CN1527877A

CN1527877A - Detergent composition comprising bacillus subtilis pectate lyases

Info

Publication number: CN1527877A
Application number: CNA028099362A
Authority: CN
Inventors: M・B・埃斯克隆; M·B·埃斯克隆; 骋; M·许莱因; 尼尔森; V·S·尼尔森; 反; J·斯梅茨
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2001-05-14
Filing date: 2002-05-14
Publication date: 2004-09-08
Anticipated expiration: 2022-05-14
Also published as: DE60231487D1; ATE425242T1; CN100552024C

Abstract

Detergent composition comprising a surfactant and a pectate lyase (EC 4.2.2.2) enzyme en-coded by a DNA sequence endogeneous to a strain of Bacillus subtilis or a stabilized variant thereof provides superior cleaning and stain removal.

Description

The detergent composition that comprises the Bacillus subtilus pectate lyase

Technical field

The present invention relates to a kind of detergent composition that comprises pectate lyase (EC 4.2.2.2) or its stabilized variants, described pectate lyase separates from Bacillus subtilus (Bacillus subtilis) bacterial strain, preferably clones from the Bacillus subtilus bacterial strain; The invention still further relates to the pectate lyase of the dna sequence encoding in the plasmid that is present in Bacillus subtilus DSM14218 bacterial strain, a kind of genus bacillus (Bacillus) expression vector and a kind of method that produces pectate lyase in the genus bacillus host cell also are provided.

Background of invention

The pectin polymkeric substance is the important composition composition of plant cell wall.Pectin is the mixed polysaccharide that a kind of main chain is made up of with polygalacturonic acid glycan (level and smooth district) and rhamnosyl galacturonic acid glycan (hair shape district) alternative, and described level and smooth district is the straight-chain polymer of 1, the 4 α-D-galacturonic acid that connects.The galacturonic acid residue can be on carboxylic group by esterification to some extent, normally in nonrandom mode with the complete esterification of polygalacturonic acid block.

Polygalacturonase can be classified according to its preferred substrate (pectin of height esterification or the pectin and the polygalacturonic acid (pectic acid) of low esterification) and reaction mechanism (β-elimination or hydrolysis).Polygalacturonase can mainly be the inscribe effect, and promptly the random site cutting polymer produces the oligomer mixture in chain, and perhaps they can be circumscribed effects, attack and produce monomer or dimer from an end of polymkeric substance.Several pectinase activities that act on the level and smooth district of pectin have been comprised in the enzyme classification that enzyme nomenclature (1992) provides, for example pectate lyase (EC 4.2.2.2), pectin lyase (EC4.2.2.10), polygalacturonase (EC 3.2.1.15), circumscribed polygalacturonase (EC3.2.1.67), circumscribed polygalacturonic acid lyase (EC 4.2.2.9) and circumscribed poly α-galacturonic acid Glycosylase (EC 3.2.1.82).

WO99/27083 discloses the pectate lyase of a kind of clone from Bacillus licheniformis (Bacilluslicheniformis).WO99/27084 discloses the pectate lyase of clone's self-adhesion agar genus bacillus (Bacillus agaradhaerens), salt tolerant genus bacillus (Bacillus halodarans) and other genus bacillus.

Cloned a kind of pectate lyase (Nasser etc. (1993) FEBS335:319-326) from Bacillus subtilus.Described pectate lyase needs divalent cation reaching maximum activity, and calcium ion is tool activation.

JP2000-262292A disclose a kind of bacillus protopectinase of encoding gene, a kind ofly comprise the transformant of described gene and use this transformant refining to fiber.

US6,172,030 disclose a kind of detergent composition that contains the genus bacillus protopectinase, and described protopectinase has 7 or higher reaction optimal pH to protopectin-and polygalacturonic acid substrate.

The purpose of this invention is to provide a kind of high performance pectate lyase that in detergent composition, has.

Summary of the invention

The inventor has found a kind of new having pectate lyase (EC 4.2.2.2) active polypeptide and successfully clone and give expression to this pectate lyase in the genus bacillus host.

Correspondingly, first aspect of the present invention relates to a kind of detergent composition, said composition includes tensio-active agent and by pectate lyase (EC 4.2.2.2) or its stabilized variants of the endogenous dna sequence encoding of Bacillus subtilus bacterial strain, preferably by the pectate lyase or its fixed point stabilized variants that obtain from the dna sequence encoding of Bacillus subtilus DSM 14218, for example by the polypeptide of the dna sequence encoding of the 1-1197 position that comprises SEQ ID NO:1 or by the fixed point stabilized variants of the pectate lyase of the dna sequence encoding of the 1-1197 position that comprises SEQ ID NO:1.

Second aspect of the present invention relates to detergent composition provided by the present invention comes clean textile, dish or crust to obtain the method for good clean-up performance.

Third aspect of the present invention relates to detergent composition provided by the present invention at clean fabric and/or fabric decontaminating and/or fabric whiteness maintenance and/or fabric-softening and/or fabric color manifests and/or cleaning of hard surfaces such as floor, wall, bathroom tile and analogue and/or hand washing and machine washing dish and/or oral cavity and/or the tooth purposes in using.

It is one of following that another aspect, (EC4.2.2.2) the active polypeptide that the present invention relates to have pectate lyase, this polypeptide are selected from: (a) by the polypeptide of the dna sequence encoding of the 1-1197 position of SEQ ID NO:1; (b) cell of the dna sequence dna of the 1-1197 position by will comprising SEQ ID NO:1 is cultivated the polypeptide that produces under the condition that can express this sequence; (c) (a) or fixed point variant (b), this variant has obtained stabilization by changing one, two, three, four or five amino acid residue for other amino-acid residue.

On the one hand, the present invention relates to a kind of enzyme preparation of pectate lyase of the present invention, the polynucleotide molecule of a kind of separated coding pectate lyase of the present invention, a kind of expression vector, a kind of method that can express culturing cell and a kind of production pectate lyase of the present invention of pectate lyase of the present invention of comprising again.

Detailed Description Of The Invention

In order to reach purpose of the present invention, term used herein " acquisition certainly " or " can obtain certainly " refer to that the specific thus source of enzyme produces when relevant with a specific source, or are produced or can be produced by them by the cell of the gene that inserts this source.

A kind of enzyme represented in term " wild-type enzyme ", and described enzyme is naturally occurring microorganism, as the fungi found at nature or the endogenous enzyme of bacterium.

A kind of modified enzyme that can produce enzyme variants of the present invention of term used herein " parent enzyme " expression.Parent enzyme can be the enzyme of a kind of separation from natural origin, or a kind of active enzyme of feature of the enzyme of having been modified in advance but still having kept being discussed.Parent's pectate lyase of the present invention can be the pectate lyase of wild-type.

Term " enzyme variants " expression aminoacid sequence is compared the enzyme that is comprising difference with the aminoacid sequence of parent enzyme.Compare with parent enzyme, this difference comprises replacement, disappearance and/or inserts.

Pectate lyase of the present invention can obtain from the Bacillus subtilus bacterial strain.

In a preferred embodiment, pectate lyase of the present invention obtains from Bacillus subtilus.The inventor is used for the budapest treaty of microbial preservation of patented procedure April 5 calendar year 2001 according to international recognition, (the Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH at Germany microbial preservation center, Mascheroder Weg lb, D-38124Braunschweig, the Germany) preservation this Bacillus subtilus bacterial strain, preserving number is DSM 14218.

In literary composition of the present invention, term " enzyme preparation " means conventional enzymic fermentation product, and it may separate, and also purifying is from a microbial species, and such goods comprise some kinds of different enzymic activitys usually; Perhaps mean the single component enzyme, preferably by using conventional recombinant technology to derive from the mixture of the enzyme of bacterium or fungi strain, these enzymes have carried out fermentation independently and carried out separation possibly can be from different bacterial classifications with purifying and they, preferred fungi or bacteria culture; Perhaps mean tunning as the microorganism of host cell expression reorganization pectate lyase, but this microorganism can produce other enzyme simultaneously, for example xyloglucanase enzymes, proteolytic enzyme or cellulase, they are the natural tunnings of this microorganism, and this term will mean the conventional enzyme complex that produces of corresponding thus natural microbial like this.

In literary composition of the present invention, linearity or ring-shaped DNA molecule represented in term " expression vector ", wherein comprises the fragment of the desired polypeptides of encoding and be operatively coupled on one with it to be used from the additional clip that it is transcribed.Described additional clip can comprise promotor and terminator sequence, also can choose wantonly to comprise one or more replication orgin, one or more selective marker, enhanser, polyadenylation signal etc.Expression vector derives from plasmid or viral DNA usually, maybe can contain the two element.Expression vector of the present invention can be any expression vector that can carry out the recombinant DNA operation easily, and the host cell that this carrier will import is depended in the selection of carrier usually.Therefore, carrier can be an autonomously replicationg vector, i.e. the carrier that exists with the outer entity of karyomit(e), and it duplicates and is independent of THE REPLICATION OF CHROMOSOME, as plasmid.Selectively, carrier can be a kind of like this carrier, when it is imported in the host cell, can be incorporated in the genome of host cell and with the karyomit(e) of being integrated and duplicates.

Be used for this paper term " recombinant expressed " relevant or " recombinant expressed " and be limiting according to the standard definition of this area with polypeptide or protein expression.The expression vector that recombinant expression of proteins has just been described above using usually carries out.

Term " isolating ", when being used for polynucleotide molecule, represent that this polynucleotide molecule has been moved apart its natural genotypic environment, therefore do not contain other external source or unwanted encoding sequence, and this molecule exists with a kind of form that is suitable for genetically engineered protein production system.This isolating molecule is the molecule that is separated with their natural surroundings, comprises cDNA and genomic clone.Isolated DNA molecule of the present invention does not contain other gene associated therewith under normal conditions, but can comprise naturally occurring 5 ' and 3 non-translational regions, as promotor and terminator.The evaluation of associated area it will be apparent to those skilled in the art that (referring to for example, Dynan and Tijan, natural 316:774-78,1985).Term " isolating polynucleotide " also can be called " clone's polynucleotide ".

When being used for protein/polypeptide, term " isolating " represents that this protein is present under the situation of non-its natural surroundings.Under preferred form, isolating protein does not contain other protein in fact, especially other homologous protein (promptly " homologous impurity " sees below).Preferably with purity in the form more than 40%, more preferably purity provides protein in the form more than 60%.

Even more preferably provide described protein with highly purified form, promptly measure this proteinic purity more than 80%, more preferably more than 95%, even more preferably more than 99% by SDS-PAGE.

Term " isolating protein/polypeptide " also can be called " protein/polypeptide of purifying ".

Term " homology impurity " means the impurity (as other polypeptide of non-polypeptide of the present invention) of the homologous cell of any cell that derives from initial acquisition polypeptide of the present invention.

It is particular source or produced by following cell thus that the term that used herein with specific microbial source is relevant " obtain from " means these polynucleotide and/or polypeptide, has inserted the gene that comes from this source in the wherein said cell.

When relating to dna fragmentation, term " is operably connected " and represents that these fragments are aligned to the form that can bring into play its intended purposes synergistically, arrives terminator as transcriptional start in promotor and by encode fragment.

Term " polynucleotide " expression thymus nucleic acid or Yeast Nucleic Acid base from 5 ' to 3 ' strand or the dichain polymer read.Polynucleotide comprise RNA and DNA, and can separate from natural origin and obtain or externally syntheticly to obtain or prepared by the combination of natural and synthetic molecules.

Term " complement of polynucleotide molecule " expression is compared with canonical sequence, has the opposite polynucleotide molecule of complementary base sequence and direction.For example, sequence 5 ATGCACGGG3 and sequence 5 CCCGTGCAT3 complementations.

Term " degenerate core nucleotide sequence " expression comprises the nucleotide sequence (comparing with the polynucleotide canonical sequence of coded polypeptide) of one or more degenerate codon.Degenerate codon comprises different nucleotide triplets, but the identical amino-acid residue of encoding (be GAU and GAC triplet all encode Asp).

The part of term " promotor " expression gene, described part contains provides RNA polymerase combination and initial dna sequence dna of transcribing.Promoter sequence is common, but not always, is positioned at 5 non-coding regions of gene.

The dna sequence dna of term " secretory signal sequence " presentation code polypeptide (" secretion peptide "), the Secretory Pathway that wherein said polypeptide will guide described bigger polypeptide to pass through cell as a moiety of a bigger polypeptide---cell of synthetic this polypeptide---.Usually bigger polypeptide is cut in the process by this Secretory Pathway transhipment to remove the secretion peptide.

Homology between two aminoacid sequences of term " identity " expression or the nucleotide sequence.In the present invention, the identity degree between two aminoacid sequences is measured by Needleman-Wunsch sequence alignment (align) method, and this method is applicable to albumen and dna sequence dna comparison.For protein sequence contrast, usedly to give tacit consent to such an extent that sub matrix is BLOSUM50, the point penalty of first residue is-12 in the breach, and the point penalty of other residue is-2 in the breach.Described sequence alignment can be used to carry out (W.R.Pearson and D.J.Lipman (1988) from the Align software of FASTA software package (v20u6 of version number), " improved biology sequential analysis instrument ", newspaper (PNAS) 85:2444-2448 of institute of NAS; And W.R.Pearson (1990) " carries out fast and the comparison of sensitive sequence with FASTP and FASTA ", Enzymology method (Methods in Enzymology), 183:63-98).

Identity degree between two nucleotide sequences can by with the identity matrix as giving tacit consent to such an extent that sub matrix utilizes above-mentioned same algorithm and software package to determine.The point penalty of first residue of breach (gap) is-16, and the point penalty of other residue in the breach is-4.

Enzyme variants

In the context of the invention, a kind of enzyme represented in term " enzyme variants ", and this enzyme comprises the aminoacid sequence different with the aminoacid sequence of standard enzyme.Described difference comprises replacement, disappearance and/or the insertion of comparing with standard enzyme.

In the context of the present invention, adopt the particular number of the amino acid residue position in the cell wall degrading enzyme, particularly pectate lyase.For example, by comparing, can clearly the amino acid position numbering be distributed to any amino-acid residue in any pectate lyase, as long as the aminoacid sequence of this pectate lyase is known with the aminoacid sequence of known pectate lyase.

Use derives from the coding scheme of the aminoacid sequence (being disclosed in SEQ ID NO:2) of the pectate lyase of the polynucleotide sequence coding that exists in the plasmid of Bacillus subtilus strain DSM 14218, from many other aminoacid sequences of pectate lyase, can disclose the position of an amino-acid residue in a pectate lyase by comparison clearly.

Describing that the present invention produces or during the multiple pectate lyase variant considered, adopting following nomenclature to be easy to reference:

Replace:

[primary amino acid; The position; The amino acid of replacing]

Correspondingly, symbol S72I means 72 Serine and is replaced by Isoleucine.

Multiple mutation is separated by adding labelled notation ("+"), and as M169I+F198V, representative is 169 and 198 sudden changes of using Isoleucine (I) to replace methionine(Met) (M) and replace phenylalanine (F) with Xie Ansuan (V) respectively.

Disappearance:

The disappearance of 195 glycine is expressed as: Gly195 ^*Or G195 ^*

Correspondingly, the disappearance of an above amino-acid residue is expressed as 195 and 196 glycine and leucic disappearance

Gly195 ^*+ Leu196 ^*Or G195 ^*+ L196 ^*

Insert:

The insertion of extra amino-acid residue is expressed as insert Methionin behind G195:

Gly195GlyLys or G195GK;

Perhaps, when inserting more than one amino-acid residue, during as insertion Lys and Ala behind G195, this insertion is expressed as:

Gly195GlyLysAla or G195GKA

In these cases, be numbered inserting amino-acid residue by next in the location number of the amino-acid residue before the amino-acid residue that lowercase is added to insertion.In above example, therefore sequence 194-196 incites somebody to action:

From 194 195 196

A-G-L

Change to 194 195 195a 195b 196

A-G-K-A-L

When inserting the amino-acid residue identical with existing amino-acid residue, degeneracy has obviously appearred in nomenclature.For example, if in above example, insert glycine after glycine, then this will be expressed as G195GG.Right

From 194 195 196

A-G-L

To 194 195 195a 196

A-G-G-L

Or the in fact same variation of 194 194a 195 196 can also be expressed as A194AG.

These examples are apparent to those skilled in the art, so representation G195GG is intended to comprise these degeneracy representations of equal value with the corresponding representation that is used for this type of insertion.

Unless otherwise prescribed, all positions of mentioning in this article by the pectate lyase numbering system all refer to above-mentioned numbering, and the aminoacid sequence of the pectate lyase of the polynucleotide sequence coding that exists in the plasmid with respect to Bacillus subtilus DSM 14218 (being disclosed in SEQ ID NO:2) and determining.

