔRG 312
R310A/ΔRG 208
E360Q/ΔRG 162
Q391E/ΔRG 127
W439R/ΔRG 312
K444R/ΔRG 112
N471D/ΔRG 292
G476D/ΔRG 296

Any one of the mutant enzymes exhibited higher amylase productivity than ΔRG, indicating that mutation heightened productivity of α-amylase in recombinant Bacillus subtilis . In particular, the productivity of each of E130V/ΔRG, A186V/ΔRG, E212D/ΔRG, Y243C/ΔRG, N277S/ΔRG and W439R/ΔRG was found to be at least 3 times greater than that of ΔRG and above all, A186V/ΔRG exhibited eminently high productivity of almost 5 times greater than that of ΔRG.

Example 5

Evaluation of Amylase Productivity-2

In a similar manner to the methods described in Examples 1, 2 and 3, each of an enzyme having Pro 18 of ΔRG/MT substituted with Thr (which will hereinafter be abbreviated as “P18T/ΔRG/MT”), an enzyme having Gln 86 substituted with Glu (“Q86E/ΔRG/MT”), an enzyme having Glu 130 substituted with Val (“E130V/ΔRG/MT”), an enzyme having Ala 186 substituted with Asn (“A186N/ΔRG/MT”), an enzyme having Tyr 243 substituted with Ser (“Y243S/ΔRG/MT”), an enzyme having Asn 277 substituted with Phe (“N277F/ΔRG/MT”), and an enzyme having Asn 471 substituted with Glu (“N471E/ΔRG/MT”) was assayed for amylase productivity. As a control, ΔRG/MT was employed. The results are shown in Table 4.

TABLE 4
             Relative amylase
Enzyme       productivity (%)
ΔRG/MT       100
P18T/ΔRG/MT  200
Q86E/ΔRG/MT  144
E130V/ΔRG/MT 344
A186N/ΔRG/MT 344
Y243S/ΔRG/MT 189
N277F/ΔRG/MT 256
N471E/ΔRG/MT 211

It was recognized that any one of the above-described mutant enzymes exhibited high amylase productivity compared with ΔRG/MT, and in particular, the productivity of each of E130V/ΔRG/MT and A186N/ΔRG/MT was at least 3 times greater than that of ΔRG/MT.

Example 6

Evaluation of Amylase Productivity-3

In accordance with the methods employed in Examples 1, 2 and 3, each of an enzyme having Asp 128 of K38AMY substituted with Val (which will hereinafter be abbreviated as “D128V”), an enzyme having Gly 140 substituted with Ser (“G140S”), an enzyme having Ser 144 substituted with Pro (“S144P”), an enzyme having Arg 168 substituted with Gln (“R168Q”), an enzyme having Asn 181 substituted with Val (“N181V”), an enzyme having Glu 207 substituted with Asp (“E207D”), an enzyme having Phe 272 substituted with Ser (“F272S”), an enzyme having Ser 375 substituted with Pro (“S375P”), an enzyme having Trp 434 substituted with Arg (“W434R”), and an enzyme having Glu 466 substituted with Asp (“E466D”) was assayed for amylase productivity. As a control, K38AMY was employed. The results are shown in Table 5.

TABLE 5
       Relative amylase
Enzyme productivity (%)
K38AMY 100
D128V  325
G140S  209
S144P  197
R168Q  264
N181V  207
E207D  109
F272S  175
S375P  115
W434R  124
E466D  212

It was recognized that compared with the wild type K38AMY, any one of the mutant enzymes exhibited high amylase productivity and in particular, D128V exhibited high productivity at least 3 times greater than that of K38AMY.

Example 7

Evaluation of Amylase Productivity-4

A mutant enzyme S144P/N181V (which will hereinafter be abbreviated as “SPNV”) having, among the mutants shown in Example 6, S144P and N181V in combination was assayed for amylase productivity in accordance with the method as described in Example 3. As a control, K38AMY, S144P and N181V were employed. The results are shown in Table 6.

TABLE 6
       Relative amylase
Enzyme productivity (%)
K38AMY 100
S144P  197
N181V  207
SPNV   257

As a result, as shown in Table 6, a further improvement in amylase productivity was brought about by combined use.

Example 8

Evaluation of Amylase Productivity-5

In accordance with the methods as described in Examples 1, 2 and 3, each of an enzyme obtained by substituting Arg 168 of the gene of a heat-resistance improved enzyme LA-K38AMY/NFQV with Gln (which will hereinafter be abbreviated as “R168Q/LA-K38AMY/NFQV”), an enzyme obtained by substituting Glu 466 of the above-described gene with Asp (“E466D/LA-K38AMY/NFQV”), and an enzyme having double mutations of Example 6 introduced into the gene (“SPNV/LA-K38AMY/NFQV”) was assayed for amylase productivity. As a control, LA-K38AMY/NFQV was employed. The results are shown in Table 7.

