Compositions based on α-amylase polypeptide from bacillus, type 195, and use thereof

FIELD: chemistry.

SUBSTANCE: disclosed is isolated α-amylase from Bacillus sp. 195, which is a shortened form which ends with a residue 492, 504 or 509 SEQ ID NO:3, given in the description. Described is a detergent additive containing said α-amylase in amount of 0.02-200 mg per g of detergent additive. Said detergent additive can be presented in form of dust-free granulated material, micro-granulated material, stabilised liquid, gel or protected enzyme.

EFFECT: invention enables to obtain a modified enzyme with higher catalytic activity compared to the parent enzyme.

4 cl, 10 dwg, 3 ex

 

The technical field to which the invention relates

The present invention describes compositions and methods of using α-amylase enzyme derived fromBacillussp. 195.

Background of invention

Starch is a mixture of amylose (15-30 weight. %) and amylopectin (70-85 weight. %). Amylose consists of linear chains of α-1,4-linked glucose units with a molecular weight (MV) of from about 60,000 to about 800000. The amylopectin is a branched polymer containing α-1,6 branch point every 24-30 glucose units; MV may reach 100 million.

Sugar from starch, in the form of a concentrated syrup, dextrose, currently produced by the process of enzymatic catalysis, which includes: (1) liquefaction (or decrease viscosity) solid starch with α-amylase to obtain a dextrin with an average degree of polymerization 7-10, and (2) saccharification received the liquefied starch (i.e. hydrolyzed starch) amyloglucosidase (also called glucoamylase or HA). The resulting syrup is characterized by high glucose. A large part produced on a commercial scale glucose syrup then subjected to enzymatic isomerization to a mixture of dextrose/fructose, known as Izosimov.

As is known, α-amylase (EC 3.2.1.1) hydrolyzing starch, glycol the n and related polysaccharides by cleavage of internal α-1,4-glucosidic ties randomly. This enzyme has many important areas of its application, in particular, sugar, brewing, alcohol and textile industries. While α-amylase extracted from a large number of bacteria, fungi, plants and animals used as a source. The most important α-amylase in the context of industrial applications, include those of α-amylase, which produce a species ofBacilli.

For many years the α-amylase enzymes used for various purposes, including starch liquefaction, rasshifrovka threads, modified starch in paper and pulp-and-paper industry and in brewing. These enzymes can also be used in the means for washing dishes and for washing to remove starchy colored substances.

One of the α-amylasesBacillus, which was subjected to sequencing, is an enzyme of theBacillussp. 195 (BAA). This enzyme consists of two domains: a catalytic domain similar to those in α-amylases animals, and domain, which contains two krahmalsoderzhaschih motif (see J. Sumitani et al., "New type of starch-binding domain: the direct repeat motif in the C-terminal regionof Bacillussp. 195 a-amylase contributes to starch binding and raw starch degrading,"Biochem J.350: 477-484 (2000)). In this work, Sumitani et al. (Sumitani et al., (2000)) were found three active forms of the gene products in the culture supernatant ofStreptomyces lividan, where specified in culture gene productBacillussp. 195 expressively in heterologous variant. These three are the shape of the size 69 kDa form with a size of 60 kDa and shape size of 50 kDa. The shape of the size 69 kDa, in all probability, is a full-sized Mature protein with a molecular weight equivalent to its estimated value, calculated on the basis of the nucleotide sequence of the full length gene. Form size of 60 kDa, apparently, corresponds to the natural enzyme fromBacillussp. 195 and, presumably, can be obtained by proteolytic processing, carried out on site between two kramarevsky motifs at the C-end. This form is characterized by decreased activity in relation to the binding and degradation of raw starch in comparison with the form of 69 kDa. While the shape of the size of 50 kDa in General not able to bind or degrade insoluble starches.

Amylase used in the treatment of textile materials preparations for washing and cleaning compositions for rasshifrovka fibers, in baking, when converted and processed starch. Thus, there is an urgent need for the identification of α-amylases, which can be obtained within a more easy process with a reduced cost in order to achieve the profitability of the agricultural production, reduce the cost of delivery from the production premises and with greater activity products.

A BRIEF DESCRIPTION of the INVENTION

Accordingly, one aspect of the present invention relates to α-amylase fromBacillussp. 195, which can be obtained in large quantities and with less cost in response to the needs of industrial production. These options can be used in many compositions and methods of using α-amylase.

One of the objects of the present invention refers to the nucleic acid, in one of the alternative approaches to optimized nucleic acid shown in Fig. 2 (SEQ ID NO: 2). Another aspect of the present invention relates to gene α-amylase, operatively associated with a nucleic acid sequence that encodes a signal peptide of α-amylase fromBacillus licheniformisor its truncated, processionary polypeptide.

In another aspect, the present invention relates to a nucleic acid that encodes a truncated form of the polypeptide shown in Fig. 4, where the specified truncation, or the processing may be implemented by any residue of 491 amino acids (e.g. amino acid 492, 494, 504, 509, after any krahmalsoderzhashchego domain and so on).

Another aspect of the present invention refers to the full sized the th polypeptide, it is shown in Fig. 4, or any truncated by carboxylic product, where the specified localized truncation after residue 491.

Another variant of implementation of the present invention relates to a vector operatively linked to nucleic acid encoding these polypeptides.

Another aspect of the present invention refers to the selected cell host containing any of the foregoing nucleic acids or vectors comprising these nucleic acids. Selected a host cell can be a prokaryotic or eukaryotic cell. Selected a host cell can be a bacterial cell(for example,B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus. B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. thuringiensis, Streptomyces lividans, S. murinusorEscherichia coli).

Another aspect relates to a detergent additive comprising the polypeptide according to the present description, where the specified detergent additive does not necessarily has the form prepareimage granules, micro-granulate, a stabilized liquid, gel or protected enzyme. The specified polypeptide in the detergent additive may be a truncated polypeptide according to the above description. Detergent additive may contain from about 0.02 mg to about 200 mg of the polypeptide per gram of the detergent to the where it is refuelled. The specified detergent additive may further include an enzyme selected from the group consisting of protease, lipase, peroxidase, oxidase, amiloliticescoe enzyme, cellulase, polyesterase and any combination thereof.

Another aspect relates to detergent compositions comprising any of the above detergent additives. Detergent composition can optionally include one or more of these ingredients: surfactant, bleaching or bleach, detergent filler, polymer, stabilizer, air conditioning for fabric, foam, suppressor of education soapy foam, anti-corrosion agent, a dye, a perfume, a means for suspension of polluting material, the inhibitor tarnishing, optical brighteners or bactericidal substance. Detergent composition can include or include in addition an additional enzyme, where the specified enzyme is a protease, a lipase, a peroxidase, an oxidase, an amylolytic enzyme, a cellulase, polyesters or any combination.

Another aspect relates to detergent compositions for manual or automatic dishwashing, comprising the polypeptide according to the present description.

Another aspect relates to a method of washing dishes, where the method includes the use of the of emergent for manual or automated washing according to the present description for washing one or more units, if necessary. This method of washing dishes include the addition of a detergent for washing dishes in such quantity that the rinsing liquid contains a polypeptide according to the present description, in the amount of from about 0,01 ppm to about 4 ppm

Another aspect relates to a detergent composition for washing clothes, including a detergent additive according to the present description. Another aspect relates to a method for cleaning fabrics, including leaching of contaminated tissue in a solution of detergent compositions according to the present invention. Consider the method also relates to a polypeptide according to the present description, where the specified polypeptide used in the solution in an amount of from about 0.01 to about 2 ppm in solution.

BRIEF DESCRIPTION of FIGURES

Fig. 1 a-b. The nucleotide coding sequence for α-amylase fromBacilussp. 195 (access number AV). The nucleotide sequence encoding a signal peptide amy195 underlined. The STOP codon is shown in bold. SEQ ID NO:1.

Fig. 2. The nucleotide coding sequence for α-amylase fromBacilussp. 195, after optimization of codons. The nucleotide sequence encoding the Mature protein amyl95 is after the nucleotide sequence that encodes a signal peptide for α-amylase fromB. licheniformis(LAT) (SEQ ID NO: 2). Nucleotide sequence that is midna sequence, encoding a signal peptide LAT underlined. The stop codon is shown in bold. Optimization of amino acid codons conducted using the GeneArt® (GeneArt GmbH, Germany).

Fig. 3. Polypeptide sequence Amy195 (SEQ ID NO: 3). The signal sequence comprises residues 1-46 (underlined). The Mature protein Amy195 starts at residue 47. The codons encoding in bold and underlined residues, replaced the stop codon with the aim of obtaining genetically protestirovannyx forms. Thus, Y511, K521 and V526, using the numbering according to Fig. 3, represents the last amino acid residues in genetically protestirovannyx forms.

Fig. 4. Amino acid sequence Amy195 shown in the form of a heterologous protein fusion with the signal sequence LAT (SEQ ID NO: 4). Capital letters in carboxykinase parts shown krahmalnaya domains belonging to the family of CBD-25. Capital letters (residues 1-29) on aminocore marked signal sequence amylase derived fromB. licheniformis.Capital letters indicate the catalytic domain of the enzyme, including subdomains, a, b and C, which, presumably, will cover the remains of about plots 30-105 and 208-300 for the subdomain And approximately residues 106-207 for the subdomain In; and about the remains 301-492 for subdomain With Val492 represents the last amino acid residue in proteoliticeski truncated form (using the numbering, shown in Fig. 4). It should be noted that the subdomain And is interrupted in the shown linear sequence of the polypeptide.

Fig. 5. A schematic relationship of nucleic acid encoding α-amylase fromBacillussp. 195, with a nucleic acid that encodes a signal sequence of the LAT and the termination sequence of the LAT in the pHPLT vector. These plasmid pHPLT-known in the art (see, for example, U.S. patent No. 5871550 and 6562612, as well as the publication of U.S. patent 20060014265). The pHPLT vector built with the achievement of gene expression amy195 in nine strains ofB. subtiliswith deletional protease (see US20050202535A1).

Fig. 6. Shows the test results on the functioning of the enzyme Amy195 as a function of pH and protein concentration. Investigated the fraction shown in the form of e-pool in Fig. 10. The test is carried out in 96-cell tablet. In each cell is placed tissue samples in one quarter inch (0,00635 m), contaminated colored rice starch (Testfabrics Inc., CS28-colored rice starch). Buffer: 25 mm HEPES, pH 8.0 25 mm CAPS, pH of 10.3 add to each cell. The tablet is subjected to pre-incubation at 40°C. the Reaction start adding enzyme Amy195 to final concentrations from 0 ppm to 2 ppm Tablet incubated at 40°C for 10 minutes with shaking at speed 750 rpm in the apparatus Eppendorf/Thermomix. After incu is then supernatant liquid is transferred into a new 96-cell tablet and measure the absorbance at a wavelength of 488 nm in the device for analysis of tablets Molecular Devices, model Spectra Max 190. Based on these results, build a graph using the program GRAFIT software Erithicus. To build the chart using isothermal algorithm Langmuir, which has the same form as the algorithm Michaelis-Menten available in the software. Each expressed protein Amy195 contains the signal peptide from the LAT, but is removed during secretion and is not present in the Mature protein Amy195.

Fig. 7. Test operation of all proteolytic fragments shown in Fig. 10. Carry out the specified test and the results depicted in the graph, as described in example 3, in accordance with the conditions described for Fig. 6, at pH 8. The data show that all factions operate as well or even better than OxAm (Genencor International, Inc.).

Fig. 8. Carry out electrophoresis in LTO-polyacrylamide gel and detected the expression of kinetically protestirovannyx molecules Amy195. When processing identifies the C-terminal residues 494, 504 and 509, according to the numbering system shown in Fig. 4. The expression in cultures carried out in accordance with the description in example 2, and the concentration estimate using OxAm as standard density.

Fig. 9. The operation in this embodiment ol the use of genetically protestirovannyx forms of amylase Amy195. The test operation is performed using the culture supernatant, which was not subjected to further purification. Test procedures and build a graph based on the data described in example 3 under the conditions described for Fig. 6, at pH 8.0. The data show that versions of the molecules perform better than OxAm.

Fig. 10.Analysis of the fractions of β-cyclodextrine column that contains proteolytic fragments Amy195. The fractions indicated by the symbols "wl" ("wash 1", allerona from the column using 25 mm bis-Tris-propane, pH 8.5, 2 mm CaCl2); "w2" ("rinse 2", allerona additional aliquot of the same buffer); and "(e-pool" (faction, erwerbende 50 mm β-cyclodextrin in the same buffer and applied on the gel three different concentrations). Matrix for β-cyclodextrine speakers are synthesized in the laboratory according to the standard Protocol of β-cyclodextrin (Sigma Aldrich Cat. No. c4767) and using epoxyoctadecane Sepharose-6B (GE Healthcare, N.J., Cat. No. 17-0480-01).

DETAILED description of the INVENTION

The present invention relates to compositions containing α-amylase fromBacillussp. 195, and to methods of their use. Also describes variations of the methods of obtaining α-amylase and its heterologous forms by modifying polypeptide posledovatelno the Mature α-amylase.

Contamination of clothes and dishes vary greatly in composition and, therefore, their ability to remove. A relatively small number of amylases, currently available on the market, can be used for cleaning clothes, and washing dishes. Retrieved fromBacillussp. 195 α-amylase does not show high identity regarding any of the bacterial amylases used in the present time. Thus, one aspect of the present invention refers to the use of wild-type protein as a skeleton to identify its variants, where this protein has the best performance with a view to its use in compositions for washing dishes and Laundry done, for example, by reducing dependence on Ca2+, stability improvements, LAS, improve range of pH, improve temperature range, increased specific activity, etc.

1.Definitions and abbreviations

As described in the present text of the detailed description of the present invention may use the following abbreviations and definitions. It should be borne in mind that the text of the description form of the singular include the multiple options, unless the context clearly requires otherwise. So, for example, the reference enzyme includes many such enzymes, and a link to the dosage includes the link is on one or more dosages and cash equivalents, well-known experts in this field, etc.