Term " washing composition stability " or " storage stability " mean the stability of albumen in the preparation that comprises washing composition (as anion surfactant).The feature of anion surfactant is the combination of anionic group and hydrophobic tail.When with protein binding, therefore positively charged amino-acid residue (as Methionin or arginine) and hydrophobic region may become interaction point.Similarly, flexible especially zone open by the kinetics mode so that be embedded in the amino acid of active site of protein usually can be by approaching.These described amino-acid residues are hydrophobic typically, and are therefore attractive to the afterbody of this tensio-active agent.Chemical interaction between enzyme and the tensio-active agent will make enzyme deactivation very for certain.Therefore improve washing composition or storage stability means under certain detergent concentration and temperature, higher enzymic activity will be retained (higher residual activity) after after a while.Correspondingly, thermostability and washing composition stability are two separate features of albumen or enzyme.

Polynucleotide

In the preferred embodiment of the invention, isolating polynucleotide of the present invention under moderate stringent condition at least can to zone or its complementary sequence hybridization of similar size among the SEQ ID NO:1.

Specifically, polynucleotide of the present invention are under moderate stringent condition at least, preferred heights stringent condition (stating as follows) down can with following probe hybridization, described probe is to comprise the denatured double stranded dna probe of sequence shown in full length sequence shown in the SEQ ID NO:1 or the SEQ ID NO:1 1-1197 position or be any probe of the subsequence that contains about 100 base pairs of growing up at least among the SEQ ID NO:1.Be used for determining that the suitable experiment condition of hybridizing between nucleotide probe and homologous dna or the RNA sequence comprises under moderate or height stringent condition: the filter membrane pre-soaking that will contain the dna fragmentation that needs hybridization or RNA is in 5 * SSC (sodium chloride/sodium citrate, Sambrook etc., 1989) in 10 minutes, and in having 5 * SSC, 5 * Denhardt ' s solution (Sambrook etc., 1989), salmon sperm dna (the Sambrook etc. of the supersound process of 0.5%SDS and 100 μ g/ml sex change, 1989) this filter membrane of prehybridization in the solution, containing (the Feinberg that 10ng/ml causes at random then, A.P. and Vogelstein, B. (1983) analytical biochemistry (Anal.Biochem.) 132:6-13); ³²The P-dCTP mark (specific activity is higher than 1 * 10 ⁹Cpm/ μ g) in the same solution of probe about 45 ℃ hybridization 12 hours.Filter membrane is washed twice in 2 * SSC, 0.5%SDS subsequently, 30 minutes, at least 60 ℃ of wash temperatures (moderate stringent condition), more preferably at least 65 ℃ (moderate/height stringent condition), even more preferably at least 70 ℃ (height stringent condition), even more preferably at least 75 ℃ (high stringent condition).

With x-ray film detect under these conditions with oligonucleotide probe hybridization on molecule.

As previously mentioned, separation polynucleotide of the present invention comprise DNA and RNA.The method of DNA isolation and RNA is well known in the art.The DNA and the RNA of available methods known in the art clones coding goal gene from Gene Bank or DNA library.

Identify with for example hybridization or PCR method subsequently and separate to encode and have the polynucleotide of the active polypeptide of pectate lyase of the present invention.

Polypeptide and polynucleotide counterpart (directly to homologue (ortholog) or symbiosis homologue (paralog)) have been the present invention further provides from the different bacterium bacterial strain.Interested especially is from the Bacillus subtilus bacterial strain, for example the pectate lyase polypeptide of strain DSM 14218.

Information provided by the invention and composition combined with conventional clone technology can clone species homologue with the active polypeptide of pectate lyase of the present invention.For example, dna sequence dna of the present invention can be used to clone from the chromosomal DNA of expressing this proteinic cell type.The probe of the available disclosed sequences Design that Clicks here is differentiated the suitable source of DNA by surveying the RNA trace.Chromosomal DNA from positive cell line prepares the library subsequently.The dna sequence dna of the present invention that coding has an active polypeptide of the pectate lyase separation that can in all sorts of ways subsequently, such as using probe to survey by disclosed sequences Design in this specification sheets and claims, or use based on open sequence one or overlap the degeneracy probe more and survey.Dna sequence dna of the present invention also can adopt polymerase chain reaction or PCR (Mullis, U.S. Patent number 4,683,202), uses based on the primer of the open sequences Design of this paper and clones.In other method, available DNA library transforms or transfection host cell, and use the expression of antibody (mono-clonal or polyclonal antibody) the testing goal DNA at pectate lyase, described enzyme is pressed method described in material and method and embodiment clone from Bacillus subtilus, as DSM 14218, and express and purifying; Perhaps can be by the expression of the activation measurement testing goal DNA relevant with having the active polypeptide of pectate lyase.

Polypeptide

The sequence of amino acid 1-399 position is the ripe pectin acid cleavage enzyme sequence that comprises the catalytic activity structural domain of enzyme of the present invention among the SEQ ID NO:2.

The present invention also provide with SEQ ID NO:2 in the polypeptide of amino acid 1-399 position and the basic homologous pectate lyase polypeptide of species homologue (directly to homologue or symbiosis homologue) thereof.The sequence of amino acid 1-399 position or it directly has 98.5% to homologue or symbiosis homologue among term used herein " basic homology " expression polypeptide and the SEQ ID NO:2, and preferably at least 99%, more preferably at least 99.5% sequence identity.Sequence identity percentage is measured by aforesaid Needleman-Wunsch sequence alignment method.

For basic homologous protein and polypeptide, it is characterized in that having one or more amino acid and replace, lack or insert.These changes are preferably less the sort of of character, promptly conserved amino acid replace (seeing Table 2) and other can remarkably influenced protein or the folding or active replacement of polypeptide; Little disappearance, normally 1 to about 30 amino acid whose little disappearances; And little amino or C-terminal extension, be no more than the little connection peptides of about 20-25 residue or be beneficial to the little extension (affinity tag) of purifying such as N-terminal methionine residues, length, as poly Histidine, albumin A (Nilsson etc., EMBO J, 4:1075,1985; Nilsson etc., Enzymology method, 198:3,1991).Usually consult, Ford etc., protein expression and purifying (Protein Expression and Purification) 2:95-107,1991, the document is incorporated herein by reference.The coding affinity tag DNA can from product vendor (as, Pharmacia Biotech, Piscataway, NJ; New EnglandBiolabs, Beverly MA) locates to buy.

Yet, even if the preferably less change of character of above-mentioned variation, these changes also can be the bigger changes of character, extend to be fused to as amino or C-terminal and of the present inventionly have on the active polypeptide of pectate lyase as reaching 300 amino acid or longer big polypeptide.

Table 1

Conserved amino acid is replaced

Alkalescence: arginine

Methionin

Histidine

Acid: L-glutamic acid

Aspartic acid

Polarity: glutamine

L-asparagine

Hydrophobicity: leucine

Isoleucine

Xie Ansuan

Aromatic series: phenylalanine

Tryptophane

Tyrosine

Little: glycine

L-Ala

Serine

Threonine

Methionine(Met)

Except 20 kinds of standard amino acids, also available non-standard amino acid (as 4-Hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline and Alpha-Methyl Serine) is replaced the amino-acid residue of polypeptide of the present invention.Also the non-conserved amino acid of available limited quantity, non-genetic code amino acids coding and alpha-non-natural amino acid are replaced amino-acid residue." alpha-non-natural amino acid " modified behind protein synthesis, and/or on their side chains the chemical structure that is different from the standard amino acid side chain arranged.Alpha-non-natural amino acid can chemosynthesis, or preferably buys and obtain, and comprises pipecolic acid, Thiazolidinecarboxylacid acid, dehydroproline, 3-and 4-methylproline and 3,3-dimethyl proline(Pro).

Indispensable amino acid in the pectate lyase polypeptide of the present invention can be differentiated by means known in the art, as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, science (Science) 244:1081-1085,1989).In one technology of back, import single alanine mutation in each residue position of molecule, the biologic activity (being the pectate lyase activity) that detects the mutating molecule that is produced is to differentiate the amino-acid residue that molecular activity is played a crucial role.Also can consult Hilton etc., journal of biological chemistry (J.Biol.Chem) 271:4699-4708,1996.Enzyme or the interactional avtive spot of other biology also can be by carrying out the physical analysis of structure and the contact site amino acid of inferring suddenlyd change determining to using such as technical measurements such as nucleus magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, consult, for example, de Vos etc., science 255:306-312,1992; Smith etc., molecular biology magazine (J.Mol.Biol.) 224:899-904,1992; Wlodaver etc., FEBSLett.309:59-64,1992.The identity of indispensable amino acid also can be inferred by the homology of analyzing the polypeptide relevant with polypeptide of the present invention.

Available known sudden change, reorganization and/or reorganization method and relevant screening method subsequently carry out a plurality of amino acid and replace and detect, these methods for example have Reidhaar-Olson and Saner (science 241:53-57,1988), Bowie and Sauer (institute of NAS reports 86:2152-2156,1989), WO95/17413 or the disclosed technology of WO95/22625.Briefly, these authors disclose reorganization/reorganization of making two or more positions in the polypeptide that different sudden changes take place simultaneously at random or carrying out different sudden changes (WO95/17413, WO95/22625), subsequently the selection function polypeptide, then with the true preface of mutagenesis polypeptide to measure the method that the admissible replacement in each position is composed.Other available method comprises that phage display is (as Lowman etc., biological chemistry 30:10832-10837,1991; Ladner etc., U.S. Patent number 5,223,409; Huse, WIPO publication WO 92/06204) and regional directed mutagenesis (Derbyshire etc., gene 46:145,1986; Ner etc., DNA 7:127,1988).

Above disclosed mutagenesis/reorganization method can combine with high-throughput, automatically screening method, to measure the activity of the mutagenesis polypeptide of cloning in the host cell.The mutagenized dna molecule of coding active polypeptide can reclaim from host cell, and checks order fast with state-of-the-art facility.These methods can be determined the importance of each amino-acid residue in the desired polypeptides fast, and can be used for the polypeptide of unknown structure.

In preferred embodiments, the invention provides the variant of the endogenous pectate lyase of Bacillus subtilus DSM14218 or other Bacillus subtilus bacterial strain, this variant is the fixed point variant, have therein one, two, three, four or the five amino acid residue be changed into other amino-acid residue, the enzyme variants that is therefore obtained has the stability of increase in detergent composition.

The example of the variant of the pectate lyase of SEQ ID NO:2 includes, but are not limited to, and those are disclosed variant in following embodiment 6 and scourability Embodiment C.

Immune cross-reactivity

Be used to measure the polyclonal antibody of immune cross-reactivity, particularly the monospecific polyclonal antibody can utilize the active enzyme of pectate lyase that has of purification to prepare.More particularly, can be with reference to N.Axelsen etc., quantitative immunoelectrophoresis guide (A manual of Quantitative Immunoelectrophoresis), Blackwell Science Press, 1973, the 23 chapters; Or A.Johnstone and R.Thorpe, practical immunochemistry (Immunochemistry in Practice), Blackwell Science Press, the described method of 1982 (specifically being the 27-31 page or leaf), by immune rabbit (or other rodent), obtain antiserum(antisera) at pectate lyase of the present invention.The immunoglobulin (Ig) of purifying can obtain from antiserum(antisera), for example by the heavy ((NH of salt ₄) ₂SO ₄), then the dialysis and on as DEAE-Sephadex, carry out ion exchange chromatography.Proteinic immunochemistry is identified and can be utilized the Outcherlony bidirectional diffusion to analyze (O.Ouchterlony, experiment immunization learns to do volume (Handbook ofExperimental Immunology) (D.M.Weir volume), Blackwell Science Press, 1967, the 655-706 page or leaf), and crossed immunoelectrophoresis (N.Axelsen etc., the source is the same, the 3rd and 4 chapters) or rocket immunoelectrophoresis (N.Axelsen etc., the 2nd chapter) carry out.

Carrier

As described further below, can transform host of the present invention with the carrier of the dna sequence dna that comprises the pectate lyase of encoding.Preferably, this carrier is integrated into host genome, and more preferably this carrier increases on host genome.

In another embodiment preferred of the present invention, this carrier preferably exists with multiple copied plasmid form with expression plasmid.

Genus bacillus expression vector of the present invention carries the dna sequence dna of the coding pectate lyase of insertion.Preferably, the expressed sequence box of this carrier comprises the control region from genus bacillus, and more preferably, this control region is host's an endogenous control region.

Express pectate lyase

Recombinant expression vector

The recombinant vectors that comprises the DNA construct of code book invention enzyme can be any carrier that can carry out the recombinant DNA operation easily.The selection of carrier often depends on that it is with the host cell that is imported into.Carrier is imported host cell often be called transformed host cells.This conversion is meant to use and as protoplastis, natural competent cell, transfection, joint, electroporation or any other equivalent method DNA is imported host cell.Therefore, carrier can be an autonomously replicationg vector, promptly has, duplicates the carrier that is independent of THE REPLICATION OF CHROMOSOME as the outer entity of karyomit(e), as plasmid.Perhaps, carrier can be such one type, when it is imported in the host cell, will partly or entirely be incorporated in the host cell gene group and with its karyomit(e) of integrating and duplicates.

Described carrier is preferably such expression vector, and the dna sequence dna of the pectate lyase of the present invention of wherein encoding is operationally transcribed other required fragment with this DNA and is connected.In general, expression vector maybe can contain both elements from plasmid or viral DNA.Term " is operably connected " and refers to all fragments so that it can be arranged by their mode of the collaborative performance function of intended purposes, as transcribes and cause and continue dna sequence dna by the CBD that encodes in promotor.

Promotor can be any dna sequence dna that shows transcriptional activity in selected host cell, and can come the homology of own coding host cell or the gene of heterologous protein.

The example that is applicable to the promotor of bacterial host cell comprises the promotor of following gene: bacstearothermophilus (Bacillus stearothermophilus) maltogenic amylase gene, Bacillus licheniformis (Bacillus licheniformis) alpha-amylase gene, bacillus amyloliquefaciens (Bacillusamyloliquefaciens) alpha-amylase gene, the P of subtilis (Bacillus subtilis) alkaline protease gene or bacillus pumilus (Bacillus pumilus) xylosidase gene or lambda particles phage _ROr P _LPromotor or colibacillary Lac, TrpOr TacPromotor.Selectively, can design integrating vector, so that only when it suitably is integrated in the host genome (as the downstream of resident (resident) promotor), the DNA of coding pectate lyase just can functional expression.

If desired, the dna sequence dna of code book invention pectate lyase can also be operably connected on the suitable terminator.

Recombinant vectors of the present invention also can comprise the dna sequence dna that can carrier be duplicated in the host cell of being discussed.

But described carrier also can comprise selective marker, for example its product can remedy the gene of host cell defective, or coding as to microbiotic as the resistance of kantlex, paraxin, erythromycin, tsiklomitsin, spectinomycin etc. or to the gene of the resistance of heavy metal or weedicide.

In order to instruct pectate lyase of the present invention to enter the Secretory Pathway of host cell, can in recombinant vectors, provide secretory signal sequence (being also referred to as leader sequence, preceding former sequence or presequence).The dna sequence dna that secretory signal sequence can be connected the pectate lyase of encoding in proper reading frame.Secretory signal sequence generally is positioned at 5 ' end of the dna sequence dna of this enzyme of coding.Described secretory signal sequence can be usually the gene that maybe can come own coding another kind of secretory protein relevant with this pectate lyase.

Be respectively applied for the dna sequence dna that connects coding pectate lyase of the present invention, promotor and randomly terminator and/or secretory signal sequence, or assemble these sequences and their inserted the method that contains the appropriate carrier of duplicating or integrate information needed well known to those skilled in the art (referring to for example by suitable pcr amplification scheme, people such as Sambrook, the preceding book that draws).

Host cell

Import in the host cell the cloned DNA molecule can with host's homology or the allos discussed.If with described host cell homology, promptly by the natural generation of this host cell, then this dna sequence dna is general operationally is connected with another promoter sequence of existing in non-its natural surroundings, perhaps is connected with another secretory signal sequence and/or terminator sequence suitably the time.Term " homologous " is intended to comprise the natural dna sequence dna that is present in the enzyme in the host organisms of being discussed of coding.Term " allogenic " is intended to dna sequence dna itself that comprise that described host cell is not expressed.Therefore dna sequence dna can derive from another organism or synthetic sequence.

The host cell that has imported cloned DNA molecule of the present invention or recombinant vectors can be any cell that can produce desired enzyme, comprises bacterium, yeast, fungi and higher eucaryotic cells.

