Highly productive alpha-amylases

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

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.

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.

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.

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.

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.

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

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%).

Bacillus sp. KSM-AP1378

2

Met Lys Leu His Asn Arg Ile Ile Ser Val Leu Leu Thr Leu Leu Leu

-30 -25 -20

Ala Val Ala Val Leu Phe Pro Tyr Met Thr Glu Pro Ala Gln Ala His

-15 -10 -5 -1 1

His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His Leu

5 10 15

Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala Asn

20 25 30

Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp Lys

35 40 45

Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp

50 55 60 65

Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr

70 75 80

Arg Ser Gln Leu Gln Gly Ala Val Thr Ser Leu Lys Asn Asn Gly Ile

85 90 95

Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp Gly

100 105 110

Thr Glu Met Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn Gln

115 120 125

Glu Ile Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp Phe

130 135 140 145

Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr His

150 155 160

Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys Ile

165 170 175

Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Ile

180 185 190

Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met Asp

195 200 205

His Pro Glu Val Ile Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr Thr

210 215 220 225

Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile

230 235 240

Lys Tyr Ser Tyr Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr Thr

245 250 255

Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Ala

260 265 270

Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val Phe

275 280 285

Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly Gly

290 295 300 305

Tyr Phe Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys His

310 315 320

Pro Ile His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly

325 330 335

Glu Ala Leu Glu Ser Phe Val Gln Ser Trp Phe Lys Pro Leu Ala Tyr

340 345 350

Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly

355 360 365

Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ser Met Lys Ser Lys

370 375 380 385

Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Tyr Ala Tyr Gly Thr Gln

390 395 400

His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu Gly

405 410 415

Asp Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly

420 425 430

Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys His Lys Ala Gly Gln

435 440 445

Val Trp Arg Asp Ile Thr Gly Asn Arg Ser Gly Thr Val Thr Ile Asn

450 455 460 465

Ala Asp Gly Trp Gly Asn Phe Thr Val Asn Gly Gly Ala Val Ser Val

470 475 480

Trp Val Lys Gln

485

3

gtatgcgaaa cgatgcgcaa aactgcgcaa ctactagcac tcttcaggga ctaaaccacc 60

ttttttccaa aaatgacatc atataaacaa atttgtctac caatcactat ttaaagctgt 120

ttatgatata tgtaagcgtt atcattaaaa ggaggtattt g atg aga aga tgg gta 176

Met Arg Arg Trp Val

-20

gta gca atg ttg gca gtg tta ttt tta ttt cct tcg gta gta gtt gca 224

Val Ala Met Leu Ala Val Leu Phe Leu Phe Pro Ser Val Val Val Ala

-15 -10 -5 -1

gat gga ttg aac ggt acg atg atg cag tat tat gag tgg cat ttg gaa 272

Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr Glu Trp His Leu Glu

1 5 10 15

aac gac ggg cag cat tgg aat cgg ttg cac gat gat gcc gca gct ttg 320

Asn Asp Gly Gln His Trp Asn Arg Leu His Asp Asp Ala Ala Ala Leu