Unless specifically provided otherwise, all technical and scientific terms in the context of the present description have the meanings commonly accepted and well-known experts in this field. Below are the following terms.

1.1Reduction

The following abbreviations have the indicated value, if from the above description, the discussion requires otherwise.

deoxyribonucleic acid MWU
AEthe alcohol ethoxylate
AEOthe alcohol ethoxylate
AEOSalexisoliviat alcohol
ASAEalexisoliviat alcohol
Amy 195α-amylase fromBacillussp. 195
AOSα-reincorporate
ASAlkylsulfate
CBD-25a family of proteins with carbohydrate binding domain 25
cDNAComplementary DNA
CMC (CMC)carboxymethylcellulose
DNA
DTMPAdiethylenetriaminepentaacetic acid
ECThe Committee on nomenclature of enzymes
EDTA (EDTA)ethylenediaminetetraacetic acid
EMPAEidgenoessische Matenalprufungs - und Forschungs Anstalt (Federal laboratory Switzerland for testing and evaluation materials)
EOethylene oxide (polymer fragment)
F&HCmeans to care for fabrics and for other household needs
goals
HA (GA)glucoamylase
IPTGisopropyl-β-D-thiogalactoside
kDakilodaltons
LASline Las
LATrefers to the amylaseB.lichemformis (for example, the signal or the termination sequence of the amylase fromB.lichemformis)
MWmolecular weight
Modified Wohlgemuth units; 1,6×l0-5mg/MWU= unit of activity
NOBSnonanoyloxybenzenesulfonate
NTAnitriloside acid
OxAmPurastar HPAM 5000L (Genencor International, Inc.)
PEGThe polyethylene glycol
pIisoelectric point
PVA (PVA)poly(vinyl alcohol)
PVP (PVP)poly(vinyl pyrrolidone)
RNAribonucleic acid
SASalkanesulfonyl
LTO-PAGthe polyacrylamide gel electrophoresis-sodium dodecyl sulfate
sp.
TAEDtetraacetylethylenediamine
w/vweight/volume
w/wweight/weight

1.2Definitions

the Term "amylase" or "amylolytic enzyme" in the context of the present description include any amylase, such as glucoamylase, α-amylase, β-amylase, α-amylaseBacillussp. wild-type, such as α-amylase fromIn.licheniformisandB. subtilis.The term "amylase" means an enzyme which, among other properties capable of catalyzing the decomposition of starch. Amylase is hydrolase, which cleaves α-D-(1→4) O-glycosidic bonds in starch. Mainly α-amylase (EC 3.2.1.1; α-D-(1→4)-glucan-glucanohydrolase) is defined as a release that cleave α-D-(1→4) O-glycosidic bonds in the starch molecule in a random order. While eksperimenta amylolytic activity, such as β-amylase (EC 3.2.1.2; α-D-(1→4)-glucan-maltogenase) and some are specific to a particular product amylase type maltogenic α-amylase (EC 3.2.1.133), break down the starch molecule with unrestored end of the substrate. β-amylase, α-glucosidase (EC 3.2.1.20; α-D-glucoside-glucohydrolase), glucoamylase (EC 3.2.1.3; α-D-(1→4)-glucan-glucohydrolase) and specific product amylase can produce maltooligosaccharide specific length of starch.

The terms "variant amylase, variant α-amylase", "variant polypeptide α-amylase" and "variant of the enzyme in the context of the present description denotes the protein α-amylase fromBacillussp. 195, which has been modified, for example, by using the signal consequently the security of the other α-amylases and which contains the optimized sequence. In the context of the present description, the terms "source enzymes", "source sequence", "the original wild-type polypeptide", "α-amylase protein wild-type" and "source polypeptides" refers to enzymes and polypeptides, which were obtained from variant forms of the polypeptides of α-amylase. The original enzyme can be an enzyme wild-type or α-amylase, which was previously constructed by recombinant technology. Thus, α-amylase polypeptide can be a recombinant engineered enzyme. Variant α-amylase may also be a fusion protein containing a heterologous polypeptide α-amylase. For example, the protein α-amylase may include the signal peptide of α-amylaseB. licheniformis(LAT), coupled with a Mature protein of other α-amylase fromBacillus. The term "variant" may be used interchangeably with the term "mutant". Variants include polypeptides and nucleic acids. Options include insertion; these options may also include additional substitution, insertion, transverse, truncation and/or inversion of one or several positions. Options may include sequences that are complementary to sequences that are capable of hybridisierung with nucleotide sequences according to the present description. For example, the variant sequence complementary to sequences capable of hybridisierung in severe conditions (i.e. the conditions comprising 50°C and washing with the use of 0.2 × SSC {I × SSC = 0.15 M NaCl, 0.015 g M Na3citrate, pH 7.0}) to the nucleotide sequences according to the present description. The term "variant" may also include sequences that are complementary to sequences that are capable of hybridisierung in very hard conditions (i.e. at a temperature of 65°C with washing in 0.1 × SSC {I × SSC = 0.15 M NaCl, 0.015 g M Na3citrate, pH 7.0}) to the nucleotide sequences according to the present description.

The terms "α-amylaseBacillussp. 195", "α-amylase Amy 195" or "Amy 195" in the context of the present description denotes nucleic acid (Fig. 1)encoding a protein according to Fig. 3, or a synthetic nucleic acid sequence according to Fig. 2, which also encodes a protein according to Fig. 4. It can include any truncated form (e.g., truncated after residue 492 in the natural, recombinant or synthetic version of the enzyme form of the enzyme without the signal sequence, or the shape of the enzyme with a heterologous signal sequence and the truncated by carboxylic). In addition, these terms can cover any derivative sequence according to Fig. 3 and with testwuide her DNA sequence, contains amino acid substitutions, deletions, insertions or extensions of amino acids at the N - or C-Termini, which are not found in nature.

The term "isolated" refers to a sequence that is at least in large part, free, from at least one other component with which this sequence is associated and found in nature.

The term "purified" refers to a material that is relatively clean state, i.e. at least 90% purity or which is characterized, at least about 95% pure or at least about 98% purity.

The term "thermostable" in the context of the present description refers to the ability of the enzyme to retain activity after exposure to elevated temperatures. thermostability of the enzyme, such as α-amylase determined by its half-life. The half-life (t1/2) represents the time in minutes, hours, or days, during which lost half of the enzyme activity under certain conditions. The value of the half-life is calculated when measuring the residual activity of α-amylase.

The expression "the range of pH" refers to the ability of the enzyme to exhibit catalytic activity in conditions ranging from acidic to basic, with coverage of 5 or more pH units.

In the context of the present description, the term "pH stable" refers to the ability of the enzyme to retain activity in a wide range of pH values.

In the context of the present description, the term "amino acid sequence" is used synonymously with the term "polypeptide" and/or the term "protein". In some cases, the term "amino acid sequence" is used synonymously with the term "peptide". In some cases, the term "amino acid sequence" is used synonymously with the term "enzyme".

In the context of the present description, the term "nucleotide sequence" or "nucleic acid sequence" refers to an oligonucleotide sequence or polynucleotide sequence and its variant, homologues, fragments and derivatives (such as their part). The specified nucleotide sequence may be of genomic, synthetic or recombinant and may be double-stranded and single-stranded representing semantic or antisense chain. In the context of the present description, the term "nucleotide sequence" includes genomic DNA, cDNA, synthetic DNA and RNA.

The term "homolog" in the context of the present description refers to a structure with a certain degree of identity with the target amino acid sequence is mi and target nucleotide sequences. Homologous sequence includes an amino acid sequence that is at least 75%, 80%, 85%, or 90% identical or at least on 95%, 96%, 97%, 98% or 99% identical to the target sequence. Typically, the homologues include the same active sites that target amino acid sequence.

In the context of the present description, the term "hybridization" refers to the way in which one strand of nucleic acid joins with a complementary chain during mating grounds, and how amplification carried out in the framework of the polymerase-cableway reaction (PCR). Variant form of nucleic acid to α-amylase may exist as single-stranded or double-stranded DNA or RNA, heteroduplex RNA/DNA or copolymer RNA/DNA.

In the context of the present description, the term "copolymer" refers to single-stranded nucleic acid, which includes ribonucleotides and deoxyribonucleotides. Nucleic acid for α-amylase may even be optimized codons for additional strengthening of the expression.

In the context of the present description, the term "synthetic" defines the conditions under which it was achieved production ofin vitroby chemical or enzymatic synthesis. The term includes, without limitation, variant forms of the nucleic KIS is from α-amylase, which is obtained by optimizing the use of codons in the organism-owners, such as, without limitation,Pichia, Streptomyces,Trichoderma reeseiandHansenula.

In the context of the present description, the term "transformed cell" includes cells that have been genetically altered through the use of recombinant DNA technology. The transformation is typically carried out by embedding one or more nucleotide sequences in a cell. Integrated nucleotide sequence may be a heterologous nucleotide sequence (i.e. a sequence that is not native to the cell, subject to transformation, such as a sequence encoding a fusion protein).

In the context of the present description, the term "operatively linked" refers to the fact that the components described are in a relationship permitting them to function in a specified manner. Regulatory sequence operatively linked with a coding sequence, are legirovanie so that expression of the coding sequence is achieved under conditions compatible with the control sequences.

In the context of the present description, the term "biologically active" refers to sequences that have similar structural is voyta (but not necessarily in the same degree) and/or similar regulatory function (but not necessarily to the same degree), and/or similar biochemical function (but not necessarily to the same extent, as a natural sequence.

2.Nucleic acids and encoded them polypeptides

The nucleic acid sequence ofBacillussp. 195 may be operatively associated with various promoters and regulators in vector and expressed in different cells masters. The sequence of nucleic acid, including 2103 residue described and deposited in GenBank with the access number No. AB006823(seeFig. 1A-B). The polypeptide sequence encoded by the nucleic acid sequence of 2103 residues described and deposited in GenBank with the access number BAA22082.1 and includes 700 amino acids in length (Fig. 3). The first 46 amino acids form a signal peptide. The splitting is carried out after the residue 46 (Ala46).

When expression in cellsB.subtilisthere are three proteoliticeski protestirovannyx form of the protein identified in the analysis in the gel. All these forms have the same aminocore, but differ in their carboxilic. The shape of the size of 49.5 kDa ending balance Val492 (sequence shown in Fig. 4), i.e. in her proteolytic cleavage occurs after residue 492. Two other longer forms, size 69 kDa and 60 kDa, respectively, contain one and two krahmalsoderzhaschih domain, as described the in Sumitani et al. (Sumitani et al., (2000)). Genetically truncated at the C-end of the form is obtained using products containing to-end balances Tyr494, Lys504 and Val509. All of these products with truncation, obtained by recombinant technology, are expressed at a high level in nine strains ofB. subtiliswith deletional protease (see US20050202535A1) under the control of the LAT promoter and signal sequence, as shown in Fig. 8.

2.1Protein fusion and recombinant proteins

One aspect of the present invention relates to proteins merge using the signal sequence of amylases from other microorganisms, such as yeast or other bacteria attached to the Mature protein ofBacillussp. 195. Namely, the first 46 amino acids that form the signal sequence according to Fig. 3, which can be removed and replaced with a signal sequence from another organism or variant signal sequence from another organism. For example, the LAT sequence (underlined and shown in capital letters) may be substituted for the first 46 amino acids, as shown in Fig. 4.

Other examples include, without limitation, the signal sequence for α-amylase fromB. subtilis(amyE) for expression inB. subtilisaprE promoter ofB. subtilisand signal sequences, are also eligible the for expression in B. subtilis.In addition, in the present invention the possibility of testing the expression inStreptomycessp. when using promoters fromStreptomycesand signal sequences from CelA.

3.The method of production and purification of proteins

Methods production and purification of proteins that are secreted into the culture medium fromBaallus,known in this field, as is known, the appropriate cell hosts for the production of α-amylases. Representative methods for the production of α-amylases described below.

3.1Materials and methods for productionα-amylases

The DNA sequence encoding the α-amylase Amy195 or a version thereof obtained by the above in the present description means or by any alternative methods known in this field, can be expressed with the formation of the enzyme form, using an expression vector which typically includes control sequences encoding the corresponding promoter, operator, binding site of the ribosome, the signal sequence of translation initiation and optional gene-repressor or different genes-activators.

For example,Bacillussp. 195 can be cultivated at 30°C, as described by Kawaguchi et al. (Kawaguchi et al., "Purification and some properties of a Haim-sensitive α-amylases from newly isolatedBacillussp. 195,"Bios. Bitechnol. Biochem56: 1792-1796 (1992)). Alternatively, the gene encoding α-amylase, operatively associated with the vector, can be transfirieran in another organism, such asStreptomyces lividansTK-24, followed by cultivation in suitable conditions, as described in the literature (J. Sumitani et al., "New type of starch-binding domain: the direct repeat motif in the C-terminal regionoi Bacillussp. 195 α-amylase contributes to starch binding and raw starch degrading,"Biochem. J.350: 477-484 (2000)).

The recombinant expression vector containing the DNA sequence, which encodes the α-amylase Amy195 or her option, may be any vector, which may be in a convenient embodiment, subjected to treatments on the basis of recombinant DNA, and the choice of such a vector is basically dependent on the host cell into which it should be entered. For example, the vector may be an autonomously can replicate the vector, i.e. a vector which exists in the form of extrachromosomal unit, replication which does not depend on the replication of the chromosome, i.e. it can be a plasmid, bacteriophage or extrachromosomal element, a mini-chromosome, or to artificial chromosome. Alternatively, the specified vector can be represented as a vector, which, for its introduction into an isolated host cell integrates into the genome of the host cell and is replicated along with one or more chromosomes, in which it is integrated. Integrated the ing gene can also be amplified with the formation of multiple copies of a gene in the chromosome due to the use of amplificare design, created by selection on antibiotics or if you are using another factor selective pressure, such as mandatory regulatory gene, or by complementaly-type dose-effect relation to the binding of gene-specific metabolic pathways.