The example that can produce the bacterial host cell of enzyme of the present invention by cultivation is a gram positive bacterium, as Bacillus strain, as Alkaliphilic bacillus (B.alkalophilus), bacillus amyloliquefaciens (B.amyloliquefaciens), bacillus brevis (B.brevis), bacillus lautus (B.lautus), bacillus lentus (B.lentus), Bacillus licheniformis (B.licheniformis), Bacillus circulans (B.circulans), Bacillus coagulans (B.coagulans), bacillus megaterium (B.megatherium), bacstearothermophilus (B.stearothermophilus), subtilis (B.subtilis) or bacillus thuringiensis (B.thuringiensis), lactobacillus (lactobacillus) bacterium pearl, streptococcus bacterial strain (Streptococcus), or streptomycete bacterial strain (Streptomyces), especially muta lead mycillin (S.lividans) or mouse ash streptomycete (S.murinus); This host cell can be gram negative bacterium such as intestinal bacteria (Echerichiacoli).

The conversion of bacterium can be carried out (referring to people such as Sambrook, on seeing) with protoplast transformation, electroporation, joint or use experience attitude cell by known methods.

When at bacterium such as expression in escherichia coli enzyme, this enzyme generally resides in the tenuigenin with the insolubility particle and (is called inclusion body), or can be directed to periplasmic space by the bacterium secretion sequence.In the previous case, with lysis, reclaim described particle, sex change makes enzyme folding again by the dilution denaturing agent thereafter.Under the latter event, this enzyme can reclaim from periplasmic space by following operation: destroy cell as ultrasonic or osmotic shock, the content of described periplasmic space is discharged, reclaim this enzyme again.

When expressing this enzyme in gram positive bacterium such as bacillus or streptomyces bacterial strain, this enzyme can reside in the tenuigenin, or is directed in the outer substratum of born of the same parents by the bacterium secretion sequence.

The example that can produce the fungal host cells of enzyme of the present invention by cultivation is as Aspergillus (Aspergillus) or Fusarium (Fusarium) bacterial strain, especially Aspergillus awamori (A.awamori), Aspergillus nidulans (A.nidulans), aspergillus niger (A.niger), aspergillus oryzae (A.oryzae) and sharp sickle spore (F.oxysporum), with trichoderma bacterial strain (Trichoderm), preferred trichoderma harziarum (T.harzianum), Trichodermareesei (T.reesei) and viride (T.viride).

The fungal cell can transform by following process, and described process comprises protoplastis formation and protoplast transformation and subsequently with known in fact mode regenerative cell's wall.The Aspergillus bacterial strain is found in EP238023 (Novo Nordisk A/S) as the purposes of host cell, and its content is quoted as a reference in the lump at this.

The example that can produce the yeast source host cell of enzyme of the present invention by cultivation is as Hansenula bacterial strain (Hansenula sp.); Genus kluyveromyces bacterial strain (Kluyveromyces sp.), especially Kluyveromyces lactis (K.lactis) and kluyveromyces marxianus (K.marcianus); Pichia bacterial strain (Pichia sp); Yeast belong bacterial strain (Saccharomyces), especially saccharomyces carlsbergensis (S.carlsbergensis), yeast saccharomyces cerevisiae (S.cerevisae), Crewe be yeast (S.kluyveri) and grape sweat yeast (S.uvarum) not; Schizosaccharomyces bacterial strain (Schizosaccharomyces sp.) is especially trembled wine fission yeast (S.pombe); With Ye Shi yeast belong bacterial strain (Yarrowia sp.), especially separate fat Ye Shi yeast (Y.lipolytica).

The example that can produce the plant origin host cell of enzyme of the present invention by cultivation is as the vegetable cell from potato (Solanum tuberosum) or tobacco (Nicotiana tabacum).

Produce the method for pectate lyase

On the other hand, the present invention also relates to a kind of method of producing enzyme preparation of the present invention, this method is included under the condition that allows this enzyme generation, and cultivation can produce the microorganism of this pectate lyase, and reclaims this enzyme from culture.Cultivation can adopt conventional fermentation process to carry out, and as cultivating on growth medium under the agitation condition of guaranteeing enough ventilations in shaking bottle or fermentor tank, produces this pectate lyase thereby induce.Growth medium can comprise conventional nitrogenous source, as peptone, yeast extract or casamino acids; The conventional carbon source of reduction such as glucose or sucrose; With inductor such as polygalacturonase or composite plant substrate such as cavings (as wheat bran or rice shell).Recovery can be adopted routine techniques, for example can be by centrifugal or filtering separation biomass and supernatant liquor, if reclaim supernatant liquor or purpose enzyme lysing cell in cell, can be further purified or by the described method crystallization of WO97/15660 by EP 0406314 described method afterwards.

Further, the invention provides a kind of method of producing separation enzyme of the present invention, wherein under the condition that allows this enzyme to produce, cultivate the suitable host cell of the dna sequence dna that has transformed this enzyme of encoding, and from culture, reclaim the enzyme that obtains.

As definition herein, isolated polypeptide (as enzyme) is not contain the polypeptide of other polypeptide substantially, as presses SDS-PAGE and measure, at least about 20% pure, preferably pure at least about 40%, more preferably from about 60% is pure, even more preferably from about 80% pure, most preferably from about 90% is pure, most preferably from about 95% pure again.

Term " isolated polypeptide " also can be described as " polypeptide of purifying ".

When the expression vector of the dna sequence dna that will contain this enzyme of encoding is transformed in the heterologous host cell, just might allos recombinant production enzyme of the present invention.

Thus, might prepare the pectate lyase composition of highly purified or single component, it is characterized in that not containing homology impurity.

Herein, homology impurity is meant any impurity (for example other polypeptide except that enzyme of the present invention) of the homologous cell that derives from initial acquisition enzyme of the present invention.

Among the present invention, the homology host cell can be the bacterial strain of Bacillus subtilus.

The substratum that is used to cultivate transformed host cells can be any conventional substratum that is suitable for the host cell growth discussed.Expressed pectate lyase can be secreted in the substratum easily, can reclaim by well-known method then, described method comprises by centrifugal or filtration isolated cell from substratum, utilize the protein component in salt such as the ammonium sulfate precipitation substratum again, next carry out chromatography, as ion exchange chromatography, affinity chromatography etc.

The present invention also relates to transgenic plant, plant part or vegetable cell, be encoded the dna sequence dna of pectate lyase of the present invention of described transgenic plant, plant part or vegetable cell transforms, so that can express and produce this enzyme of recyclable amount.This enzyme can reclaim from plant or plant part.

Described transgenic plant can be dicotyledons or monocotyledons, are called for short dicotyledonous or unifacial leaf.Monocotyledonous example is a herbage, as English grass (bluegrass, annual bluegrass belongs to (Poa)), and fodder grasses such as fescue grass, rye grass, temperate zone herbage belongs to (Agrostis) as cutting Gu Ying; And cereal grass, as wheat, oat, rye, barley, paddy rice, Chinese sorghum and corn.

The example of dicotyledons is tobacco, leguminous plants such as lupine, potato, beet, pea, Kidney bean and soybean, and the plant of Cruciferae (Brassicaceae), as Cauliflower, oilseed rape and close relative model animals Arabidopis thaliana (Arabidopsis thaliana).

The example of plant part is stem, callus, leaf, root, fruit, seed and stem tuber.In literary composition of the present invention, special plant tissue also is considered to plant part as chloroplast(id), apoplast, plastosome, vacuole, peroxysome and tenuigenin.And, any vegetable cell, no matter which kind of is tissue-derived, also is considered to plant part.

The offspring who also comprises these plants, plant part and vegetable cell within the scope of the present invention.

Can come the transgenic plant or the vegetable cell of construction expression enzyme of the present invention with methods known in the art.In brief, described plant or vegetable cell pass through in the expression construct introduced plant host genome with one or more code book invention enzyme, and the plant or the vegetable cell breeding of the change of gained are made up for transgenic plant or vegetable cell.

Easily, expression construct is a DNA construct, wherein comprise the gene of code book invention enzyme and be operatively coupled on it together in selected plant or plant part, expressing the needed suitable regulating and controlling sequence of this gene.In addition, this expression construct can comprise selective marker that is used to identify the host cell that wherein has been integrated into expression construct and the necessary dna sequence dna of plant (latter is depended on used DNA introduction method) that the construct importing is come into question.

The selection of regulating and controlling sequence such as promotor, terminator sequence and optional signal or transit sequence according to as when, where wish and how to express enzyme and determine.For example, the expression of gene of the enzyme of the present invention of encoding can be composing type or induction type or can be growth, stage or tissue-specific, and gene product is orientable to particular organization or plant part, as seed or leaf.As Tague etc., plant physiology (Plant Phys.), 86,506,1988 have described such regulating and controlling sequence.

For constitutive expression, can use 35S-CaMV promotor (Franck etc., 1980. cells (cell) 21:285-294).Organ specific promoters can be for example from storage gully (sink) tissue such as seed, potato tuber and fruit (Edwards and Coruzzi, 1990, genetics annual review (Annu.Rev.Genet.) .24:275-303) or metabolism gully tissue such as meristematic tissue (Ito etc., 1994. promotor molecular biology of plants (Plant Mol.Biol.) .24:863-878), seed specific promoters is as the gluten from paddy rice, prolamine, sphaeroprotein or albumin promoter (Wu etc., plant and stechiology (Plant and Cell Physiology) Vol.39, No.8pp.885-889 (1998)), unknown seed protein gene (the Conrad U. etc. that come from legumin B4 and broad bean, plant physiology magazine (Journal of Pplant Physiology) Vol.152, No.6pp.708-711 (1998)) broad bean promotor is from the promotor (Chen etc. of seed oil body protein.Plant and stechiology vol.39, No.9 pp.935-941 (1998)), from the storage protein napA promotor of colea (Brassica napus), or other any seed specific promoters known in the art, described in WO91/14772.In addition, described promotor also can be the leaf specificity promoter as from the rbcs promotor of paddy rice or tomato (Kyozuka etc., plant physiology (Plant Physiology) Vol.102, No.3 pp.991-1000 (1993)), chlorella virus VITAMIN B4 methyl transferase gene promotor (Mitra, A. and Higgins, DW, molecular biology of plants (Plant Molecular Biology) Vol.26, No.1pp.85-93 (1994)) or from the aldP gene promoter of paddy rice (Kagaya etc., molecule and General Genetics (Molecular and GeneralGenetis) Vol.248, No.6pp.668-674 (1995)) or the promotor of wound-induced such as potato pin2 promotor (Xu etc., molecular biology of plants (Plant Molecular Biology) Vol.22, No.4pp.573-588 (1993)).

Can utilize promotor to strengthen element and in plant, obtain higher expression of enzymes amount.For example, promotor strengthen element can be inserted in promotor and the nucleotide sequence of this enzyme of encoding between intron.As Xu etc., preceding quoted passage chapter discloses and has used first intron of rice actin 1 gene to strengthen expression.

Any other parts of selectable marker gene and expression construct can be from this area existing the selection.

Can comprise that agriculture bacillus mediated conversion, virus-mediated conversion, microinjection, microparticle bombardment, biological bombardment transform and electroporation (Gasser etc., science, 244,1293 according to routine techniques known in the art; Potrykus, biology/technology (Bio/Techn.) .8,535,1990; Shimamoto etc., nature (Natare), 338,274,1989), in DNA construct introduced plant genome.

At present, (summary is seen Hooykas and Schilperoort in the transgenosis of agrobacterium tumefaciens (Agrobacterium tamefaciens) mediation, 1992. molecular biology of plants .19:15-38) be the prefered method that produces the transgenosis dicotyledons, but also can utilize its transforming monocots, although to usually preferred other method for transformation of these monocotyledonss.At present, produce embryo (Christou, 1992. plant magazines (Plant J.) .2:275-281 that the monocotyledonous prefered method of transgenosis is microparticle bombardment (bag is by the small gold grain or the tungsten particle of transfering DNA) embryo callus or growth; Shimamoto, 1994. current biotechnology viewpoint (Carr.Opin.Biotechnol.) .5:158-162; Vasil etc., 1992. biologies/technology (Bio/Technology) 10:667-674).The method of alternative in addition transforming monocots is based on the method for protoplast transformation, and this method is described in Omirulleh S, etc., molecular biology of plants Vol.21, No.3pp.415-428 (1993).

After conversion, pick out the transformant of having introduced expression construct and according to the method well-known in the art whole plant of regenerating.

Enzyme composition

More on the one hand, the present invention relates to comprise enzyme composition with the active enzyme of aforesaid pectate lyase.

Enzyme composition of the present invention is except comprising pectate lyase of the present invention, the enzyme that can also comprise one or more other type, for example hemicellulase such as zytase and mannase, cellulase or endo-beta-1,4-glucanase component, chitinase, lipase, esterase, polygalacturonase, xyloglucanase enzymes, at, phytase, oxydo-reductase (peroxidase, haloperoxidase, oxydase, laccase), proteolytic enzyme, amylase, reductase enzyme, phenol oxidase, lignoenzyme, Starch debranching enzyme, pectate lyase, the pectin acetylase, polygalacturonase, the phammogalacturonane enzyme, pectin lyase, pectin methylesterase, cellobiohydrolase, trans-glutaminases, or their mixture.

Enzyme composition can prepare according to methods known in the art, can be liquid state or drying composition.For example, this enzyme composition can be granular or microgranular.The enzyme that is included in the composition can come stabilization with methods known in the art.

Purposes

Pectate lyase has the potential purposes in many industry and Application Areas.Yet pectate lyase particularly suitable of the present invention is made the composition of detergent composition.The following example that provides is the preferable use of enzyme composition of the present invention.Based on the known technology of this area, other condition of the consumption of enzyme composition of the present invention and use said composition can be determined.

Pectate lyase of the present invention is the basal component of detergent composition of the present invention, calculates with the weight of pure enzyme in composition, and preferably this enzyme accounts for 0.0001% to 2%, more preferably accounts for 0.0005% to 0.1%, most preferably accounts for 0.001% to 0.02%.

Except the enzyme core that comprises catalyst structure domain, pectate lyase of the present invention also can comprise cellulose binding domain (CBD), and the enzyme core (catalytic activity structural domain) of this cellulose binding domain and this enzyme is operably connected.Cellulose binding domain (CBD) can be used as the integral part of this codase, maybe the CBD in another source can be imported in this pectate lyase, thereby produce the enzyme heterozygote.In this article, term " cellulose binding domain " should be by Peter Tomme etc. (in " enzymatic degradation of insoluble carbohydrate " (Enzymatic Degradation of InsolubleCarbohydrates) book, " cellulose binding domain: classification and characteristic ", John N.Saddler and Michael H.Penner (volume), ACS Symposium Series, No.618,1996) definition is understood.This definition is classified as 10 families (I-X) with more than 120 kinds of cellulose binding domains, and confirms in such as plurality of enzymes such as cellulase, zytase, mannase, arabinofuranosidase, acetylase and chitinases CBD is arranged.Also found the CBD that exists with non-water-disintegrable polysaccharide conjugated protein form in algae such as red algae Porphyra purpurea, consulted Tomme etc., the source is the same.Yet most CBD is from cellulase and zytase, and CBD can be present in proteinic N and C-terminal or portion within it.The enzyme heterozygote is known in the art, consult WO90/00609 and WO95/16782, at least one fragment of coding DNA by will containing cellulose binding domain and by or the DNA construct of the pectate lyase DNA sequences encoding that is not attached thereto by joint be converted in the host cell, and cultivate this host cell to express fusion gene, can prepare the enzyme heterozygote.The enzyme heterozygote can be described with following formula:

CBD-MR-X wherein CBD is corresponding to the N-end or the C-stub area of the aminoacid sequence of cellulose binding domain at least; MR is region intermediate (joint), can be key or short linking group, and preferably about 2-100 carbon atom is long, and more preferably 2-40 carbon atom is long; MR or preferably have about 2-100 amino acid, more preferably 2-40 amino acid; And X is the N-end or the C-stub area of pectate lyase of the present invention.

Purposes in detergent industry

In washing and wearing process, the dyestuff on painted fabric or the clothes can ooze out from fabric usually, and this makes fabric seem outmoded and fades.Remove fiber on the fabric face and can partly recover the primary colors and the outward appearance of fabric.Term used herein " color clarification " is meant by cycles of washing repeatedly and part is recovered the primary colors of fabric or clothes.

Term " goes balling-up " and is meant the fiber bobbles of removing on the fabric face.

Term " soak solution " is meant the aqueous solution that can soak clothing before carrying out conventional washing process.Soak solution can contain conventional one or more compositions that use in washing or the laundry processes.

Term " washings " is meant the aqueous solution of washing clothes therein, the mixing effect of chemistry that this clothes washing normally carries out by hand or carries out in washing machine and machinery.The aqueous solution of the normally Powdered or liquid detergent compositions of washings.

Term " rinsing liquid " is meant and is used for immersion usually immediately or handles the aqueous solution of clothes with rinsing clothes (promptly removing the detergent solution on the clothing basically) after clothes washing.Can contain fabric-conditioning or softening compositio in the rinsing liquid.