20 25 30

agt gat gct ggt att aca gct att tgg att ccg cca gcc tac aaa ggt 368

Ser Asp Ala Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Tyr Lys Gly

35 40 45

aat agt cag gcg gat gtt ggg tac ggt gca tac gat ctt tat gat tta 416

Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu

50 55 60

gga gag ttc aat caa aag ggt act gtt cga acg aaa tac gga act aag 464

Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys

65 70 75 80

gca cag ctt gaa cga gct att ggg tcc ctt aaa tct aat gat atc aat 512

Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys Ser Asn Asp Ile Asn

85 90 95

gta tac gga gat gtc gtg atg aat cat aaa atg gga gct gat ttt acg 560

Val Tyr Gly Asp Val Val Met Asn His Lys Met Gly Ala Asp Phe Thr

100 105 110

gag gca gtg caa gct gtt caa gta aat cca acg aat cgt tgg cag gat 608

Glu Ala Val Gln Ala Val Gln Val Asn Pro Thr Asn Arg Trp Gln Asp

115 120 125

att tca ggt gcc tac acg att gat gcg tgg acg ggt ttc gac ttt tca 656

Ile Ser Gly Ala Tyr Thr Ile Asp Ala Trp Thr Gly Phe Asp Phe Ser

130 135 140

ggg cgt aac aac gcc tat tca gat ttt aag tgg aga tgg ttc cat ttt 704

Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp Arg Trp Phe His Phe

145 150 155 160

aat ggt gtt gac tgg gat cag cgc tat caa gaa aat cat att ttc cgc 752

Asn Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu Asn His Ile Phe Arg

165 170 175

ttt gca aat acg aac tgg aac tgg cga gtg gat gaa gag aac ggt aat 800

Phe Ala Asn Thr Asn Trp Asn Trp Arg Val Asp Glu Glu Asn Gly Asn

180 185 190

tat gat tac ctg tta gga tcg aat atc gac ttt agt cat cca gaa gta 848

Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe Ser His Pro Glu Val

195 200 205

caa gat gag ttg aag gat tgg ggt agc tgg ttt acc gat gag tta gat 896

Gln Asp Glu Leu Lys Asp Trp Gly Ser Trp Phe Thr Asp Glu Leu Asp

210 215 220

ttg gat ggt tat cgt tta gat gct att aaa cat att cca ttc tgg tat 944

Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His Ile Pro Phe Trp Tyr

225 230 235 240

aca tct gat tgg gtt cgg cat cag cgc aac gaa gca gat caa gat tta 992

Thr Ser Asp Trp Val Arg His Gln Arg Asn Glu Ala Asp Gln Asp Leu

245 250 255

ttt gtc gta ggg gaa tat tgg aag gat gac gta ggt gct ctc gaa ttt 1040

Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val Gly Ala Leu Glu Phe

260 265 270

tat tta gat gaa atg aat tgg gag atg tct cta ttc gat gtt cca ctt 1088

Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu Phe Asp Val Pro Leu

275 280 285

aat tat aat ttt tac cgg gct tca caa caa ggt gga agc tat gat atg 1136

Asn Tyr Asn Phe Tyr Arg Ala Ser Gln Gln Gly Gly Ser Tyr Asp Met

290 295 300

cgt aat att tta cga gga tct tta gta gaa gcg cat ccg atg cat gca 1184

Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala His Pro Met His Ala

305 310 315 320

gtt acg ttt gtt gat aat cat gat act cag cca ggg gag tca tta gag 1232

Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Glu Ser Leu Glu

325 330 335

tca tgg gtt gct gat tgg ttt aag cca ctt gct tat gcg aca att ttg 1280

Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala Tyr Ala Thr Ile Leu

340 345 350

acg cgt gaa ggt ggt tat cca aat gta ttt tac ggt gat tac tat ggg 1328

Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr Gly Asp Tyr Tyr Gly

355 360 365

att cct aac gat aac att tca gct aaa aaa gat atg att gat gag ctg 1376

Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp Met Ile Asp Glu Leu

370 375 380

ctt gat gca cgt caa aat tac gca tat ggc acg cag cat gac tat ttt 1424

Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe

385 390 395 400

gat cat tgg gat gtt gta gga tgg act agg gaa gga tct tcc tcc aga 1472

Asp His Trp Asp Val Val Gly Trp Thr Arg Glu Gly Ser Ser Ser Arg