In the vector, the DNA sequence must be operatively linked to a suitable promoter sequence. The specified promoter may be any DNA sequence which shows transcriptional activity in the selected cell the owner and may be obtained from genes encoding proteins homologous or heterologous to the cells of the host. Representative promoters for transcription of the DNA sequence encoding the α-amylase Amy 195 or her option, especially in a bacterial host cell, are the promoter of the lac-operon fromE. colithe promoters of the gene dagA and agarases or elA fromStreptomyces coelicolorthe promoters of the gene of α-amylase fromBacillus licheniformis(amyL), the promoters of the gene maltogenic amylase (amyM) fromBacillus stearothermophilus,the promoters of α-amylase (amyQ) fromBacillus amyloliquefaciensthe promoters of genes xylA and xylB fromBacillus subtilisand so on.For transcription in a fungal organism-host as examples of suitable promoters can specify the promoters obtained from the genes encoding, respectively, the amylase fromAspergillus oryzaeTKA, aspartic proteinase fromRhizomucor mieheineutral α-amylase fromAspergillus niger, kislotostabilen α-amylasesfrom A. nigerthe glucoamylase of theA. nigerthe lipase fromRhizomucor mieheialkaline protease fromA. oryzae, triosephosphate fromA. oryzaeor acetamides fromA. nidulans. In the case when the gene encoding a variant polypeptide α-amylase, is expressed in bacterial cells such asE. colican be selected an appropriate promoter, for example, the promoter from bacteriophage comprising the T7 promoter and the promoter of phage lambda. Examples of suitable promoters for expression in yeast include, without limitation, promoters Gal 1 and Gal 10 ofSaccharomyces cerevisiaeand the promoters of thePichia pastorAOX1 or AOX2. For expression in cellsTrichoderma reeseican be used promoter CBHII (cellobiohydrolase II).

The expression vector may also comprise a suitable transcription terminator and, in the case of eukaryotic polyadenylation sequence, operatively linked to a DNA sequence that encodes the α-amylase Amy 195 or its variants. Sequence termination and polyadenylation can be appropriately selected from the same sources as the promoter.

The specified vector may also comprise a DNA sequence enabling the vector to replicate in the cell host. Examples of such sequences I have are originy replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.

The specified vector may also include breeding marker, i.e. a gene the product of which complements a defect existing in the selected cell host, such asdalgenes fromB. subtilisorB.licheniformis,or gene, which imparts resistance to antibiotics, such as, for example, a gene giving resistance to ampicillin, kanamycin, chloramphenicol or tetracycline. Moreover, the vector may include selection markers forAspergillussuch asamdS, argB, niaDandxxsC,the token that defines the resistance to hygromycin, or the selection may be accompanied by a concomitant transformation, known in this area. See, for example, an application for an international patent PCT WO 91/17243.

Although intracellular expression or solid state fermentation can be useful in a number of respects, for example, in cases when the host cells used some bacteria or fungi, one aspect of the invention relates to the expression of α-amylase Amy 195 or its variants in the culture medium. Basically, the α-amylase signal includes a sequence of aminocore that allows it to achieve its secretion into the culture medium. If desired, the signal peptide may be replaced by another sequence, for example, replacing DNA sequences encoding appropriate signal is AUX polypeptide. The signal sequence of α-amylases in the typical case are characterized by the presence of three domains, N-terminal domain of the H-domain and C-terminal domain, where the length typically ranges from 18 to 35 residues, but they can be longer, as shown in the example signal sequence Amy 195.

The specified expression vector typically includes components of the cloning vector, such as, for example, an item that allows Autonomous replication of the vector in the selected organism, the host, and one or more detectable phenotypic markers for breeding purposes. This expression vector normal includes a control nucleotide sequence such as a promoter, operator, binding site of the ribosome, the signal sequence of translation initiation and, optionally, a gene-repressor and one or more gene-activators. Additionally, the expression vector can include a sequence encoding a amino acid sequence, which can affect the variant α-amylase in the host cell organelle, such as peroxisome or a specific compartment of the host cell. Such acting sequence include, without limitation, the sequence SKL. For expression under control of the control sequences sequence nucleic sour the s variant α-amylase operatively associated with a control sequence in an appropriate way, with regard to expression. The part of the representative vector is shown in Fig. 5.

The procedures used for ligating design DNA that encodes a variant α-amylase, the promoter, terminator and other elements, respectively, and for the purposes of their incorporation into appropriate vectors containing the information necessary for replication, are well-known experts in this field (see, for example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nded., Cold Spring Harbor, 1989, and 3rded., 2001).

Isolated cell containing either the structure of DNA or the expression vector, can be successfully used as a host cell in the procedure of recombinant production of α-amylase Amy 195 or its variants. This cell can be transformed design DNA that encodes the enzyme, by integrating design DNA (in the form of one or more copies) in the host chromosome. This integration is basically considered an advantage, because in this case the DNA sequence, more likely to be stably maintained in the cell. Integration of structures of DNA into the host chromosome can be carried out by conventional methods, for example, by homologous or heterologous recombination. Alternatively, the specified cell can be transformed with the above expression vector in the conditions of use of different types of host cells.

Examples of suitable bacterial organisms-hosts include Gram-positive bacteria, such as species ofBacillaceae,includingBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, B. megaterium BacillusandBacillus thuringiensis;the speciesStreptomycessuch asStreptomyces murinus;the lactic acid bacteria, includingLactococcusspp., such asLactococcus lactis; Lactobacillusspp., includingLactobacillus reuteri; Leuconostocspp.;Pediococcusspp. andStreptococcusspp. Alternatively, the strains for use as the host body can be selected from Gram-negative bacteria belonging toEnterobacteriaceaeincludingE. colior belonging to aPseudomonadaceae.

Suitable yeast organism, the host may be selected from the relevant biotech relation to the type of yeast, which includes, without limitation, the following types of yeast, asPichiasp.,Hansenulasp. orKluyveromyces, Yarrowinia, Schizosaccharomycesor kind belonging to aSaccharomyces,includingSaccharomyces cerevisiaeor kind belonging to aSchizosaccharomycessuch as, for example,S. pombe. The strain of methylotrophic species of the yeastPichia pastoriscan also be used as the host body. Alternatively, the organism-owner may apply toHansenula. Suitable organisms-owners among filamentous fungi include species ofAspergillus,for example,, Aspergills niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamoriorAspergillus nidulans.Alternatively, as the host body can be used strains ofFusariumfor example, Fusarium oxysporum,orRhizomucorsuch asRhizomucor miehei. Other suitable strains include strains of the speciesThermomycesandMucor. Additionally, as the host body can be usedTrichoderma reesei. A suitable procedure for transformation of host cellsAspergillusincludes, without limitation, the procedure described in EP 238023.

According to another aspect, the present invention proposes a method of obtaining α-amylase Amy 195 or its variant comprising culturing the host cell described above under conditions suitable for production of the enzyme and recovering the enzyme from the cells and/or culture medium.

The medium used for culturing cells, can be any standard environment suitable for growing under consideration of the host cell and achieve the expression of α-amylase Amy 195 or its variants. Suitable environment and components of the media are available from commercial sources or can be obtained in accordance with known procedures (for example, described in catalogues of the American type culture Collection (American Type Culture Collection).

According to another aspect, an enzyme secreted from the host cells, sportsouth in the product created on the basis of full of broth. According to the methods of the present invention, the receipt of spent fermentation broth from the recombinant microorganism can be achieved using any method of cultivation, known in this area, which leads to expression of α-amylase. In this regard, the fermentation should be understood as a process comprising culturing in katalozhnyh flasks, low - or large-scale fermentation (including continuous fermentation, periodic fermentation, periodic fermentation with water or solid state fermentation) in laboratory or industrial fermentors performed in a suitable medium and under conditions that allow to Express or to highlight the amylase. The term "exhaust full fermentation broth" in the context of the present description is defined as nefrackzionirovannam the contents of the fermentation material, which includes culture medium, extracellular proteins (e.g. enzymes) and cell biomass. It should be understood that the term "full exhaust fermentation broth also covers cell biomass, which was lysed or subjected permeabilization using methods known in this field.

An enzyme secreted from the host cells may be recovered from the culture cf is built using well-known procedures, which include the separation of the cells from the medium by centrifugation or filtration and sedimentation proteinopathy components of the environment with the use of a salt such as ammonium sulphate, followed by procedures chromatography, such as ion exchange chromatography, affinity chromatography, etc.

In one aspect the present invention relates to polynucleotide in the vector, which is operatively connected with a control sequence and which is capable of expression of the coding sequence in a cell host, which is an expression vector. When this control sequence can be modified, for example by additional regulatory elements of transcription, in order to make the level of transcription under the control of the control sequence more responsive to the modulators of transcription. These control sequences can, in particular, include the promoters.

Cell host can be cultured in appropriate conditions which allow the expression of α-amylase Amy 195 or its variants. The expression of the enzymes may be constitutive, so that they can be produced on a regular basis, or induced, where this expression requires a stimulus for initiation. In the case of inducible expression productively can be initiated in the event that when, for example, adding an inducing substance in the culture medium, for example, dexamethasone or IPTG or Sepharose. The polypeptides may also be obtained in recombinant form in a cell-free systemin vitrofor example, in the system, including rabbit reticulocytes TnT™ (Promega).

In the case of α-amylase Amy 195 or its variants expressing their master-organism can be cultured in appropriate for this organism the host environment, in aerobic conditions. Shake or combination of methods of mixing or aeration in this case can be useful when the output is carried out when appropriate to this the host body temperature, for example, in the temperature range from about 25°C to about 75°C (for example, at a temperature of 30°C - 45°C), depending on the needs of the host body and the conditions associated with the production of the desired variant α-amylase. The cultivation can be carried out for from about 12 to about 100 hours or more (including any period of time, in hours, for example, from 24 to 72 hours). In a typical case, the culture broth has a pH from about 5.5 to about 8.0 and is also determined by the culture conditions required for the body of the host in relation to the production of variant α-amylase.

3.2The materials and methods used is for protein purification

Methods of fermentation, separation and concentration are well known in this field and can use standard techniques to obtain the concentrated α-amylase Amy 195 or its variant in solution.

After fermentation receive the fermentation broth of microbial cells and various suspended solids, including residual materials fermentation, remove using standard separation techniques, obtaining a solution with amylase. Basically, using filtration, centrifugation, filtration, vacuum filtration in a rotating drum, ultrafiltration, centrifugation with subsequent procedure ultrafiltration, extraction or chromatography, or the like

It is advisable that the concentration of the solution containing the α-amylase Amy 195 or her option, in order to optimize the recovery process. The use of concentrated solutions requires an increase in the incubation time for collecting the purified precipitate the enzyme.

The solution containing the enzyme concentrate in the framework of standard methods for the concentration to obtain the desired level of enzyme. The concentration of the solution containing the enzyme can be achieved under any of the following methods. Representative purification methods include, without limitation, vacuum filtration in the rotating drum and/or ultrafiltration.

The solution containing the enzyme, concentrated to produce a concentrated solution of the enzyme, so that the enzymatic activity of the specified concentrated α-amylase Amy 195 or its variant in the containing solution was, at least about 4 g/l (in particular, at least about 4.8 g/l or at least 5.6 g/l or even higher). In some embodiments, purification of the indicated concentration can be increased even up to about 25 g/L.

The term "precipitating agent" in the context of cleaning refers to the compound that is effective in the direction of deposition of α-amylase Amy 195 or her version of the concentrated solution of the enzyme in solid form, regardless of the nature, i.e. crystalline, amorphous, or a mixture thereof.

The deposition can be carried out using, for example, the precipitating agent-based metal halide. The precipitating agents based on metal halide include, without limitation, alkali metal chlorides, alkali metal bromides and mixtures of two or more of these metal halides. Representative halides of metals include sodium chloride, potassium chloride, sodium bromide, potassium bromide and mixtures of two or more of these metal halides. Such precipitating agent-based halide metals, such as sodium chloride, may also be used as a con is Hervanta.

The precipitating agent-based metal halide is used in an amount which is effective for the deposition of α-amylase Amy 195 or its variants. Choosing the lowest effective amount, and the optimal number of metal halide, which is effective to cause precipitation of the enzyme, as well as the deposition conditions in order to achieve maximum recovery, including incubation time, pH, temperature and concentration of the enzyme can easily be determined by a specialist after standard testing procedures.

Basically, at least from about 5% (weight/volume) to about 25% (weight/volume) of metal halide added to the concentrated enzyme solution and usually at a concentration equal to at least 8% (weight/volume). Basically, add no more than 25% (weight/volume) of the metal halide to a solution of concentrated enzyme and usually not more than about 20% (weight/volume). The optimum concentration of precipitating agent on the basis of the halide of the metal will depend, among other factors, on the nature of the specific variant α-amylase Amy 195 and its concentration in the concentrated solution of the enzyme.

Another alternative method of deposition of the enzyme is the use of organic compounds. Representative organic compounds used is as precipitating agents, include 4-hydroxybenzoic acid, salts of alkaline metal and 4-hydroxybenzoic acid, a complex of alkyl esters of 4-hydroxybenzoic acid, and mixtures of two or more of these organic compounds. Adding these organic precipitating agents can be administered before, simultaneously with or after addition of the precipitating agent on the basis of the halide of the metal, and you may practice adding both precipitating agents based on organic compounds and metal halide, sequentially or simultaneously.