The clothing that available the inventive method is handled can be conventional rinsable clothing.The major portion of preferred clothing is by cotton, mixes continuous or natural or artificial fiber is plain (as derives from the cellulosic fibre that contains xylan, as from paper pulp) or fabric weaving or nonwoven made of its mixture, comprise knitted fabrics, woven fabrics, denim, yarn and towelling.The example of mixture is that cotton or regenerated fiber/viscose fiber and one or more are followed mixtures of material, and the described material of following for example is the fiber (as regenerated fiber/viscose fiber, ramie, flax/linen thread and yarn, jute, cellulose acetate fiber, lyocell) of wool, synthon (as tynex, acrylic fibre, trevira, polyvinyl alcohol fiber, thermovyl, Saran, polyurethane fibre, polyurea fiber, Kevlar) and cellulose.

Be surprisingly found out that detergent composition of the present invention can provide the color clarification really, goes balling-up and remove the function of earth spot from the hemicellulose component of fabric fibre by the pectate lyase hydrolysis.

The spot that removal comes from plant, timber, the spot based on mould dirt, mud and fruit is one of current the most difficult cleaning task, and this trend is particularly more arranged in cold washing.These spots contain mainly the complex mixture based on the filamentary material of carbohydrate and their derivative (fiber and cell wall constituent) usually.The food stain usually is difficult to be removed effectively from contaminated thing.Removal is challenging especially from high painted or " doing " spot in fruit and/or the vegetables juice.The specific example of such spot comprises orange juice, tomato juice, banana, mango or Cauliflower spot.In fact, the pectin polymkeric substance is the important component of plant cell wall.On fabric, dish and crust, can find to contain the spot thing of pectin usually.

Found that in the cleaning that comprises clothing, dish and crust detergent composition of the present invention can provide good cleaning and soil removability.Although wish without being limited by theory, it is believed that the pectate lyase that is included in the detergent composition of the present invention with pectin and pectic acid in the ten minutes hydrolysis spot effectively, thereby the removal of spot is worked.As mentioned above, in fact in plant cell wall composition, all there are this pectin and pectic acid composition from the spot (as apple, banana, tomato, orange, mango, avocado and careless spot) of plant and/or fruit.Pectin and pectic acid composition also are widely used in food and the aesthetic nursing industry, as ice-creams, jam, shampoo and lipstick.

In addition, also find when detergent composition of the present invention is mixed with laundry composition, can provide the function of fabric-softening.Although wish without being limited by theory, it is believed that the pectin that exists and pectic acid composition because their acid properties will be securely in conjunction with calcium ion and positively charged ion, make fabric coarse in textile stains.Remove these pectin and pectic acid composition and be considered to stop those calcium ions and cationic combination, therefore can provide certain bating effect.

Open and the embodiment of washing composition

Surfactant system

Detergent composition of the present invention comprises surfactant system, and wherein tensio-active agent can be selected from non-ionic and/or anionic and/or cationic and/or amphoteric and/or zwitterionic and/or semi-polar tensio-active agent.

Tensio-active agent exists with the amount of 0.1% to 60% weight usually.

Tensio-active agent preferably be mixed with composition in the compatible form of enzyme component that exists.Tensio-active agent most preferably is mixed with the form that can promote or not reduce the stability of any enzyme in the composition at least in liquid or gelatinous composition.

Used preferred system comprises one or more nonionic as herein described and/or the anion surfactants as tensio-active agent according to the present invention.More preferably, composition of the present invention comprises high-caliber anion surfactant, promptly reaches 30% in free acid, preferred 10-25% weight.Wherein used preferred anionic tensio-active agent is alkylbenzene sulfonate, alkyl-sulphate and the alkyl ethoxy sulfate of sour form as described below.In an embodiment preferred again, high anionic surfactant compositions of the present invention also contains amine oxide tensio-active agent and/or amidoamines such as amido propyl group diethylamine.Find, comprise laundry, wash the dishes and the cleaning of crust in, detergent composition of the present invention (also comprising high-caliber anion surfactant) can provide good cleaning and soil removability.Although without wishing to be bound by theory, think anion surfactant can help solubilising enzyme of the present invention want the fruitlet glue fragment of hydrolysis and stop they on the surface of cleaning again the deposition.

Spendable suitable anion surfactant is an alkyl sulfonate surfactants, and it comprises C ₈-C ₂₀The linear ester class of carboxylic acid (being lipid acid), this tensio-active agent is by gaseous state SO ₃Sulfonation forms, referring to " american petroleum chemist association magazine " (" The Journal of the American OilChemists Society "), 52 (1975), pp.323-329.Suitable raw material comprises the natural fat material derived from Tallow, beef, palm wet goods.

Preferred alkyl sulfonate surfactants especially in laundry applications, comprises the alkyl sulfonate surfactants of following structural formula:

R wherein ³Be C ₈-C ₂₀Alkyl, preferred alkyl or its combination; R ⁴Be C ₁-C ₆Alkyl, preferred alkyl or its combination; M is a positively charged ion, and itself and alkyl ester sulfonic acid form water-soluble salt.The salifiable positively charged ion of suitable shape comprises metal such as sodium, potassium and lithium and replacement or unsubstituted ammonium cation, such as Monoethanolamine MEA BASF, diethanolamine and trolamine.Preferred R ³Be C ₁₀-C ₁₆Alkyl, and R ₄Be methyl, ethyl or sec.-propyl.Especially preferred is methyl ester sulfonate, wherein R ³Be C ₁₀-C ₁₆Alkyl.

Other suitable anion surfactant comprises alkyl sulfate surfactant, and this tensio-active agent is general formula R OSO ₃The water-soluble salt of M or acid, wherein R C preferably ₁₀-C ₂₄Alkyl, preferred alkyl or contain C ₁₀-C ₂₀The hydroxyalkyl of alkyl composition, more preferably C ₁₂-C ₁₈Alkyl or hydroxyalkyl, and M is H or positively charged ion, for example the ammonium of alkali metal cation (for example sodium, potassium, lithium) or ammonium or replacement (for example methyl-, dimethyl-and trimethyl ammonium positively charged ion and quaternary ammonium cation such as tetramethyl-ammonium and lupetidine positively charged ion and derived from quaternary ammonium cation of alkylamine such as ethamine, diethylamine, triethylamine and composition thereof etc.).Common C ₁₂-C ₁₆Alkyl chain is preferred for cold washing (for example being lower than about 50 ℃), and C ₁₆-C ₁₈Alkyl chain is preferred for high-temperature wash (for example being higher than about 50 ℃).

Other can be used for cleaning purpose anion surfactant also can be included in the laundry detergent composition of the present invention.They can comprise soap salt (ammonium salt that comprises for example sodium, potassium, ammonium and replacement as single-, two-and triethanolamine salt), C ₈-C ₂₂Uncle's type or secondary type sulfonated alkane, C ₈-C ₂₄Ethylenic sulfonate, by sulfonation poly carboxylic acid by the sulfonation reaction preparation of the pyrolysis product of alkaline earth metal citrate (for example, british patent specification 1,082,179 is disclosed), C ₈-C ₂₄Alkyl polyglycol ether sulfate (it contains maximum 10 moles oxyethane); Monoesters class (the especially saturated and unsaturated C of alkyl glycerol sulfonate, fatty acyl group glycerol sulfonate, fatty oil base glycerol vitriol, alkyl phenol epoxy ethane ether salt, sulfonated alkane, alkylphosphonic, isethionate such as acyl-hydroxyethyl sulfonate, N-acyl taurine salt, amber alkyl amide salts and sulfosuccinate, sulfosuccinic ester ₁₂-C ₁₈Monoesters) and the diester class of sulfosuccinic ester (especially saturated and unsaturated C ₆-C ₁₂Diester), the vitriol of the vitriol of acyl sarcosinate, alkyl polysaccharide such as alkylpolyglucoside (compound of nonionic non-sulfuric acidization is stated as follows), chain primary alkyl sulfate and alkyl polyethoxye carboxylate salt are such as general formula R O (CH ₂CH ₂O) _k-CH ₂(wherein R is C to the salt of COO-M+ ₈-C ₂₂Alkyl, k are from 1 to 10 integers, and M is the positively charged ion that forms soluble salt.Resinous acid and hydrogenated resin acid such as rosin, staybelite and in tallol or also be suitable derived from the resinous acid and the hydrogenated resin acid of tallol.

Alkylbenzene sulfonate is very preferred.Especially preferred is straight chain (linearity) alkylbenzene sulfonate (LAS), and wherein alkyl group preferably comprises 10 to 18 carbon atoms.

Other example is described in " tensio-active agent and washing composition " (Surface Active Agents andDetergents) (Vol.I and II, Schwartz, Perrry and Berch).Multiple this type of tensio-active agent also is disclosed in US 3,929 prevailingly, 678 (23 hurdles, 58 row are to 29 hurdles, and 23 row are introduced into as a reference in this article).

Other preferred anionic surfactants tensio-active agent comprises alkyl alkoxylated sulfate surfactant.Relevant example is general formula R O (A) _mSO ₃The water-soluble salt of M or acid, wherein R is unsubstituted C ₁₀-C ₂₄Alkyl or have C ₁₀-C ₂₄The hydroxyalkyl of alkyl composition, preferred C ₁₂-C ₂₀Alkyl or hydroxyalkyl, more preferably C ₁₂-C ₁₈Alkyl or hydroxyalkyl, A is oxyethyl group or propoxy-unit, and m is greater than 0, usually between about 0.5 to about 6, more preferably between about 0.5 to about 3, M is H or positively charged ion and can is the ammonium cation of metallic cation (for example sodium, potassium, lithium, calcium, magnesium etc.), ammonium or replacement for example.Alkyl ethoxylated sulfate and alkyl propoxylated sulphates are also included among the present invention.The object lesson of the ammonium cation that replaces comprise methyl-, dimethyl-, trimethylammonium-ammonium cation and quaternary ammonium cation such as tetramethyl-ammonium and lupetidine positively charged ion and derived from those materials of alkylamine such as ethamine, diethylamine, triethylamine and composition thereof or the like.Exemplary tensio-active agent is C ₁₂-C ₁₈Alkyl polyethoxylated (1.0) vitriol (C ₁₂-C ₁₈E (1.0) M), C ₁₂-C ₁₈Alkyl polyethoxylated (2.25) vitriol (C ₁₂-C ₁₈M) and C (2.25) ₁₂-C ₁₈Alkyl polyethoxylated (3.0) vitriol (C ₁₂-C ₁₈E (3.0) M) and C ₁₂-C ₁₈Alkyl polyethoxylated (4.0) vitriol (C ₁₂-C ₁₈E (4.0) M), wherein M is selected from sodium and potassium aptly.When this analog anion surfactants was included in wherein, laundry detergent composition of the present invention comprised about 1% usually to about 40%, preferred about 3% this analog anion surfactants to about 20% weight.

The polyethylene oxide of alkylphenol, poly(propylene oxide) and polybutylene oxide condenses are suitable as the nonionogenic tenside in the surfactant system of the present invention, preferred polyethylene oxide condensation compound.These compounds comprise the condensation product of alkylphenol and oxirane, and wherein the alkyl of alkylphenol has about 6 to 14 carbon atoms in the straight or branched configuration, preferred about 8 to 14 carbon atoms.In preferred embodiments, oxyethane with respect to every mole of alkylphenol with about 2 to about 25 moles, more preferably from about 3 exist to about 15 moles amount.Such ionic surfactant pack of buying is drawn together Igepal ^TMCO-630 is sold by GAF Corporation; And Triton ^TMX-45, X-114, X-100 and X-102 are by Rohm ﹠amp; Haas Company sells.Usually these tensio-active agents are called alkyl phenolic alkoxy thing (for example alkylphenol ethoxylate).

Uncle's type and secondary type fatty alcohol and about 1 condensation product to about 25 moles of ethylene oxide are suitable as the nonionogenic tenside in the surfactant system of the present invention.The alkyl chain of Fatty Alcohol(C12-C14 and C12-C18) can be straight or branched, uncle's type or secondary type, and comprises about 8 usually to about 22 carbon atoms.Preferably its alkyl group contain have an appointment 8 to about 20 carbon atoms, more preferably from about 10 to the alcohols of about 18 carbon atoms by every mole with about 2 condensation products to about 10 moles oxyethane.With respect to every mole alcohol, about 2 to about 7 moles oxyethane, most preferably 2 to 5 moles oxyethane is present in the described condensation product.Such examples of nonionic surfactants that can buy comprises Tergitol ^TM15-S-9 (C ₁₁-C ₁₅The condensation product of straight chain alcohol and 9 moles of ethylene oxide), Tergitol ^TM24-L-6NMW (C ₁₂-C ₁₄The condensation product of the narrow molecular weight distributions of primary alconol and 6 moles of ethylene oxide), the both is sold by Union Carbide Corporation; Neodol ^TM45-9 (C ₁₄-C ₁₅The condensation product of straight chain alcohol and 9 moles of ethylene oxide), Neodol ^TM23-3 (C ₁₂-C ₁₃The condensation product of the oxyethane of straight chain alcohol and 3.0 moles), Neodol ^TM45-7 (C ₁₄-C ₁₅The condensation product of straight chain alcohol and 7 moles of ethylene oxide), Neodol ^TM45-5 (C ₁₄-C ₁₅The condensation product of straight chain alcohol and 5 moles of ethylene oxide), it is sold by Shell Chemical Company; Kyro ^TMEOB (C ₁₃-C ₁₅The condensation product of pure and mild 9 moles of ethylene oxide), by The Procter ﹠amp; Gamble Company sells; With Genapol LA 050 (C ₁₂-C ₁₄The condensation product of pure and mild 5 moles of ethylene oxide), sell by Hoechst.The preferred HLB scope of above-mentioned product is 8-11, most preferably 8-10.

Also can be used as surfactant system of the present invention nonionogenic tenside be US4,565, disclosed alkyl polysaccharide in 647, its have comprise about 6 to about 30 carbon atoms, preferred about 10 to the hydrophobic grouping of about 16 carbon atoms with contain have an appointment 1.3 to about 10, preferred about 1.3 to about 3,1.3 polysaccharide most preferably from about, for example poly-glycosides hydrophilic radical to about 2.7 sugar units.Any reducing sugar that comprises 5 or 6 carbon atoms all can use, for example, glucose, semi-lactosi, and the galactosyl part can be replaced with glucosyl part (hydrophobic grouping randomly is connected on the positions such as 2-, 3-, 4-, produces glucose or semi-lactosi with respect to glucoside or galactoside thus).Between sugar key can be for example 1 position of additional sugar unit and before between 2-, 3-, 4-and/or the 6-position of sugar unit.

Preferred alkylpolyglycosides has following general formula:

R ²O (C _nH _2nO) _t(glycosyl) _xR wherein ²Be selected from alkyl, alkyl phenyl, hydroxyalkyl, hydroxyalkyl phenyl and composition thereof, wherein alkyl comprises about 10 to about 18, and preferred about 12 to about 14 carbon atoms; N is 2 or 3, preferred 2; T is 0 to about 10, preferred 0; And x is about 1.3 to about 10, and preferred about 1.3 to about 3, most preferably from about 1.3 to about 2.7.Glycosyl is preferably derived from glucose.In order to prepare these compounds, at first form alcohol or alkyl polyethoxye alcohol, form glucoside (being connected on 1) with glucose or source of glucose reaction then.Additional glycosyl units can its 1 and before state 2-, 3-, 4-and/or the 6-position of glycosyl units, form between the preferred main 2-position and connect.

Oxyethane and the condensation product of the hydrophobic basic cpd that is formed by the condenses of propylene oxide and propylene glycol be also suitable to be used as other nonionic surfactant system of the present invention.The hydrophobic part of these compounds preferably has about 1500 to about 1800 molecular weight and water insoluble.In this hydrophobic part, add polyoxyethylene and partly be tending towards improving the whole water-soluble of molecule, and the fluid characteristics of product is maintained about 50% the level that polyoxyethylene content accounts for the condensation product gross weight at most, promptly be equivalent to and be no more than about 40 moles ethylene oxide condensation.The example of this compounds comprises some Pluronic that can buy that is sold by BASF ^TMTensio-active agent.

Also suitable nonionogenic tenside as nonionic surfactant system of the present invention be the condensation product of the reaction product of oxyethane and propylene oxide and quadrol.The hydrophobic part of these products is made up of the reaction product of quadrol and excessive propylene oxide, and molecular weight is about 2500 to about 3000 usually.The degree of this hydrophobic part and ethylene oxide condensation makes condensation product contain to have an appointment 40% polyoxyethylene and molecular weight to about 80% weight to be about 5,000 to about 11,000.The example of this class nonionogenic tenside comprises some Tetronic that can buy that is sold by BASF ^TMCompound.

