KR19980702783A - Improved Laundry Detergent Composition Including Amylase - Google Patents

Abstract

The present invention relates to novel alpha-amylase mutants obtained from the DNA sequences of naturally occurring alpha-amylases or recombinant alpha-amylases described. In general, the mutant alpha-amylase may modify in vitro a precursor DNA sequence encoding a naturally occurring alpha-amylase or recombinant alpha-amylase to delete one or more oxidizable amino acid residues in the amino acid sequence of the precursor alpha-amylase or Or by substitution (substitution). These mutant alpha-amylases have been modified for oxidative stability and / or pH performance and / or heat resistance compared to precursors. Also described are detergents and starch liquefaction compositions comprising mutant amylases as well as methods of using mutant amylases. More specifically the mutant alpha-amylase is the preferred mutant alpha-amylase obtained at the MET197 or MET15 position or from Bacillus lycaniformis modified with other alpha-amylase residues obtained from TRP138 residues or other microbial sources (Bacillus, Aspergillus). -Amylase.

Description

Improved Laundry Detergent Composition Including Amylase

Alpha-amylase (alpha-1,4-glucan-4-glucanohydrolase, EC3.2.1.1) hydrolyzes alpha-1,4-glucosidic bonds inside starch molecules very randomly, resulting in lower molecular weight Produces maltose-dextrin. Alpha-amylase is quite commercially available, resulting in starch processing; Alcohol production; As a cleaning agent for detergent substrates; And initial stages of starch desizing processing in the textile industry. One wave amylase is a wide variety of microorganisms including Bacillus and Aspergillus and, for example, B. aureus. Lyconiformis (B. licheniformis), b. Amylolycapacitances (B. amylolichefacience), B. B. subtillis, or b. Produced by amylase, which is produced in the largest amount in bacterial sources such as B. Stearothermophillus. Recently, the preferred enzymes used commercially are b. It is obtained from Lycan Formemis because of their performance at higher heat and thicker pH and weakly alkaline pH.

Conventionally, research has been conducted using recombinant DNA techniques to determine which amino acid residues play an important role in the catalytic activity of amylases and / or to modify which amino acids in the active sites of many amylases. [Vihinen, M et al. (1990) J. Biochem. 107: 267-272; Holm, L. et al. (1990) Protein Engineering 3: 181-191; Takase, K. et al. (1992) Biochemica et Biophysica Acta. 1120: 280-288; Matsui, I. et al. (1992) Febs Letters Vol. 310, No. 3, pp. 216-218; 'which residues are important for thermal stability, Suzuki, Y. et al. (1989) J. Biol. Chem. 264: 18933-18938; And rain. One group is used in the introduction of mutations in several histidine residues in B. licheniformis amylase, the principle of substitution at histidine residues compared to other similar Bacillus amylases. Since Lycanformyl amylase (known as heat resistance) contains more histidine, it has been found that replacing histidine will therefore affect the heat resistance of the enzyme. Declerck, N, and his colleagues (1990) J. Boil. Chem. 265: 15481-15488; FR 2 665 178-A1; Joyet, P. and his colleagues (1992) Bio / Technology 10: 1579-1583]

It has been found that alpha-amylases are inactivated by hydrogen peroxide and other oxidants between pH 4-10.5 as described in the Examples herein. Commercially, alpha-amylase enzymes can be used under different, dramatically different conditions, for example under high pH and low pH conditions, depending on the commercial method used. For example, alpha-amylase can be used in the process of liquefaction of starch, which is preferably carried out at low pH (pH 5.5). Amylases, on the other hand, can often be used in commercial dish protection detergents or laundry detergents which contain oxidizing agents such as bleaches or peracids and which are more used in alkaline conditions.

In order to modify the stability or activity of amylases under various conditions, for example, selective substitution, substitution or deletion of oxidizable amino acids such as methionine, tryptophan, tyrosine, histidine or cysteine resulted in a variety of enzyme properties compared to its precursors. It turns out that it is modified. Currently commercially available amylases are not acceptable (ie, unstable) under a variety of conditions, resulting in the need for amylases with modified stability and / or activity. Such stability (performance against oxidation, temperature or pH) as compared to wild type or precursor enzymes can be achieved by sufficiently maintaining the activity of the enzyme. This property can be influenced by the introduction of mutations in the case of maintaining heat resistance or by changing oxidative stability or vice versa. Moreover, when one or more oxidizable amino acids and other amino acids in the alpha-amylase precursor sequence are substituted or when one or more oxidizable amino acids are deleted, the modified enzyme activity at any pH is present outside the optimum pH of the alpha-amylase precursor. will be. In other words, the mutant enzyme of the present invention can be modified in accordance with the pH performance because the oxidative stability of the enzyme is enhanced.

[Summary of invention]

The present invention provides a mutated DNA sequence encoding alpha-amylase, wherein the mutated DNA sequence is a sequence of a mutated DNA sequence obtained from an alpha-amylase precursor by replacing (substituting) or deleting one or more oxidizable amino acids. It relates to laundry detergent compositions comprising novel alpha-amylase mutants that are expression products. In one embodiment of the invention said mutant may be obtained by substituting one or more methionine residues and other amino acids in the precursor of alpha-amylase. In other embodiments of the invention said mutant comprises replacing only one or more tryptophan residues or with one or more methionine residues in an alpha-amylase precursor. In general, alpha-amylase mutants modify in vitro a DNA sequence precursor encoding a naturally occurring alpha-amylase or recombinant alpha-amylase that encodes the deletion or substitution of one or more amino acid residues in the amino acid sequence precursor. Obtained.

Preferably, substitution or deletion of one or more amino acids in the amino acid sequence is possible when one or more of the methionine, tryptophan, cysteine, histidine or tyrosine residues is most preferably substituted or deleted. The oxidizable amino acid residue may be replaced by any of 20 naturally occurring amino acids. If the intended effect is to modify the oxidative stability of the precursor, then the amino acid residues are amino acids that are not oxidizable (eg, alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, leucine, lysine, phenylalanine). , Proline, serine, threonine or valine) or other oxidizable amino acids (e.g., cysteine, methionine, tryptophan, tyrosine or histidine; shown in order from least oxidized to less oxidized). Similarly, if the desired effect is to modify heat resistance, 20 other naturally occurring amino acids may be substituted. (Ie cysteine may be substituted with methionine)

Preferred laundry detergents are methionine residues. Mutants including those substituted with any of the methionine residues (+8, +15, +197, +256, +304, +366 and +438) found in Lyconformis alpha-amylase. Most preferably the substituted methionine is b. Lyconformyl alfa-amylase is located at +197 or +15. Preferred substituent amino acids that replace methionine at the +197 position are alanine (A), isoleucine (I), threonine (T) or cysteine (C). Although other substituent amino acids not listed below may be useful, preferred substituent amino acids at +15 are leucine (L), threonine (T), asparagine (N), aspartate (D), serine (S), valine ( V) and isoleucine (I). Two particularly preferred variants of the invention are M 197 T and M 15 L.

Another embodiment of the present invention comprises tryptophan residues. It relates to a laundry detergent (see FIG. 2) comprising a mutant comprising a substitution with any of the tryptophan residues found in Lyconformis alpha-amylase. Preferably the replaced tryptophan is b. Lycanformis alpha-amylase's +138 position. Mutants at the tryptophan residue (substitution) may be generated on their own or together with mutations at other oxidizable amino acid residues. In particular, it may be advantageous to modify one or more tryptophan residues by replacing them with one or more methionine residues. (E.g., double mutations at the +138 / +197 position)

In general, the alpha-amylase mutants included in the laundry detergents of the present invention have a degree of oxidative stability in the presence of hydrogen peroxide and other oxidants such as, for example, bleach or peracids, or more particularly oxidants such as chloramine-T. Modified. Mutant enzymes with enhanced oxidative stability will be useful for prolonging shelf life and enhancing compatibility with bleach, perborate, percarbonate or peracid of amino acids used in wash products. Thus, preferred embodiments of the present invention include laundry detergents comprising the alpha-amylase variant of the present invention and further comprising a bleach or peroxide compound. Particularly preferred embodiments of the invention are laundry detergents comprising a mutation α-amylase according to the invention having a pH of at least about 10 and more preferably between about 10 and about 12. Also preferred are granular laundry detergents having a pH between RK DIR 10-DIR 12 and further containing a bleach or peroxide compound.

In addition, the mutant enzymes according to the invention are surprisingly characterized by a much better activity in the neutral pH range compared to wild-type or less progressive amylases. Thus, another preferred embodiment of the present invention comprises a laundry detergent composition comprising a mutant alpha-amylase having a pH between about 5.0 and about 10.0, more preferably between 6.0 and about 10.0. Most preferred embodiments are liquid laundry detergents having a pH between about 6.0 and about 10.0.

Similarly, it may be useful for industrial processes required to effectively and rapidly deplete enzymatic activity. The mutant enzymes of the present invention also have a broad pH range such that mutants such as M15L show stability in low pH starch liquefaction and mutants such as M197T show stability under high pH wash product conditions. In addition, the mutant of the present invention may be thermal stability is changed to enhance the stability of the mutant at high or low temperature. Any change in the enzymatic properties of the mutant may be beneficial, depending on its intended purpose and for the manufacture of a laundry detergent comprising mutant alpha-amylase relative to its precursor.

Preferred laundry detergents of the present invention are b. Reikoni Formis, B. Amyloliques, and b. It is obtained from Bacillus species, such as Mophilus as stearate and most preferably Bacillus Lycoformis.

A first aspect of the invention is a b. There is provided a laundry detergent comprising a novel form of alpha-amylase normally produced by Lycoenformis. This novel form, represented by type A 4, has an additional four alanine residues at the N-terminus of secretory amylase. (See FIG. 4B) A mutant or derivative of the A 4 form of alpha-amylase is included in the present invention. A mutant or derivative of the A 4 form includes one or more alpha-amylase-containing additional mutants of one or more A 4 types, such as, for example, one or more oxidizable amino acid mutations (by substitution, substitution or deletion). I mean.