A more detailed description of these methods see, for example, in U.S. patent No. 5281526. Basically, organic precipitating agents are selected from the group consisting of alkali metal salts and 4-hydroxybenzoic acid, such as sodium or potassium salts, linear or branched alkyl complex esters of 4-hydroxybenzoic acid, where the alkyl group contains from 1 to 12 carbon atoms, and mixtures of two or more of these organic compounds. Organic precipitating agents can represent, for example, linear or branched alkyl complex esters of 4-hydroxybenzoic acid, where this alkyl group contains from 1 to 10 carbon atoms, and may be a mixture of two or more of these organic compounds. A representative of the organization is practical compounds include linear alkyl esters of 4-hydroxybenzoic acid, where this alkyl group contains from 1 to 6 carbon atoms, and may be a mixture of two or more of these organic compounds. Can also be used methyl ester of 4-hydroxybenzoic acid, complex propyl esters of 4-hydroxybenzoic acid, complex butyl ester of 4-hydroxybenzoic acid, complex ethyl ester of 4-hydroxybenzoic acid, and mixtures of two or more of these organic compounds. Additional organic compounds include, without limitation, methyl ester of 4-hydroxybenzoic acid (called methyl paraben), complex propyl ester of 4-hydroxybenzoic acid (called propyl-paraben), where both of these compounds are preservatives for amylase.

Adding organic precipitating agent is characterized by the advantage of defined high flexibility of deposition conditions with respect to pH, temperature, concentrations of α-amylase Amy 195 or its variant, the concentration of the precipitating agent and the incubation time.

The precipitating agent based on organic acids are used in amounts that are effective to improve the efficiency of deposition of the enzyme, which is carried out with the use of a precipitating agent-based metal halide. The selection of the least efficient number and optimal to which icesta precipitating agent based on organic compounds, and of deposition conditions to achieve maximum recovery, including the duration of incubation, pH, temperature, concentration of enzyme may be performed by the person skilled in the art given this description, after carrying out the standard testing procedures.

Basically, add at least about a 0.01% (weight/volume) precipitating agent based on organic compounds in a concentrated solution of a variant enzyme, and usually at least add approximately 0.02% (weight/volume). Basically, add no more than about 0.3% (weight/volume) precipitating agent based on an organic compound to a concentrated solution of a variant of the enzyme, and usually not more than approximately 0.2% (weight/volume).

The concentrated enzyme solution containing the precipitating agent-based metal halide and precipitating agent based on organic compounds, may be adjusted in pH, which will depend on the nature of the enzyme to be cleaned. Basically, pH is adjusted to approximately close to the isoelectric point of the amylase. The pH value adjusted to achieve a pH in the range from about 2.5 pH units below the isoelectric point (pI) to about 2.5 pH units above the isoelectric point.

The incubation time required to obtain osedc the purified enzyme depends on the nature of the specific enzyme, the concentration of the enzyme and the one or more precipitating agents and their concentrations. Basically, a time effective to precipitate the enzyme is from about 1 to about 30 hours; and usually does not exceed about 25 hours. In the presence of precipitating agent based on organic compounds the duration of the incubation period is reduced to less than about 10 hours, and in most cases is even approximately 6 hours.

In General, the temperature during incubation is from about 4°C to about 50°C. basically, this method is carried out at a temperature from about 10°to about 45°C (for example, in the temperature range from about 20°C to about 40°C). The optimum temperature for the induction of deposition varies in accordance with conditions in which there is a solution, and from the nature of the enzyme, or one or more precipitating agents.

Full recovery of the precipitate of purified enzyme and the efficiency with which this process is increasing with stirring of a solution comprising an enzyme, adding the halide of the metal and adding organic compounds. Stage mixing is carried out in the process of adding the halide of the metal and organic compounds, as well as throughout the past the subsequent incubation period. Suitable methods of mixing include mechanical stirring or shaking, vigorous aeration or any other similar procedure.

After the incubation of the purified enzyme is separated from the dissociated pigment and other impurities are collected using the standard procedures of separation, such as filtration, centrifugation, filtration, vacuum filtration in a rotating drum, ultrafiltration, filtration under pressure, the cross-membrane microfiltration, membrane microfiltration in cross flow or the like, a Further purification of the precipitate purified enzyme can be achieved when washing this precipitate with water. So, for example, the residue is purified enzyme was washed with water containing precipitating agent on the basis of the halide of the metal, or water containing a metal halide and the organic compound as precipitating agents.

During fermentation of α-amylase Amy 195 or its variant accumulates in the culture broth. For isolation and purification of the desired variant α-amylase culture broth is centrifuged or filtered to remove cells, and the resulting cell-free liquid used for cleaning enzyme. In one embodiment of the present invention specified cell-free broth absoluut using AMM sulfate is tion, approximately 70% of saturation; then the fraction of the precipitate obtained with the use of salt taken up to 70% of saturation, dissolved in buffer and applied on a column, such as column with Sephadex G-100, and then elute to restore the fraction of active enzyme. For further purification can be used a standard procedure, such as ion-exchange chromatography.

Purified enzymes are used for washing and cleaning. For example, they can be used as detergents in washing powders and to remove stains. They can then be included in the finished product, having a form or liquid (solution, suspension), or solid (granular, powder form).

A more specific example of the purification method is described in the work of Sumitani et al. (J. Sumitani et al., "New type of starch-binding domain: the direct repeat motif in the C-terminal regionof Bacillussp. 195 α-amylase contributes to starch binding and raw starch degrading,"Biochem. J.350:477-484 (2000)), and below this method is given in abbreviated form. The enzyme obtained from 4 liters of culture supernatant fromStreptomyces lividansTK24, process (NH4)2SO4at 80% saturation. Sediment recover by centrifugation at 10000 x g for 20 minutes at 4°C) and pererastayut in 20 mm Tris/HCl buffer (pH 7.0)containing 5 mm CaCl2. The solubilized sludge is then subjected to dialysis against rebuffer. Cialisovernight the sample is applied on a column of Sephacryl S-200, which was previously equilibrated with 20 mm Tris/HCl buffer (pH 7.0), 5 mm CaCl2and spend a linear elution with a speed of over 7 cm/hour in the same buffer. The fractions obtained by elution from the column, collect and evaluate their activity in the test for enzymatic activity and the use of electrophoresis in the LTO-PAG. Next, the protein is purified as follows. A column of Toyopearl HW55 (Tosoh Bioscience, Montgomeryville, PA; Cat. No. 19812) balance 20 mm Tris/HCl buffer (pH 7.0)containing 5 mm CaCl2and 1.5 M (NH4)2SO4. The enzyme elute in a linear gradient from 1.5 to 0 M (NH4)2SO4in 20 mm Tris/HCl buffer, pH 7.0, containing 5 mm CaCl2. The active fractions are collected and the enzyme precipitated (NH4)2SO4at 80% saturation. The residue is taken, pererastayut and deleteroute according to the above procedure. Cialisovernight sample applied to the column Mono Q HR5/5 (Amersham Pharmacia; Cat. No. 17-5167-01), which was pre-equilibrated to 20 mm Tris/HCl buffer (pH 7.0)containing 5 mm CaCl2with a flow rate of 60 ml/hour. The active fractions are collected and added to a 1.5 M solution (NH4)2S04. The fraction of active enzyme is subjected to re-chromatography on a column of Toyopearl HW55, as described earlier, with homogeneous enzyme, the evaluation of palamalaguti in LTO-page (see J. Sumitani et al., "New type of starch-binding domain: the direct repeat motif in the C-terminal regionof Bacillussp. 195 α-amylase contributes to starch binding and raw starch degrading,"Biochem. J.350: 477-484 (2000)), which provides a General description and discussion of this method and its variations.

For recovery in large-scale processes, this enzyme may be partially purified, as described in General terms above, by removing the cells by flocculation by polymers. Alternatively, the enzyme can be purified by filtration, followed by concentration by ultrafiltration with the use of available membranes and related equipment. However, in some embodiments, application of the enzyme does not need to be cleaned, and in this case the entire culture broth may be subjected to lizirovania and further used without additional processing. Further, the enzyme can be processed to give, for example, granules.

4.Composition for cleaning

α-amylase Amy195 and one or more of its variants possess valuable properties, that can be used in many industrial processes. These enzymes can be used as a component in detergent compositions for washing, dishwashing and cleaning of hard surfaces. They can also be introduced into the composition as part of a detergent additive as part of a detergent composition, as the hour is ü composition, designed for automatic or manual dishwashing, etc. α-amylase Amy195 and one or more of its options can be included in these funds in concentration, which is standardly used in detergents. According to the present invention, in the present detergent compositions of α-amylase may be added in an amount corresponding to within 0.00001 - 1 mg (calculated on the pure enzyme protein) of α-amylase per liter of fluid for washing/dishwashing. Representative compositions are given below.

4.1Detergent composition for washing

Accordingly, α-amylase Amyl95 or her option may typically be a component of a detergent composition in the form of only one enzyme or in combination with other enzymes, including other amylolytic enzymes. As such, it can be added to the composition of the detergent composition in the form of dust-free granulate, a stabilized liquid, or protected enzyme. Dust-free pellets can be obtained, for example, by the procedure described in U.S. patent NoNo. 4106991 and 4661452, and can optionally contain a coating applied according to known in the field methods. Examples of waxy coating materials include poly(ethylenoxide) products (polyethyleneglycol, PEG) with average molecular weight of the t 1000 to 20000; ethoxylated nonylphenols containing from 16 to 50 ethyleneoxide units, ethoxylated fatty alcohols, where part of their structure, the alcohol contains from 12 to 20 carbon atoms, and which contain from 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono -, di - and triglycerides of fatty acids. Examples of film-forming coating materials suitable for use in the methods of application using a fluidized bed, see, for example, in the patent GB no. 1483591. Liquid enzyme compositions can be stabilized, for example, by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Other stabilizers enzymes are also known in this field. Protected enzymes can be obtained according to the method described, for example, in EP 238216. The polyols has long been known as stabilizers of proteins, but also as a way of improving the solubility of proteins (see, for example, J. K. Kaushik et al., "Why is trehalose an exceptional protein stabilizer?"J. Biol. Chem.278: 26458-65 (2003) and is contained in the link operation, and Monica Conti et al., "Capillary isoelectric focusing: the problem of protein solubility,"J. Chromatography A757: 237-245 (1997)).

Detergent composition may be presented in any form, for example, in the form of powders, granules, pastes, or liquids. Liquid detergent can be the ü water, which typically contains approximately 70% water and from 0% to about 30% organic solvent. This composition can also be represented in the form of a compact gel that contains approximately 30% water.

Detergent composition comprises one or more surfactants, each of which may be anionic, nonionic, cationic or zwitterionic. Detergent typically contains from 0% to about 50% anionic surfactants, such as linear Las (LAS); α-reincorporate (AOS); alkylsulfate (sulfate fatty alcohol) (AS); alexisoliviat alcohol (AEOS or AES); secondary alkanesulfonyl (SAS); methyl ester of α-sulfo-fatty acids; alkyl - or alkenyl-succinic acid; or soap. This composition can also contain from 0% to about 40% non-ionic surfactant such as alcohol ethoxylate (AEO or AE), ethoxylates carboxylating alcohol ethoxylate of Nonylphenol, alkylpolyglycoside, alkyldimethylammonium, monoethanolamide ethoxylated fatty acids, monoethanolamide fatty acids or amide polyhydroxyalkanoic fatty acids (as described, for example in WO 92/06154).

Additionally, the detergent composition can include one or more other enzymes, such as lipase, another amylolyticus the enzyme, cutinase, protease, cellulase, peroxidase, and/or laccase, in any combination.

The specified detergent may contain from about 1% to about 65% detergent filler or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, citrate, nitryltriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTMPA), alkyl - or alkenyl-succinic acid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst). The specified detergent can also be represented in the form without filler, for example, essentially in the form of free detergent filler. While enzymes can be used in the composition is any composition that is compatible with maintaining the stability of the enzyme. These enzymes mainly can be protected from the action of harmful components through the use of well-known forms of encapsulation, for example, by granulating or sequestration in the hydrogels. Enzymes and, in particular, α-amylase, such as molecules amy195, whether or not containing krahmalnaya the documents can be used in various compositions, including compositions used for Laundry and dishwashing, in compositions used for cleaning the surface but also in the compositions used to obtain ethanol from to is making a movie or biomass.

The detergent may include one or more polymers. Examples include carboxymethyl cellulose (CMC), poly(vinyl pyrrolidone) (PVP), polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylate, such as polyacrylates, copolymers of maleic /acrylic acid copolymers loyaltybrief/acrylic acid.

The specified detergent may contain a whitening system, which, in turn, may include a source of H2O2such as perborate or percarbonate, which can be combined with a bleaching activator, forming nagkalat, such as tetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate (NOBS). Alternatively, whitening system may include peroxyacids (e.g., peroxyacids amide, amidnogo or Sultonova type). Whitening system can also be a whitening system enzyme type, for example, on the basis of perhydrol, such as the system described in International application PCT WO 2005/056783.

The enzymes in the present detergent compositions can be stabilized through the use of standard stabilizing agents, for example, polyol, such as propylene glycol or glycerin; sugar or sugar alcohol, lactic acid, boric acid or a derivative of boric acid, such as, n is the sample, ester aromatic compounds borate; and this composition may be produced in accordance with the procedure described, for example, in WO 92/19709 and WO 92/19708.

The specified detergent may also contain other standard detergent ingredients, such as fabric conditioners including clays, amplifiers flushing suppressor education soapy foam, anti-corrosion means, the material used for the suspension of polluting particles, means preventing re-deposition polluting particles, dyes, antibacterial agents, inhibitors tarnishing, optical brighteners or perfumes.

The pH (measured in water solution concentration) usually has a neutral or alkaline value, for example, a pH of from about 7.0 to about 11.0 in.