405 410 415

cct aat tca ggc ctt gcg act att atg tcg aat gga cct ggt ggt tcc 1520

Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn Gly Pro Gly Gly Ser

420 425 430

aag tgg atg tat gta gga cgt cag aat gca gga caa aca tgg aca gat 1568

Lys Trp Met Tyr Val Gly Arg Gln Asn Ala Gly Gln Thr Trp Thr Asp

435 440 445

tta act ggt aat aac gga gcg tcc gtt aca att aat ggc gat gga tgg 1616

Leu Thr Gly Asn Asn Gly Ala Ser Val Thr Ile Asn Gly Asp Gly Trp

450 455 460

ggc gaa ttc ttt acg aat gga gga tct gta tcc gtg tac gtg aac caa 1664

Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser Val Tyr Val Asn Gln

465 470 475 480

taacaaaaag ccttgagaag ggattcctcc ctaactcaag gctttcttta tgtcgcttag 1724

cttaacgctt ctacgacttt gaagcttta 1753

4

501

Bacillus sp. KSM-K38

4

Met Arg Arg Trp Val Val Ala Met Leu Ala Val Leu Phe Leu Phe Pro

-20 -15 -10

Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr

-5 -1 1 5 10

Glu Trp His Leu Glu Asn Asp Gly Gln His Trp Asn Arg Leu His Asp

15 20 25

Asp Ala Ala Ala Leu Ser Asp Ala Gly Ile Thr Ala Ile Trp Ile Pro

30 35 40

Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr

45 50 55

Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr

60 65 70 75

Lys Tyr Gly Thr Lys Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys

80 85 90

Ser Asn Asp Ile Asn Val Tyr Gly Asp Val Val Met Asn His Lys Met

95 100 105

Gly Ala Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn Pro Thr

110 115 120

Asn Arg Trp Gln Asp Ile Ser Gly Ala Tyr Thr Ile Asp Ala Trp Thr

125 130 135

Gly Phe Asp Phe Ser Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp

140 145 150 155

Arg Trp Phe His Phe Asn Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu

160 165 170

Asn His Ile Phe Arg Phe Ala Asn Thr Asn Trp Asn Trp Arg Val Asp

175 180 185

Glu Glu Asn Gly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe

190 195 200

Ser His Pro Glu Val Gln Asp Glu Leu Lys Asp Trp Gly Ser Trp Phe

205 210 215

Thr Asp Glu Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His

220 225 230 235

Ile Pro Phe Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg Asn Glu

240 245 250

Ala Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val

255 260 265

Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu

270 275 280

Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg Ala Ser Gln Gln Gly

285 290 295

Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala

300 305 310 315

His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro

320 325 330

Gly Glu Ser Leu Glu Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala

335 340 345

Tyr Ala Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr

350 355 360

Gly Asp Tyr Tyr Gly Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp

365 370 375

Met Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr

380 385 390 395

Gln His Asp Tyr Phe Asp His Trp Asp Val Val Gly Trp Thr Arg Glu

400 405 410

Gly Ser Ser Ser Arg Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn

415 420 425

Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Arg Gln Asn Ala Gly

430 435 440

Gln Thr Trp Thr Asp Leu Thr Gly Asn Asn Gly Ala Ser Val Thr Ile

445 450 455

Asn Gly Asp Gly Trp Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser

460 465 470 475

Val Tyr Val Asn Gln

480

5

30

Artificial Sequence

Synthetic DNA

5

gagtcgacca gcacaagccc atcataatgg 30

6

21

Artificial Sequence

Synthetic DNA

6

taaagcttca atttatattg g 21

7

40

Artificial Sequence

Synthetic DNA

7

gggtcgacca gcacaagccg atggattgaa cggtacgatg 40

8

29

Artificial Sequence

Synthetic DNA

8

taaagctttt gttattggtt cacgtacac 29

9

27

Artificial Sequence

Synthetic DNA

9