Embodiments of the compositions of the present invention provide laundry detergent compositions, liquids, gels or granules comprising the alpha-amylase mutants described herein. Particularly preferred are detergent compositions comprising only the mutant at the +197 position or together with other enzymes such as endoglucosidase, cellulase, protease, lipase or other amylase enzymes.

Especially preferred are laundry detergent compositions comprising M15X / W138X / M197X mutants, most preferably M15T / W138Y / M197T (TYT) mutants. In addition, the compositions of the present invention may include alpha-amylase mutants in which one or more position-specific mutations have occurred.

According to the method which is an embodiment of the present invention, the laundry detergent composition of the present invention is used in a method for washing contaminated laundry.

Other embodiments of the compositions of the present invention provide compositions useful for starch processing and in particular starch liquefaction. The starch liquefaction composition of the present invention preferably comprises alpha-amylase mutants by substitution or deletion at the M 15 position. In addition, such compositions include additional ingredients known to those skilled in the art, such as antioxidants, calcium, ions, and the like.

The present invention is a novel alpha-amylase mutant with an amino acid sequence not found in nature, wherein the amino acid residues of one or more alpha-amylase precursors, in particular alpha oxidized amino acids, are substituted with different amino acids. Pertains to amylase mutants. Mutant enzymes of laundry detergents of the present invention are modified in stability and activity, specifically, but not limited to, oxidative stability, pH-dependent performance, specific activity and / or heat resistance.

1A-1C are ratios. Representing the DNA sequence of the alpha-amylase gene obtained from Lyconformformis (NCIB 8061), by Gray, G. and his colleagues (1986) J. Bacter. 166: 635-643, SEQ ID NO: 31 and its translation product by inference.

2 is rain. SEQ ID NO: 32 is the amino acid sequence of the mature alpha-amylase enzyme obtained from Lyconformformis (NCIB 8061).

3A-3B show the alignment of the primary structure of Bacillus alpha-amylase. Said rain. Lyconformyl amylase (Am-Lich), SEQ ID NO: 33, by Gray, G. and his colleagues (1986), J. Bact. 166: 635-643; ratio. Amylolycanisms Amylase (Am-Amylo), SEQ ID NO: 34 is described by Takkinen, K. and his colleagues (1983), J. Biol. Chem. 258: 1007-1013; ratio. Am-Stearo, SEQ ID NO: 35 is described by Ihara, H and his colleagues (1985) by J. Biochem. 98: 95-103.

4A shows the amino acid sequence of the mature alpha-amylase variant M 197 T, showing SEQ ID NO: 36.

4B is rain. The amino acid sequence of the A4 type of alpha-amylase obtained from Lycoformform NCIB 8061 is shown in SEQ ID NO: 37. Numbering started at the N-terminus starting with 4 additional alanines.

Figure 5 shows pA4BL, BLA4 is ratio. Lymponiformis alpha-amylase gene, Amp ^R between PstI and SstI, is an ampicillin resistance gene obtained from pBR 322; And CAT are chloro amphenicol resistant genes obtained at pC 194.

6 is rain. Lycony Formis (SEQ ID NO: 38), B. Subtilis (SEQ ID NO: 39), rain in pA4BL. Lyconformformis (SEQ ID NO: 40) and pBLapr endocrine. Signal sequence of Lycoformforme (SEQ ID NO: 41)-indicates the junction of the mature protein.

7A shows the inactivation of some alpha-amylases. [Spezyme brand AA20 and M197L (type A4) were treated with 0.88 MH ₂ O ₂ at pH 5.0, 25 ° C.]

7B shows the inactivation of some alpha-amylases. [Spezyme brand AA20 and M197T (type A4) were treated with 0.88 MH ₂ O ₂ at pH 5.0, 25 ° C.]

7C shows the inactivation of some alpha-amylases. [Spezyme brand AA20 and M197L (type A4) were treated with 0.88 MH ₂ O ₂ at pH 5.0, 25 ° C.]

8 is a schematic diagram of a method for generating an M197X cassette mutant.

9 shows expression level of M197X variants.

Figure 10 shows the heat resistance of M197X variant treated with 5 mM CaCl ₂ for 5 minutes at pH 5.0, 95 ℃.

11A and 11B show ball activity of some amylases in automatic dish protector detergents. FIG. 11A shows the stability of some amylase in a Cascade trademark product (commercially available dish protection product), at 65 ° C., when starch is present or no starch is present. FIG. 11B shows the stability of some amylase in Sunlight brand products (commercially available tableware products) when starch is present or absent at 65 ° C. FIG.

12 shows a schematic diagram of a method for producing an M15X cassette.

13 shows expression of M15X variants.

14 shows the specific activity of M15X variants on soluble starch.

FIG. 15 shows the heat resistance of M15X variant treated with 5 mM CaCl ₂ at 90 ° C., pH 5.0 for 5 minutes.

FIG. 16 shows the wild type ratio of jet liquefaction performance at pH 5.5 and specific activity of starch and soluble substrate of M15 variant. It is expressed as a% activity function of Lyconformis.

17 is rain. Inactivation with chloramine-T treatment at pH 8.0 when Lyconformyl alpha-amylase (AA20 at 0.65 mg / ml) was compared to M197A (1.7 mg / ml) and M197L (0.53 mg / ml) Indicates.

18 is rain. Inactivation with chloramine-T treatment at pH 4.0 when Lyconformis alpha-amylase (AA20 at 0.22 mg / ml) was compared to M197A (4.3 mg / ml) and M197L (0.53 mg / ml) Indicates.

19 is rain. Lyamineformas alpha-amylase (AA20 at 0.75 mg / ml) compared to the double variants M197T / W138F (0.64 mg / ml) and M197T / W138Y (0.60 mg / ml) Reaction in the case of T treatment is shown.

FIG. 20 shows the results of testing the stability of various alpha-amylase multiple mutants included in automatic dish detergent (ADD) formulations at elevated temperatures from room temperature to 65 ° C. FIG.

FIG. 21 shows the stability (as compared to the wild type) of the amylase mutants of the seedlings measured by% residual activity over time for 0-30 days at room temperature.

FIG. 22 shows the stability (as compared to wild type) of some amylase mutants in automatic dishwashing detergent at 38 ° C. (100 ° F.) at 80% relative humidity for 0-30 days.

FIG. 23 shows the pH activity graphical form of any amylase on Phadebas substrate at 25 ° C. and neutral and alkaline pH.

24 shows the stability of peracetic acid versus any amylase over time at 52 ° C. and pH 9.3.

FIG. 25 shows reflectance (delta form) vs. teratyl amylase at 40 ° C. compared to Termamyl amylase. FIG. The relative washing ability of amylase (TYT) according to the invention in liquid laundry detergent in terms of ppm of amylase added is indicated.

FIG. 26 shows reflectance (delta) vs. The relative washing ability of amylase (TYT) according to the invention in liquid laundry detergent in terms of ppm of amylase added is indicated.

27 is reflectance (delta) vs. The cleaning performance of amylase according to the present invention, which is commercially available in terms of added amylase ppm, is shown.

The amylase used for starch liquefaction may be inactivated due to the activity present in the starch slurry. (See US Patent Applications 07 / 785,624 and 07 / 785,623 and US Patent 5,180,669, issued January 19, 1993 and incorporated herein by reference.) Furthermore, such as bleach- or peracid-containing detergents Amylase can be partially or completely inactivated when amylase is used when an oxidant is present. Accordingly, the present invention focuses on modifying the sensitivity of amylases to oxidizers added to laundry detergents. Mutant enzymes in laundry detergents of the present invention may also modify the pH properties and / or heat resistance by enhancing the oxidative stability of the enzyme at low or high pH.

Alpha-amylases as used herein include recombinant amylases as well as naturally occurring amylases. Preferred amylases of the present invention are: Ricanformis or b. Alpha-amylase obtained from Stearmophilus, the ratios described herein. Aspergillus [i.e., A4 type, as well as the alpha-amylase obtained from Lyconformformis. Oraijae and A. Fungal alpha-amylase, such as those obtained from A. niger.

Recombinant alpha-amylase refers to alpha-amylase wherein the DNA sequence encoding the naturally occurring alpha-amylase is modified to produce a mutant DNA sequence encoding the substitution, insertion or deletion of one or more amino acids in the alpha-amylase sequence. Suitable modification methods are described herein and also described in U.S. Pat. Patents 4,760,025 and 5,185,258, which are incorporated herein by reference.

Homology is observed between almost all endo-amylases, from plants to mammals and bacterias. Appl. By Nakajima, R. T. and his colleagues. Microbiol. Biotechnol. (1983), 23: 355-360; Rogers, J. C. (1985) Biochem. Biophys. Res. Commun, 128: 470-476] As shown in FIG. 3, there are four particularly high homology regions of the Bacillus amylase, and the underlined part of FIG. 3 is a high homology region. Moreover, sequence alignment can be used to map the relationship between Bacillus endo-amylase. J. Molec. By Feng, D. F. and Doolittle, R. F. (1987). Evol. 35: 351-360] b. Steamorphus and b. The relative sequence homology between Lycanformyl amylase is about 66%, as measured by Holm, L. and his colleagues (1990), based on Protein Engineering 3 (3) pp 181-191. ratio. Lycony Formis and B. The homology of the sequences between amylolyases amylase was about 81%, measured by Holm, L. and his colleagues. In addition to the importance of homology between sequences, it is generally understood that structural homology is also important in comparing amylases or other enzymes. For example, structural homology between fungal amylase and bacteria (bacillus) amylase has been suggested and fungal amylase is therefore within the scope of the present invention.