TABLE 7
                     Relative amylase
Enzyme               productivity (%)
LA-K38AMY/NFQV       100
R168Q/LA-K38AMY/NFQV 304
E466D/LA-K38AMY/NFQV 264
SPNV/LA-K38AMY/NFQV  154

As a result, it was recognized that any one of the mutant enzymes obtained in this Example exhibited high amylase productivity at least about 1.5 times greater than that of LA-K38AMY/NFQV and in particular, R168Q/LA-K38AMY/NFQV exhibited about 3 times greater productivity.

Example 9

Evaluation of Amylase Productivity-6

In accordance with the methods as described in Examples 1, 2 and 3, each of an enzyme obtained by substituting Asp 128 of the gene of a heat-resistance improved enzyme LA-K38AMY/QEYK/NFQV with Val (which will hereinafter be abbreviated as “D128V/LA-K38AMY/QEYK/NFQV”) and an enzyme having double mutations of Example 6 introduced into the gene (“SPNV/LA-K38AMY/QEYK/NFQV”) was assayed for amylase productivity. As a control, LA-K38AMY/QEYK/NFQV was employed. The results are shown in Table 8.

TABLE 8
                          Relative amylase
Enzyme                    productivity (%)
LA-K38AMY/QEYK/NFQV       100
D128V/LA-K38AMY/QEYK/NFQV 602
SPNV/LA-K38AMY/QEYK/NFQV  427

As a result, it was recognized that any one of the mutant enzymes obtained in this Example exhibited markedly high amylase productivity compared with LA-K38AMY/QEYK/NFQV and in particular, D128V/LA-K38AMY/QEYK/NFQV exhibited drastic increase (about 6 times) in productivity.

Example 10

Evaluation of Amylase Productivity-7

Into D128V/LA-K38AMY/QEYK/NFQV which was recognized to show a drastic increase in productivity among the mutant enzymes shown in Example 9, a mutation for heightening oxidant resistance by substituting Met 107 with Leu (this mutation will hereinafter be abbreviated as “M107L”) was introduced in accordance with the methods as described in Examples 1 and 2 (“ML/DV/LA-K38AMY/QEYK/NFQV”).

Then, the gene of the mutant enzyme ML/DV/LA-K38AMY/QEYK/NFQV was assayed for amylase productivity in accordance with the method of Example 4. As a control, D128V/LA-K38AMY/QEYK/NFQV was employed. The results are shown in Table 9.

TABLE 9
                          Relative amylase
Enzyme                    productivity (%)
D128V/LA-K38AMY/QEYK/NFQV 100
M107L/D128V/LA-           115
K38AMY/QEYK/NFQV

The relative amylase productivity of the mutant enzyme ML/DV/LA-K38AMY/QEYK/NFQV was 115%, indicating that introduction of M107L mutation for reinforcing oxidant resistance did not adversely affect high productivity of amylase in recombinant Bacillus subtilis.

Example 11

Evaluation of Amylase Productivity-8

In accordance with the methods as described in Examples 1, 2 and 3, an enzyme obtained by substituting AsP 128 of the gene of heat-resistance-improved enzyme QEYK/NFQV with Gln (the resultant enzyme will hereinafter be abbreviated as “D128Q/QEYK/NFQV”) was assayed for amylase productivity. As a control, QEYK/NFQV was employed. The results are shown in Table 10.

TABLE 10
                Relative amylase
Enzyme          productivity (%)
QEYK/NFQV       100
D128Q/QEYK/NFQV 247

It was recognized that the mutant enzyme exhibited productivity of at least 2 times greater than that of QEYK/NFQV.

Example 12

Solubility Assay

After storage of each of the mutant enzyme preparations as shown in Table 11 at 4° C. for 1 week, the precipitate formed by centrifugation (13000 rpm, 10 minutes, 4° C.) was separated. The precipitate was suspended in the same volume, as that before centrifugation, of a Tris-HCl buffer (pH 7.0) containing of 2 mM CaCl 2 . The resulting suspension was diluted about 500-folds with the same buffer to dissolve the former in the latter and enzymatic activity in the resulting solution was measured. The supernatant was diluted in a similar manner and enzymatic activity in it was also measured. Solubility of each of the mutant enzymes was evaluated by comparing the enzymatic activity in each of the precipitate solution and supernatant with that of the preparation before storage at 4° C. The results are shown collectively in Table 11.