ccagatctac ttaccatttt agagtca 27

10

34

Artificial Sequence

Synthetic DNA

10

atttgccaaa tgacgggcag cattggaatc ggtt 34

11

34

Artificial Sequence

Synthetic DNA

11

aaccgattcc aatgctgccc gtcatttggc aaat 34

12

34

Artificial Sequence

Synthetic DNA

12

tttgaatggc atttgtcaaa tgacggggaa ccac 34

13

33

Artificial Sequence

Synthetic DNA

13

acaaggagtc agttggaagg tgccgtgaca tct 33

14

21

Artificial Sequence

Synthetic DNA

14

cgaaaccaag taatatcagg t 21

15

27

Artificial Sequence

Synthetic DNA

15

aatacccatt ccgattttaa atggcgc 27

16

33

Artificial Sequence

Synthetic DNA

16

gattgggatc agtcatgyca gcttcagaac aaa 33

17

33

Artificial Sequence

Synthetic DNA

17

aaattcaccg gaaaggtatg ggactgggaa gta 33

18

20

Artificial Sequence

Synthetic DNA

18

tcatccagat gtaatcaatg 20

19

33

Artificial Sequence

Synthetic DNA

19

cttagaaatt ggggagaatg gtatacaaat aca 33

20

33

Artificial Sequence

Synthetic DNA

20

gtgaaacata ttaaatgcag ctatacgaga gat 33

21

33

Artificial Sequence

Synthetic DNA

21

aacaccacag gtaaagaaat gtttgcagtt gca 33

22

21

Artificial Sequence

Synthetic DNA

22

agaattttgg caaaatgacc t 21

23

26

Artificial Sequence

Synthetic DNA

23

ttgctgcaat ccataactat ttaaat 26

24

33

Artificial Sequence

Synthetic DNA

24

cttgctgcaa tcgaaagyta tttaaataaa aca 33

25

33

Artificial Sequence

Synthetic DNA

25

ggctattttg atatggcaaa tattttaaat ggt 33

26

21

Artificial Sequence

Synthetic DNA

26

tctgacaagg cagcaaggtt a 21

27

27

Artificial Sequence

Synthetic DNA

27

gatccacttc tggaagcacg tcaaacg 27

28

27

Artificial Sequence

Synthetic DNA

28

gggggtaata aaagaatgta tgtcggg 27

29

26

Artificial Sequence

Synthetic DNA

29

atgtatgtcg ggcgacataa agctgg 26

30

25

Artificial Sequence

Synthetic DNA

30

gatggttggg gggatttcac tgtaa 25

31

27

Artificial Sequence

Synthetic DNA

31

ttcactgtaa acgatggggc agtttcg 27

32

21

Artificial Sequence

Synthetic DNA

32

ggtttgggtg cagcaataaa t 21

33

33

Artificial Sequence

Synthetic DNA

33

tttgaatggc atttgnnnaa tgacgggaac cac 33

34

33

Artificial Sequence

Synthetic DNA

34

aaattcaccg gaaagnnntg ggactgggaa gta 33

35

33

Artificial Sequence

Synthetic DNA

35

gtgaaacata ttaaannnag ctatacgaga gat 33

36

33

Artificial Sequence

Synthetic DNA

36

cttgctgcaa tcgaannnta tttaaataaa aca 33

37

25

Artificial Sequence

Synthetic DNA

37

gatggttggg gggaattcac tgtaa 25

38

39

Artificial Sequence

Synthetic DNA

38

ccaacgaatc gttggcaggt aatttcaggt gcctacacg 39

39

33

Artificial Sequence

Synthetic DNA

39

attgatgcgt ggacgagttt cgacttttca ggg 33

40

21

Artificial Sequence

Synthetic DNA

40

tttcgacttt ccagggcgta a 21

41

43

Artificial Sequence

Synthetic DNA

41

ggtgttgact gggatcagca atatcaagaa aatcatattt tcc 43

42

42

Artificial Sequence

Synthetic DNA

42

catattttcc gctttgcaaa tacggtntgg aacaggcgag tg 42

43

44

Artificial Sequence

Synthetic DNA

43

aatatcgact ttagtcatcc agatgtacaa gatgagttga agga 44

44

43

Artificial Sequence

Synthetic DNA

44

gacgtaggtg ctctcgaatc ttatttagat gaaatgaatt ggg 43

45

21

Artificial Sequence

Synthetic DNA

45

cgataacatt ccagctaaaa a 21

46

38

Artificial Sequence

Synthetic DNA

46

gacctggtgg ttccaagaga atgtatgtag gacgtcag 38

47

42

Artificial Sequence

Synthetic DNA

47

aatggcgatg gatggggcga tttctttacg aatggaggat ct 42

48

39

Artificial Sequence

Synthetic DNA

48

ccaacgaatc gttggcagnn natttcaggt gcctacacg 39

49

33

Artificial Sequence

Synthetic DNA

49

gttgactggg atgagcgcaa acaagaaaat cat 33

50

25

Artificial Sequence

Synthetic DNA

50

tggatgaaga gttcggtaat tatga 25

51

33

Artificial Sequence

Synthetic DNA

51

agtcatccag aggtcgtaga tgagttgaag gat 33