The nonionogenic tenside that is preferably used as surfactant system of the present invention is the polyethylene oxide condensation compound of alkylphenol, uncle's type and secondary type Fatty Alcohol(C12-C14 and C12-C18) and about 1 condenses to about 25 moles of ethylene oxide, alkyl polysaccharide and composition thereof.The C that most preferably has 3 to 15 oxyethyl groups ₈-C ₁₄Alkylphenol b-oxide and C with 2 to 10 oxyethyl groups ₈-C ₁₈(preferred average out to C ₁₀) pure b-oxide and composition thereof.

Nonionogenic tenside very preferably is the polyhydroxy fatty acid amide surfactants of following formula:

R wherein ¹Be H or R ¹Be C _1-4Alkyl, 2-hydroxyethyl, 2-hydroxypropyl or its mixture, R ²Be C _5-31Alkyl, Z are polyhydroxy alkyl (having at least 3 straight-chain alkyl chains that are directly connected to the hydroxyl on the chain) or its alkoxy derivative.Preferably, in the reductive amination reaction, R ¹Be methyl, R ²It is straight chain C _11-15Alkyl or C _16-18Alkyl or alkenyl such as cocounut oil alkyl or its mixture, Z is derived from reducing sugar such as glucose, fructose, maltose or lactose.

Laundry detergent composition of the present invention also can comprise positively charged ion, both sexes, zwitter-ion, semi-polar tensio-active agent and/or cosurfactant, and those nonionic and/or anion surfactants of not describing in preamble.When being included in wherein, detergent composition of the present invention comprises 0.2% usually to about 25%, preferred about 1% this type of cats product to about 8% weight, both sexes, zwitter-ion and/or semi-polar tensio-active agent.

The positively charged ion cleansing surfactants that is suitable for laundry detergent composition of the present invention has a long chain hydrocarbon groups.The example of this cats product comprises ammonium surfactant such as alkyl trimethyl ammonium halogenide and general formula [R ²(OR ³) _y] [R ⁴(OR ³) _y] ₂R ⁵The tensio-active agent of N+X-, wherein R ²Be in alkyl chain, to contain 8 alkyl or the alkyl benzyl groups of having an appointment, each R to about 18 carbon atoms ³Be selected from-CH ₂CH ₂-,-CH ₂CH (CH ₃)-,-CH ₂CH (CH ₂OH)-,-CH ₂CH ₂CH ₂-and composition thereof; Each R ⁴Be selected from C ₁-C ₄Alkyl, C ₁-C ₄Hydroxyalkyl, by connecting two R ⁴The benzyl rings structure that group forms ,-CH ₂CHOHCHOHCOR ⁶CHOHCH ₂OH (R wherein ⁶Be molecular weight less than about 1000 any hexose or hexose polymkeric substance) and hydrogen (when the y non-zero); R ⁵With R ⁴Identical or alkyl chain, wherein total carbon atom number or R ²Add R ⁵Be no more than about 18; Each y is from 0 to about 10, and y value summation is 0 to about 15; And X is any compatible negatively charged ion.

Cats product very preferably is the water-soluble quaternary ammonium compound that is used for said composition, and this compound has general formula R ₁R ₂R ₃R ₄N ⁺X ^-(i), R wherein ₁Be C ₈-C ₁₆Alkyl, R ₂, R ₃And R ₄Each is C independently ₁-C ₄Alkyl, C ₁-C ₄Hydroxyalkyl, benzyl and-(C ₂H ₄₀) _xH, wherein the value of x is 2 to 5, and X is a negatively charged ion.R ₂, R ₃And R ₄In maximum one be benzyl.

Preferred R ₁Alkyl chain length is C ₁₂-C ₁₅, especially when this alkyl group derived from coconut or palm nuclear fat and when having mixed chain length, maybe derive when obtaining when this alkyl group synthesizes by olefinic polymerization or oxo alcohol.

Preferred R ₂, R ₃And R ₄Group is methyl and hydroxyethyl, and negatively charged ion X can be selected from halogen ion, methylsulfuric acid radical ion, acetate ion and phosphate anion.

The example that is applicable to the quaternary ammonium compound of the general formula (i) here has:

Cocounut oil trimethyl ammonium muriate or bromide;

Cocounut oil methyl dihydroxy ethyl ammonium muriate or bromide;

Decyl triethyl ammonium muriate;

Decyl dimethyl hydroxyethyl ammonium muriate or bromide;

C _12-15Dimethyl hydroxyethyl ammonium muriate or bromide;

Coco dimethyl hydroxyethyl ammonium muriate or bromide;

Myristyl trimethyl ammonium Methylsulfate;

Lauryl dimethyl hexadecyldimethyl benzyl ammonium muriate or bromide;

Lauryl dimethyl (oxyethylene group) 4 ammonium muriate or bromides;

Cholinesterase (compound of general formula (i), wherein R ₁Be

Alkyl, and R ₂, R ₃, R ₄Be methyl).

Dialkylimidazolium quinoline [compound of general formula (i)].

Other cats product that can be used for this paper is also recorded in US 4,228,044 and EP 000224 in.

Amphoterics also is suitable in the laundry detergent composition of the present invention.These tensio-active agents can broadly be described as the aliphatic derivatives of the second month in a season or tertiary amine or heterocycle and stretch aliphatic derivatives with tertiary amine, and wherein aliphatic group can be a straight or branched.One of them aliphatic substituting group comprises at least about 8 carbon atoms, and about 8 to about 18 carbon atoms usually, and at least one comprises the anionic water-soluble group, for example carboxyl, sulfonate radical, sulfate radical.Examples of amphoteric surfactants is seen US 3,929,678 (19 hurdles, 18-35 is capable).

Zwitterionics also is suitable in the laundry detergent composition.These tensio-active agents broadly can be described as the derivative of derivative, heterocyclic secondary and tertiary amine of secondary amine and tertiary amine or the derivative of quaternary ammonium, quaternary phosphine or uncle's sulphur compound.Example about zwitterionics can be referring to US 3,929,678 (the 19th hurdles, the 38th walks to the 22nd hurdle the 48th row).

Semi-polar nonionic surfactants is the specific type of nonionogenic tenside, and it comprises that containing 1 has about 10 and be selected to the moieties of about 18 carbon atoms and 2 and contain the 1 water-soluble amine oxide compound to the part of the alkyl of about 3 carbon atoms and hydroxyalkyl of having an appointment; Contain 1 have about 10 to the moieties of about 18 carbon atoms and 2 be selected from contain have an appointment 1 to the alkyl of about 3 carbon atoms and hydroxyalkyl the water soluble oxidized phosphine; With contain 1 and have about 10 to the moieties of about 18 carbon atoms be selected from and contain 1 the water-soluble sulfoxide of having an appointment to the part of the alkyl of about 3 carbon atoms and hydroxyalkyl.

Semi-polarity nonionic detergent tensio-active agent comprises the amine oxide tensio-active agent of tool following formula:

R wherein ³Be to contain have an appointment 8 alkyl, hydroxyalkyl or alkyl phenyl or its mixture to about 22 carbon atoms; R ⁴Be to contain 2 alkylidene group or hydroxy alkylidene or its mixtures of having an appointment to about 3 carbon atoms; X is 0 to about 3: each R ⁵Respectively be to contain to have an appointment 1 to the alkyl or the hydroxyalkyl of about 3 carbon atoms or contain 1 the polyethylene oxide group of having an appointment to about 3 oxyethane.R ⁵Can interconnect, for example connect into ring structure by oxygen or nitrogen-atoms.

These amine oxide tensio-active agents especially comprise C ₁₀-C ₁₈Alkyl dimethyl amine oxide and C ₈-C ₁₂Alkoxyethyl dihydroxy ethyl amine oxide.

Suitable cosurfactant is selected from primary amine and tertiary amine.

The suitable primary amine that is used for here comprises general formula R ₁NH ₂Amine, R wherein ₁Be C ₆-C ₁₂, preferred C ₆-C ₁₀Alkyl chain or general formula R ₄X (CH ₂) _n, X is-O-,-C (O) NH-or-NH-, R ₄Be C ₆-C ₁₂Alkyl chain, n are 1 to 5, preferred 3.R ₁Alkyl chain can be a straight or branched and with can be with being no more than 12, preferably disconnects less than 5 ethylene oxide moiety.

Preferably the amine according to above-mentioned general formula is positive alkylamine.Here the amine of Shi Yonging can be selected from the 1-hexylamine, 1-octylame, 1-decyl amine and lauryl amine.Other preferred primary amine comprises C ₈-C ₁₀Oxygen base propyl group amine, octyloxy propyl group amine, 2-ethylhexyl oxygen base propyl group amine, lauryl amido propylamine and amido propylamine.

The suitable tertiary amine that is used for here comprises having general formula R ₁R ₂R ₃The tertiary amine of N, wherein R ₁With

R ₂Be C ₁-C ₈Alkyl chain or

R ₃Be C ₆-C ₁₂, preferred C ₆-C ₁₀Alkyl chain, or R ₃Be R ₄X (CH ₂) n, wherein X be-O-,-C (O) NH-or-NH-, R ₄Be C ₄-C ₁₂, n is 1 to 5, preferred 2-3.R ₅Be H or C ₁-C ₂Alkyl and x are 1 to 6.

R ₃And R ₄It can be straight or branched; R ₃Alkyl chain can be with being no more than 12, and the ethylene oxide moiety that preferably is less than 5 disconnects.Preferred tertiary amine is R ₁R ₂R ₃N, wherein R ₁Be C ₆-C ₁₂Alkyl chain, R ₂And R ₃Be C ₁-C ₃Alkyl or

R wherein ₅Be H or CH ₃, and x=1-2.

The amide amine of also preferred following formula:

R wherein ₁Be C ₆-C ₁₂Alkyl; N is 2-4, and preferred n is 3; R ₂And R ₃Be C ₁-C ₄

Most preferred amine comprises 1-octylame, 1-hexylamine, 1-decyl amine, 1-lauryl amine, C _8-10Oxygen base propylamine, N-cocounut oil 1,3-diaminopropanes, cocounut oil alkyl dimethylamine, lauryl dimethylamine, lauryl two (hydroxyethyl) amine, cocounut oil two (hydroxyethyl) amine, 2 moles of propenoxylated lauryl amines, 2 moles of propenoxylated octylames, lauryl amido propyl group dimethylamine, C _8-10Amido propyl group dimethylamine and C ₁₀Amido propyl group dimethylamine.

The most preferred amine that is used in the present composition is 1-hexylamine, 1-octylame, 1-decyl amine, 1-lauryl amine.Especially expectation is 7 times of ethoxylates, lauryl amido propylamine and the cocounut oil amido propylamine of dodecyl dimethyl amine and dihydroxy ethyl cocounut oil alkylamine and oleyl amine.

Builder system

Can further comprise builder system according to composition of the present invention.The builder system of any routine all is suitable for here, comprise alumino-silicate materials, silicate, polycarboxylate and lipid acid, as materials such as edetate, metal ion chelation agent such as aminopolyphosphonic acid ester, especially diethylamine tetramethylene phosphonic acid and diethylenetriamine pentamethylenophosphonic acid(DTPP).Although because environment reason preferably phosphate washing assistant more not, it still can be with herein.

Suitable washing assistant can be an inorganic ion exchange material, normally inorganic hydrated aluminosilicate material, and the synthetic zeolite of hydration more particularly is as hydrated zeolite A, X, B, HS or MAP.

Another inorganic builders material that is fit to is a layered silicate, for example SKS-6 (Hoechst).SKS-6 is by water glass (Na ₂Si ₂O ₅) the crystal layered silicate formed.

The suitable polycarboxylate that contains a carboxylic group comprises its lactic acid, glycolic acid and ether derivant, is disclosed in belgian patent Nos.831, in 368,821,369 and 821,370.The polycarboxylate that comprises two carboxylic groups comprises the water-soluble salt of Succinic Acid, propanedioic acid, (ethylenedioxy) diacetic acid, toxilic acid, diglycollic acid, tartrate, tartronic acid and fumaric acid; and at German Offenle-enschrift 2; 446; 686 and 2; 446,487, US 3,935; disclosed ether carboxylate and at belgian patent No.840 in 257, disclosed sulfinyl carboxylicesters in 623.The polycarboxylate that contains three hydroxy-acid groups comprises especially water-soluble citrate, aconitate and citraconate and succinic acid ester derivative such as English Patent No.1,379, disclosed carboxymethyl oxygen base succinate in 241, disclosed 2-hydroxyl propoxy-succinate (lactoxysuccinates) and in Holland application 7205873 at English Patent No.1,387, disclosed oxidation polycarboxylate material such as 2-oxa--1,1 in 447,3-tricarballylic acid ester.

The polycarboxylate that comprises four carboxylic groups is included in English Patent No.1, disclosed oxygen di-succinate in 261,829, contains sulfo group substituent 1,1,2,2,-ethane tetracarboxylic ester, 1,1,3,3-propane tetracarboxylic acid ester, be included in English Patent Nos.1,398,421 and 1,398,422 and US 3,936,448 in disclosed sulfo-succinic acid ester derivative, with at English Patent No.1, disclosed sulfonation pyrolysis citrate in 082,179, and English Patent No.1,439,000 disclose and have comprised the substituent polycarboxylate of phosphino-.

Alicyclic and heterocyclic polycarboxylate comprises pentamethylene-suitable, suitable, suitable-tetracarboxylic ester, cyclopentadiene pentacarboxylic acid ester, 2,3,4, the 5-tetrahydrofuran (THF) is suitable, suitable, suitable-tetracarboxylic ester, 2,5-tetrahydrofuran (THF)-suitable-dicarboxylic ester, 2,2,5,5,-tetrahydrofuran (THF) tetracarboxylic ester, 1,2,3,4,5, the carboxymethyl derivant of 6-hexane-hexacarboxylic acid ester and polyvalent alcohol such as sorbyl alcohol, mannitol and Xylitol.Aromatic multi-carboxy acid's ester is included in English Patent No.1, disclosed mellitic acid, 1,2,4 in 425,343,5-pyromellitic acid and phthalic acid derivatives.

Preferred polycarboxylate is that each molecule comprises the nearly hydroxyl-carboxylicesters of 3 carboxylic groups, more preferably citrate in above-mentioned.

The builder system that preferably is used for the present composition comprises the mixture of water-insoluble silico-aluminate washing assistant such as zeolite A or layered silicate (SKS-6) and water-soluble carboxyl acid esters sequestrant such as citric acid.

The sequestrant that is fit to be included in the detergent composition of the present invention is quadrol-N, N '-disuccinic acid (EDDS) or its basic metal, alkaline-earth metal, ammonium or substituted ammonium salt, or their mixture.Preferred EDDS compound is free acid form and its sodium or magnesium salts.The example of these preferred EDDS sodium salts comprises Na ₂EDDS and Na ₄EDDS.The example of preferred EDDS magnesium salts comprises MgEDDS and Mg ₂EDDS.Magnesium salts most preferably is included in the present composition.

Preferred builder system comprises the mixture of water-insoluble silico-aluminate washing assistant such as zeolite A and water-soluble carboxyl acid esters sequestrant such as citric acid.

Other washing assistant material that can be formed for the part of the builder system in the granular composition comprises inorganic materials such as alkaline carbonate, supercarbonate, silicate and organic materials such as organic phospho acid ester, amino polyalkylene phosphonic acid ester and aminopolycanboxylic acid's ester.

Other suitable water-soluble organic salt is homopolymerization acid or co-polymeric acids or their salt, and wherein poly carboxylic acid comprises at least two by no more than two carboxylic groups that carbon atom is separated mutually.

GB-A-1,596,756 disclose this base polymer.The example of this type of salt is the multipolymer of the polyacrylic ester of MW 2000-5000 and they and maleic anhydride, and the molecular weight of this multipolymer is 20,000 to 70,000, especially about 40,000.

Generally include the detergent builder compound salt of 5% to 80% weight in the composition.Preferably include 5% to 30% washing assistant in the liquid washing agent.

Enzyme

Preferred detergent composition except comprising enzyme preparation of the present invention, also comprises the enzyme that other can provide clean-up performance and/or fabric maintenance benefit.

These enzymes comprise proteolytic enzyme, lipase, at, amylase, cellulase, peroxidase, oxydase (for example laccase)

Proteolytic enzyme: any proteolytic enzyme that is suitable for basic solution can use.Suitable proteolytic enzyme comprises the proteolytic enzyme from animal, plant or microorganism.The proteolytic enzyme that derives from microorganism is preferred.Also comprise mutant through chemistry or genetic modification.Described proteolytic enzyme can be serine protease, preferred alkaline microbial protease or trypsin-like proteolytic enzyme.The example of Sumizyme MP is a subtilisin, particularly from the subtilisin of genus bacillus, for example, subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (describing among the WO 89/06279).The example of trypsin-like proteolytic enzyme is that trypsin is for example from pig or ox) and as the disclosed Fusarium proteolytic enzyme of WO89/06270.