Particular forms of detergent compositions comprising α-amylase Amy195 or its variants, can be made with the formation of compositions that resembles the following:

1) Detergent composition is made in the form of a granulate having a bulk density equal to at least 600 g/l, which includes linear Las (calculated by the acid), in an amount of from about 7% to about 12%; alexisoliviat alcohol (for example, C12-18alcohol, 1-2 ethyleneoxide units (EO)or alkylsulfate (for example, C1618 ), in an amount of from about 1% to about 4%; alcohol ethoxylate (e.g., C14-15alcohol, 7 EO), in the amount of from about 5% to about 9%; sodium carbonate (i.e. Na2CO3), in an amount of from about 14% to about 20%; soluble silicate (e.g., Na2Oh , 2Si2), in an amount of from about 2% to about 6%; zeolite (e.g., NaAlSi4), in an amount of from about 15% to about 22%; sodium sulfate (i.e. Na2SO4), in an amount of from about 0% to about 6%; sodium citrate/citric acid (for example, C6H5Na3About7/C6H8O7), in the amount of from 0% to about 15%; perborate sodium (for example, NaB3H2O), in the amount of from about 11% to about 18%; TAED, in the amount of from about 2% to about 6%; carboxymethylcellulose (CMC), in the amount of from 0% to about 2%; polymers (e.g., copolymer of maleic/acrylic acid, PVP, PEG)0-3%; enzymes (calculated on the pure enzyme), and 0.0001-0.1% protein; and minor ingredients (e.g., suppressor of education soap suds, perfumes, optical brighteners, fotoatelier) 0-5%.

2) Detergent composition is made in the form of a granulate having a bulk density equal to at least 600 g/l, which includes linear Las (calculated by the acid), from about 6% to about 11%; adaxial the veils of alcohol (e.g., With12-18alcohol, 1-2 EO) or alkylsulfate (for example, C16-18), in an amount of from about 1% to about 3%; alcohol ethoxylate (e.g., C14-15alcohol, 7 EO), in the amount of from about 5% to about 9%; sodium carbonate (i.e. Na2CO3), in an amount of from about 15% to about 21%; soluble silicate (e.g., Na2Oh, 2Si2), in an amount of from about 1% to about 4%; zeolite (e.g., NaAlSi4), in an amount of from about 24% to about 34%; sodium sulfate (i.e. Na2SO4), in an amount of from about 4% to about 10%; sodium citrate/citric acid (for example, C6H5Na3About7/C6H8O7), in the amount of from 0% to about 15%; carboxymethylcellulose (CMC), in the amount of from 0% to about 2%; polymers (e.g., copolymer of maleic/acrylic acid, PVP, PEG), 1-6%; enzymes (calculated on the pure protein enzyme), and 0.0001 to 0.1%; minor ingredients (e.g., suppressor of education soapy foam, perfume) 0-5%.

3) Detergent composition is made in the form of a granulate having a bulk density equal to at least 600 g/l, which includes linear Las (calculated by the acid), in an amount of from about 5% to about 9%; alcohol ethoxylate (e.g., With12-15alcohol, 7 EO), in the amount of from about 7% to about 14%; Soaps, such as fatty acid (e.g. the R, C16-22fatty acid), in an amount of from about 1% to about 3%; sodium carbonate (i.e. Na2CO3), in an amount of from about 10% to about 17%; soluble silicate (e.g., Na2Oh, 2Si2), in an amount of from about 3% to about 9%; zeolite (as, NaAlSi4), in an amount of from about 23% to about 33%; sodium sulfate (i.e. Na2SO4), in the amount of from 0% to about 4%; perborate sodium (for example, NaB3H2O), in the amount of from about 8% to about 16%; TAED, in the amount of from about 2% to about 8%; phosphonate (for example, EDTMPA), in the amount of from 0% to about 1%; carboxymethylcellulose (CMC), in the amount of from 0% to about 2%; polymers (e.g., copolymer of maleic/acrylic acid, PVP, PEG)0-3%; enzymes (calculated on the pure protein enzyme), and 0.0001 to 0.1%; minor ingredients (e.g., suppressor of education soapy foam, perfume, optical Brightener) 0-5%.

4) Detergent composition is made in the form of a granulate having a bulk density equal to at least 600 g/l, which includes linear Las (calculated by the acid), in an amount of from about 8% to about 12%; alcohol ethoxylate (e.g., With12-15alcohol, 7 EO), in the amount of from about 10% to about 25%; sodium carbonate (i.e. Na2CO3), in an amount of from about 14% to about 22%; soluble with the Licata (for example, Na2Oh, 2Si2), in an amount of from about 1% to about 5%; zeolite (e.g., NaAlSi4), in an amount of from about 25% to about 35%; sodium sulfate (e.g.,Na2SO4), in the amount of from 0% to about 10%; carboxymethylcellulose (CMC), in the amount of from 0% to about 2%; polymers (e.g., copolymer of maleic/acrylic acid, PVP, PEG), 1-3%; enzymes (calculated on the pure protein enzyme), and 0.0001 to 0.1%; and minor ingredients (e.g., suppressor of education soapy foam, perfume) 0-5%.

5) Aqueous liquid detergent composition comprising linear Las (calculated by the acid), in an amount of from about 15% to about 21%; alcohol ethoxylate (e.g., With12-15alcohol, 7 EO or12-15alcohol, 5 EO), in the amount of from about 12% to about 18%; Soaps, such as fatty acid (e.g. oleic acid), in an amount of from about 3% to about 13%; alkenyl-succinic acid (C12-14), in the amount of from 0% to about 13%; aminoethanol, in the amount of from about 8% to about 18%; citric acid in an amount of from about 2% to about 8%; phosphonate, in the amount of from 0% to about 3%; polymers (e.g., PVP, PEG), in the amount of from 0% to about 3%; borate (e.g., In4About7), in the amount of from 0% to about 2%; ethanol, in an amount of from 0% to about 3%; propylene glycol in an amount of from about 8% is about 14; enzymes (calculated on the pure protein enzyme), and 0.0001 to 0.1%; and minor ingredients (e.g., tools, contributing to the dispersion, the suppressor education soapy foam, perfume, optical Brightener) 0-5%.

6) Aqueous structured liquid detergent composition comprising linear Las (calculated by the acid), in an amount of from about 15% to about 21%; alcohol ethoxylate (e.g., With12-15alcohol, 7 EO or12-15alcohol, 5 EO), 3-9%; Soaps, such as fatty acid (e.g. oleic acid), in an amount of from about 3% to about 10%; zeolite zeolite (NaAlSi4), in an amount of from about 14% to about 22%; potassium citrate, in an amount of from about 9% to about 18%; borate (e.g., In4About7), in the amount of from 0% to about 2%; carboxymethylcellulose (CMC), in the amount of from 0% to about 2%; polymers (e.g., PEG, PVP), in the amount of from 0% to about 3%; linked polymers, such as, for example, a copolymer of loriseacrist/acrylic acid (in a molar ratio of 25:1, MV-3800), in the amount of from 0% to about 3%, glycerin in an amount of from 0% to about 5%; enzymes (calculated on the pure protein enzyme), and 0.0001 to 0.1%; and minor ingredients (e.g., dispersing agents, suppressors education soapy foam, perfume, optical brighteners), 0-5%.

7) Detergent composition, and is prepared in the form of a granulate, possessing a bulk density equal to at least 600 g/l, which includes sulfate fatty alcohol, in an amount of from about 5% to about 10%; monoethanolamide ethoxylated fatty acids, in an amount of from about 3% to about 9%; Soaps, such as fatty acid 0-3%; sodium carbonate (e.g., Na2CO3), in an amount of from about 5% to about 10%; soluble silicate (e.g., Na2Oh, 2Si2), in an amount of from about 1% to about 4%; zeolite (e.g., NaAlSi4), in an amount of from about 20% to about 40%; sodium sulfate (e.g.,Na2SO4), in an amount of from about 2% to about 8%; perborate sodium (for example, NaB3H2O), in the amount of from about 12% to about 18%; TAED, in the amount of from about 2% to about 7%; polymers (e.g., copolymer of maleic/acrylic acid, PEG), in the amount of from about 1% to about 5%; enzymes (calculated on the pure protein enzyme), and 0.0001 to 0.1%; and minor ingredients (e.g. optical brighteners, suppressors education soapy foam, perfume), 0-5%.

8) Detergent composition is made in the form of a granulate comprising a linear Las (calculated by the acid), in an amount of from about 8% to about 14%; monoethanolamide ethoxylated fatty acids, in an amount of from about 5% to about 11%; Soaps, such as the LM is too acid, in the amount of from 0% to about 3%; sodium carbonate (e.g., Na2CO3), in an amount of from about 4% to about 10%; soluble silicate (e.g., Na2Oh, 2Si2), in an amount of from about 1% to about 4%; zeolite (e.g., NaAlSi4), in an amount of from about 30% to about 50%; sodium sulfate (e.g.,Na2SO4), in an amount of from about 3% to about 11%; sodium citrate (for example, C6H5Na3About7), in an amount of from about 5% to about 12%; polymers (e.g., PVP, copolymer of maleic/acrylic acid, PEG), in the amount of from about 1% to about 5%; enzymes (calculated on the pure protein enzyme), and 0.0001 to 0.1%; and minor ingredients (e.g., suppressor of education soapy foam, perfume), 0-5%.

9) Detergent composition is made in the form of a granulate, which includes linear Las (calculated by the acid), from about 6% to about 12%; non-ionic surfactant, in an amount of from about 1% to about 4%; Soaps, such as fatty acid, in amount from about 2% to about 6%; sodium carbonate (e.g., Na2CO3), in an amount of from about 14% to about 22%; zeolite (e.g., NaAlSi4), in an amount of from about 18% to about 32%; sodium sulfate (e.g.,Na2SO4), in an amount of from about 5% to p is IMEMO 20%; sodium citrate (for example, C6H5Na3About7), in an amount of from about 3% to about 8%; perborate sodium (for example, NaB3H2O), in the amount of from about 4% to about 9%; the bleaching activator (e.g., NOBS or TAED), in the amount of from about 1% to about 5%; carboxymethylcellulose (CMC), in the amount of from 0% to about 2%; polymers (e.g., polycarboxylate or PEG), in the amount of from about 1% to about 5%; enzymes (calculated on the pure protein enzyme), and 0.0001 to 0.1%; and minor ingredients (e.g. optical Brightener, perfume), 0-5%.

10) the Aqueous liquid detergent composition comprising linear Las (calculated by the acid), in an amount of from about 15% to about 23%; alexisoliviat alcohol (for example, C12-15alcohol, 2-3 EO), in the amount of from about 8% to about 15%; alcohol ethoxylate (e.g., With12-15alcohol, 7 EO or12-15alcohol, 5 EO), in the amount of from about 3% to about 9%; Soaps, such as fatty acid (e.g. lauric acid), in an amount of from 0% to about 3%; aminoethanol, in the amount of from about 1% to about 5%; sodium citrate, in an amount of from about 5% to about 10%; hydrotropes substance (for example, toluensulfonate sodium), in the amount of from about 2% to about 6%; borate (e.g., In4About7), in the amount of from 0% to about 2%; carboxylat is cellulose (CMC), in the amount of from 0% to about 1%; ethanol, in an amount of from about 1% to about 3%; propylene glycol in an amount of from about 2% to about 5%; enzymes (calculated on the pure protein enzyme), and 0.0001 to 0.1%; and minor ingredients (e.g. polymers, a means of facilitating dispersion, perfume, optical brighteners), 0-5%.

11) the Aqueous liquid detergent composition comprising linear Las (calculated by the acid), in an amount of from about 20% to about 32%; alcohol ethoxylate (e.g., for example, With12-15alcohol, 7 EO or12-15alcohol, 5 EO), 6-12%; aminoethanol, in the amount of from about 2% to about 6%; citric acid in an amount of from about 8% to about 14%; borate (e.g., In4About7), in an amount of from about 1% to about 3%; the polymer (for example, copolymer of maleic/acrylic acids, linked polymer, such as, for example, the copolymer loyalplayboy/acrylic acid linked polymer, such as, for example, a copolymer of lauriemittiet/acrylic acid), in an amount of from 0% to about 3%; glycerin in an amount of from about 3% to about 8%; enzymes (calculated on the pure protein enzyme), and 0.0001 to 0.1%; and minor ingredients (e.g., girotropnye substances, dispersing substances, perfume, optical brighteners), 0-5%.

12) Detergent composition is made in the form of g is of anulata, possessing a bulk density equal to at least 600 g/l, which includes an anionic surfactant (linear Las, alkylsulfate, α-reincorporate, methyl ester of α-sulfazine acid, alkanesulfonyl, soap), in the amount of from about 25% to about 40%; nonionic surfactant (e.g. alcohol ethoxylate), in the amount of from about 1% to about 10%; sodium carbonate (e.g., Na2CO3), in an amount of from about 8% to about 25%; soluble silicate (e.g., Na2O 2SiO2), in an amount of from about 5% to about 15%; sodium sulfate (e.g.,Na2SO4), in the amount of from 0% to about 5%; zeolite (NaAlSi4), in an amount of from about 15% to about 28%; perborate sodium (for example, NaB34H2O), in the amount of from 0% to about 20%; the bleaching activator (TAED or NOBS), in the amount of from 0% to about 5%; enzymes (calculated on the pure protein enzyme), and 0.0001 to 0.1%; and minor ingredients (e.g., perfume, optical brighteners), 0-3%.

13) Detergent composition corresponding to the composition of 1)to 12)above, where the linear Las, fully or partly substituted (C12-C18) alkylsulfates.

14) Detergent composition is made in the form of a granulate having a bulk density, p the ate, at least 600 g/l, which includes (C12-C18) alkylsulfate, in the amount of from about 9% to about 15%; alcohol ethoxylate in an amount of from about 3% to about 6%; amide polyhydroxyalkanoic acid, in an amount of from about 1% to about 5%; zeolite (e.g., NaAlSi4), in an amount of from about 10% to about 20%; layered disilicate (for example, SK56 from Hoechst), in the amount of from about 10% to about 20%; sodium carbonate (e.g., Na2CO3), in an amount of from about 3% to about 12%; soluble silicate (e.g., Na2O 2SiO2), in the amount of from 0% to about 6%; sodium citrate, in an amount of from about 4% to about 8%; percarbonate sodium, in an amount of from about 13% to about 22%; TAED, in the amount of from about 3% to about 8%; polymers (e.g., polycarboxylates and PVP), in the amount of from 0% to about 5%; enzymes (calculated on the pure protein enzyme), and 0.0001 to 0.1%; and minor ingredients (e.g. optical Brightener, fotoatelier, odorant, suppressor education soapy foam), 0-5%.