	TABLE 11
	Residual activity (%) after
	storage at 4° C.
	Enzyme	Supernatant	Precipitate
	ΔRG	55	40
	ΔRG Gln86 → Glu	83	11
	ΔRG Pro260 → Glu	70	18
	ΔRG Lys269 → Gln	74	27
	ΔRG Asn471 → Asp	74	23
	ΔRG Lys484 → Gln	71	24

As a result, when an improved α-amylase (ΔRG) having heat resistance improved by deleting Arg 181 and Gly 182 was stored at 4° C. for one week, precipitation of the enzyme was recognized and only about half of the activity remained in the supernatant. On the other hand, the mutant enzymes obtained by introducing a further mutation in ΔRG-LAMY showed a high activity residual ratio in the supernatant, indicating an improvement in solubility by mutation. In particular, the enzyme having Gln 86 substituted with Glu showed the highest enzyme solubility and 80% of the enzyme remained in the supernatant under the conditions of this Example.

Example 13

Detergent Composition for Automatic Dish Washer

A detergent composition for automatic dish washer having the composition as shown in Table 12 was prepared, followed by incorporation therein of various mutant enzymes obtained in the productivity increasing method. As a result, the highly productive mutant enzymes exhibited similar or superior detergency to the control enzyme when they were equal in activity.

TABLE 12
Composition of detergent         (%)
Pluronic L-61                    2.2
Sodium carbonate                 24.7
Sodium bicarbonate               24.7
Sodium percarbonate              10.0
No. 1 sodium silicate            12.0
Trisodium citrate                20.0
Polypropylene glycol             2.2
“Silicone KST-04” (product of Toshiba Silicone) 0.2
“Sokalan CP-45” (product of BASF) 4.0

Capability of Exploitation Industry

By using the mutant α-amylases according to the present invention, α-amylases are available at a high yield from recombinant microorganisms, making it possible to largely reduce the cost of their industrial production. The mutation for productivity increase in the present invention does not adversely affect biochemical properties of the enzymes so that highly productive liquefying alkaline α-amylases having heat resistance, chelating agent resistance and oxidant resistance and being useful as enzymes for a detergent can be produced.