Preferably the proteolytic enzyme that can buy comprises that commodity are called Alcalase, Savinase, Primase, Durazym, Esperase (Novozymes A/S, Denmark sells); Maxatase, Maxacal, Maxapem, Properase, Purafect, Purafect OxP, (Genencor InternationalInc. sale); Opticlean and Optimase (those that Solvay Enzymes sells).In the weight of zymoprotein in composition, the proteolytic enzyme that is blended in the detergent composition of the present invention can account for 0.00001% to 2%, preferably account for 0.0001% to 1% of composition weight, more preferably account for 0.001% to 0.5% of composition weight, even more preferably account for 0.01% to 0.2% of composition weight.

Lipase:Any lipase that is applicable to basic solution all can use.Suitable lipase comprises that those derive from the lipase of bacterium or fungi.Also comprise mutant through chemistry or genetic modification.

The example of useful lipase comprises the lipase from Humicola lanuginosa, described in EP 258 068 and EP 305 216, lipase from Rhizomucor miehei, as described in EP238 023, lipase from candiyeast Pseudomonas (Candida), antarctic candida (C.antarctica) lipase for example, antarctic candidia lipase A or B described in EP214 761, lipase from Rhodopseudomonas (Pseudomonas), for example from Pseudomonas alcaligenes (P.alcaligenes) or pseudomonas pseudoalcaligenes (P.pseudoalcaligenes) (seeing for example EP 218 272), pseudomonas cepacia (P.cepacia) (seeing for example EP 331376), (GB 1 for Pseudomonas stutzeri (P.stutzeri), 372,034), the lipase of Pseudomonas fluorescens (P.fluorescens), lipase from bacillus, for example from subtilis (people (1993) such as Dartois, biological chemistry and biophysics journal (Biochemica et Biophysica acta), 1131,253-260), the lipase of bacstearothermophilus (JP 64/744992) and bacillus pumilus (B.Pumilus) (WO 91/16422).

In addition, some clones' lipase also can use, be included in Yamaguchi etc., (1991), gene (Gene) 103, disclosed penicillium cammenberti (Penicilliumcamembertii) lipase among the 61-67, geotrichum candidum (Geotricum candidum) lipase (Schimada, Y. etc., (1989), journal of biological chemistry (J.Biochem.)., 106,383-388), with various Rhizopuses (Rhizopus) lipase such as R.delemar lipase (Hass, M.J etc., (1991), gene 109,117-113), snow-white head mold (R.niveus) lipase (Kugimiya etal., (1992), biological chemistry biotechnology bio-science (Biosci.Biotech.Biochem.) 56,716-719) and Rhizopus oryzae (R.oryzae) lipase.

The lipolytic enzyme of other type such as at also can be used, for example, disclosed in WO 88/09367 derived from pseudomonas mendocina (Pseudomonas mendocina), or derived from the at (for example disclosed in WO 90/09446) of fusarium solani (Fusarium solani pisi).

Particularly suitable lipase is lipase such as M1 Lipase ^TM, Luma fast ^TMAnd Lipomax ^TM(Genencor), Lipolase ^TMWith Lipolase Ultra ^TM(NovozymesA/S) and lipase P " Amano " (Amano Pharmaceutical Co.Ltd.).

In the weight of zymoprotein in composition, the lipase that is blended in the detergent composition of the present invention accounts for 0.00001% to 2% usually, preferably account for 0.0001% to 1% of composition weight, more preferably account for 0.001% to 0.5% of composition weight, even more preferably account for 0.01% to 0.2% of composition weight.

Amylase: any amylase (a and/or b) that is applicable to basic solution all can use.Suitable amylase comprises that those derive from the amylase of bacterium or fungi.Also comprise mutant through chemistry or genetic modification.Amylase for example comprises, for example from the α-Dian Fenmei of the specific bacterial strain of Bacillus licheniformis, detailed content is referring to GB 1,296,839.The amylase that can buy is Duramyl ^TM, Termamyl ^TM, Fungamyl ^TMAnd BAN ^TM(can from Novozymes A/S), Rapidase ^TMAnd Maxamyl ^TM(can from Genencor).

In the weight of zymoprotein in composition, the amylase that is blended in the detergent composition of the present invention accounts for 0.00001% to 2% usually, preferably account for 0.0001% to 1% of composition weight, more preferably account for 0.001% to 0.5% of composition weight, even more preferably account for 0.01% to 0.2% of composition weight.

Cellulase: any cellulase that is suitable for basic solution all can use.Suitable cellulase comprises the cellulase that derives from bacterium or fungi.Also comprise through chemistry or genetic modification or mutant.The plain enzyme of suitable fibers is disclosed in the following document: at US 4,435,307 and WO91/17243 in the fungal cellulase that produces from Humicola insolens is disclosed, in WO96/34108 and WO 96/34092, disclose from the bacterium of bacillus and had a liking for soda cellulose enzyme (BCE 103), at WO 94/21801, US 5,475,101 and US 5, EG III cellulase from trichoderma (Trichoderma) is disclosed in 419,778.Particularly suitable cellulase is the cellulase that helps the color maintenance.This cellulase example is the cellulase that is disclosed among the european patent application No.0 495 257.The cellulase that can buy comprises the Celluzyme that is produced by Humicola insolens bacterial strain ^TMAnd Carezyme ^TM(Novozymes A/S), KAC-500 (B) ^TM(Kao Corporation) and Puradax ^TM(Genencor International).

In the weight of zymoprotein in composition, the cellulase that is blended in the detergent composition of the present invention accounts for 0.00001% to 2% usually, preferably account for 0.0001% to 1% of composition weight, more preferably account for 0.001% to 0.5% of composition weight, even more preferably account for 0.01% to 0.2% of composition weight.

Peroxidase/oxydase: peroxidase is used in combination with hydrogen peroxide or hydrogen peroxide cource (for example percarbonate, perborate or persulphate).Oxydase and combination with oxygen are used.Two types enzyme all is used for " solution bleaching ", prevents that promptly the textile dye on the DYED FABRICS is transferred on another fabric when two kinds of fabrics wash in washings (preferably containing just like disclosed toughener in WO94/12621 and WO 95/01426) together.Suitable peroxidase/oxydase comprises the peroxidase/oxydase that derives from plant, bacterium or fungi.Also comprise mutant through chemistry or genetic modification.

In the weight of zymoprotein in composition, the peroxidase and/or the oxydase that are blended in the detergent composition of the present invention account for 0.00001% to 2% usually, preferably account for 0.0001% to 1% of composition weight, more preferably account for 0.001% to 0.5% of composition weight, even more preferably account for 0.01% to 0.2% of composition weight.

The mixture of above-mentioned enzyme all is included in herein, particularly the mixture of proteolytic enzyme, amylase, lipase and/or cellulase.

In the weight of zymoprotein in composition, the enzyme of the present invention or other any enzyme that are blended in the detergent composition account for 0.00001% to 2% usually, preferably account for 0.0001% to 1% of composition weight, more preferably account for 0.001% to 0.5% of composition weight, even more preferably account for 0.01% to 0.2% of composition weight.

SYNTHETIC OPTICAL WHITNER

The selectable in addition detergent component that is included in the detergent composition of the present invention comprises SYNTHETIC OPTICAL WHITNER.These bleach additive compositions can comprise one or more oxygen bleaching agents with according to the SYNTHETIC OPTICAL WHITNER of selecting and one or more different bleach-activating agents.If exist, the oxygen bleaching compound exists with about 1% to about 25% level usually.Be preferred for SYNTHETIC OPTICAL WHITNER in the present composition and be the Cyclam that the transition metal complex of most ring rigid ligand of the following stated, the particularly Cyclam of dichloro-dimethyl ethylidene bridge joint close manganese and/or Dichlorodiethyl ethylidene bridge joint and close manganese; And hydrogen peroxide releasing agent and the amino caproyl oxygen of bleach-activating agent 4-[N-(nonanoyl) base]-combination of benzene sulfonic acid sodium salt (NACA-OBS).

Have now found that the detergent composition of the present invention that also comprises SYNTHETIC OPTICAL WHITNER comprise laundry, wash the dishes and the cleaning of crust in outstanding cleaning and detergency ability can be provided.Without wishing to be bound by theory, but think the further oh group in oxidation pectin/pectic acid composition of SYNTHETIC OPTICAL WHITNER, make them more solvable and be easier to remove.

Being surprised to find bleach system can make the cleaning efficiency of pectate lyase maximize.In addition, also be surprised to find the detergent composition that comprises pectate lyase of the present invention and bleach system and provide good cleaning action owing to the synergy of bleach system and pectate lyase, wherein bleach system provides the whiteness hold facility of outstanding cleansing power, soil removability and clothing, and the pectin composition of pectate lyase degraded dirt and/or can be in conjunction with the pectin composition on the fabric of dirt in clothing.These bleach systems-enzyme mixed system provides outstanding cleaning performance, particularly to the coloured spot and the health dirt of food.In addition, when being mixed with clothing and/or fabric care compositions, composition of the present invention provides collaborative whiteness hold facility.

Without wishing to be bound by theory, but think that the natural pectin in the primary wall that is present in many common fruits and vegetables base dirts and cotton fabric can attract and keep here the dirt residue, particularly high painted dirt composition.Remove pectin composition by pectate lyase and these chromoplastids can be exposed to bleach system.This synergistic reason is considered to: these bleach systems are by bleaching strong colored component to remove food stains and health dirt, and the pectin composition of pectate lyase degraded dirt and/or be present in pectin composition on the fabric in clothing, thereby the pectin composition on the fabric can combine with these dirts or interact and makes described dirt be difficult to removal.

The bleaching components that is used for here can be any SYNTHETIC OPTICAL WHITNER that can be used for detergent composition, comprises oxygen bleaching agent and other SYNTHETIC OPTICAL WHITNER known in the art: be a hydration or the four hydration perborate and the percarbonate of 400-800 micron as particle diameter.

Be applicable to that SYNTHETIC OPTICAL WHITNER of the present invention can be activatory or non-activated SYNTHETIC OPTICAL WHITNER.

A kind of available oxygen bleaching agent comprises percarboxylic acids SYNTHETIC OPTICAL WHITNER and its salt.The suitable example of this type of SYNTHETIC OPTICAL WHITNER comprises monoperphthalic acid magnesium hexahydrate, metachloroperbenzoic acid magnesium, 4-amino in the ninth of the ten Heavenly Stems-4-oxo Perbutyric Acid and diperoxy dodecanedioic acid.US 4,483, and 781, disclose these SYNTHETIC OPTICAL WHITNER among US 740,446, EP 0 133 354 and the US 4,412,934.SYNTHETIC OPTICAL WHITNER very preferably is also included within US4, and disclosed 6-amino in the ninth of the ten Heavenly Stems-6-oxo is crossed oxy hexanoic acid in 634,551.

Spendable other kind SYNTHETIC OPTICAL WHITNER comprises halogen bleaching agent.The example of hypohalite SYNTHETIC OPTICAL WHITNER comprises for example TCCA (Trichloroisocyanuric acid), dichloroisocyanuric acid sodium and potassium and N-chlorine and N-bromoalkane sulphonamide.These materials account for the 0.5-10% weight of finished product usually, preferred 1-5% weight.

The hydrogen peroxide releasing agent can be used in combination with bleach-activating agent; bleach-activating agent such as tetra acetyl ethylene diamine (TAED); (NOBS is at US 4,412 for nonanoyl oxygen base benzene sulfonate; open in 934); 3,5-trimethylammonium-hexsanol oxygen base benzene sulfonate (ISONOBS, open in EP 120 591) or penta-acetyl glucose (PAG); they form the peracid as active bleaching thing after crossing hydrolysis, thereby bleaching effect is improved.In addition, fit closely bleach-activating agent is C8 (6-decoyl amino-caproyl) oxygen base benzene sulfonate, C9 (6-nonanoyl amino caproyl) oxygen base benzene sulfonate and C10 (the amino caproyl of 6-caprinoyl) oxygen base benzene sulfonate or and composition thereof.Other hydrophobicity bleach-activating agent comprises, but is not limited to the amino hexylyloxy of 4-[N-(nonanoyl)]-benzene sulfonic acid sodium salt (NACA-OBS) (an one example is in U.S. Patent No. 5,523,434 in open), 12 carbonic acyl radical oxygen base benzene sulfonate (LOBS or C ₁₂-OBS), 10-undecylene acyloxy benzene sulfonate (UDOBS or the undersaturated C in 10 places in the position ₁₁-OBS) and caprinoyl aminobenzoic acid (DOBA).Same suitable activator is an acylated citrate esters, as disclosed acylated citrate esters in EP 624 154.

SYNTHETIC OPTICAL WHITNER (comprising peroxy acid) and bleach system (comprising bleach-activating agent and peroxy bleaching compound) useful in detergent composition of the present invention are open in WO95/10592.

Hydrogen peroxide also can exist by adding the enzyme system (being enzyme and substrate thereof) that can produce hydrogen peroxide in beginning or in washing and/or rinse cycle.This kind of enzyme system is disclosed in the European patent application EP 0 537 381.

Other SYNTHETIC OPTICAL WHITNER of non-oxygen bleaching agent also is known in the art and can utilizes at this.A kind of significant especially non-oxygen bleaching agent comprises photoactivation SYNTHETIC OPTICAL WHITNER such as sulfonation phthalocyanine phthalocyanine zinc and/or aluminium.These materials can deposit on the substrate in washing process.Under the radiation of light, in oxygen, by the suspended garment drying, this sulfonation phthalocyanine phthalocyanine zinc will be activated, and cause substrate to be bleached as by day.

Preferred Phthalocyanine Zinc and photoactivation bleaching method are disclosed in US 4,033,718.Usually, detergent composition comprises about 0.025% sulfonation phthalocyanine phthalocyanine zinc to about 1.25% weight.SYNTHETIC OPTICAL WHITNER also can comprise manganese and cobalt catalyst, and for example manganese-based catalyst is disclosed in United States Patent(USP) Nos. 5,576,282,5,246,621,5,244,594,5,194,416,5,114,606 and the open text Nos.549 of european patent application, 271 A1,549,272 A1,544,440 A2 and 544,490 A1; For example the cobalt bleaching catalyst is disclosed in for example United States Patent(USP) Nos. 5,597,936,5,595,967,5,703,030 and M.L.Tobe, " basic hydrolysis of transition metal complex " (" Base Hydrolysis of Transition-Metal Complexes "), Adv.Inorg.Bioinorg.Mech., (1983), 2, the 1-94 page or leaf.

The composition here can comprise suitably that also the transition metal complex of most ring rigid ligand is as bleaching catalyst.Usage quantity is a catalytically effective amount, suitably about 1ppb or more, and for example nearly to about 99.9%, more generally about 0.001ppm or more, preferably about 0.05ppm is about 500ppm extremely.

Be applicable to that the transition metal bleach catalyzer with big ring rigid ligand in the present composition can illustrate by following non-limitative example:

Two chloro-5,12-dimethyl-1,5,8,12-four azabicyclos [6.6.2] n-Hexadecane closes manganese (II)

Two chloro-4,11-dimethyl-1,4,8,11-four azabicyclos [6.6.2] n-Hexadecane closes manganese (II)

Two chloro-5,12-diethyl-1,5,8,12-four azabicyclos [6.6.2] n-Hexadecane closes manganese (II)

Two chloro-4,11-diethyl-1,4,8,11-four azabicyclos [6.6.2] n-Hexadecane closes manganese (II)

Two water-5,12-dimethyl-1,5,8,12-four azabicyclos [6.6.2] n-Hexadecane closes manganese (II)-hexafluorophosphate

One water-hydroxyl-5,12-dimethyl-1,5,8,12-four azabicyclos [6.6.2] n-Hexadecane closes manganese (III)-hexafluorophosphate

Two water-5,12-dimethyl-1,5,8,12-four azabicyclos [6.6.2] n-Hexadecane closes manganese (III)-a tetrafluoro borate

Two chloro-5,12-dimethyl-1,5,8,12-four azabicyclos [6.6.2] n-Hexadecane closes manganese (II)-hexafluorophosphate

Two chloro-5,12-di-n-butyl-1,5,8,12-four azabicyclos [6.6.2] n-Hexadecane closes manganese (II)

Two chloro-5,12-dibenzyl-1,5,8,12-four azabicyclos [6.6.2] n-Hexadecane closes manganese (II)

Two chloro-5-normal-butyl-12-methyl isophthalic acids, 5,8,12-four azabicyclos [6.6.2] n-Hexadecane closes manganese (II)

Two chloro-5-n-octylcyclam 2-methyl isophthalic acids, 5,8,12-four azabicyclos [6.6.2] n-Hexadecane closes manganese (II)

Two chloro-5-normal-butyl-12-methyl isophthalic acids, 5,8,12-four azabicyclos [6.6.2] n-Hexadecane closes manganese (II).