15) Detergent composition is made in the form of a granulate having a bulk density equal to at least 600 g/l, which includes (C12-C18) alkylsulfate, in the amount of from about 4% to about 8%; alcohol ethoxylate in an amount of from about 11% to about 15%; soap, in number is a number from about 1% to about 4%; zeolite MAR or zeolite A, in the amount of from about 35% to about 45%; sodium carbonate (Na2CO3), in an amount of from about 2% to about 8%; soluble silicate (e.g., Na2O 2SiO2), in the amount of from 0% to about 4%; percarbonate sodium, in an amount of from about 13% to about 22%; TAED, 1-8%; carboxymethylcellulose (CMC), in the amount of from 0% to about 3%; polymers (e.g., polycarboxylates and PVP), in the amount of from 0% to about 3%; enzymes (calculated on the pure protein enzyme), and 0.0001 to 0.1%; and minor ingredients (e.g. optical Brightener, phosphonate, perfume), 0-3%.

16) Detergent compositions as described above in paragraphs (1)-15), which contain stabilized or encapsulated nagkalat or as an additional component or as a substitute for already defined above bleaching systems.

17) Detergent compositions as described above in paragraphs 1), 3), 7), 9) and 12), where the specified perborate replaced by percarbonates.

18) Detergent compositions as described above in paragraphs 1), 3), 7), 9), 12), 14) and 15), which additionally contain manganese catalyst. Specified manganese catalyst is, for example, one of the compounds described in the manual of manganese catalysts ("Efficient manganese catalysts for low-temperature bleaching,"Nature369:637-639(1994)).

19) Detergent composition, zgodovina in the form of a nonaqueous detergent liquid, comprising a liquid nonionic surfactant, such as, for example, linear alkoxycarbonyl primary alcohol, water system (e.g., phosphate), one or more enzymes and alkaline substance. The specified detergent may also include anionic surfactant and/or a whitening system.

α-amylase Amy 195 or its variant can be introduced into the composition in concentrations that are typically used in detergents. According to the present invention, the specified enzyme can be added to the detergent composition in an amount corresponding to within 0.00001-1.0 mg (calculated on the pure protein of the enzyme α-amylase Amy 195 or its variant, per liter of the cleaning liquid.

In another embodiment of the present invention, other enzymes, such as 2,6-β-D-fructosidase, can be included in detergent compositions containing α-amylase Amy 195 or her option, to use for removal/cleaning of biofilm present in the household and/or industrial textiles /Laundry.

The specified detergent composition may also be made, for example, in the form of a detergent composition, suitable for hand or machine (automatic) washing, including the composition of the additive for washing, suitable for pre-treatment fabrics spots, and composition with added softener for washing fabrics, or may be made in the form of a detergent composition for use in cleaning hard surfaces in the home, or can be produced in a form suitable for manual or automated washing.

In a particular aspect of the present invention, the specified detergent composition can include 2,6-β-D-fructosidase, in addition to α-amylase Amy 195 or its variant, and one or more other suitable cleaning enzymes, such as protease, lipase, cutinase, carbohydrase, cellulase, pectinase, mannanase, arabinose, galactose, another amylolytic enzyme, xylanase, oxidase, laccase, and/or peroxidase and/or combinations thereof.

Mainly the properties of one or more selected enzymes must be compatible with the selected to work the detergent (for example, the optimal pH value, compatibility with other enzymatic and non-enzymatic ingredients and the like), and these one or more enzymes must be present in effective amounts.

Protease: suitable proteases include the enzymes of animal, plant, or microbial origin. This also includes chemically modified or genetically engineered protein mutant form, as well as the versions of the in vivo protein. This protease may be a serine protease or metalloprotease, such as alkaline microbial protease, trypsinogen protease or chymotrypsinogen protease. Examples of alkaline proteases include subtilisin, especially those subtilisin derived fromBacillusfor example, subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (see, for example, WO 89/06279). Examples trypsinogen proteases include trypsin (e.g., trypsin porcine or bovine origin) and protease fromFusarium(see, for example, WO 89/06270 and WO 94/25583). Examples suitable for use proteases include, without limitation, the variants described in WO 92/19729, WO 98/20115, WO 98/20116 and WO 98/34946. Commercially available protease enzymes include, without limitation: Alcalase®, Savinase®, Primase™, Duralase™, Esperase®, and Kannase™ (Novo Nordisk A/S); Maxatase®, Maxacal™, Maxapem™, Properase®, Purafect®, Purafect OxP™, FN2™, and FN3™(Genencor International, Inc.).

Lipase: suitable lipases include lipases bacterial or fungal origin. Chemically modified, proteoliticeski modified or genetically engineered protein ligands are also included in the present invention. Examples suitable for use lipases include, without limitation, lipase fromHumicola(synonym ofThermomyces)for example,H. lanuginosa (T. lanuginosus)(see, for example, EP 258068 and EP 305216), fromH. insolens/i> (see, for example, WO 96/13580); lipase fromPseudomonas(for example, fromP. alcaligenesorP. pseudoalcaligenes;see, for example, EP 218272),P. cepacia(see, for example, EP 331376),P. stutzeri(see, for example, GB 1372034),P. fluorescens,the strain ofPseudomonassp. SD 705 (see, for example, WO 95/06720 and WO 96/27002),P. wisconsmensis (see e g,WO 96/12012); lipase fromBacillus(for example, fromB. subtilis;see, e.g., Dartois et al.Biochemica et Biophysica Acta,1131: 253-360 (1993)),B. stearothermophilus(see, for example, JP 64/744992) orC. pumilus(see, for example, WO 91/16422). Additional options lipases suitable for use in compositions according to the present invention include lipases described, for example, in the following patents: WO 92/05249, WO 94/01541, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079, WO 97/07202, EP 407225 and EP 260105. Some commercially available lipase enzymes include Lipolase® and Lipolase Ultra ® (Novo Nordisk A/S).

Polyesters: in the composition can be introduced suitable polyesterase, such as the enzymes described in WO 01/34899 and WO 01/14629.

Amylase: these compositions can be combined with other amylases, such as the enhanced α-amylase non-industrial nature. These amylase may include commercially available amylase, such as, without limitation, Duramyl®, Termamyl®, Fungamyl® and BAN™ (Novo Nordisk A/S); Rapidase®, Purastar® (from Genencor International, Inc.).

Cellulase: cellulase can also be added to the data to the positions. Suitable cellulase include the enzymes of bacterial or fungal origin. The present invention also encompasses chemically modified or genetically engineered mutant form of the protein. Suitable include cellulase cellulase of the generaBacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium,for example, fungal cellulase derived fromHumicola insolens, Myceliophthora thermophilaandFusarium oxysporumas described, for example, in U.S. patents NoNo. 4435307; 5648263; 5691178; 5776757; and in the patent WO 89/09259. Representative of cellulase, which can be considered for use include cellulase, which can be used to care for colored fabrics. Examples of such cellulases include cellulase described, for example, in EP 0495257, EP 0531372, WO 96/11262, WO 96/29397 and WO 98/08940. Other examples are cellulase variants such as cellulase described in WO 94/07998; WO 98/12307; WO 95/24471; PCT/DK98/00299; EP 531315; U.S. patent NoNo. 5457046; 5686593 and 5763254. Commercially available cellulase include Celluzyme®, Carezyme® (Novo Nordisk A/S); Clazinase® and Puradax HA® (Genencor International, Inc.); and KAC-500(B)™ (Kao Corporation).

Peroxidase/oxidase: suitable peroxidase/oxidase considered in the context of use in the present compositions include the enzymes of plant, bacterial or fungal origin. In the present invention discusses the use of chemically modi is tirovannyh or genetically engineered mutant forms of the protein. Examples suitable for use peroxidases include peroxidase fromCoprinus,for example, fromC.Cinereus,and their variants, such as described in WO 93/24618, WO 95/10602 and WO 98/15257. Commercially available peroxidases include, for example, Guardzyme™ (Novo Nordisk A/S).

One or more detergent enzymes may be included in a detergent composition by adding separate additives containing one or more enzymes, or when making joint supplements that contain all of these enzymes. Detergent additive, for example, a separate additive or a combined additive, can be manufactured, for example, in the form of granules, liquids, suspensions, etc. Representative detergent composition additives include, without limitation, granulates, in particular, dust-free granules, liquids, in particular a stabilized liquid, or suspension.

Dust-free pellets can be obtained, for example, by the procedure described in U.S. patent NoNo. 4106991 and 4661452, and can optionally contain a coating applied according to known in the field method. Examples of waxy coating materials include poly(ethylenoxide) products (for example, polyethylene glycol, PEG) with average molecular weight of from 1000 to 20000; ethoxylated nonylphenols containing from 16 to 50 euteleostei units; ethoxylated fatty the pirates, where the alcohol contains from 12 to 20 carbon atoms and which contains from 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono -, di - and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by the fluidized bed technique, see, for example, in GB 1483591. Liquid enzyme preparations may be, for example, stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes can be obtained by the method described in EP 238216.

Detergent composition can be provided in any suitable form, for example, in the form of bars, tablets, powder, granules, paste or liquid. Liquid detergent may be aqueous, typically containing up to about 70% water and from 0% to about 30% organic solvent. Can also be considered a compact gel detergents containing about 30% or less water. Detergent composition can optionally include one or more surfactants, which may be non-ionic, including propolene, and/or anionic and/or cationic and/or zwitterionic. These surfactants can be represented in a wide range of concentrations, from about 01% wt. to about 60 wt.%.

The specified detergent, when its inclusion in the composition of the described composition contains typically from about 1% to about 40% anionic surfactants, such as linear Las, α-reincorporate, alkylsulfate (sulfate fatty alcohol), amoxicullin alcohol, secondary alkanesulfonyl, methyl ester of α-sulfazine acid, alkyl - or alkenyl-succinic acid or soap.

The specified detergent, when its inclusion in the composition of the described composition typically contains from about 0.2% to about 40% non-ionic surfactants such as ethoxylates alcohol ethoxylates of Nonylphenol, alkylpolyglycoside, alkyldimethylammonium, monoethanolamide ethoxylated fatty acids, monoethanolamide fatty acid amide polyhydroxyalkanoic acid or N-acyl-N-alkyl derivative of glucosamine ("glocality").

The specified detergent may contain from 0% to about 65% detergent filler or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitriloside acid, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid, alkyl - or alkenyl-succinic acid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst).

The specified detergent includes one or a number of the polymers. Representative polymers include carboxymethyl cellulose (CMC), poly(vinyl pyrrolidone) (PVP), poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylate, for example, polyacrylates, copolymers of maleic/acrylic acid copolymers lauriemittiet/acrylic acid.

One or more of the enzymes included in detergent compositions can be stabilized by using a standard stabilizing agents, such as, for example, polyol (for example, propylene glycol or glycerol), sugar or sugar alcohol, lactic acid, boric acid or a derivative of boric acid (for example, an ester of an aromatic borate) or phenyl derivative Bronevoy acid (for example, 4-formylphenylboronic acid). This composition can be made according to the procedure described in WO 92/19709 and WO 92/19708.

According to the present invention used in detergent compositions, one or more enzymes, in particular, variants of the enzymes can be added in an amount corresponding to from about 0.01 to about 100 mg of enzyme protein per liter of wash liquid (in particular, from about 0.05 to about 5.0 mg of enzyme protein per liter of wash liquid and from 0.1 to about 1.0 mg of enzyme protein per liter of wash liquid is).

4.2Composition for cleaning

In the detergent composition of drugs, α-amylase Amy 195 and/or its variant is usually used in the form of liquid compositions containing propylene glycol. The specified enzyme is subjected to solubilization, such as propylene glycol, by mixing 25 vol.% in a solution of propylene glycol containing 10% of calcium chloride.

α-amylase Amy 195 and/or its variant according to the present description can be made in the form detergent compositions suitable for use in washing or cleaning compositions for other applications. They can be represented in the form of powders, gels or liquids. These compositions can include one enzyme or its combination with other amylolytic enzymes and/or other used for the purification of enzymes or activating whitening enzymes and other components that are commonly used in compositions for cleaning.

Thus, the detergent composition for washing may include a surfactant. The specified surfactant may be anionic, nonionic, cationic, amphoteric or a mixture of these types. This detergent can contain from 0 wt.% to about 90 wt.% non-ionic surfactants, such as ethoxyline is installed/propoxycarbazone lineannotation alcohols with low foaming tendency, or does not form the foam.

Detergent composition may contain a detergent filler in the form of salts of inorganic and/or organic type. These detergent fillers can be divided into phosphorus-containing and not containing phosphorus types. This detergent composition typically contains from about 1% to about 90% detergent filler. Examples of phosphorus-containing inorganic alkaline detergent fillers, in case of their presence in the composition include water-soluble salts, in particular, pyrophosphates, orthophosphate and polyphosphates alkali metal. Example phosphorus-containing organic alkaline detergent filler, in case of its presence in the composition includes soluble salts of phosphonates. Examples not containing phosphorus inorganic fillers, when present in the composition include water-soluble carbonates, borates or silicates of the alkali metal, and various types of water-insoluble crystalline or amorphous aluminosilicates, the most prominent representatives of which are zeolites.

Examples of suitable organic fillers include alkali metal, ammonium and substituted ammonium; citrates; succinate; malonate; sulfonates of fatty acids; carboxymethyloxysuccinic; polyacetate ammonium; carboxylates; polycarboxylate; aminopolycarboxylate the ATA; polyaminocarboxylic and polyhydroxyalkanoate.