Alpha-amylase mutants have an amino acid sequence obtained from the amino acid sequence of the precursor alpha-amylase. Alpha-amylase precursors include natural occuring alpha-amylases and recombinant alpha-amylases (as defined above). The amino acid sequence of the alpha-amylase mutant is obtained from the precursor alpha-amylase sequence by replacing, deleting or inserting one or more amino acids of the precursor amino acid sequence. Modifying the precursor DNA sequence encoding the amino acid sequence of precursor alpha-amylase is in itself better than manipulating the precursor alpha-amylase enzyme. Suitable methods for engineering precursor DNA sequences are described herein and in U.S. Pat. Methods described in patents 4,760,025 and 5,185,258.

The positions of the specific residues corresponding to M197, M15 and W138 of Bacillus lycoformis alpha-amylase, ie methionine, histidine, tryptophan, cysteine and tyrosine positions, for substitution or deletion were identified herein. The position number of the amino acids (ie +197) is the number added to the mature Bacillus lycan Formosa alpha-amylase sequence shown in FIG. 2. However, the present invention is not limited to mutations of specific mature alpha-amylases (B. lycoformforms) but specifically identified. The range extends to precursor alpha-amylases containing amino acid residues that are at the same position as Lycanformis alpha-amylase residues. If the precursor alpha-amylase residue is homologous (ie, coincides with a position in the primary or tertiary structure) or is non. If the position or specific moiety of the Lycoformis alpha-amylase residue is similar (ie, the same or similar in ability to bind, react or interact chemically or structurally), the precursor alpha-amylase residue (amino acid) is ratio. The same as Lycanformis alpha-amylase residues.

To form homology in the primary structure, the amino acid sequence of the precursor alpha-amylase is non. Lycanformis alpha-amylase primary sequence, specifically as shown in FIG. 3, can be directly likened to a combination of residues known as constants in all known alpha-amylases. The same residues can also be determined in tertiary sphere: porcine pancreatic alpha-amylase [Buisson, G. and his colleagues. (1987) EMBO J. 6: 3909-3916; Taka-Aspergillus Oraijae-Amylase A [Matsuura, Y. and his colleagues, (1984) J. Biochem. (Tokyo) 95: 697-702; And a. Niger's acid alpha-amylase (Boel, E. and his colleagues, (1990) Biochemistry 29: 6244-6249) has been reported for the crystal structure. (The two structures of the former are similar to each other.) A common super-secondary structure was found between glucanases [MacGregor, E.A. And Svensson, B. (1989) Biochem. J. 259: 145-152]. The structure of Bacillus alpha-amylase is unknown, although the structure of Steamophilus glucinase is based on the enzyme structure of taka-amylase A.

Three highly conserved regions are shown in FIG. 3, which represent many residues (His105; Arg 229; Asp 231; His 235; Glu 261 and Asp 328) that are thought to be part of the active-position in Lyconformis. Containing the part numbered). Matsrra, Y. and his colleagues (1984) J. Biochem. (Tokyo) 95: 697-702; Buisson, G. and his colleagues (1987) EMBO J. 6: 3909-3916; Vihinen, M. and his colleagues (1990) J. Biochem. 107: 267-272]

As used herein, an expression vector refers to a DNA construct containing a DNA sequence operably linked to a suitable control sequence capable of carrying out the expression of said DNA in a suitable host. Such control sequences can include a promoter that performs transcription, any operator sequence that regulates such transcription, a sequence that encodes an appropriate mRNA ribosomal-binding site, and a sequence that can control termination of transcription and translation. Preferred promoters are b. Subtilis arpE promoter. The vector can be a plasmid, phage granules or simply a potential gene insertion sequence. Once the vector is transformed into a suitable host, the vector can replicate and function independently of the host's genes, or in some cases be incorporated into the host genes. In the present specification, plasmids and vectors can sometimes be used interchangeably and plasmids are the form of the vector most commonly used in the present invention. However, the present invention is intended to include other forms of expression vectors that have the same function and are known or will be known in the art.

Host strains (or host cells) useful in the present invention generally include transformable microorganisms capable of expressing alpha-amylase as prokaryotic or eukaryotic cell hosts. In particular, host strains of the same species or the same gene from which the alpha-amylase can be obtained, such as Bacillus strains, are suitable. Preferably alpha-amylase negative Bacillus strain (deleted gene) and / or Bacillus subtilis strain BG 2473 ( amyE, apr, Bacillus strains that do not produce alpha-amylases and proteases such as npr) are used. Host cells are transformed or transfected with vectors constructed by recombinant DNA techniques. Such transformed host cells can replicate a vector encoding alpha-amylase and its variants (mutants) or can express the desired alpha-amylase.

Preferably the mutant of the invention may be secreted into the medium during the fermentation process. It can be secreted by some signal sequence such as aprE signal peptide.

Many of the alpha-amylase mutants of the invention are useful for preparing various detergent compositions, in particular some dish protection cleaning compositions, in particular cleaning compositions containing known oxidants, such as, for example, bleaches or peroxide compounds. The alpha-amylase of the present invention may be formulated in a known powdered, liquid or gel form detergent having a pH between 4.5 and about 12.0, preferably between about 5.0 and about 10.0 and most preferably between about 6.0 and about 10.0. . Further preferred embodiments include laundry detergents having a pH between about 10.0 and about 12.0, wherein bleach is present in the composition. Suitable granular amylase-containing compositions are described in U.S. Pat. It may be prepared as described in patent applications 07 / 429,881, 07 / 533,721 and 07 / 957,973. The cleaning composition, which is a detergent, may also contain other enzymes such as known proteases, lipases, cellulase, endoglycosidase or other amylases, as well as adjuvants, stabilizers or other excipients known to those skilled in the art. . The enzymes may be provided in a co-granule or blended mix or in other ways known to those skilled in the art. Moreover, it is contemplated by the present invention that multiple mutants may be useful for washing or other methods. For example, mutant enzymes that change positions +15 and +197 can enhance performance useful for wash products, or multiple mutations that change positions +197 and +138 can improve performance. have. Particularly preferred mutant enzymes for washing products, in particular for dish protection preparations, include M15T / M197T; M15S / M197T; W138Y / M197T; M15S / W138Y / M197T; And M15T / W138Y / M197T.

Other embodiments of the present invention include the combination of the mutant alpha-amylase enzymes described herein with other enzymes (ie proteases, lipases, cellulase, etc.), preferably oxidatively stable proteases. Suitable proteases that are stable to oxidation are described in U.S. Pat. Genetically engineered proteases as described in heading 34606, as well as commercially available enzymes such as, for example, DURAZYM (Novo Nordisk), MAXAPEM (Gist-brocades) and PURAFECT OXP (Genencor International, Inc.). . Such protease mutants (oxidative stable proteases), specifically b. Suitable methods for preparing methionine-substituted mutants at the same position as M222 in amyloliase are described in U.S. Pat. No. 34606. Suitable methods for determining equivalent positions in other subtilisins are described in U.S. Pat. 34606, EP 257,446 and USSN 212,291.

Abbreviations used herein, specifically three or one letter words for amino acids, are described in Dale, J.W., Molecular Genetics of Bacteria, John Wiley Sons, (1989) Appendix B.

Example 1

ratio. Substitution of Methionine Residues of B. licheniformis alpha-amylase

Alpha-amylase gene (FIG. 1) is a ratio obtained from the National Collection of Industrial Bacteria, Aberdeen, Scotland (Gray, G. and his colleagues (1986) J. Bacteriology 166: 635-643). It was cloned in Lycoformform NCIB8061. A 1.72 kb PstI-SstI fragment encoding the last 3 residues of the signal sequence; Fully mature protein and terminator sites were subcloned with M13MP18. The synthetic terminator was inserted between the BclI and SstI positions using a synthetic oligonucleotide cassette of the form

[SEQ ID NO: 1]

SEQ ID NO: 1 is non. It is designed to contain the B. amylolichefacience subtilisin transcription terminator. Wells and his colleagues (1983) Nucleic Acid Research 11: 7911-7925]

In essence, site-directed mutagenesis by oligonucleotides includes Zoller, M. and his colleagues (1983) Meth. Enzymol. The method of 100: 468-500 is used: in short. 5′-phosphorylated oligonucleotides to introduce the desired mutants on the M13 1-stranded DNA template using oligonucleotides described in Table 1 to replace each of the seven methionines found in Lycanformis alpha-amylase. Primers are used. Each mutant oligonucleotide was also inserted at the restriction endonuclease site to screen for relevant mutations.

TABLE 1

ratio. Mutant oligonucleotides for substituting methionine residues of Lycanformis alpha-amylase.

[SEQ ID NO: 2]

[SEQ ID NO: 3]

[SEQ ID NO: 4]

[SEQ ID NO: 5]

[SEQ ID NO: 6]

[SEQ ID NO: 7]

[SEQ ID NO: 8]

[SEQ ID NO: 9]

* Bold letters indicate base changes introduced by oligonucleotides.

Codon changes were expressed in the form of M8A, and methionine (M) at +8 position was changed to alanine (A).

* Underlined parts indicate restriction endonuclease action sites introduced by oligonucleotides.

Heterogeneous heteroprints. The clones were used to transfect E. Coli mutL cells (Karmer and his colleagues (1984), Cell 38: 879), after plaque-purification and clones were analyzed by restriction analysis of RF1. . Dideoxy sequencing confirmed the presence of RF1 [Sanger and his colleagues (1977), Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467] each of the PstI-SstI fragments is composed of 2. Subcloned into plasmid pA4BL, an E. coli vector.