Froth suppressor

Another selectable components is a froth suppressor, for example siloxanes and silicon-dioxide-mixture of siloxanes.Siloxanes can be represented by the alkylation silicone materials usually, and silicon-dioxide uses with form in small, broken bits usually, for example various types of aerosils and xerogel and water drain silica.These materials can mix with the form of particulate, therein froth suppressor can advantageously discharge be mixed into water miscible or water is dispersible, in fact in the carrier of impermeable on-surface-active detergent.

Alternatively, froth suppressor can dissolve or be dispersed in the liquid vehicle and by spraying on one or more other compositions and use.

Preferred silicone foam control agent is disclosed in US 3,933,672.Other useful especially froth suppressor has self-emulsifying siloxane foams inhibitor, and is open in German patent application DTOS 2,646,126.An example of this compound is DC-544, is siloxane-glycol copolymer, can buy from DowCorning.

Particularly preferred Foam Control is the froth suppressor system that comprises the mixture of silicone oil and 2-alkyl-alkanol.2-alkyl-the alkanol that is fit to is 2-butyl-octanol, and it can be bought and obtain, and commercial name is Isofol 12R.

This froth suppressor system is open in European patent application EP 0 593 841.

Particularly preferred silicone foam control agent is open in european patent application No.92201649.8.Described composition can comprise siloxanes/silica mixture and pyrogene atresia silicon-dioxide such as Aerosil ^R

Above-mentioned froth suppressor is generally 0.001% to 2% weight of composition, preferred 0.01% to 1% weight.

Other composition

Other composition that can be used in the detergent composition is as dirt suspension agent, dirt releasing agent, white dyes, abrasive material, bactericide, tarnish inhibitor, tinting material and/or capsulation or the spices of capsulation not.

Particularly suitable capsulation material is a water-soluble capsule, and this capsule is made up of polysaccharide and polyol, as GB1, and disclosing in 464,616.

Other water-soluble capsulation material that is fit to comprises dextrin, as at US 3,455, and disclosing in 838, it is derived from the not gelling starch ester of the dicarboxylic acid that replaces.These acid-ester dextrin preferred preparation is from the starch of starch such as waxy corn, wax kind jowar, sago, cassava and potato.The suitable example of described capsulation material comprises the N-Lok that National Starch produces.N-Lok capsulation material is made up of modified corn starch and glucose.Starch is by adding simple function substituted radical such as the modification of octenyl Succinic anhydried.

Here anti-redeposition agent of Shi Heing and dirt suspension agent comprise derivatived cellulose such as methylcellulose gum, carboxymethyl cellulose and hydroxy ethyl cellulose and all-or altogether-poly-poly carboxylic acid or their salt.It is polyacrylic ester and the copolymer of maleic anhydride and acrylic acid that washing assistant is described that this base polymer is included in previous crops, and the multipolymer of EMA acid anhydride and ethene, methylvinylether or methacrylic acid, and maleic anhydride accounts at least 20 moles of % of multipolymer.These materials are usually with 0.5% to 10% weight of composition, and more preferably 0.75% to 8%, most preferably 1% to 6% weight is used.

Preferred white dyes is anionic in nature, its example is 4,4 '-two-(2-diethanolamino-4-phenylamino-s-triazine-6-base is amino) toluylene-2,2 ' disulfonic acid disodium, 4,4 '-two-(2-morpholino-4-phenylamino-s-triazine-6-base is amino)-toluylene-2,2 '-the disulfonic acid disodium, 4,4 '-two-(2,4-diphenylamino-s-triazine-6-base is amino) toluylene-2,2 '-the disulfonic acid disodium, 4 ', 4 " two-(2; 4-diphenylamino-s-triazine-6-base is amino) toluylene-2-sodium sulfonate; 4; 4 '-two-(2-phenylamino-4-(N-methyl-N-2-hydroxyethyl amino)-s-triazine-6-base is amino) toluylene-2; 2 '-the disulfonic acid disodium; 4,4 '-two-(4-phenyl-2,1,3-triazole-2-yl)-toluylene-2,2 '-the disulfonic acid disodium, 4,4 '-two-(2-phenylamino-4-(1-methyl-2-hydroxyethyl amino)-s-triazine-6-base is amino) toluylene-2,2 '-the disulfonic acid disodium, 2-(diphenylethyllene-4 " (naphtho--1 ', 2 ': 4; 5)-1; 2,3-triazole-2 " sodium sulfonate and 4,4 '-two-(2-sulfo group styryl) biphenyl.

Other available polymeric material has polyoxyethylene glycol, particularly molecular weight 1000-10000, more preferably 2000 to 8000 and most preferably from about 4000 polyoxyethylene glycol.These materials are with 0.20% to 5%, and more preferably 0.25% to 2.5% weight is used.These polymkeric substance and prerequisite to homopolymerization-or copolymerization-multi-carboxylate for viscosity when transition metal impurity exists, protein and oxidable dirt on whiteness safeguard, the raising of fabric ash deposition and clean-up performance is valuable.

Available dirt releasing agent normally has the different ethylene glycol of arranging and/or the multipolymer or the terpolymer of propylene glycol unit and terephthalic acid in the present composition.The example of this polymkeric substance is disclosed in US 4,116, and 885 and 4,711,730 and EP 0 272 033.Particularly preferred polymkeric substance according to EP 0 272 033 has following general formula: (CH ₃(PEG) ₄₃) _0.75(POH) _0.25[T-PO) _2.8(T-PEG) _0.4] T (POH) _0.25((PEG) ₄₃CH ₃) _0.75Wherein PEG is-(OC ₂H ₄) O-, PO is (OC ₃H ₆O) and T be (pOOC ₆H ₄CO).

In addition very usefully as dimethyl terephthalate (DMT), sulfoisophthalic acid dimethyl ester, ethylene glycol and 1, the modified poly ester of the randomcopolymer of 2-propylene glycol, mainly by the sulfosalicylic acid ester and secondly by ethylene glycol and/or 1, the monoesters of 2-propylene glycol constitutes end group.Target is to obtain a kind of two ends all by the end capped polymkeric substance of sulfosalicylic acid ester group, and in the present context, " mainly " is meant that most of described multipolymer is by sulfosalicylic acid ester group end-blocking.Yet some multipolymer is not by complete end-blocking, so their end group can be by ethylene glycol and/or 1, and the monoesters of 2-propylene glycol constitutes, promptly " secondly " constitute by such material.

Here the polyester of Xuan Zeing comprises 1 of the dimethyl terephthalic acid of about 46% weight, about 16% weight, the 2-propylene glycol, the ethylene glycol of about 10% weight, the dimethyl methyl yl benzoic acid of about 13% weight and the sulfoisophthalic acid of about 15% weight, and its molecular weight is about 3,000.Polyester and their preparation method are referring to disclosing among the EP 311 342.

Tenderizer

Fabric softener also can be mixed in the laundry detergent composition of the present invention.These tenderizers can be inorganic or organic types.Inorganic tenderizer can give an example just like at GB-A-1 400898 and US5, disclosed smectic clays (smectite clay) in 019,292.The organic fabric tenderizer be included among GB-A1514 276 and the EP 0 011 340 disclosed water-insoluble tertiary amine and in EP-B-0 026 528 disclosed they with single C ₁₂-C ₁₄The associating of quaternary ammonium salt and in EP 0 242 919 disclosed pair of long-chain acid amides.The organic composition of other available fabric-softening system is included in disclosed high molecular weight polyethylene oxide material in EP 0 299 575 and 0 313146.

The level of smectic clays normally 5% to 15%, more preferably 8% to 12% weight, this material joins in the remaining ingredient of preparation with the dry mixed composition.The level of mixing of organic fabric tenderizer such as water-insoluble tertiary amine or two long-chain acid amides materials is 0.5% to 5% weight, be generally 1% to 3% weight, and the adding level of high molecular weight polyethylene oxide material and water-soluble cationic material is 0.1% to 2%, is generally 0.15% to 1.5% weight.These materials join in the spraying drying part of composition usually, although may be more suitable in some cases they are added to wherein with the dry mixed particle form, or they are sprayed onto on other solid ingredient of composition with fusing solution form.

The polymeric dye transfer inhibitor

Detergent composition of the present invention also can comprise 0.001% to 10%, and is preferred 0.01% to 2%, more preferably the polymeric dye transfer inhibitor of 0.05% to 1% weight.Described polymeric dye transfer inhibitor is mixed into usually in the detergent composition and transfers on other fabric of washing simultaneously from colored fabric in order to suppress dyestuff.These polymkeric substance can be before the fugitive dye that washes out from DYED FABRICS has an opportunity to contact other fabric the washing in conjunction with or absorb this dyestuff.

Particularly suitable polymeric dye transfer inhibitor is multipolymer, the polyvinyl pyrrolidone polymers of polyamines N-oxidation thing polymkeric substance,-vinyl-pyrrolidone and N-vinyl imidazole, Ju Yi Xi oxazolidinone and polyvinyl imidazol or their mixture.

Add these polymkeric substance and also can improve the performance of enzyme of the present invention.

Detergent composition of the present invention can be liquid, pulpous state, gel, bar-shaped or particle form.

Dustless particle for example can adopt at US 4,106,991 and 4,661,452 (both is NovoIndustri A/S) disclosed method production, but and optional nature ground add dressing by means known in the art.The example of wax coating material be molecular-weight average be 1000 to 20000 poly-(oxyethane) products (polyoxyethylene glycol, PEG); Ethoxylized nonylphenol with 16 to 50 ethylene oxide units; Ethoxylized fatty alcohol with 15 to 80 ethylene oxide units, wherein alcohol comprises 12 to 20 carbon atoms; Fatty Alcohol(C12-C14 and C12-C18); Lipid acid; With the list of lipid acid-and two-and Witepsol W-S 55.The example that is applicable to the film formation coating material of fluidization provides in GB 1483591.

Particulate composition of the present invention also can be taked " fine and close form ", that is, they can have the density higher than common granulated detergent, promptly 550 arrives 950g/l; In the case, granular detergent composition of the present invention can comprise more common granulated detergent " mineral filler salt " in a small amount; Typical filling salt is the vitriol and the muriate of alkaline-earth metal, typically is sodium sulfate; " densification " washing composition comprises 10% filling salt at the most usually.Liquid composition of the present invention is " fine and close form " also, and in the case, liquid detergent composition of the present invention can comprise more common liquid washing agent water in a small amount.Usually, the water-content of spissated liquid washing agent account for detergent composition weight less than 30%, more preferably less than 20%, most preferably less than 10%.

Composition of the present invention can for example be mixed with hand washing and machine washing laundry detergent composition (comprising laundry additive composition and the pretreated composition that is suitable for polluting fabric), fabric sofetening composition that adds during rinsing and the composition that is used for average family cleaning crust and washes the dishes and operate.

Following embodiment is intended to illustrate the present composition, but not is intended to the scope of the invention is limited or defines.In detergent composition, the meaning of the component identifier of writing a Chinese character in simplified form is as follows:

LAS: straight chain C ₁₂Sodium alkyl benzene sulfonate

TAS: tallow alkyl sodium sulfate

XYAS:C _1x-C _1ySodium alkyl sulfate

ABEY: with the C that is mainly of the ethylene oxide condensation of average Y mole _1A-C _1BStraight chain primary alcohol

XYEZS: every mole of C with the ethylene oxide condensation of average Z mole _1x-C _1ySodium alkyl sulfate

Non-ionic surfactant: average degree of ethoxylation is 3.8 and average degree of propoxylation is 4.5 C ₁₃-C ₁₅The blended ethoxylated/propoxylated fatty alcohol is sold by BASFGmbH, and commodity are called PlurafaxLF404

CFAA:C ₁₂-C ₁₄Alkyl N-methyl glucose amide

TFAA:C ₁₆-C ₁₈Alkyl N-methyl glucose amide

QAS:R ₂.N+ (CH ₃) ₂(C ₂H ₄OH), R wherein ₂=C ₁₂-C ₁₄

DTPA: ethylidene pentaacetic acid

SADS: formula 2-R.C ₄H ₇.-1,4-(SO ₄) ₂-(R=C wherein _10-18) C _14-22Alkyl two sodium sulfate

MES:x-sulfo group C ₁₈Fatty acid methyl ester

Soap: derived from straight-chain alkyl carboxylic acid's sodium of 80/20 mixture of butter and coco-nut oil fatty acid

Silicate: amorphous silicic sodium (SiO ₂: Na ₂O ratio=2.0)

NaSKS-6: formula d-Na ₂Si ₂O ₅Crystalline multilayer silicate

Carbonate: anhydrous sodium carbonate

MA/AA:1: 4 toxilic acids/acrylic copolymer, molecular-weight average is about 80,000

Zeolite A: the formula Na of basic granules size in the 1-10 micrometer range ₁₂(AlO ₂SiO ₂) ₁₂27H ₂The hydrated sodium aluminosilicate of O

Citrate trianion: two hydration trisodium citrates

PB1: anhydrous sodium perborate monohydrate SYNTHETIC OPTICAL WHITNER

The percarbonate empirical formula is 2Na ₂CO ₃3H ₂O ₂Anhydrous SPC-D SYNTHETIC OPTICAL WHITNER

TAED: acetylethylenediamine

The amino hexylyloxy of NACA-OBS:4-[N-(nonanoyl)]-benzene sulfonic acid sodium salt

NOBS: the ninth of the ten Heavenly Stems acyloxy Phenylsulfonic acid sodium salt

CMC: Xylo-Mucine

HEDP: hydroxyl ethane dimethylene phosphonic acids

DETPMP: diethylenetriamine five (methylene phosphonic acid), to sell by Monsanto, commodity are called Dequest2060

TEPAE: ethylidene five amine ethoxylates

PVP: polyvinyl pyrrolidone polymers

PVPVI: poly-(4-vinylpridine)-N-oxide compound/vinyl imidazole and vinylpyrrolidone copolymers

PVNO: molecular-weight average is polyvinylpyridine-N-oxide compound of 50,000

Whitening agent: 4,4 '-two (2-sulfo group styryl) biphenyl disodium and/or 4,4 '-two (4-phenylamino-6-morpholino-1,3,5-triazines-2-yl) toluylene-2,2 '-the disulfonic acid disodium

EDDS: the quadrol-N of sodium-salt form, N '-disuccinic acid [S, S] isomer

Froth suppressor: MPT (fusing point) is 50 ℃ 25% paraffin, 17% water drain silica, 58% paraffin oil

Particle froth suppressor: the starch of 12% siloxanes/silicon-dioxide, 18% Stearyl alcohol, 70% particle form

Vitriol: anhydrous sodium sulphate

HMWPEO: high-molecular weight polyethylene oxide

Enzyme: above-described proteolytic enzyme, amylase, cellulase, lipase in the specification sheets

Metal catalyst: the Cyclam of Dichlorodiethyl ethylidene bridge joint closes manganese

Amine: C _8-10Amido propyl group diethylamine

SRP: the end capped polyester of negatively charged ion

STPP: Tri sodium Phosphate

Supercarbonate: sodium bicarbonate

PAAC: five ammino Cobaltous diacetate (III) salt

Paraffin: paraffin oil, to sell by Wintershall, commodity are called Winog70

BTA: benzotriazole

Triacetate: sodium acetate trihydrate

PEGX: molecular weight is about the polyoxyethylene glycol of X

SKTP: Tri sodium Phosphate potassium

SLF18: available from the low foam type tensio-active agent of Olin Corporation

ACNI: formula C _9/11H _19/23EO ₈The alkyl-blocked nonionogenic tenside of-cyclohexyl acetal

PA30: the molecular weight available from BASF is about 8,000 polyacrylic acid ester homopolymer

Coagel pre-composition: available from the aqueous solution of the 5% active coagel DKP of 3V Inc.

BTA: benzotriazole

MEA: monoethanolamine

The enzyme component comprises in an embodiment with the percentage ratio that pure enzyme accounts for total composition weight.

Following non-limiting examples is used for the present invention is illustrated.

Material and method

Bacterial strain and donor biology

Bacillus subtilus DSM 14218 comprises the plasmid of the DNA (SEQID NO:1) that contains code book invention pectate lyase.This gene also can be cloned from strains A TCC 23857 (Bacillus subtilus PL2306) or this DNA can come from same strains A TCC 23857D (both can obtain from ATCC, LGC Promochem AB, PO Box 1737, SE-501 17 Boras, Sweden).

Bacillus subtilus PL 1801.This bacterial strain is Bacillus subtilus DN 1885 (Diderichsen, B., the Wedsted with ruined apr and npr gene, U., Hedegaard, L., Jensen, B.R., Sj ф holm, C. (1990), coding comes from the clone of aldB of the extracellular enzyme alpha-acetolactate decarboxylase of bacillus brevis, bacterium magazine (J.Bacteriol.), 172,4315-4321), this destruction betides in the transcriptional units of known Bacillus subtilus cellulose gene, causes producing the cellulase negative cells.Basically as (Eds.A.L.Sonenshein, J.A.Hoch and Richard Losick (1993) Bacillus subtilus and other gram positive bacterium (Bacillus substillis and other Gram-PositiveBacteria), AAM p.618) describedly carries out this gene disruption.