51 1 1786 DNA Bacillus sp. KSM-AP1378 sig_peptide (155)..(247) 1cagcgtgata atataaattt gaaatgaaca cctatgaaaa tatggtagcg attgcgcgac 60gagaaaaaac ttgggagtta ggaagtgata ttaaaggatt ttttttgact tgttgtgaaa 120acgcttgcat aaattgaagg agagggtgct tttt atg aaa ctt cat aac cgt ata 175 Met Lys Leu His Asn Arg Ile -30 -25att agc gta cta tta aca cta ttg tta gct gta gct gtt ttg ttt cca 223Ile Ser Val Leu Leu Thr Leu Leu Leu Ala Val Ala Val Leu Phe Pro -20 -15 -10tat atg acg gaa cca gca caa gcc cat cat aat ggg acg aat ggg acc 271Tyr Met Thr Glu Pro Ala Gln Ala His His Asn Gly Thr Asn Gly Thr -5 -1 1 5atg atg cag tat ttt gaa tgg cat ttg cca aat gac ggg aac cac tgg 319Met Met Gln Tyr Phe Glu Trp His Leu Pro Asn Asp Gly Asn His Trp 10 15 20aac agg tta cga gat gac gca gct aac tta aag agt aaa ggg att acc 367Asn Arg Leu Arg Asp Asp Ala Ala Asn Leu Lys Ser Lys Gly Ile Thr25 30 35 40gct gtt tgg att cct cct gca tgg aag ggg act tcg caa aat gat gtt 415Ala Val Trp Ile Pro Pro Ala Trp Lys Gly Thr Ser Gln Asn Asp Val 45 50 55ggg tat ggt gcc tat gat ttg tac gat ctt ggt gag ttt aac caa aag 463Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln Lys 60 65 70gga acc gtc cgt aca aaa tat ggc aca agg agt cag ttg caa ggt gcc 511Gly Thr Val Arg Thr Lys Tyr Gly Thr Arg Ser Gln Leu Gln Gly Ala 75 80 85gtg aca tct ttg aaa aat aac ggg att caa gtt tat ggg gat gtc gtg 559Val Thr Ser Leu Lys Asn Asn Gly Ile Gln Val Tyr Gly Asp Val Val 90 95 100atg aat cat aaa ggt gga gca gac ggg aca gag atg gta aat gcg gtg 607Met Asn His Lys Gly Gly Ala Asp Gly Thr Glu Met Val Asn Ala Val105 110 115 120gaa gtg aac cga agc aac cga aac caa gaa ata tca ggt gaa tac acc 655Glu Val Asn Arg Ser Asn Arg Asn Gln Glu Ile Ser Gly Glu Tyr Thr 125 130 135att gaa gca tgg acg aaa ttt gat ttc cct gga aga gga aat acc cat 703Ile Glu Ala Trp Thr Lys Phe Asp Phe Pro Gly Arg Gly Asn Thr His 140 145 150tcc aac ttt aaa tgg cgc tgg tat cat ttt gat ggg aca gat tgg gat 751Ser Asn Phe Lys Trp Arg Trp Tyr His Phe Asp Gly Thr Asp Trp Asp 155 160 165cag tca cgt cag ctt cag aac aaa ata tat aaa ttc aga ggt acc gga 799Gln Ser Arg Gln Leu Gln Asn Lys Ile Tyr Lys Phe Arg Gly Thr Gly 170 175 180aag gca tgg gac tgg gaa gta gat ata gag aac ggc aac tat gat tac 847Lys Ala Trp Asp Trp Glu Val Asp Ile Glu Asn Gly Asn Tyr Asp Tyr185 190 195 200ctt atg tat gca gac att gat atg gat cat cca gaa gta atc aat gaa 895Leu Met Tyr Ala Asp Ile Asp Met Asp His Pro Glu Val Ile Asn Glu 205 210 215ctt aga aat tgg gga gtt tgg tat aca aat aca ctt aat cta gat gga 943Leu Arg Asn Trp Gly Val Trp Tyr Thr Asn Thr Leu Asn Leu Asp Gly 220 225 230ttt aga atc gat gct gtg aaa cat att aaa tac agc tat acg aga gat 991Phe Arg Ile Asp Ala Val Lys His Ile Lys Tyr Ser Tyr Thr Arg Asp 235 240 245tgg cta aca cat gtg cgt aac acc aca ggt aaa cca atg ttt gca gtt 1039Trp Leu Thr His Val Arg Asn Thr Thr Gly Lys Pro Met Phe Ala Val 250 255 260gca gaa ttt tgg aaa aat gac ctt gct gca atc gaa aac tat tta aat 1087Ala Glu Phe Trp Lys Asn Asp Leu Ala Ala Ile Glu Asn Tyr Leu Asn265 270 275 280aaa aca agt tgg aat cac tcc gtg ttc gat gtt cct ctt cat tat aat 1135Lys Thr Ser Trp Asn His Ser Val Phe Asp Val Pro Leu His Tyr Asn 285 290 295ttg tac aat gca tct aat agt ggt ggc tat ttt gat atg aga aat att 1183Leu Tyr Asn Ala Ser Asn Ser Gly Gly Tyr Phe Asp Met Arg Asn Ile 300 305 310tta aat ggt tct gtc gta caa aaa cac cct ata cat gca gtc aca ttt 1231Leu Asn Gly Ser Val Val Gln Lys His Pro Ile His Ala Val Thr Phe 315 320 325gtt gat aac cat gac tct cag cca gga gaa gca ttg gaa tcc ttt gtt 1279Val Asp Asn His Asp Ser Gln Pro Gly Glu Ala Leu Glu Ser Phe Val 330 335 340caa tcg tgg ttc aaa cca ctg gca tat gca ttg att ctg aca agg gag 1327Gln Ser Trp Phe Lys Pro Leu Ala Tyr Ala Leu Ile Leu Thr Arg Glu345 350 355 360caa ggt tac cct tcc gta ttt tac ggt gat tac tac ggt ata cca act 1375Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp Tyr Tyr Gly Ile Pro Thr 365 370 375cat ggt gtt cct tcg atg aaa tct aaa att gat cca ctt ctg cag gca 1423His Gly Val Pro Ser Met Lys Ser Lys Ile Asp Pro Leu Leu Gln Ala 380 385 390cgt caa acg tat gcc tac gga acc caa cat gat tat ttt gat cat cat 1471Arg Gln Thr Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe Asp His His 395 400 405gat att atc ggc tgg acg aga gaa ggg gac agc tcc cac cca aat tca 1519Asp Ile Ile Gly Trp Thr Arg Glu Gly Asp Ser Ser His Pro Asn Ser 410 415 420gga ctt gca act att atg tcc gat ggg cca ggg ggt aat aaa tgg atg 1567Gly Leu Ala Thr Ile Met Ser Asp Gly Pro Gly Gly Asn Lys Trp Met425 430 435 440tat gtc ggg aaa cat aaa gct ggc caa gta tgg aga gat atc acc gga 1615Tyr Val Gly Lys His Lys Ala Gly Gln Val Trp Arg Asp Ile Thr Gly 445 450 455aat agg tct ggt acc gtc acc att aat gca gat ggt tgg ggg aat ttc 1663Asn Arg Ser