[Plasmid pA4BL]

U.S., which is incorporated herein by reference. In the following manner described in patent application 860,468, a silent PstI position was introduced into codon +1 (the first amino acid present after the signal cleavage site) of the aprE gene obtained from pS168-1. Stahl, M.L. And Ferrari, E. (1984) J. Bacter. 158: 411-418] and then the aprE promoter and signal peptide sites were cloned from the pJH101 plasmid like the HindIII-PstI fragment and then [Ferrari, F.A. And his colleagues (1983) J. Bacter. 154: 153-1515, and subcloned with pUC18-induced plasmid JM102. Ferrari, E. and Hoch, J.A. (1989) Bacillus, ed. C.R. Harwood, Plenum Pub., Pp 57-72]. ratio. PstI-SstI fragments obtained from Lycoformamis alpha-amylase were added to generate pA4BL with aprE signal peptide-amylase linkage site as shown in FIG. 6 (FIG. 5).

[ratio. Transformation to B.subtillis]

pA4BL is E. Replicate in E. coli. It is a plasmid that can be integrated into the subtilis chromosome. Plasmids containing different variants are compared. After transformation into subtilis [Anagnostopoulis, C. and Spizizen, J. (1961) J. Bacter. 81: 741-746], which was integrated into the aprE position of the chromosome by the Cambell-type mechanism. Young, M. (1984) J. Gen. Microbiol. 130: 1613-1621] The Bacillus subtilis strain BG 2473 is an amylase ( amyE) and 2 proteases ( apr, npr) is an I 168 derivative deleted. Stahl, ML and Ferrari, E., J. Bacter. 158: 411-418 and US Pat. No. 5,264,366, incorporated herein by reference, after transformation of sacU32 (Hy) [Henner, DJ and his colleagues (1988) J. Bacter. 170: 296-300] Mutations were introduced by PBS-1 mediated transduction. Hoch, JA (1983) 154: 1513-1515.

ratio. N-terminal analysis of amylase expressed in pA4BL in the subtilis revealed that the amylase was processed and had four external alanines at the N-terminus of the secreted amylase protein (type A). The external residues do not have any significant and detrimental effects on the heat resistance or activity of A 4 type and in some cases may enhance performance. In the subsequent experiments, correctly ordered forms of Lyconformyl amylase and variant M197T were prepared in a very similar construction. (See Figure 6)

In particular, subcloning from pA4BL (FIG. 5) to M13BM20, ie, the 5 'end of the A 4 construct, onto the EcoRI-SstII fragment to obtain the coding strand template for the mutant oligonucleotides described below. I was. Boehringer Mannheim

[SEQ ID NO: 10]

5'-CAT CAG CGT CCC ATT AAG ATT TGC AGC CTG CGC AGA CAT GTT GCT-3 '

This primer removed the codons of the four outer N-terminal alanines (the correct form screened due to the absence of the PstI position). The EcoRI-SstII fragment was subcloned back into the pA4BL vector, resulting in plasmid pBLapr. The M197T substituent on the SstII-SstI fragment was then separated from pA4BL (M197T) and inserted into the complementary pBLapr vector to generate plasmid pBLapr (M197T). ratio. N-terminal analysis of amylase expressed in pBLapr in subtilis revealed that the enzyme was cleared. It was found that it has the same N-terminus as observed in Lycanformis alpha-amylase.

Example 2

Oxidative Susceptibility of Methionine Variants

Non-Spezyme brand AA20 products such as those sold by Genencor International. Lyconformis alpha-amylase was rapidly inactivated with hydrogen peroxide. (FIG. 7) Rain several methionine variants. The crude supernatant was isolated by expression in subtilis shake flask medium and dissolved in ammonium sulfate. The amount of ammonium sulphate in 20% saturated ammonium sulphate supernatant was increased to saturation to 70% and the amylase precipitated and then resuspended. The variant was then exposed to 0.88 M hydrogen peroxide at pH 5.0, 25 ° C. When substituted at the +197 position (M197L) to show resistance to peroxide oxidation; Six methionine variants of Lyconformyl alfa-amylase were continuously oxidized to peroxides. (See FIG. 7)

However, analysis as described in more detail below showed that if the variant could be sensitive to oxidation at pH 5.0, 25 ° C., the properties could be modified and enhanced under different conditions. (E.g. liquefaction)

Example 3

Construction of all possible variants at position 197

As summarized in FIG. 8, cassette mutagenesis generated all M197 variants (M197X) in A4 form.

1) Site directed mutagenesis (by primer extension in M13) was used to prepare M197A using the mutant oligonucleotides as follows.

[SEQ ID NO: 11]

The EcoRV position (codon 200-201) can also be inserted by rearranging the ClaI positions (codons 201-202).

2) Another silence restriction site (BstBI) was then inserted across codons 186-188 using primers LAAM 12 (Table II).

3) The resulting M197A (BstBI ⁺ , EcoRV ⁺ ) variants were subcloned (PstI-SstI fragments) into plasmid pA4BL, and the resulting plasmids were cleaved with BstBI and EcoRV to elute large vector-containing fragments on an agarose gel. electroelution).

4) Synthetic primers LAAM14-30 (Table II) were each annealed with a common primer LAAM13 (Table II), mostly complementary. The resulting cassettes encoded all remaining naturally occurring amino acids at the +197 position and individually bound them to the vector fragments prepared above.

TABLE II

Synthetic oligonucleotides used in cassette mutagenesis to generate M197X variants.

[SEQ ID NO 12]

[SEQ ID NO: 13]

[SEQ ID NO: 14]

[SEQ ID NO: 15]

[SEQ ID NO: 16]

[SEQ ID NO: 17]

[SEQ ID NO: 18]

[SEQ ID NO: 19]

[SEQ ID NO: 20]

[SEQ ID NO: 21]

[SEQ ID NO 22]

[SEQ ID NO: 23]

[SEQ ID NO: 24]

[SEQ ID NO: 25]

[SEQ ID NO 26]

[SEQ ID NO: 27]

SEQ ID NO: 28

SEQ ID NO: 29

[SEQ ID NO: 30]

It was designed to destroy the EcoRV position by the combination of the cassettes. The specific positions of the plasmids obtained from E. coli transformants were lost and screened. Moreover, the common bottom strand of the cassette contained a frame shift and encoded the NsiI position, so that the transformants derived from the chain were screened and separated using the properties of specific NsiI positions. And in some cases could not be expected to express active amylase.

Those that were shown to be positive by restriction assays were confirmed by sequencing and ratios in shake-flask medium to express. Transform into subtilis. (FIG. 9) Thereafter, the soluble substrate assay was used to determine the specific activity of the M197A mutant of the seedlings. The data obtained using the following assay is shown in Table III.

[Soluble substrate assay]

The method is described in Megazyme (Aust.) Pty. Ltd. A rate-dependent assay developed on the basis of the endpoint assay kit supplied by. Each glass bottle containing the substrate (p-nitrophenyl maltoheptaside, BPNPG 7) was dissolved in 10 ml of sterile water and then assay buffer (50 mM maleate buffer, pH 6.7, 5 mM calcium chloride, 0.002%). Dilution 1-4 times with Tween20). The assay was performed by adding 10 μl amylase to a 790 μl substrate in a 25 ° C. cuvette. The rate of hydrolysis could be measured by the change in absorbance at 410 nm after being left for 75 seconds. The speed of this assay could be expressed as a linear straight line up to 0.4 absorbance units / min.

The amylase protein concentration was determined using Bradford, M. (1976) Anal. Biochem. It was measured by a standard Bio-Rad assay (Bio-Rad Laboratories) based on the method of 72: 248.

[Starch Hydrolysis Assay]

Standard methods were used to assay the activity of alpha-amylase, AA20 of the Spezyme brand. The method is described in detail in Example 1 of USSN 07 / 785,624 and incorporated herein by reference. Natural starch formed blue by iodine, but did not form blue when starch was hydrolyzed to shorter dextrin molecules. The substrate is 5 mg / liter of soluble Lintner starch in phosphate buffer (42.5 mg / liter potassium dihydrogen phosphate, 3.16 mg / liter sodium hydroxide) in pH 6.2. The samples were added to 25 mM calcium chloride and then negatively tested at 30 ° C. to measure activity over time. The activity was expressed as liquefon (LU) per g or ml by the following formula.

[Wherein, LU = liquefon unit, V = sample volume (5 ml), t = dextrinization time (minutes), D = dilution factor = dilution volume / ml or dilution volume of the added enzyme / g]

TABLE III

Example 4

Characteristics of variant M 15 L

The variant M 15 L obtained per previous example did not increase amylase activity (Table III) and was subsequently inactivated by hydrogen peroxide. (FIG. 7) However, the zet-liquefaction performance of starch, especially at low pH as shown in Table IV, was significantly increased.

Starch liquefaction is typically performed using a Hydroheater M103-M steam injection machine with a 2.5 liter delay coil mounted behind a mixing chamber and a terminal back pressure valve. Starch was supplied to the injector by a Moyno pump and the steam was supplied by a 150 psi steam line to 90-100 psi. The temperature detector was installed just behind the Hydroheater injector and just in front of the back pressure valve.

The starch slurry was obtained in a corn wet mill and used within two days. The starch solids content was distilled out with demineralized water until the desired amount and the pH of the starch was adjusted to 2% NaOH or saturated Na ₂ CO ₃ . Typical liquefaction conditions are as follows:

Starch = 32-35% solids

Calcium = 40-50 ppm (30 ppm added)

pH = 5.0-6.0

Alpha-Amylase = 12-14 LU / g Starch Anhydrous

Starch was introduced into the injection machine at a rate of about 350 ml / min. The injection machine temperature was maintained at 105-107 ° C. The starch sample was sent to a second liquefaction step at 95 ° C. in an injection vessel and left for 90 minutes.

After the second liquefaction step, the dextrose equivalent (DE) of the sample was measured and tested for the presence of raw starch [both of the above methods were used in both Standard Analytical Methods of the Member Companies of the Corn Refiners Association, Inc. By the method described in the sixth edition.] The degree of liquefaction of starch was immediately measured. Starches generally treated under the conditions given above and at pH 6.0 will be liquefied starch which is about 10 DE and does not contain raw starch. The liquefaction test results using the mutants of the invention are shown in Table IV.