Other suitable organic fillers include high molecular weight polymers and copolymers, which are known to possess the properties of the filler, for example, suitable polyacrylic acid, primulina acid and copolymers of polyacrylic/polimolekuly acid, and their salts.

The specified cleaning composition may contain bleaching agents, chlorine/bromine-type or oxygen type. Examples of inorganic bleach chlorine/bromine type include lithium hypochlorite, sodium or calcium and hypobromite of these metals, and chlorinated trisodium phosphate. Examples of organic bleach chlorine/bromine type include heterocyclic N-bromo and N-chloro-imides, such as trichloroisocyanurate, Tripolitania dobromyslova and dichloroisocyanurate acids and their salts with solubilities in water, cations such as potassium and sodium. Acceptable connections, as.

Composition for cleaning may contain oxygen bleach, for example, in the form of inorganic percale, optionally in the presence of such predecessor of bleach or in the form of peroxynitrate connection. Typical examples of suitable peroxide bleaches are perborate alkali metal, as t is trihydrate, and monohydrate, percarbonate, prsilikat and perphosphate alkali metal. Representative activator materials are TIED and glycerol triacetate. Enzyme activation system whitening may also be present in the composition, such as perborate or percarbonate, triacetate of glycerol and perhydrol (see, for example, WO 2005/056783).

Composition for cleaning may be subject to stabilisation using standard stabilizing means, suitable for one or more enzymes used, for example, polyol, such as propylene glycol, a sugar or sugar alcohol, lactic acid, boric acid or a derivative of boric acid (for example, an ester of an aromatic borate).

Composition for cleaning may also contain other conventional detergent ingredients, for example, deflocculation, filter material, means of reducing foaming, anti-corrosion agents, means for suspension of mud particles, airing, means preventing re-deposition of mud particles, dehydrating, dyes, antibacterial agents, fluorescent agents, thickeners and fragrances.

Below the present invention will be described with reference to the detailed variations in its implementation, although it should be understood that the description can be entered in various modifications.

4.3Methods of assessment detergent compositions

There are many tests to evaluate the cleaning properties of α-amylase. Below is a description of examples of such testing cleaning performance of the considered agents, which include the following.

The term "sample" is a piece of material, such as fabric, which contains a deposited spot. This material may be a fabric made of cotton, polyester or a mixture of natural and synthetic fibers. Such a sample may also be paper, such as filter paper or nitrocellulose, or a piece of hard material, such as ceramics, metal or glass. For the case of amylase, the specified spot is starch-based, but may also include blood, milk, coloring matter, pollution grass, tea, wine, spinach, gravy, chocolate products, cheese, clay, pigment, oil, or a mixture of these compounds.

The term "reduced sample" is a slice of the sample, which was obtained using a stamping device with one aperture or which was obtained using the standard production stamping device with 96 holes, where the nature of such a stamp with lots of holes corresponds to a 96-microtiter cell tablets, or the specified slice is brazza was obtained from the core sample in any other way. Consider the sample may be a tissue sample, paper, metal or other suitable material. The "reduced sample" may contain stain, applied before or after it is put in a cell for 24-, 48 - or 96-microtiter cell tablet. Specified reduced the sample can also be obtained when applying the stain on a small piece of material. For example, this reduced the sample can be a piece of fabric with the marked spot with a diameter of 5/8" or 0.25" (0,015875 m or 0,00635 m). Standard production stamp is designed so that it could deliver to 96 samples simultaneously to all the cells in 96-cell tablet. The specified device can deliver more than one sample per cell by simply drawing on the same 96-cell plate several times. Stamping device with multiple holes can be considered for simultaneous delivery of samples to the tablet in any format, including, without limitation, the 24-cell, 48-cell and 96-cell-based tablets. In another possible method, contaminated investigated the platform can be a ball made of any metal, plastic, glass, ceramic or any other suitable material, which cover contaminating the substrate. One or more paritycheck material balls are placed in a cell 96-, 48 - or 24-cell tablet or tablets of a larger format, containing a suitable buffer and enzyme. In this case, the supernatant examined for the presence of released contaminants are material, either by direct absorbance measurement, or after the secondary reaction staining. Analysis of the released contaminant material may also be carried out in the framework of the mass-spectral analysis. Another test within microscreening can be carried out when the shipping and the respective setting of the sample, for example, cotton fabric, dyed Indigo dye, a cell, a multiple-cell tablet, with the addition of particles, such as sand or larger particles, such as garnet, selected to particles 6-8 or 9 size, followed by shaking the tablet so as to cause the grinding samples added particles. The specified test has been used for the evaluation of cellulases for use for washing stones. The efficiency of the enzyme can be assessed either on the development of painting (for example, on release of the dye Indigo, which is dissolved in dimethyl sulfoxide, and the subsequent absorption measurement with the length And600nm) in the reaction buffer or by measuring the reflectivity of a polished sample.

In the case when, for example, the raw samples of type kmch (BMI) (Kromolice/ink) are washed in detergent without bleach a large part of the ink is released even without the addition of protease. Add protease leads to a small increase in the release of ink, quantification of which may be difficult on the background of existing material. The present invention relates to Protocol processing, which allows you to control the degree of fixation of the dye spot. In result, it becomes possible to obtain samples, which, for example, release varying the amount of the coloring matter when washing in the absence of the investigated enzyme. The use of fixed samples leads to a sharp increase in the ratio signal/noise in tests with washing. In addition, by varying the degree of fixation can create colored spots, which give the best results under different conditions cleaning.

Samples containing spots with known "strength" on the materials of different types available in the commercial version (EMPA, St. Gallen, Switzerland; wfk-Testgewebe GmbH, Krefeld, Germany; and Center for Test Materials, Vlaardingen, The Netherlands) and/or can be obtained from a practicing specialist in this area (Morris and Prato,Textile Research Journal52(4): 280286 (1982)). Other analyzed samples include, but are not limited to, one or more spots such as CPM (BMI), i.e. from blood/milk/ink on cotton fabric, dyed spinach cotton fabric or painted grass chlorine is cheboman fabric, as well as cotton fabric, dyed chocolate/milk/soot.

Spot type CPM can be applied on cotton adding 0,0003%-0,3% hydrogen peroxide. Other combinations include pollution grass or spinach, plotted together with 0,001%-1% glutaraldehyde, gelatin, Kumasi-dye, in combination with 0,001%-1% glutaraldehyde, or chocolate, milk and soot, together with 0,001%-1% glutaraldehyde.

The specified pattern can also be subjected to agitation during incubation with the enzyme and/or detergent composition. The results of leaching depends on the orientation of the samples in the cells (horizontal or vertical), in particular, in the case of a 96-cell tablet. This indicates that mixing during incubation was insufficient. Although there are many ways to ensure adequate mixing during incubation, can be constructed holder, in which microtiter tablet is fixed between the two tablets of aluminium in the form of a "sandwich". This can be done simply, for example, by placing an adhesive film on the cells of the tablet and then placing the two aluminum plates 96-cell tablet using any type of suitable commercially available clamps. And then this design may be included in commercially available shaker is and the incubation period. Installation work of the shaker at the speed of approximately 400 rpm leads to a very efficient mixing, despite the fact that such holder effectively prevents leakage of fluid or contamination of adjacent cells.

Trinitrobenzenesulfonic acid (TNBC) (TNBS) can be used to quantify the concentration of amino groups in the wash liquid. This definition is a measure of the quantity of protein that was removed from the sample (see, for example, Cayot and Tainturier,Anal. Biochem.249: 184-200 (1997)). However, if the detergent or enzyme sample leads to the formation of unusually small peptide fragments (e.g., as a result of the presence of peptidases in the sample), there can be obtained a more pronounced signal TNBC, i.e. more "noise".

Another way of assessing the quality of washing the stains of the blood/milk/ink or stains other nature based on the release of the coloring matter. Proteolysis of protein in the samples leads to the release of particles of the coloring matter, which can be quantified by measuring the absorption of wash liquid. Absorption can be determined by using to measure any wavelength in the range from 350 to 800 nm. The specified absorption is measured at a wavelength of 410 nm or 620 nm. Rinsing fluid can also be analyzed to determine the quality of the and remove stains, containing grass, spinach, gelatin or Kumasi-dye. Suitable conditions for the evaluation of such stains include, in the case of spinach or other herb, measurement at a wavelength of 670 nm, and in the case of gelatin or Kumasi-dye at a wavelength of 620 nm. So, for example, selected aliquot of wash liquid (in a typical case, for example, 100-150 ál of cells to 96-cell tablet) and placed in a cell or multiple-cell cell microplate. Then this aliquot is placed in a spectrophotometer to determine the absorbance at the appropriate wavelength.

This system can also be used to assess the effectiveness of the composition with the enzyme and/or detergent composition when washing dishes, for example, using spots of blood/milk/ink on a suitable substrate, such as a tissue sample, ceramic or plastic.

In one aspect of the spot type kmch fixed on cotton fabric, adding a 0.3% hydrogen peroxide to the sample CPM/cotton for 30 minutes at 25°C, or adding of 0.03% hydrogen peroxide to the sample CPM/cotton for 30 minutes at 60°C. the smaller the sample size of 0.25" (0,00635 m) cut out from a sample CPM/cotton and placed in a cell 96-microtiter cell tablet. In each cell make known mixture of detergent compositions and enzyme, such as a variant of the aircraft is OK. After sealing the top of the microtiter tablet adhesive film, specified microtiter tablet is clamped between two aluminum plates and subjected to stirring in the corner shaker with a speed of about 250 rpm for 10-60 minutes. After this period of time supernatant transferred into a cell of a new microtiter tablet and measure the absorption coloring matter at a wavelength of 620 nm. Similarly, the above procedure can be used for testing spots spinach or grass stains, printed on cotton fabric, adding 0.01% glutaraldehyde to the sample of spinach/cotton or to sample the grass/cotton for 30 minutes at 25°C. the same procedure can be carried out with samples containing spots of chocolate, milk and/or soot.

EXAMPLES

EXAMPLE 1

The expression inB. subtilis

The design depicted in Fig. 5, is subjected to transformation in 9 strains ofB. subtiliswith deletional protease (degUHy32, oppA, ΔspoII3501, amyE::xylRPxylAcomK - ermC, ΔaprE, ΔnprE, Δepr, ΔispA, Δbpr, Δvpr, ΔwprA, Δmpr-ybfJ, ΔnprB) (see US20050202535A1). The culture of this strain grown in the following medium (which contains per liter): 10 g Soytone, 75 g glucose, 7.2 g of urea, 40 mm MOPS, 4 mm Tricine, 3 mm dibasic potassium phosphate, 21,4 mm KOH, 50 mm NaCl, 276 µm potassium sulfate, 528 µm chloride MAGN what I 50 μm dihydrate of trisodium citrate, 100 μm dihydrate calcium chloride, 14 μm of iron sulfate heptahydrate, is 5.9 μm, dihydrate manganese sulfate, of 5.7 μm monohydrate zinc sulfate, 2.9 μm chloride dihydrate copper and 4.2 μm uranyl cobalt, and 4.5 μm dihydrate of sodium molybdate. For a final volume of 1l of all the components, except Soytone, mix 500 ml, sterile filtered and added to equal parts 2 x Soytone, which was previously sterilized by autoclaving. Trace elements and citrate can be obtained from concentrated solutions 100X or 1000X. Buffers, potassium hydroxide, sodium chloride, potassium sulfate and magnesium chloride, and trace elements can be obtained from 10X concentrated solutions. After mixing of all components, the pH of the mixture is brought to a value of 7.3. Before using the specified environment, add 20 mm of calcium chloride.

This culture expresses the enzyme in the form of various protestirovannyx forms. Obviously Mature form (without signal sequence) was observed in the field marker size 69 kDa, while conducting electrophoresis in a 10% gel LTO-PAG. There are also two shorter forms.

The activity of α-amylase Amy195 fractionary, always working culture broth affinity resin based on β-cyclodextrin-sepharose, then the specified resin sobiraut washed with 25 mm bis-Tris-propane buffer (pH 8.5), containing 2 mm of calcium chloride (Cl2), followed by elution of the washed resin with the same buffer with the addition of 50 mm β-cyclodextrin. As a result of processing the culture broth β-cyclodextrine resin from the broth partially removed kind of molecules with a size of 60 kDa (approximately 50%) and completely removed type of molecules the size of 69 kDa. Washing the resin with buffer gives almost pure protein 60 kDa; elution buffer containing β-cyclodextrin, gives protein size 69 kDa in the presence of impurities, about 25%, protein 60 kDa. The specified component appreciate when conducting electrophoresis in LTO-SDS page, where the results of this analysis are illustrated in Fig. 10. The content of the enzyme in the fractions appreciate gueldenstaedtia using drugs OxAm amylase (Genencor International, Inc.), which performs the function of the normal protein. N-terminal analysis of the dark bands on the line marked "wl" in Fig. 10, gives the sequence "AAPGPKDATA" (SEQ ID NO: 5). Mass spectral analysis in combination with the definition of the N-terminal sequence allows the identification of a protein that has the sequence shown in the upper part of Fig. 4 (i.e., without the signal sequence and C-terminal extensions, reflecting krahmalnaya motifs). The analyses carried out show that this molecular fragment consists of the of Aminov α-amylase And, B and C.

EXAMPLE 2

Expression of the catalytic domain in genetically protestirovanny form Amu

Gene Amy 195 subjected to processing in three different sites in order to analyze the expression of truncated forms, as well as for the purpose of testing the efficiency of washing. The specified truncation/processing carried out according to standard techniques known to experts in this field, amino acid residues with non-494, 504 and 509, where the numbering used in the polypeptide corresponds to the sequence shown in Fig. 4. These plasmids, containing versions of the genes subjected to transformation in nine strains ofBacillus subtiliswith deletional protease (degUHy32, oppA, ΔspoII3501, amyE::xylRPxylAcomK - ermC, ΔaprE, ΔnprE, Δepr, ΔispA, Δbpr, Δvpr, ΔwprA, Δmpr-ybfJ, ΔnprB). Next, cells were cultured in flasks with septum volume of 250 ml containing 50 ml of enriched environment, with the addition of 10 or 30 mm Cl2during 64 hours at 37°C with shaking at 250 rpm Cultural supernatant analyze, conducting electrophoresis in LTO-SDS page, and amylase content assessed by the method of gueldenstaedtii.