Gly Thr Val Thr Ile Asn Ala Asp Gly Trp Gly Asn Phe 460 465 470act gta aac gga ggg gca gtt tcg gtt tgg gtg aag caa taaataagga 1712Thr Val Asn Gly Gly Ala Val Ser Val Trp Val Lys Gln 475 480 485acaagaggcg aaaattactt tcctacatgc agagctttcc gatcactcat acacccaata 1772taaattggaa gctt 1786 2 516 PRT Bacillus sp. KSM-AP1378 2Met Lys Leu His Asn Arg Ile Ile Ser Val Leu Leu Thr Leu Leu Leu -30 -25 -20Ala Val Ala Val Leu Phe Pro Tyr Met Thr Glu Pro Ala Gln Ala His-15 -10 -5 -1 1His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His Leu 5 10 15Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala Asn 20 25 30Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp Lys 35 40 45Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp50 55 60 65Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr 70 75 80Arg Ser Gln Leu Gln Gly Ala Val Thr Ser Leu Lys Asn Asn Gly Ile 85 90 95Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp Gly 100 105 110Thr Glu Met Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn Gln 115 120 125Glu Ile Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp Phe130 135 140 145Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr His 150 155 160Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys Ile 165 170 175Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Ile 180 185 190Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met Asp 195 200 205His Pro Glu Val Ile Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr Thr210 215 220 225Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile 230 235 240Lys Tyr Ser Tyr Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr Thr 245 250 255Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Ala 260 265 270Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val Phe 275 280 285Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly Gly290 295 300 305Tyr Phe Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys His 310 315 320Pro Ile His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly 325 330 335Glu Ala Leu Glu Ser Phe Val Gln Ser Trp Phe Lys Pro Leu Ala Tyr 340 345 350Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly 355 360 365Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ser Met Lys Ser Lys370 375 380 385Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Tyr Ala Tyr Gly Thr Gln 390 395 400His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu Gly 405 410 415Asp Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly 420 425 430Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys His Lys Ala Gly Gln 435 440 445Val Trp Arg Asp Ile Thr Gly Asn Arg Ser Gly Thr Val Thr Ile Asn450 455 460 465Ala Asp Gly Trp Gly Asn Phe Thr Val Asn Gly Gly Ala Val Ser Val 470 475 480Trp Val Lys Gln 485 3 1753 DNA Bacillus sp. KSM-K38 sig_peptide (162)..(224) 3gtatgcgaaa cgatgcgcaa aactgcgcaa ctactagcac tcttcaggga ctaaaccacc 60ttttttccaa aaatgacatc atataaacaa atttgtctac caatcactat ttaaagctgt 120ttatgatata tgtaagcgtt atcattaaaa ggaggtattt g atg aga aga tgg gta 176 Met Arg Arg Trp Val -20gta gca atg ttg gca gtg tta ttt tta ttt cct tcg gta gta gtt gca 224Val Ala Met Leu Ala Val Leu Phe Leu Phe Pro Ser Val Val Val Ala -15 -10 -5 -1gat gga ttg aac ggt acg atg atg cag tat tat gag tgg cat ttg gaa 272Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr Glu Trp His Leu Glu1 5 10 15aac gac ggg cag cat tgg aat cgg ttg cac gat gat gcc gca gct ttg 320Asn Asp Gly Gln His Trp Asn Arg Leu His Asp Asp Ala Ala Ala Leu 20 25 30agt gat gct ggt att aca gct att tgg att ccg cca gcc tac aaa ggt 368Ser Asp Ala Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45aat agt cag gcg gat gtt ggg tac ggt gca tac gat ctt tat gat tta 416Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60gga gag ttc aat caa aag ggt act gtt cga acg aaa tac gga act aag 464Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys65 70 75 80gca cag ctt gaa cga gct att ggg tcc ctt aaa tct aat gat atc aat 512Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys Ser Asn Asp Ile Asn 85 90 95gta tac gga gat gtc gtg atg aat cat aaa atg gga gct gat ttt acg 560Val Tyr Gly Asp Val Val Met Asn His Lys Met Gly Ala Asp Phe Thr 100 105 110gag gca gtg caa gct gtt caa gta aat cca acg aat cgt tgg cag gat 608Glu Ala Val Gln Ala Val Gln Val Asn Pro Thr Asn Arg Trp Gln Asp 115 120 125att tca ggt gcc tac acg att gat gcg tgg acg ggt ttc gac ttt tca 656Ile Ser Gly Ala Tyr Thr Ile Asp Ala Trp Thr Gly Phe Asp Phe Ser 130 135 140ggg cgt aac aac gcc tat tca gat ttt aag tgg aga tgg ttc cat ttt 704Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp Arg Trp Phe His Phe145 150 155 160aat ggt gtt gac tgg gat cag cgc tat caa gaa aat cat att ttc cgc 752Asn Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu Asn His Ile Phe Arg 165 170 175ttt gca aat acg aac tgg aac tgg cga gtg gat gaa gag aac ggt aat 800Phe Ala Asn Thr Asn Trp Asn Trp Arg Val Asp Glu Glu Asn Gly Asn 180 185 190tat gat tac ctg tta gga tcg aat atc gac ttt agt cat cca gaa gta 848Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe Ser His Pro Glu Val 195 200 205caa gat gag ttg aag gat tgg ggt agc tgg ttt acc gat gag tta gat 896Gln Asp Glu Leu Lys Asp Trp Gly Ser Trp Phe Thr Asp Glu Leu Asp 210 215 220ttg gat ggt tat cgt tta gat gct att aaa cat att cca ttc tgg tat 944Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His Ile Pro Phe Trp Tyr225 230 235 240aca tct gat tgg gtt cgg cat cag cgc aac gaa gca gat caa gat tta 992Thr Ser Asp Trp Val Arg His Gln Arg Asn Glu Ala Asp Gln Asp Leu 245 250 255ttt gtc gta ggg gaa tat tgg aag gat gac gta ggt gct ctc gaa ttt 1040Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val Gly Ala Leu Glu Phe 260 265 270tat tta gat gaa atg aat tgg gag atg tct cta ttc gat gtt cca ctt 1088Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu Phe Asp Val Pro Leu 275 280 285aat tat aat ttt tac cgg gct tca caa caa ggt gga agc tat gat atg 1136Asn Tyr Asn Phe Tyr Arg Ala Ser Gln Gln Gly Gly Ser Tyr Asp Met 290 295 300cgt aat att tta cga gga tct tta gta gaa gcg cat ccg atg cat gca 1184Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala His Pro Met His Ala305 310 315 320gtt acg ttt gtt gat aat cat gat act cag cca ggg gag tca tta gag 1232Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Glu Ser Leu Glu 325 330 335tca tgg gtt gct gat tgg ttt aag cca ctt gct tat gcg aca att ttg 1280Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala Tyr Ala Thr Ile Leu 340 345 350acg cgt gaa ggt ggt tat cca aat gta ttt tac ggt gat tac tat ggg 1328Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr Gly Asp Tyr Tyr Gly 355 360 365att cct aac gat aac att tca gct aaa aaa gat atg att gat gag ctg 1376Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp Met Ile Asp Glu Leu 370 375 380ctt gat gca cgt caa aat tac gca tat ggc acg cag cat gac tat ttt 1424Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe385 390 395 400gat cat tgg gat gtt gta gga tgg act agg gaa gga tct tcc tcc aga 1472Asp His Trp Asp Val Val Gly Trp Thr Arg Glu Gly Ser Ser Ser Arg 405 410 415cct aat tca ggc ctt gcg act att atg tcg aat gga cct ggt ggt tcc 1520Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn Gly Pro Gly Gly Ser 420 425 430aag tgg atg tat gta gga cgt cag aat gca gga caa aca tgg aca gat 1568Lys Trp Met Tyr Val Gly Arg Gln Asn Ala Gly Gln Thr Trp Thr Asp 435 440 445tta act ggt aat aac gga gcg tcc gtt aca att aat ggc gat gga tgg 1616Leu Thr Gly Asn Asn Gly Ala Ser Val Thr Ile Asn Gly Asp Gly Trp 450 455 460ggc gaa ttc ttt acg aat gga gga tct gta tcc gtg tac gtg aac caa 1664Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser Val Tyr Val Asn Gln465 470 475 480taacaaaaag ccttgagaag ggattcctcc ctaactcaag gctttcttta tgtcgcttag 1724cttaacgctt ctacgacttt gaagcttta 1753 4 501 PRT Bacillus sp. KSM-K38 4Met Arg Arg Trp Val Val Ala Met Leu Ala Val Leu Phe Leu Phe Pro -20 -15 -10Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr-5 -1 1 5 10Glu Trp His Leu Glu Asn Asp Gly Gln His Trp Asn Arg Leu His Asp 15 20 25Asp Ala Ala Ala Leu Ser Asp Ala Gly Ile Thr Ala Ile Trp Ile Pro 30 35 40Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr 45 50 55Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr60 65 70 75Lys Tyr Gly Thr Lys Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys 80 85 90Ser Asn Asp Ile Asn Val Tyr Gly Asp Val Val Met Asn His Lys Met 95 100 105Gly Ala Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn Pro Thr 110 115 120Asn Arg Trp Gln Asp Ile Ser Gly Ala Tyr Thr Ile Asp Ala Trp Thr 125 130 135Gly Phe Asp Phe Ser Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp140 145 150 155Arg Trp Phe His Phe Asn Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu 160 165 170Asn His Ile Phe Arg Phe Ala Asn Thr Asn Trp Asn Trp Arg Val Asp 175 180 185Glu Glu Asn Gly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe 190 195 200Ser His Pro Glu Val Gln Asp Glu Leu Lys Asp Trp Gly Ser Trp Phe 205 210 215Thr Asp Glu Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His220 225 230 235Ile Pro Phe Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg Asn Glu 240 245 250Ala Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val 255 260 265Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu 270 275 280Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg Ala Ser Gln Gln Gly 285 290 295Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala300 305 310 315His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro 320 325 330Gly Glu Ser Leu Glu Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala 335 340 345Tyr Ala Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr 350 355 360Gly Asp Tyr Tyr Gly Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp 365 370 375Met Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr380 385 390 395Gln His Asp Tyr Phe Asp His Trp Asp Val Val Gly Trp Thr Arg Glu 400 405 410Gly Ser Ser Ser Arg Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn 415 420 425Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Arg Gln Asn Ala Gly 430 435 440Gln Thr Trp Thr Asp Leu Thr Gly Asn Asn Gly Ala Ser Val Thr Ile 445 450 455Asn Gly Asp Gly Trp Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser460 465 470 475Val Tyr Val Asn Gln 480 5 30 DNA Artificial Sequence Synthetic DNA 5gagtcgacca gcacaagccc atcataatgg 30 6 21 DNA Artificial Sequence Synthetic DNA 6taaagcttca atttatattg g 21 7 40 DNA Artificial Sequence Synthetic DNA 7gggtcgacca gcacaagccg atggattgaa cggtacgatg 40 8 29 DNA Artificial Sequence Synthetic DNA 8taaagctttt gttattggtt cacgtacac 29 9 27 DNA Artificial Sequence Synthetic DNA 9ccagatctac ttaccatttt agagtca 27 10 34 DNA Artificial Sequence Synthetic DNA 10atttgccaaa tgacgggcag cattggaatc ggtt 34 11 34 DNA Artificial Sequence Synthetic DNA 11aaccgattcc aatgctgccc gtcatttggc aaat 34 12 34 DNA Artificial Sequence Synthetic DNA 12tttgaatggc atttgtcaaa tgacggggaa ccac 34 13 33 DNA Artificial Sequence Synthetic DNA 13acaaggagtc agttggaagg tgccgtgaca tct 33 14 21 DNA Artificial Sequence Synthetic DNA 14cgaaaccaag taatatcagg t 21 15 27 DNA Artificial Sequence Synthetic DNA 15aatacccatt ccgattttaa atggcgc 27 16 33 DNA Artificial Sequence Synthetic DNA 16gattgggatc agtcatgyca gcttcagaac aaa 33 17 33 DNA Artificial Sequence Synthetic DNA 17aaattcaccg gaaaggtatg ggactgggaa gta 33 18 20 DNA Artificial Sequence Synthetic DNA 18tcatccagat gtaatcaatg 20 19 33 DNA Artificial Sequence Synthetic DNA 19cttagaaatt ggggagaatg gtatacaaat aca 33 20 33 DNA Artificial Sequence Synthetic DNA 20gtgaaacata ttaaatgcag ctatacgaga gat 33 21 33 DNA Artificial Sequence Synthetic DNA 21aacaccacag gtaaagaaat gtttgcagtt gca 33 22 21 DNA Artificial Sequence Synthetic DNA 22agaattttgg caaaatgacc t 21 23 26 DNA Artificial Sequence Synthetic DNA 23ttgctgcaat ccataactat ttaaat 26 24 33 DNA Artificial Sequence Synthetic DNA 24cttgctgcaa tcgaaagyta tttaaataaa aca 33 25 33 DNA Artificial Sequence Synthetic DNA 25ggctattttg atatggcaaa tattttaaat ggt 33 26 21 DNA Artificial Sequence Synthetic DNA 26tctgacaagg cagcaaggtt a 21 27 27 DNA Artificial Sequence Synthetic DNA 27gatccacttc tggaagcacg tcaaacg 27 28 27 DNA Artificial Sequence Synthetic DNA 28gggggtaata aaagaatgta tgtcggg 27 29 26 DNA Artificial Sequence Synthetic DNA 29atgtatgtcg ggcgacataa agctgg 26 30 25 DNA Artificial Sequence Synthetic DNA 30gatggttggg gggatttcac tgtaa 25 31 27 DNA Artificial Sequence Synthetic DNA 31ttcactgtaa acgatggggc agtttcg 27 32 21 DNA Artificial Sequence Synthetic DNA 32ggtttgggtg cagcaataaa t 21 33 33 DNA Artificial Sequence Synthetic DNA 33tttgaatggc atttgnnnaa tgacgggaac cac 33 34 33 DNA Artificial Sequence Synthetic DNA 34aaattcaccg gaaagnnntg ggactgggaa gta 33 35 33 DNA Artificial Sequence Synthetic DNA 35gtgaaacata ttaaannnag ctatacgaga gat 33 36 33 DNA Artificial Sequence Synthetic DNA 36cttgctgcaa tcgaannnta tttaaataaa aca 33 37 25 DNA Artificial Sequence Synthetic DNA 37gatggttggg gggaattcac tgtaa 25 38 39 DNA Artificial Sequence Synthetic DNA 38ccaacgaatc gttggcaggt aatttcaggt gcctacacg 39 39 33 DNA Artificial Sequence Synthetic DNA 39attgatgcgt ggacgagttt cgacttttca ggg 33 40 21 DNA Artificial Sequence Synthetic DNA 40tttcgacttt ccagggcgta a 21 41 43 DNA Artificial Sequence Synthetic DNA 41ggtgttgact gggatcagca atatcaagaa aatcatattt tcc 43 42 42 DNA Artificial Sequence Synthetic DNA 42catattttcc gctttgcaaa tacggtntgg aacaggcgag tg 42 43 44 DNA Artificial Sequence Synthetic DNA 43aatatcgact ttagtcatcc agatgtacaa gatgagttga agga 44 44 43 DNA Artificial Sequence Synthetic DNA 44gacgtaggtg ctctcgaatc ttatttagat gaaatgaatt ggg 43 45 21 DNA Artificial Sequence Synthetic DNA 45cgataacatt ccagctaaaa a 21 46 38 DNA Artificial Sequence Synthetic DNA 46gacctggtgg ttccaagaga atgtatgtag gacgtcag 38 47 42 DNA Artificial Sequence Synthetic DNA 47aatggcgatg gatggggcga tttctttacg aatggaggat ct 42 48 39 DNA Artificial Sequence Synthetic DNA 48ccaacgaatc gttggcagnn natttcaggt gcctacacg 39 49 33 DNA Artificial Sequence Synthetic DNA 49gttgactggg atgagcgcaa acaagaaaat cat 33 50 25 DNA Artificial Sequence Synthetic DNA 50tggatgaaga gttcggtaat tatga 25 51 33 DNA Artificial Sequence Synthetic DNA 51agtcatccag aggtcgtaga tgagttgaag gat 33