TABLE IV

Example 5

Construction of M15X Variants

Natural ratios by the cassette mutation generation method outlined in FIG. 12, which are generally the steps described in Example 3 above. All M15 (M15X) variants were generated in Lyconformis.

1) Directed mutagenesis (via primer expansion in M13) was used to introduce specific restriction sites flanked by M15 codons that facilitate insertion of mutagenic cassettes. In particular, the BstB1 position of codons 11-13 and the Msc1 position of codons 18-20 were introduced using the following two oligonucleotides.

[SEQ ID NO: 48]

[SEQ ID NO: 49]

2) The vector used for the M15X cassette mutation generation method was constructed by subcloning the Sfil-SstII fragment with plasmid pBLapr in the mutated amylase (BstB1 +, Msc1 +). The resulting plasmid was then cleaved in BstB1 and Msc1 and the large vector fragments were isolated by electroelutation from polyacrylamide gels.

3) Mutagenesis Cassettes were made of M197X variants. Synthetic oligomers, each encoding a codon 15 substitution, was annealed to a common bottom primer. As the cassette properly binds to the vector, the Msc1 position was destroyed in view of the loss of the Msc1 position to screen positive transformants. The basal primer contained a specific SnaB1 position in view that the transformant derived from the basal primer was removed by screening for the SnaB1 position. The primer also inhibited the expression of amylase and contained a frameshift induced in a common basal chain.

The synthetic cassette is listed in Table V and the general cassette mutagenesis is shown in FIG. 12.

TABLE V

Synthetic oligonucleotides used in cassette mutagenesis to generate M15X variants.

[SEQ ID NO: 50]

[SEQ ID NO: 51]

SEQ ID NO: 52

[SEQ ID NO: 53]

[SEQ ID NO: 54]

[SEQ ID NO: 55]

[SEQ ID NO 56]

[SEQ ID NO: 57]

SEQ ID NO: 58

[SEQ ID NO: 59]

[SEQ ID NO: 60]

* Underlined indicates changes in amino acid codon at position 15.

Conservative substitution occurs in some cases to prevent the introduction of new restriction sites.

Example 6

Bench liquefaction using the M15X variant

11 alpha-amylase variants obtained by substitution in M15 prepared as in Example 5 using a bench liquefaction system during pH 5.5, liquefaction were compared to AA20 (available from Genencor International, Inc.) under the Sqezyme brand. Activity was assayed. The bench scale liquefaction system is a stainless steel coil (0.25 inch in diameter) with a static mixing element about 7 inches in length about 12 inches from the front end and a 30 psi back pressure valve at the rear end. 350 ml). The coils were immersed except for their respective end portions in a glycerol-water bath equipped with a heating portion controlled by a thermostat maintained at a temperature of 105-106 ° C.

The enzyme containing starch slurry, which is kept in suspension by stirring, is about 70 ml / min by means of a metering pump controlled by a piston. Was introduced. The starch was recovered from the end of the coil and left to stand (95 ° C. for 90 minutes). Immediately after the above standing, the DE of the liquefied starch was measured as in Example 4. The results are shown in FIG.

Example 7

Features of the M197X Variants

As can be seen in FIG. 9, it can be seen that the amylase activity (measured in the soluble substrate assay) expressed by the M197X variant (type A4) is widely distributed. The amylase was partially purified from the supernatant by precipitation with 2 volumes of ethanol and then resuspended. It was then screened for heat resistance by heating to 95 ° C. for 5 minutes in 10 mM acetate buffer, pH 5.0 in the presence of 5 mM calcium chloride (FIG. 10); After warming, A4 wild type activity remained 28%. In M197W and M197P, no active protein could be recovered from the supernatant. Sequencing revealed that the M197H variant contained N190K, which is the second mutation. M197L was tested separately and found to be the most inferior variant of heat resistance. This indicates that the correlation between amylase activity and heat resistance expression is very large. Lyconformyl amylase was limited at residues that can be regulated at position 197 to enhance or maintain heat resistance. : Under these conditions, cysteine and threoine were preferably at maximum heat resistance, while the stability of alanine and isoleucine was transient. Moreover, different substituents at +197 had other advantages, such as modified performance on pH or modified oxidative stability. For example, it can be seen that the M197L variant was easily inactivated by air oxidation and enhanced heat resistance. In other words, compared to the M197L variant, both M197T and M197A not only had high heat resistance (Figure 10) but also high activity (Table III), while maintaining resistance to inactivation by peroxides at pH 5-10. (Figure 7)

Example 7

Stability and Performance in Detergent Formulations

The stability of M197T (Type A4), M197T and M197A (Type A4) was measured with an automatic dish protection detergent (ADD) substrate. A 2 ppm Savinase brand product (protease commercially available from Novo Industries, commonly used in ADD form) was added to two commercially available bleach-containing ADDs: a product of the Cascade brand (Procter and Gamble, Ltd.). .) And Sunlight brand (Unilever) and amylase variant inactivation process and Termamyl brand (Novo Nordisk, A / S commercially available heat-resistant alpha-amylase) was left at 65 ℃. The concentration of the ADD product used in both cases was equal to the 'pre-soak' state: 14 gm of product per liter of water (hardness of 7 grams per gallon).

As can be seen in FIGS. 11A and 11B, both variants of M197T were more stable than the Termamyl brand product and M197A (type A4), which were inactivated after the first assay was performed. The benefit of this stability is observed when starch is present or absent as measured by the following method. After stirring, preheated amylase in vitro was added to 5 ml of ADD and Savinase branded products, and then activity was measured as a function of time using a soluble substrate assay. In the starch tube there was spaghetti starch baked on the side (140 ° C., 60 minutes). The results are shown in FIGS. 11A and 11B.

Example 9

Features of the M15X Variant

All M15X variants were propagated in Bacillus subtilis and the expression level was observed as shown in FIG. 13. Amylase was separated and then partially separated by dissolution in 20-70% ammonium sulfate. The specific activity of these variants on soluble substrates was measured as in Example 3. (FIG. 14) Most of the M15X amylases were greater than the specific activity of the Spezyme brand AA20. A benchtop heat stability assay was performed on the variants by heating amylase at 90 ° C. for 5 minutes in 50 mM acetate buffer, pH 5 in the presence of 5 mM CaCl ₂ . (FIG. 15) The assay was performed on most of the variants as in AA20 of the Spezyme brand. Variants exhibiting adequate stability in the assay (suitable stability means having at least about 60% heat resistance of the Spezyme trademark AA20 product) were tested for specific activity against starch and liquefaction performance at pH 5.5. The most interesting of these mutations are shown in FIG. 16. The liquefaction performance at pH 5.5 with M15D, N and T together with L was superior to that of the Spezyme brand AA20 product and the specific activity in the soluble substrate and starch hydrolysis assay was increased.

In general, substitution of methionine at position 15 could provide variants with low pH-liquefaction performance and / or increased specific activity.

Example 10

Tryptophan's susceptibility to oxidation

Chloramine-T (sodium N-chloro-p-toluenesulfonimide) is a selective oxidizing agent that oxidizes methionine to methionine sulfoxide at neutral or alkaline pH. At acidic pH, chloramine-T is changed to methionine and tryptophan. (Schechter, Y., Burnstein, Y. and Patchornik, A. (1975) Biochemistry 14 (20) 4497-4503) FIG. 17 shows the ratio at pH 8.0. Inactivation of Lycanformis alpha-amylase by chloramine-T is shown. (AA20 = 0.65 mg / ml, M197A = 1.7 mg / ml, M197L = 1.7 mg / ml) The data indicate that inactivation of alpha-amylase can be prevented by changing methionine at position 197 to leucine or alanine. In other words, as shown in FIG. 18 (FIG. 18: AA20 = 0.22 mg / ml, M197A = 4.3 mg / ml, M197L = 0.53 mg / ml; 200 mM NaAcetate pH 4.0) At pH 4.0, the inactivation of M197A and M197L It proceeds, but it requires more than the equivalent of chlormine-T. This suggests that tryptophan residues are involved in the phenomenon of inactivation by chloramine-T. Moreover, amino acid sequencing by tryptic mapping indicated that tryptophan at position 138 was oxidized by chloramine -T. To demonstrate this (although no data), site-directed mutations were performed on tryptophan 138 as follows.

Preparation of Alpha-amylase Double Mutants W138 and M197

Seed variants W138 (F, Y and A) were prepared by double mutation with M197T. The double mutants were prepared by the methods described in Examples 1 and 3 (as described in Example 3). Generally, rain. A single stranded negative strand was prepared from the M13MP18 clone of the 1.72 kb coding sequence (PstI-SstI) of the Lycanformis alpha-amylase M197T mutant. Site-engineered mutagenesis was performed using the following primers, basically by Zoller, M. and his colleagues, except that T4 gene 32 protein and T4 polymerase were used in place of Clenu. It was carried out by the method. All of these primers contained specific mutations as well as desired mutations in order to identify clones in which the appropriate mutation occurred.

SEQ ID NO: 42

Substitution of Tryptophan 138 with Phenylalanine.

SEQ ID NO: 43

Substitution of tyrosine for tryptophan 138.

[SEQ ID NO: 44]

Substitution of Allycin for Tryptophan 138. : This primer also engineered specific positions downstream and upstream at position 138.

Mutants were identified by restriction analysis and W138F and M138Y were verified by DNA sequencing. It was found that a nucleotide deletion occurred between the BspEI and XmaI positions, which are the specific positions of the W138A sequence, but the rest of the gene was sequenced correctly. The SstII / SstI fragment, 1.37 kb, containing W138X and M197T mutations was transferred from M13MP18 to the expression vector pBLapr to produce pBLapr (W138F, M197T) and pBLapr (W138Y, M197T). The fragment containing the specific positions BspEI and XmaI positions were cloned into pBLapr (BspEI, XmaI, M197T) as they are useful for cloning cassettes containing other amino acids substituted at the 138 position.