It was shown that the expression of amylase based protestirovannyx genes is approximately 2 times higher than the expression of the same domain in the full-size gene of the wild type. These results, proillyustriroval is installed in Fig. 8, show that processionary gene has the best performance for the expression of the protein.

EXAMPLE 3

Test clean

All fractions identified in the gel, then analyze format 96-cell test using the sample rice starch, dyed orange dye CS28. This test is carried out in 25 mm HEPES (pH 8.0)and 25 mm CAPS (pH 10,3) buffers.

The cleaning characteristics of all types Amy195 allocated in the framework of example 1 is tested in conditions that simulate the washing using to estimate the concentration of amylase as a function. The results of the faction "e-pool" for Fig. 10 shown in Fig. 6. Cleaning efficiency is determined by the amount of dye released in supernatant liquid, when carrying out measurements on a spectrophotometer at a wavelength of 488 nm. For more information on the procedure of this test is shown in U.S. patent No. 7122334. This enzyme is extremely effective in testing conditions at pH 8.0, but also demonstrates the amazing properties on stain removal at a pH of 10.3. All major protein bands for each protein lines in the gel (Fig. 10) demonstrate the presence of cleaning, and the best results were obtained in the band on "wl". All cleaning activity is shown in Fig. 7 in testing conditions at pH 8.0. Protestirovanny form that ends with the amino acid OST the rigid 492, according Fig. 4, shows the best properties (see Fig. 7, "•")than the form, keeping one krokhmalskii domain (see Fig. 7, "□"). The results of this test show that the α-amylase Amy 195 has a high efficiency for removing stains from textile samples.

EXAMPLE 4

Product characteristics derived from genetically processioning gene

Products genetically processioning gene obtained by the above procedure of example 2, test for evaluating the effectiveness of washing in the same manner as described with respect to proteolytic enzymes in the framework of example 3, above. Samples of rice CS28 incubated with a range of concentrations of the catalytic fragment Amy195. Washing efficiency is assessed according to the released in the supernatant paint, conducting measurements at a wavelength of 488 nm. All three fragments genetically processioning gene exhibit good characteristics based on the results of leaching, as illustrated in Fig. 9.

The specified test for the evaluation of the sample can be modified in several ways for different purposes. Specified 96-cell test is very suitable as a large-scale test on the cleaning efficiency when measuring the absorption of the supernatant after incubation of the enzyme with the samples, whereas, for example, the 24-cell is the tablet samples introduced into cells, can be used for washing larger samples, in which case as of the evaluation measures can be used reflectance determined by known in the field procedure. The results of both measurements, determine the absorbance of the supernatant and determine the reflection coefficient of the sample show an almost perfect correlation.

The correlation of the reflectance of the washed sample with absorption of supernatant was high; the coefficient of determination r2has a value of 0.99. This test can be, in principle, be scaled up to 384-cell tablet. The specified test can be conducted with any contaminated sample and, in addition CS28 of the sample can also be tested CS26, CS27 and CS29 samples (for example, corn starch, potato starch and starch from tapioca, respectively; Testfabrics, Inc., West Pittiston, PA) to demonstrate the effectiveness of the determination performed according to the procedure of example 3. This test can also be used to estimate detergent compositions and conducted at different temperatures and different pH values. These tests were taken and accordingly adapted from U.S. patent No. 7122334.

Everything listed in the description of the links included in this application fully, for all targets.

1. The selected α-amyl is a from Bacillus sp.195, representing a shortened form ending balance 492, 504 or 509 SEQ ID NO:3.

2. Detergency additive containing a shortened form of the α-amylase according to claim 1 in an amount of from 0.02 mg to 200 mg per gram of the detergent additive, in the form of dust-free granulate, micro-granulate, a stabilized liquid, gel or protected enzyme.

3. Detergent additive according to claim 2, characterized in that the truncated form of α-amylase has a molecular weight of 49 kDa and 69 kDa, in the analysis by electrophoresis in a 10% gel LTO-PAG.

4. Detergent additive according to claim 2, further comprising an enzyme selected from the group consisting of protease, lipase, peroxidase, oxidase, amylase enzyme, cellulase, polyesterase and any combination thereof.



 

Same patents:

FIELD: medicine.

SUBSTANCE: Bacillus amyloliquefaciens strain, Russian National Collection of Industrial Microorganisms B-10291 is produced by the multistage selection on a medium containing rifampicin that is followed by the selection of the most productive rifampicin-resistant clones. The strain produces alpha amylase of activity 900 GOST units/ml.

EFFECT: strain exhibits a lower level of foaming that allows reducing an amount of an antifoam additive used during fermentation more than in 3 times.

3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: strain of mycelial fungus Aspergillus oryzae All Russian collection microorganisms F-3927 D - producer of acid α-amylase can be used in various areas of industry and agriculture for saccharifying starch-containing raw material.

EFFECT: increase of acid α-amylase output.

4 ex

FIELD: chemistry, organic.

SUBSTANCE: invention relates to biotechnology and may be used for production of enzyme preparations of thermostable α-amylase. The invention enables one to increase output of thermostable α-amylase.

EFFECT: increase of thermostable α-amylase output.

1 tbl, 4 ex

FIELD: microbiological industry, in particular production of citric acid and acid-resistant α-amylase and glucoamylase enzymes.

SUBSTANCE: claimed method includes starch and maize flour hydrolysis in ratio of 1:7-1:40 with bacterial α-amylase enzyme preparation at elevated temperature and pressure to obtain value of carbon source dextrose equivalent not more than 30; addition of nitrogen organic source and mineral salts such as copper (II), zinc, cobalt (II) sulfates in amount of (0.5-1.0)x10-3, (1.5-3.5)x10-3, (1.5-4.5)x10-3 g/dm3, respectively, iron (II) to starch and maize flour hydrolyzate followed by medium fermentation with acid-forming fungi Aspergillus niger. Than hydrolyzate is diluted with sterile water to obtain fermentable sugar content of 120-140 g/dm3 and carbon and nitrogen organic source in ratio of (50-60):1, respectively. Method of present invention males in possible to convert sugar to citric acid with yield of 79.3-83.1 % and to obtain acid-resistant α-amylase and glucoamylase enzymes having activities of 2.50-3.50 U A-C/cm3 and 80.2-147.4 U Gl-C/cm3, respectively.

EFFECT: method for production of citric acid, α-amylase and glucoamylase with increased yield.

1 tbl, 4 ex

FIELD: molecular biology, biochemistry, enzymes.

SUBSTANCE: based on analysis of results in determination of three-dimensional structure of α-amylase from Bacillus invention proposes modifications of amino acid sequence of native enzyme distinguishing by enhanced stability form as compared with the parent form. Prepared variants of α-amylase comprise at least one amino acid replace at positions k40, V74, H103, S141, T142, F188, H220, N234, K249, D261, L268, V279, N342, H344, G397, A403, K425, S442, S479, S493, T494, S495, A496, A498, Q500, K520, A555 and/or N595. Acquired resistance of mutant forms of α-amylase to external effect and retention of enzymatic activity of enzyme provides possibility for effective replacement of native enzyme. Invention can be used in food industry, in particular, in bread baking.

EFFECT: improved and valuable properties of enzyme.

2 cl, 1 dwg, 1 tbl, 7 ex

The invention relates to biotechnology

The invention relates to the microbiological industry, and relates to a method of obtaining from starch-containing raw materials citric acid and acid enzymes:-amylase and glucoamylase

The invention relates to the microbiological industry, and relates to a method of obtaining from starch-containing raw materials citric acid and acid enzymes:-amylase and glucoamylase

The invention relates to biotechnology and can be used in alcohol, starch and syrup, brewery, bakery, confectionery, fruit, textile industries that require enzymes that are resistant to high temperatures

FIELD: chemistry.

SUBSTANCE: fused proteins contain an endoglucanase nucleus amino acid sequence having at least 95% identity to SEQ ID NO:2, fused with an amino acid sequence containing a linker and a cellulose-binding domain (CBD), having at least 95% identity to SEQ ID NO:15. Such fused proteins can be obtained via a recombinant technique using suitable polynucleotides, expression vectors and host cells.

EFFECT: invention provides cellulase, having low activity with respect to restaining, and can be used to treat cellulose material; disclosed fused proteins and enzyme preparations based thereon can be used to prepare detergent compositions or for improving quality of animal feed.

26 cl, 8 dwg, 10 tbl, 10 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to a detergent composition which contains glycosyl hydrolase selected from GH 5, 12, 44 or 74 families, and carrier particles which contains an agent having a positive effect. The invention also relates to a method of processing and/or cleaning affected places with the detergent composition.

EFFECT: higher efficiency of delivering the agent having positive effect.

15 cl, 28 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a cleaning, bleaching or disinfecting composition which is a stabilised oxidase composition containing said oxidase and a stabiliser and at least one substrate for said oxidase, where said stabiliser contains at least one oxidase inhibitor, where said stabiliser is selected from thiosulphate and 2-amino-2-methyl-1-propanol. In certain especially preferable versions of the invention, said oxidase is selected from glucose oxidase, sorbitol oxidase, choline oxidase, hexose oxidase and alcohol oxidase. The invention also relates to a method of forming a bleaching product in a detergent solution, involving a step for adding said composition to said detergent solution, wherein the preferred version, said bleaching product is a peroxide or a bleaching system which can be activated using peroxide.

EFFECT: ensuring stability a H2O2-generating system during storage and production of bleaching agents when diluting a detergent in a detergent solution.

15 cl, 8 tbl, 9 ex, 3 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to a method of cleaning at least part of a surface and/or fabric, involving: optional steps for washing and/or rinsing the surface and/or fabric; bringing the surface and/or fabric into contact with a washing solution containing a perhydrolase enzyme and a substrate for said enzyme, where the initial pH of the washing solution is alkaline and the amount of the perhydrolase enzyme and substrate is sufficient to lower the pH of the washing solution to 6.5 or lower; and optional washing and/or rinsing the surface and/or fabric, where said contact takes place during the washing cycle, and where lowering of the pH of the washing solution improves efficiency of the component of the washing solution.

EFFECT: improved method for bleaching textile.

7 cl, 5 ex, 5 dwg

FIELD: chemistry.

SUBSTANCE: composition contains more than 5% anionic surfactant, less than 25% nonionic surfactant, a light-sensitive ingredient and an inorganic mother-of-pearl agent. The light-sensitive ingredient is selected from a group comprising enzymes, dyes, vitamins, aromatising agents and mixtures thereof.

EFFECT: inorganic mother-of-pearl agent improves stability of light-sensitive ingredients in the detergent composition.

20 cl, 14 ex

FIELD: chemistry.

SUBSTANCE: invention discloses a composition which contains a fabric toning agent and a bacterial alkaline enzyme which exhibits endo-beta-1,4-glucanase activity (E.C.3.2.1.4). The fabric toning agent meets Testing method 1 requirements given in the description. Described also is a method of cleaning and/or processing a surface or fabric using the said composition.

EFFECT: composition ensures better removal of yellow stains and improved deposit of the agent with improvement of the outer appearance of the fabric.

18 cl, 16 ex

FIELD: chemistry.

SUBSTANCE: cleaning compositions contain organic catalysts having improved enzyme compatibility and having the following formulae: where each R1 independently denotes a branched alkyl group selected from a group comprising 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, isodecyl, isotridecyl and isopentadecyl, or a linear alkyl group containing 11-18 carbon atoms. The composition also contains one or more auxiliary ingredients.

EFFECT: cleaning compositions with efficient bleaching at low water temperature.

15 cl, 16 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to a composition which contains lipase, and a bleaching catalyst which can accept an oxygen atom from a peroxy acid and transfer the oxygen atom to an oxidisable substrate.

EFFECT: less bad smell which arises when using lipase enzymes in detergents.

13 cl, 4 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: detergent composition contains a polymer combined with a surfactant or an inorganic detergent component and auxiliary ingredients. The fat purification efficiency index of the detergent composition is at least equal to 10. The polymer is a random grafted copolymer which has a hydrophilic skeleton and hydrophobic side chains, obtained by grafting (a) polyethylene oxide; (b) vinyl ester of acetic and/or propionic acid; and/or C1-4alkyl ester of acrylic or methacrylic acid; and (c) modifying monomers. The detergent compositions preferably additionally contain lipase enzyme.

EFFECT: improved removal of fat contaminants and stains with reduced amounts of conventional surfactants or inorganic detergent components.

22 cl, 6 ex

FIELD: chemistry.

SUBSTANCE: compositions contain certain versions of lipase and a photo-bleaching agent based on xanthene dye, a photoinitiator and their mixtures.

EFFECT: more efficient cleaning, leading to minimal undesirable bad smells.

11 cl, 3 tbl, 16 ex

FIELD: gene and protein engineering, in particular substances for detergents and cleaning compositions.

SUBSTANCE: invention relates to mutant forms of subtilisin Bacillus, obtained by certain combination of replacement in origin native enzyme amino acid sequence. Such mutant forms are differ from precursor subtilisin by increased cleaning effect. According to present invention all subtilisin variants are characterized either by residue substitution in position accepted to 232 position of Bacillus amyloliquifaciens amino acid sequence with valine, or by residue substitution in position accepted to 212 position of said subtilisin natural form with proline. To produce disclosed new form of enzyme respective mutant DNA sequences have been expressed in host cells, preferably in strain Bacillus cells with decreased level of proteases activity. Subtilisin variants of present invention are useful as components of any cleaning composition allowing for protease including.

EFFECT: new materials for detergents and cleaning compositions of improved effect.

6 cl, 5 dwg, 6 tbl, 3 ex

Up!