Claims

1. A mutant α-amylase which is derived from an α-amylase consisting of an amino acid sequence represented by SEQ ID No. 2 by substitution or deletion of at least one amino acid residue corresponding to any one of Pro18, Gln86, Glu130, Asn154, Arg171, Ala186, Glu212, Val222, Tyr243, Pro260, Lys269, Glu276, Asn277, Arg310, Glu360, Gln391, Trp439, Lys444, Asn471, and Gly476 of the amino acid sequence.

2. A mutant α-amylase according to claim 1, wherein the substitution or deletion of at least one amino acid residue is substitution of the amino acid residue corresponding to Pro18 with Ser or Thr, the amino acid residue corresponding to Gln86 with Glu, the amino acid residue corresponding to Glu130 with Val or Gln, the amino acid residue corresponding to Asn154 with Asp, the amino acid residue corresponding to Arg171 with Cys or Gln, the amino acid residue corresponding to Ala186 with Val or Asn, the amino acid residue corresponding to Glu212 with Asp, the amino acid residue corresponding to Val222 with Glu, the amino acid residue corresponding to Tyr243 with Cys or Ser, the amino acid residue corresponding to Pro260 with Glu, the amino acid residue corresponding to Lys269 with Gln, the amino acid residue corresponding to Glu276 with His, the amino acid residue corresponding to Asn277 with Ser or Phe, the amino acid residue corresponding to Arg310 with Ala, the amino acid residue corresponding to Glu360 with Gln, the amino acid residue corresponding to Gln391 with Glu, the amino acid residue corresponding to TRp4439 with Arg, the amino acid residue corresponding to Lys444 with Arg, the amino acid residue corresponding to Asn471 with Asp or Glu, or the amino acid residue corresponding to Gly476 with Asp.

3. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Pro18 with Ser or Thr.

4. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Gln86 with Glu.

5. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Glu130 with Val or Gln.

6. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Asn154 with Asp.

7. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Arg171 with Cys or Gln.

8. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Ala186 with Val or Asn.

9. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Glu212 with Asp.

10. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Val222 with Glu.

11. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Tyr243 with Cys or Ser.

12. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Pro260 with Glu.

13. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Lys269 with Gln.

14. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Glu276 with His.

15. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Asn277 with Ser or Phe.

16. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Arg310 with Ala.

17. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Glu360 with Gln.

18. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Gln391 with Glu.

19. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Trp439 with Arg.

20. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Lys444 with Arg.

21. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Asn471 with Asp or Glu.

22. The mutant α-amylase according to claim 2, wherein the substitution or deletion of at least one amino acid residue is the amino acid residue corresponding to Gly476 with Asp.

23. A composition comprising the mutant α-amylase of claim 1 and a carrier.

24. A composition comprising the mutant α-amylase of claim 2 and a carrier.

25. A method of making a detergent, comprising adding the mutant α-amylase of claim 1 to one or more detergent components.

26. A method of making a detergent, comprising adding the mutant α-amylase of claim 2 to one or more detergent components.