Single mutation at amino acid position 138

The general method described in the examples above was carried out to prepare lumps of W138 single variants (F, Y, L, H and C).

The 1.24 kb Asp718-SstI fragment containing the M197T mutation in the plasmid pBLapr (W138X, M197T) of Example 7 was replaced with a wild-type fragment of 197 methionine to allow pBLapr (W138F), pBLapr (W138Y) and pBLapr (BspEI, XmaI). ) Was generated.

The variants W138L, W138H and W138C were combined with a synthetic cassette using the following primers to prepare pBLapr (BspEI, XmaI) vectors:

[SEQ ID NO: 45]

Substitution of leucine for tryptophan 138.

CC GGA GAA CAC CTA ATT AAA GCC CTA ACA CAT TTT CAT TTT C

[SEQ ID NO: 46]

Substitution of Tryptophan 138 with Histidine.

CC GGA GAA CAC CTA ATT AAA GCC CAC ACA CAT TTT CAT TTT C

[SEQ ID NO: 47]

Substitution of Tryptophan 138 with Cysteine.

CC GGA GAA CAC CTA ATT AAA GCC TGC ACA CAT TTT CAT TTT C

The double mutants, M197T / W138F and M197T / W138Y, were reacted with chloramine-T to compare with wild type. (AA20 = 0.75 mg / ml, M197T / W138F = 0.64 mg / ml, M197T / W138Y = 0.60 mg / ml; 50 mM NaAcetate pH 5.0) The results shown in FIG. 19 show chloramine-T as a mutation in tryptophan 138. It is shown that variants with more resistance to can be obtained.

Example 11

Preparation of Multiple Mutations

The following multiple mutants were prepared by the methods described in Examples 1, 3, 5 and 10. : M15T / M197T; M15S / M197T; W138Y / M197T; M15S / W138Y / M197T and M15T / W138Y / M197T. Some of the multiple mutations have already been exemplified, for example, W138Y / M197T was prepared and tested in Example 10. The multiple mutations were identified by restriction analysis.

A number of multiple mutations within the scope of the present invention have been further tested for their performance as wash products (automatic dish protection detergent) additives. These tests are detailed below.

[Stability test]

An automatic dishwashing detergent (ADD) solution was prepared containing 4000 ppm of perborate and TAED in a hardness of 7 gpg of water. Some amylase mutants were added to the ADD solution to give a variant with a rate of 0.4 when assayed by the Ceralpha method. Megazyme (Austr.) Pty. Ltd., Parramatta, NSW, Australia] one set of samples was left at room temperature (21-23 ° C.) for 30 minutes. After the heated enzyme was added, the second set of samples was warmed from room temperature to about 65 ° C., and then the activity of the amylase mutant was measured, and the activity and relative activity when the enzyme was added were measured. . (% Relative activity)

Results shown in FIG. 20 are ratios. Methionine at position +197 of Lycanformis alpha-amylase indicates that the stability of the formulation consisting of ADD, perborate, TAED has been modified.

[Starch Hydrolysis Assay]

A 4000 ppm solution of automatic dishwashing detergent solution (ADD) containing perborate and TAED in water of 7 gpg hardness was prepared and then cooked elbow macaroni was added. The amylase mutant was added to the ADD solution and the final concentration was 5 ppm of active enzyme. The tubes were warmed at 50 ° C. for 30 minutes and then the concentration of reducing sugars released by the dinitrosalicylic acid method was measured against the glucose standard curve. The results are shown in Table VI.

Table VI

The results shown in Table VI above were calculated as M15T / M197T; M15S / M197T; W138Y / M197T; M15S / W138T / M197T and M15T / W138Y / M197T exhibited better performance in the absence of this enzyme and compared to the wild type alpha-amylase superstructure.

[Oatmeal staining]

The dishes were soiled with seasoned oatmeal dough and dried overnight at 37 ° C. The dishes were placed in an ASKO Model 770 dishwasher and washed by circulation with Quick Wash at 45 ° C. using 10 g of an automatic dishwashing detergent containing 5% perborate, 3% TAED and 11 mg of amylase enzyme. The plates were weighed before, after, and after they were cleaned to determine the average percentage of contaminants removed from the dishes. The results are shown in Table VII below.

TABLE VII

The data show that mutant enzymes offer more benefits than those provided by the wild type. Wild type amylase in oatmeal removal provided more than 20% washing performance over ADD without amylase.

Example 12

Tableware protection washing composition

Wild type and mutant amylase 1% (W / W) granules were prepared by Kortex Automatic Dishwasher Detergent with 5% (w / w) sodium perborate monohydrate and 3% (w / w) TAED. The preparation was left for 4 weeks at room temperature (21-23 ° C.) or 38 ° C., 80% relative humidity. The results are shown in FIGS. 21 and 22.

The data indicate that activity decreases with longer storage time in the detergent, which indicates wild-type amylase activity measured by the Ceralpha method. At room temperature, the mutant enzymes were completely stable. At 38 ° C. and 80% relative humidity all mutants were more stable than wild type.

The advantage of the automatic dishwashing detergent formulation containing the mutant amylase is that in the presence of perborate and TAED the mutants are significantly more stable than the wild type, providing significant performance in removing stains by starch foods during washing. have.

Example 13

Oxidation Stable Protease / Oxidation Stable Amylase

1) Enzyme granules containing wild type protease and protease stable to wild type amylase or bleach (GG36-M222S) were prepared in U.S. A buffer containing 0.1 M sodium borate pH 10.2 and 0.005% Tween 80 at a concentration of 12.5 mg for amylase stable for bleach, containing amylase (AA20-M15T / W138Y / M197T), which was prepared by the method described in heading 34606. Dissolved in. To 9 ml of the solution was added 1 ml of distilled water or 1 ml of 30% hydrogen peroxide. The solution was allowed to warm at 25 ° C. for 30 minutes and then the protease and amylase activity in each solution was measured and expressed as% of original activity. The data is shown in Table VIII below.

TABLE VIII

The data show a combination of bleach-stable amylase mutants and bleach-stable protease mutants, wherein the two mutants in which the mutations occurred in amino acid residues susceptible to oxidation have poor resistance to inactivation by bleach. It provides a combined advantage of proteases and amylases in the formulation. After 30 minutes, the bleach-stable amylase and bleach-stable protease mostly retained initial activity in the bleach, whereas the combination of wild-type enzymes lost more than 80% of the initial activity during the same time.

Example 14

Contrast Active Forms of Amylase Variants

Aspects of the activity of amylase variants were obtained. Two Phadebas tablets (Phadebas Amylase Test Kit, Pharmacia Diagnostics) were prepared in 12 milliliter buffers (0.05 M borate / 0.05 M potassium phosphate / 0.005 M calcium chloride) in each vial at pH approximately 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0. ) Was determined to determine the relative hydrolytic activity of each tested enzyme to starch. When the two tablets were added, the resulting pHs were 6.21, 7.0, 7.68, 8.75, 9.72 and 10.63. Each vial was mixed by magnetic stirring, and upon mixing, an aliquot of 200 μL was pipetted into a 96 Well Costar polystyrene plate. 9.9 mg / ml Termamy1 (wild-type Bacillus Lycoformforms alpha amylase, Novo Nordisk, Denmark), 5.2 mg / ml Spezyme AA20 (Bacillus Lycoformis alpha amylase, Genencor International, Inc., South San Francisco, Ca.) or Enzyme samples were obtained in a solution containing 4.1 mg / ml of mutant amylase (M15T / W138Y / M197T) according to the invention. Each sample was diluted to 1/5000 in amylase assay buffer (50 mM acetate buffer, pH 6.7, 5 mM CaCl ₂ , 0.002% Tween 20). 10 μL of the dilution enzyme was added to the substrate solution with a multichannel pipette. The reaction mixture was mixed at 1100 rpm for 30 minutes using an Ika-Schuttler MTS 2 plate shaker. The reaction was terminated by filtering insoluble substrate particles from a supernatant containing enzyme and dissolved blue dye using a 0.45 micron hydrophilic 96 well filtration plate (Mass., Bedford, Millipore, Multiscreen Filtration System). Samples were separated with a multichannel pipette. PH was measured using 200 μL of each control sample containing a substrate free of enzymes.

From the filtered supernatant, an aliquot of 100 μL of each sample was removed and placed in another 96 well Costar polystyrene plate and pipetted again with a multichannel pipette. Then absorbance at 620 nm was measured. The results are shown in FIG. 23. Shown is a graph of incubation time. As shown in Figure 23, the mutant enzyme according to the present invention had a predominant activity at about 8.5 in the pH range of 6 or more compared to Termamyl or Spezyme AA20.

Example 15

Oxidative Stability of Amylase Against Peracetic Acid

12.3 μL of amylase was diluted in an Ependorf tube containing 0.007.7 M calcium chloride at pH 10.0, 25 ° C. and 977.7 μL of 0.1 M CHES (2-n-cyclohexylaminoethane-sulfonic acid) solution. The samples were vortexed to mix and 10 μL aliquots were separated to determine initial activity against maltoheptaside. The remaining 980 μL was mixed with 32% peracetic acid. During the test, the samples were incubated in the heating block using glycerol as the conduction fluid. The temperature in the reaction tube was 52 ° C. The cap of the tube was sealed during the reaction. At 52 ° C., the buffer had a pH of about 9.3, which was determined by Methods of Enzymology, vol. As reported in 87 (1982), it was consistent with pH fluctuations for CHES buffer at high temperatures.

10 ml were taken after the sample was stirred to measure the respective activity at 0 minutes, 15 minutes, 30 minutes, 45 minutes and 60 minutes. Results are compared to relative activity (based on the amount of hydrolyzed starch released into solution) vs. A graph of pH is shown in FIG. 24. As shown in Figure 24, the mutant enzyme according to the present invention was surprisingly high in initial activity compared to Termamyl when incubated at pH 9.3 with an oxidizing agent.

Example 16

Comparison of Cleaning Performance of Amylase in Liquid Laundry Detergents

The washing performance of the mutant amylase according to the invention under conventional liquid laundry detergent conditions was tested in comparison to commercial wild type amylase. Cotton Swatch PEPD 7435WRL (Scientific Services S / D, Inc., Sparrow Bush, N.Y.) stained with cornstarch / India ink was used to test the wash performance of amylase additives.

Grease release TIDE concentrated detergent (Proctor Gamble, Cincinnati Ohio) was heated at 90 ° C. for 1 hour to inactivate some of the enzymes present (eg proteases or lipases). The amylase used was Termamyl 60T amylase (wild-type Bacillus Lycoformis amylase, Novo, Nordisk, Denmark) and amylases of the present invention, namely M15T / W138Y / M197T.

In a final wash solution (Termamyl or M15T / W138Y / M197T) 1.0 g of liquid detergent and amylase 0.05, 0.1, 0.5, or 1.0 ppm were measured and then discarded. No control was added to amylase. Wash tests were performed on a Model 7243S Terg-o-tometer (United States Testing Co., Hoboken, NJ). A wash consisting of distilled water and 10 ml concentrated water was added and the final concentration was 100 ppm Ca ²⁺ ions and 50 ppm Mg ²⁺ . 1 L of wash was added to each Terg-o-timer pot. After the wash was brought to an appropriate temperature, the stirrer was turned on and at the same time detergent and amylase were added to the wash. After dissolving for 3 minutes, Swatch was added to the wash solution. After stirring for 15 minutes at 100 rpm, the stirrer was turned off and the swatches were separated from the Terg-o-tometer pot and then rinsed in the washing machine with cooling water for 10 minutes. After rinsing, the starch swat was separated and then compressed to dryness. After that, the wash was measured by a reflectometer. It measured at 40 degreeC and 55 degreeC. Changes in reflectivity (normalized to detergent) vs. 25 and 26 are graphs of the amylase ppm added, respectively.

As shown in FIG. 25 and FIG. 26, the mutant amylase of the present invention was superior in performance to the commercially available amylase in washing ability.

Example 17

Amylase Washing Performance of the Invention in Liquid Detergents

The washing performance of the mutant amylase according to the present invention was tested with two commercially available liquid detergent compositions, SA8 (Michigan, Ada, Amway Corp.) and Purex (Arizona, Phoenix, Dial Corp.). Mutant amylase (M15T / W138Y / M197T) was added to bring final concentrations in the wash solution to 0.0, 0.05, 0.1, 0.5, 1.0 and 5.0 ppm. 0.75 g / L of SA8 was added to the washing liquid to adjust the pH of the washing solution to 6.5 and the temperature to 55 deg. Purex was added to the wash solution at 1.0 g / L to adjust the pH of the wash solution to 9.0 and the temperature to 40 ° C. The washing method was the same as in Example 16. The change in reflectance (normalized to detergent) for Swatch was measured. The results are shown in FIG. 27.

As can be seen in FIG. 27, the addition of amylase significantly improved the washing ability of the detergent.

[Sequence list]

(1) General Information:

(i) Applicants: Barnett, Christopher

Madson, Colin

Fauer, Scott Dee.

(ii) Name of the Invention: Improved Cleaning Composition

(iii) SEQ ID NO: 68

(iv) your address;

(A) Recipient: Genenko International

(B) Distance: 180 Kimball Way

(C) City: South San Francisco

(D) State: California

(E) Country name: United States of America

(F) ZIP Code: 94080

(v) computer readable form:

(A) Medium form: floppy disk

(B) Computer: IBM PC Compatibility

(C) operating system: PC-DOS / MS-DOS

(D) Software: PatentIn Release # 1.0,

Version # 1.25

(vi) Current application data:

(A) Application number:

(B) filing date:

(C) classification:

(viii) information about agents;

(A) Statement: Horn, Margaret A.

(B) Registration Number: 33,401

(C) Related / List Number: GC220-3

(ix) telecommunication information

(A) Phone number: (415) 742-7536

(B) Telefax number: (415) 742-7217

(2) Information about sequence 1

(i) sequence characteristics

(A) Length: 56 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 1:

[SEQ ID NO: 1]

GATCAAAACA TAAAAAACCG GCCTTGGCCC CGCCGGTTTT TTATTATTTT TGAGCT56

(2) information on sequence 2

(i) sequence characteristics

(A) Length: 29 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 2:

[SEQ ID NO: 2]

TGGGACGCTG GCGCAGTACT TTGAATGGT29

(2) information on sequence 3

(i) sequence characteristics

(A) Length: 34 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 3:

[SEQ ID NO: 3]

TGATGCAGTA CTTTGAATGG TACCTGCCCA ATGA34

(2) information on sequence 4

(i) sequence characteristics

(A) Length: 36 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 4:

[SEQ ID NO: 4]

GATTATTGT TGTATGCCGA TATCGACTAT GACCAT36

(2) information on sequence 5

(i) sequence characteristics

(A) Length: 26 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 5:

[SEQ ID NO: 5]

CGGGGAAGGA GGCCTTTACG GTAGCT26

(2) information on sequence 6

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 6:

[SEQ ID NO: 6]

GCGGCTATGA CTTAAGGAAA TTGC24

(2) information on sequence 7

(i) sequence characteristics

(A) Length: 23 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 7:

[SEQ ID NO: 7]

CTACGGGGAT GCATACGGGA CGA23

(2) information on sequence 8

(i) sequence characteristics

(A) Length: 35 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 8:

[SEQ ID NO: 8]

CTACGGGGAT TACTACGGGA CCAAGGGAGA CTCCC35

(2) information on sequence 9

(i) sequence characteristics

(A) Length: 36 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 9:

[SEQ ID NO: 9]

CCGGTGGGGC CAAGCGGGCC TATGTTGGCC GGCAAA36

(2) information on sequence 10

(i) sequence characteristics

(A) Length: 45 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 10:

[SEQ ID NO: 10]

CATCAGCGTC CCATTAAGAT TTGCAGCCTG CGCAGACATG TTGCT45

(2) Information about sequence 11

(i) sequence characteristics

(A) Length: 36 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 11:

[SEQ ID NO: 11]

GATTATTTGG CGTATGCCGA TATCGACTAT GACCAT36

(2) Information about sequence 12

(i) sequence characteristics

(A) Length: 21 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 12:

[SEQ ID NO 12]

GGGAAGTTTC GAATGAAAAC G21

(2) information on sequence 13

(i) sequence characteristics

(A) Length: 38 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 13:

[SEQ ID NO: 13]

GTCGGCATAT GCATATAATC ATAGTTGCCG TTTTCATT38

(2) Information about sequence 14

(i) sequence characteristics

(A) Length: 41 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 14:

[SEQ ID NO: 14]

CGAATGAAAA CGGCAACTAT GATTATTTGA TCTATGCCGA C 41

(2) information on sequence 15

(i) sequence characteristics

(A) Length: 41 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 15:

[SEQ ID NO: 15]

CGAATGAAAA CGGCAACTAT GATTATTTGT TCTATGCCGA C 41

(2) Information about sequence 16

(i) sequence characteristics

(A) Length: 41 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 16:

[SEQ ID NO: 16]

CGAATGAAAA CGGCAACTAT GATTATTTGG TTTATGCCGA C41

(2) Information about sequence 17

(i) sequence characteristics

(A) Length: 41 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 17:

[SEQ ID NO: 17]

CGAATGAAAA CGGCAACTAT GATTATTTGA GCTATGCCGA C 41

(2) Information about sequence 18

(i) sequence characteristics

(A) Length: 41 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 18:

[SEQ ID NO: 18]

CGAATGAAAA CGGCAACTAT GATTATTTGC CTTATGCCGA C41

(2) Information about sequence 19

(i) sequence characteristics

(A) Length: 41 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 19:

[SEQ ID NO: 19]

CGAATGAAAA CGGCAACTAT GATTATTTGA CATATGCCGA C 41

(2) Information about sequence 20

(i) sequence characteristics

(A) Length: 41 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 20:

[SEQ ID NO: 20]

CGAATGAAAA CGGCAACTAT GATTATTTGT ACTATGCCGA C 41

(2) Information about sequence 21

(i) sequence characteristics

(A) Length: 41 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 21:

[SEQ ID NO: 21]

CGAATGAAAA CGGCAACTAT GATTATTTGC ACTATGCCGA C 41

(2) Information about sequence 22

(i) sequence characteristics

(A) Length: 41 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 22:

[SEQ ID NO 22]

CGAATGAAAA CGGCAACTAT GATTATTTGG GCTATGCCGA C 41

(2) Information about sequence 23

(i) sequence characteristics

(A) Length: 41 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 23:

[SEQ ID NO: 23]

CGAATGAAAA CGGCAACTAT GATTATTTTGC AATATGCCGA C 41

(2) Information about sequence 24

(i) sequence characteristics

(A) Length: 41 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 24:

[SEQ ID NO: 24]

CGAATGAAAA CGGCAACTAT GATTATTTGA ACTATGCCGA C

(2) information on sequence 25

(i) sequence characteristics

(A) Length: 41 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 25:

[SEQ ID NO: 25]

GCAATGAAAA CGGCAACTAT GATTATTTGA AATATGCCGA C 41

(2) Information about sequence 26

(i) sequence characteristics

(A) Length: 41 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 26:

[SEQ ID NO 26]

CGAATGAAAA CGGCAACTAT GATTATTTGG ATTATGCCGA C 41

(2) Information about sequence 27

(i) sequence characteristics

(A) Length: 41 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 27:

[SEQ ID NO: 27]

CGAATGAAAA CGGCAACTAT GATTATTTGG AATATGCCGA C 41

(2) information on sequence 28

(i) sequence characteristics

(A) Length: 41 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 28:

SEQ ID NO: 28

CGAATGAAAA CGGCAACTAT GATTATTTGT GTATTGCCGA C 41

(2) Information about sequence 29

(i) sequence characteristics

(A) Length: 41 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 29:

SEQ ID NO: 29

CGAATGAAAA CGGCAACTAT GATTATTTGT GGTATGCCGA C 41

(2) Information about sequence 30

(i) sequence characteristics

(A) Length: 41 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 30:

[SEQ ID NO: 30]

CGAATGAAAA CGGCAACTAT GATTATTTGA GATATGCCGA C 41

(2) Information about sequence 31

(i) sequence characteristics

(A) Length: 1968 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 31:

[SEQ ID NO: 31-1]

[SEQ ID NO: 31-2]

GTGTCATCAG CCCTCAGGAA GGACTTGCTG ACAGTTTGAA TCGCATAGGT AAGGCGGGGA1860

TGAAATGGCA ACGTTATCTG ATGTAGCAAA GAAAGCAAAT GTGTCGAAAA TGACGGTATC1920

GCGGGTGATG AATCATCCTG AGACTGTGAC GGATGAATTG AAAAAGCT1968

(2) information on sequence 32

(i) sequence characteristics

(A) Length: 483 amino acids

(B) form: amino acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: protein

(xi) Sequence description: SEQ ID NO: 32:

[SEQ ID NO: 32-1]

[SEQ ID NO: 32-2]

(2) Information about sequence number 33

(i) sequence characteristics

(A) Length: 511 amino acids

(B) form: amino acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: protein

(xi) Sequence description: SEQ ID NO: 33:

[SEQ ID NO: 33-1]

[SEQ ID NO: 33-2]

(2) Information about sequence 34

(i) sequence characteristics

(A) Length: 520 amino acids

(B) form: amino acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: protein

(xi) Sequence description: SEQ ID NO: 34:

[SEQ ID NO: 34-1]

[SEQ ID NO: 34-2]

(2) Information about sequence 35

(i) sequence characteristics

(A) Length: 548 base pairs

(B) form: amino acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: protein

(xi) Sequence description: SEQ ID NO: 35:

[SEQ ID NO: 35-1]

[SEQ ID NO: 35-2]

[SEQ ID NO: 35-3]

(2) information on sequence 36

(i) sequence characteristics

(A) Length: 483 amino acids

(B) form: amino acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: protein

(xi) Sequence description: SEQ ID NO: 36:

[SEQ ID NO: 36-1]

[SEQ ID NO: 36-2]

(2) Information about sequence 37

(i) sequence characteristics

(A) Length: 487 base pairs

(B) form: amino acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: protein

(xi) Sequence description: SEQ ID NO: 37:

[SEQ ID NO: 37-1]

[SEQ ID NO: 37-2]

(2) Information about sequence 38

(i) sequence characteristics

(A) Length: 32 base pairs

(B) form: amino acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: protein

(xi) Sequence description: SEQ ID NO: 38:

[SEQ ID NO: 38]

(2) Information about sequence 39

(i) sequence characteristics

(A) Length: 33 base pairs

(B) form: amino acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: protein

(xi) Sequence description: SEQ ID NO: 39:

[SEQ ID NO: 39]

(2) Information about sequence 40

(i) sequence characteristics

(A) Length: 35 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: protein

(xi) Sequence description: SEQ ID NO: 40:

[SEQ ID NO 40]

(2) Information about sequence 41

(i) sequence characteristics

(A) Length: 32 base pairs

(B) form: amino acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: protein

(xi) Sequence description: SEQ ID NO: 41:

[SEQ ID NO: 41]

(2) Information about sequence 42

(i) sequence characteristics

(A) Length: 33 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 42:

SEQ ID NO: 42

CACCTAATTA AAGCTTTCAC ACATTTTCAT TTT33

(2) Information about sequence 43

(i) sequence characteristics

(A) Length: 33 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 43:

SEQ ID NO: 43

CACCTAATTA AAGCTTACAC ACATTTTCAT TTT33

(2) Information about sequence 44

(i) sequence characteristics

(A) Length: 66 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 44:

[SEQ ID NO: 44]

CCGCGTAATT TCCGGAGAAC ACCTAATTAA AGCCGCAACA CATTTTCATT TTCCCGGGCG60

CGGCAG66

(2) Information about sequence 45

(i) sequence characteristics

(A) Length: 42 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 45:

[SEQ ID NO: 45]

CCGGAGAACA CCTAATTAAA GCCCTAACAC ATTTTCATTT TC 42

(2) Information about sequence 46

(i) sequence characteristics

(A) Length: 42 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 46:

[SEQ ID NO: 46]

CCGGAGAACA CCTAATTAAA GCCCACACAC ATTTTCATTT TC 42

(2) Information about sequence 47

(i) sequence characteristics

(A) Length: 42 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 47:

[SEQ ID NO: 47]

CCGGAGAACA CCTAATTAAA GCCTGCACAC ATTTTCATTT TC 42

(2) Information about sequence 48

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 48:

[SEQ ID NO: 48]

GATGCAGTAT TTCGAACTGG TATA24

(2) Information about sequence 49

(i) sequence characteristics

(A) Length: 26 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 49:

[SEQ ID NO: 49]

TGCCCAATGA TGGCCAACAT TGGAAG26

(2) information on sequence 50

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 50:

[SEQ ID NO: 50]

CGAATGGTAT GCTCCCAATG ACGG24

(2) Information about sequence 51

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 51:

[SEQ ID NO: 51]

CGAATGGTAT CGCCCCAATG ACGG24

(2) Information about sequence 52

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 52:

SEQ ID NO: 52

CGAATGGTAT AATCCCAATG ACGG24

(2) Information about sequence 53

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 53:

[SEQ ID NO: 53]

CGAATGGTAT GATCCCAATG ACGG24

(2) Information about sequence 54

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 54:

[SEQ ID NO: 54]

CGAATGGTAT CACCCCAATG ACGG24

(2) Information about sequence 55

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 55:

[SEQ ID NO: 55]

CGAATGGTAT AAACCCAATG ACGG24

(2) Information about sequence 56

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 56:

[SEQ ID NO 56]

CGAATGGTAT CCGCCCAATG ACGG24

(2) Information about sequence 57

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 57:

[SEQ ID NO: 57]

CGAATGGTAT TCTCCCAATG ACGG24

(2) Information about sequence 58

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 58:

SEQ ID NO: 58

CGAATGGTAC ACTCCCAATG ACGG24

(2) Information about sequence 59

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 59:

[SEQ ID NO: 59]

CGAATGGTAT CTTCCCAATG ACGG24

(2) Information about sequence 60

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 60:

[SEQ ID NO: 60]

CGAATGGTAT TGTCCCAATG ACGG24

(2) Information about sequence 61

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 61:

SEQ ID NO: 61

CGAATGGTAT CAACCCAATG ACGG24

(2) Information about sequence 62

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 62:

SEQ ID NO: 62

CGAATGGTAT GAACCCAATG ACGG24

(2) Information about sequence 63

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 63:

SEQ ID NO: 63

CGAATGGTAT GGTCCCAATG ACGG24

(2) Information about sequence 64

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 64:

SEQ ID NO: 64

CGAATGGTAT ATTCCCAATG ACGG24

(2) Information about sequence 65

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 65:

[SEQ ID NO: 65]

CGAATGGTAT TTTCCCAATG ACGG24

(2) Information about sequence 66

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 66:

SEQ ID NO: 66

CGAATGGTAC TGGCCCAATG ACGG24

(2) Information about sequence 67

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 67:

[SEQ ID NO: 67]

CGAATGGTAT TATCCCAATG ACGG24

(2) Information about sequence 68

(i) sequence characteristics

(A) Length: 24 base pairs

(B) form: nucleic acid

(C) Number of chains: 1 chain

(D) Distribution state: linear

(ii) Molecular type: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 68:

[SEQ ID NO: 68]

CCGTCATGG GACTACGTAC CATT24

Claims (10)

ratio. Methionine and B. at M + 197 position in B.licheniformis. Expression of a mutated DNA sequence encoding alpha-amylase obtained from precursor alpha-amylase by replacing one or more methionine or tryptophan at the M + 15 or W + 138 position in B.licheniformis alpha-amylase An improved bleach-containing wash composition comprising the addition of the product, Mutant Alpha-Amylase, to a laundry detergent composition is an improvement. The improved laundry detergent composition of claim 1, wherein the cleaning composition is a liquid composition. The method of claim 1, wherein the mutant alpha-amylase is M15T / M197T; M15S / M197T; W138Y / M197T; Improved cleaning composition selected from the group comprising M15S / W138Y / M197T and M15T / W138Y / M197T. The mutant protease of claim 1, wherein the mutant protease is an expression product of a mutated DNA sequence encoding a protease obtained from a precursor protease by replacing methionine at the B. amylolichefacience protease M + 222 position. Further comprising an improved laundry detergent composition. 5. The improved laundry detergent composition of claim 4, wherein the mutant protease comprises a substituent selected from the group of amino acids comprising alanine, cysteine and serine. The group according to claim 4, wherein the group comprises an alpha-amylase mutant selected from the group comprising M15T / M197T, M15S / M197T, W138Y / M197T, M15S / W138Y / M197T and M15T / W138Y / M197T and M222C, M222S and M222A. An improved laundry detergent composition comprising a protease mutant selected from. 3. The improved cleaning detergent composition of claim 2, wherein the pH of the liquid detergent is between about 6.0-10.0. 2. The improved laundry detergent composition of claim 1, wherein said laundry detergent contains a bleach. The improved laundry detergent composition of claim 8, wherein the pH of the laundry detergent is at least about 10. 10. 10. The improved laundry detergent composition of claim 9, wherein said laundry detergent is granular.