Press Yasbin, R.E., Wilson, G.A. and Young, F.E. the conversion and the transfection of (1975) Bacillus subtilus lysogenic strain: the selective induction that proves prophage in the competent cell. bacteriology magazine, (J.Bacteriol.) 121:296-304) described method preparation and transformed competence colibacillus cell.

Conventional molecular biology method

Unless otherwise mentioned, all DNA operation and transform the molecular biology method that all adopts standard (Sambrook etc. (1989) molecular cloning experiment guide, (Molecular cloning:Alaboratory manual) cold spring harbor laboratory, cold spring port, NY; Ausubel, F.M. etc. (volume) " up-to-date molecular biology manual " (Current Botocots in Molecular Biology) .John Wileyand Sons, 1995; Harwood, C.R., and Cutting, S.M. (volume) " genus bacillus molecular biology method " (Molecular Biological Methods for Bacillus) .John Wiley and Sons, 1990).

The enzyme (as can be from New England Biolabs, Inc obtains restriction enzyme, ligase enzyme etc.) that is used for DNA operation by manufacturer specification.

The genomic dna preparation

(American type culture collection, detailed description USA) are used as the propagation of the Bacillus subtilus bacterial strain of donor biology in the liquid medium within 3 to press ATCC.37 ℃ with the 300rpm incubation after 18 hours, reclaims cell, presses [Pitcher, D.G., Saunders, N.A., Owen, R.J such as Pitcher; With the quick extracting bacterial genomes of guanidine thiocyanate DNA; Applied microbiology communication (lett ApplMicrobiol) 1989,8:151-156] the method isolation of genomic DNA.

Plasmid

pMOL995：

This plasmid is the derivative of pUB110, comprises basically to make this plasmid can be at element, the kalamycin resistance gene of Bacillus subtilus internal breeding, and strong promoter and the signal peptide of clone from bacillus amyloliquefaciens amyQ gene arranged.This signal peptide comprises a Sac II site, and this is convenient to clone the coding DNA of the protein maturation part that merges with signal peptide.This just causes preceding proteic expression, and this protein is directed to outside.

This plasmid makes up with common gene engineering method and forms, and is summarized as follows.

The description of plasmid DMOL995:

With plasmid pUB110 (McKenzie, T. etc., 1986, plasmid (Plasmid) 15:93-103) be the basis, make up pMOL995.In several clone's steps, different features is imported the restriction site NciI of pUB110.

The feature of pMOL995 (SEQ ID NO:3) is:

Base 4076-6661 and 1-1962 coding plasmid pUB110.

Base 1963-2305 coding is from the amyL gene transcription terminator of Bacillus licheniformis ATCC 14580 and single restriction enzyme site (EagI, SalI etc.) of some importings.

Base 2306-3766 (oppositely) coding is from the α-Dian Fenmei maturing part of WO95/26397 (being disclosed in the SEQ ID NO:4 of WO95/26397).

Base 3767-4705 (oppositely) coding clone is from the promotor of the α-Dian Fenmei of bacillus amyloliquefaciens and signal peptide (see WO95/10603 and as promotor) with near the single SacI that imports 4075 of the bases and XmaI site and near the single Sac II site that imports 3769 of the bases, and described site is used for subsequently cloning signal peptide meeting frame.

pMOL944：

This plasmid is the derivative of pUB110, comprises basically to make this plasmid can be at element, the kalamycin resistance gene of Bacillus subtilus internal breeding, and has strong promoter and the signal peptide of clone from the amyL of Bacillus licheniformis ATCC14580 gene.This signal peptide comprises a Sac II site, and this is convenient to clone the coding DNA of the protein maturation part that merges with signal peptide.This just causes preceding proteic expression, and this protein is directed to outside.

The structure of pMOL944:

With single restriction enzyme NciI digestion pUB110 plasmid (McKenzie, T etc., 1986, plasmid 15:93-103).To increase from plasmid pDN1981 (P.L.Jorgensen etc., 1990, gene, 96, p37-41.) the PCR fragment of the amyL promotor of Shanging digests with NciI, and inserts among the pUB110 of NciI digestion, produces plasmid pSJ2624.

Two used PCR primers have following sequence:

Primer C (SEQ ID NO:8)

5′-GTCGCCGGGGCGGCCGCTATCAATTGGTAACIGTATCTCAGC-3

Primer D (SEQ ID NO:9)

5′-GTCGCCCGGGAGCTCTGATCAGGTACCAAGCTTGTCGACCIGCAGAA

TGAGGCAGCAAGAAGAT-3′

Primer C inserts a NotI site in plasmid.

Plasmid pSJ2624 is subsequently with SacI and NotI digestion, and the new PCR fragment of the amyL promotor on pDN1981 with SacI and NotI digest amplification (described fragment is synthetic with primer E and F), this dna fragmentation is inserted the pSJ2624 of SacI-NotI digestion, thereby produce plasmid

pSJ2670。

This clone's step amyL promotor of clone for the first time replaces with the opposite same promotor of direction.Two primers that are used for pcr amplification have following sequence:

Primer E (SEQ ID NO:10)

5′-GTCGGCGGCCGCTGATCACGTACCAAGCTTGTCGACCTGCAGAATG

AGGCAGCAAGAAGAT-3′

Primers F (SEQ ID NO:11)

5′-GTCGGAGCTCTATCAATTGGTAACTGTATCTCAGC-3′

Plasmid pSJ2670 digests with restriction enzyme PstI and BclI, to insert above-mentioned plasmid with PstI and BclI digestion back with the PCR fragment of the cloned dna sequence of two primer G and H amplification own coding alkali starch enzyme SP772 (seeing WO9526397A1), thereby produce plasmid pMOL944.Two primers that are used for pcr amplification have following sequence:

Primer G (SEQ ID NO:12)

5′-AACAGCTGATCACGACTGATCTTTTAGCTTGGCAC-3′

Primer H (SEQ ID NO:13)

5′-AACTGCAGCCGCGGCACATCATAATGGGACAAATGGG-3′

Primer H inserts a SacII site in plasmid.

Substratum

TY (as Ausubel, F.M. etc. (volume .) " up-to-date molecular biology manual " .John Wiley and Sons, described in 1995).

LB agar (as Ausubel, F.M. etc. (volume .) " up-to-date molecular biology manual " .JohnWiley and Sons, described in 1995).

LBPG is supplemented with 0.5% glucose and 0.05M potassiumphosphate, the LB agar of pH7.0.

The BPX substratum is described among the EP 0 506 780 (WO 91/09129).

Terminal point lyase test (in 235nm), the pectic acid unit of activity

In order to measure β-elimination, usefulness is dissolved in the 1.0% polygalacturonic acid sodium salt (Sigma P-1879) in the 0.1M EPPS damping fluid (pH8) is measured 235nm place light absorption value as substrate increase.70 ℃ of incubations 20 minutes.Add the 0.02M H of 5 times of volumes ₃PO ₄Stopped reaction.When calculating catalytic rate, per minute 235nm place light absorption value increases by 5.2 corresponding to forming the unsaturated product of 1 μ mol (Nasuna and Starr (1966) journal of biological chemistry (J.Biol.Chem.) .Vol 241 5298-5306 pages or leaves; And Bartling, Wegener and Olsen (1995) microbiology (Microbiology) Vol 141873-881 page or leaf).

Pectic acid unit of activity is defined as under pH8.0 and 70 ℃, and per minute causes a micromole to measure cleaved products forming needed enzyme amount.

Embodiment 1

Clone Bacillus subtilus pectate lyase gene

Subclone and the ripe pectate lyase of expression in Bacillus subtilus

With the PCR primer sets that following two oligonucleotide are formed the dna sequence dna of the pectate lyase of the present invention of encoding is done pcr amplification:

SEQ ID NO:4 (primer A)

5′-CAT?TCT?GCA?G CC?GCG?GCA?GCT?GAT?TTA?GGC?CAC?CAG?ACG-3’

SEQ ID NO:5 (primer B)

5’-GTA?CCT?CGC?GA G?TCG?ACT?TCT?TAA?TTT?AAT?TTA?CCC?GCA?CCC?GC-3’

Restriction site Sac II and Sal I are added with underscore.

Above-mentioned oligonucleotide is used for the PCR reaction, and this reacts on the HiFidelity that is supplemented with each 200 μ M of dNTP, 2.6 units ^TMThe HiFidelity of Expand enzyme mixture and each 200pmol of primer ^TMCarry out in the PCR damping fluid (Boehringer Mannheim, Germany).The genomic dna that separates from the Bacillus subtilus bacterial strain is added into this PCR reaction as template.Genomic dna is by separating as mentioned above.

Carry out the PCR reaction with DNA thermal cycler (Landgraf, Germany).94 ℃ of incubations carried out 1 circulation in 1 minute; Carry out 10 circulations then, each circulation is 94 ℃ of sex change 15 seconds, anneals 60 seconds for 60 ℃, and 72 ℃ were extended 120 seconds; Carry out 20 circulations then, each circulation is 94 ℃ of sex change 15 seconds, and 120 seconds (extend step at this, each circulation increases by 20 seconds) extended in 60 ℃ of annealing in 60 seconds and 70 ℃.(Nusieve FMC) goes up electrophoretic analysis 5 μ l amplified productions at 0.7% sepharose.Size shows that for the appearance of the dna fragmentation of 1.2kb gene fragment is correctly increased.

Subclone PCR fragment

Press manufacturer specification QIA fast PCR purification kit (Qiagen, USA) 45 μ l aliquots containigs of the PCR product of the above-mentioned generation of purifying.The DNA wash-out of purifying is in the 10mM Tris-HCl of 50 μ l, pH8.5.Digest the PCR fragment of 5 μ g pMOL995 and 25 μ l purifying with Sac II and Sal I, (Nusieve FMC) goes up electrophoresis, and associated clip is downcut from glue at 0.7% sepharose, (Qiagen USA) carries out purifying with QIA PhastGel extraction agent box to press manufacturer specification.Subsequently isolating PCR dna fragmentation is connected on the pMOL995 of Sac II-Sal I digestion and purifying.Spend the night for 16 ℃ with every kind of dna fragmentation, 1U T4 dna ligase and the T4 ligase enzyme damping fluid (Boehringer Mannheim, Germany) of each 0.5 μ g and to finish connection.

Connect mixture and be used to transformed competence colibacillus Bacillus subtilus PL2306.Transformant is laid on the LBPG agar plate that contains 10 μ g/ml kantlex.37 ℃ of incubations are after 18 hours, and bacterium colony comes across on the flat board.Several clones are by analyzing from the cultured solution of broth isolated plasmid dna that spends the night.

Such positive colony is heavily rule several times on as above used agar plate, and this clone is called as MB331.MB331 is cloned among the TY that contains 10 μ g/ml kantlex, 37 ℃ of overnight incubation, and the method for recommending for the Bacillus subtilus plasmid prepares by manufacturers in second day prepares test kit #27106 separation quality grain from the 1ml cell in a small amount with Qiaprep Spin plasmid.Plasmid DNA check order and announcement and SEQ ID NO:1 in the part of pectate lyase gene of encoding mature pectate lyase dna sequence dna with identity.

Embodiment 2

Expression, purifying and evaluation Bacillus subtilus pectate lyase

In 25 * 200ml BPX substratum that will contain 10 μ g/ml kantlex in the Erlenmeyer flask of 500ml band baffle plate as the MB331 clone (preservation is DSM 14218) of acquisition as described in the embodiment 1 37 ℃, 300rpm cultivated 5 days, obtained the 4550ml cultured solution of broth.With the pH regulator to 6.1 of acetic acid with nutrient solution, then, the cationoid reagent (C521 10%) and the 60ml reagents for anion (A130 0.1%) that add 25ml in stirring are used for producing flocculation.Utilize Sorval RC 3B whizzer, at 6 ℃, 10000rpm separated the material of flocculation in centrifugal 30 minutes.The supernatant liquor cumulative volume that produces is 3750ml.

Utilize Whatman GF/D and C flint glass F filter clarified supernatant, concentrate on molecular weight cut-off is the Filtron UF film of 10kDa at last, cumulative volume is 1500ml, and pH is transferred to 6.0.

In order to obtain highly purified pectate lyase, the S-Sepharose cation-exchange chromatography is carried out in final step.1500ml solution is splined on the good 800ml of the sodium-acetate buffer balance of 50mmol pH6.0 contains in the post of S-Sepharose (Pharmacia).0.5M NaCl gradient elution is used in pectate lyase generation combination afterwards.

Identify

Pure enzyme is the single band of 44kDa on SDS-PAGE, and iso-electric point is about 7.6.

Use molar extinction coefficient 71950 (forming) when measuring proteinic concentration based on inferring amino acid from this sequence.

The activity of pectate lyase can be suppressed by EDTA.

Dsc (DSC) shows that the melting temperature (Tm) of pure enzyme in the 0.1M of pH8 Tris damping fluid is 58.8 ℃.

Embodiment 3

Clone Bacillus subtilus 168 pectate lyase genes

Subclone and the ripe pectate lyase of expression in Bacillus subtilus

SEQ ID NO:14 (primer J)

5′-CAT?TCT?GCA?G CC?GCG?GCA?GCT?GAT?TTA?GGC?CAC?CAG?ACG-3′

SEQ ID NO:15 (primer K)

5′-GTA?CCT?CGC?GA G?CGG?CCG?CTT?CTT?AAT?TTA?ATT?TAC?CCG?CAC?CCG?C-3′

Restriction site Sac II and Not I are added with underscore.

Above-mentioned oligonucleotide is used for the PCR reaction, and this reacts on the HiFidelity that is supplemented with each 200 μ M of dNTP, 2.6 units ^TMThe HiFidelity of Expan enzyme mixture and each 200pmol of primer ^TMCarry out in the PCR damping fluid (Boehringer Mannheim, Germany).The method separation is added into this PCR reaction from the genomic dna of Bacillus subtilus bacterial strain as template as mentioned above.

Carry out the PCR reaction with DNA thermal cycler (Landgraf, Germany).94 ℃ of incubations carried out 1 circulation in 1 minute; Carry out 10 circulations then, each circulation is 94 ℃ of sex change 15 seconds, anneals 60 seconds for 60 ℃, and 72 ℃ were extended 120 seconds; Carrying out 20 circulations afterwards, respectively is that 120 seconds (extend step at this, each circulation increases by 20 seconds) extended in 94 ℃ of sex change 15 seconds, 60 ℃ of annealing in 60 seconds and 70 ℃.(Agarose SIGMA) goes up electrophoretic analysis 5 μ l amplified productions at 0.7% sepharose.Size shows that for the appearance of the dna fragmentation of 1.2kb gene fragment is correctly increased.

Subclone PCR fragment

Press manufacturer specification QIA fast PCR purification kit (Qiagen, USA) 45 μ l aliquots containigs of the PCR product of the above-mentioned generation of purifying.The DNA wash-out of purifying is in the 10mM Tris-HCl of 50 μ l, pH8.5.Digest the PCR fragment of 5 μ g pMOL944 and 25 μ l purifying with Sac II and Sal I, (NuSieve FMC) goes up electrophoresis, and associated clip is downcut from glue at 0.7% sepharose, (Qiagen USA) carries out purifying with QIA PhastGel extraction agent box to press manufacturer specification.Subsequently isolating PCR dna fragmentation is connected on the pMOL944 of Sac II-Sal I digestion and purifying.Spend the night for 16 ℃ with every kind of dna fragmentation, 1U T4 dna ligase and the T4 ligase enzyme damping fluid (Boehringer Mannheim, Germany) of each 0.5 μ g and to finish connection.

Connect mixture and be used to transformed competence colibacillus Bacillus subtilus PL1801.Transformant is laid on the LBPG agar plate that contains 10 μ g/ml kantlex.37 ℃ of incubations are after 18 hours, and bacterium colony comes across on the flat board.Several clones are by analyzing from the cultured solution of broth isolated plasmid dna that spends the night.

Such positive colony is heavily rule several times on as above used agar plate, and this clone is called as MB1306.MB1306 is cloned among the TY that contains 10 μ g/ml kantlex, 37 ℃ of overnight incubation, and the method for recommending for the Bacillus subtilus plasmid prepares by manufacturers in second day prepares test kit #27106 separation quality grain from the 1ml cell in a small amount with Qiaprep Spin plasmid.Plasmid DNA check order and disclosed with SEQ ID NO:xx in the part of pectate lyase gene of encoding mature pectate lyase dna sequence dna with identity.

Embodiment 4

Express the pectate lyase of Bacillus subtilus 168

To be cloned in the 25 * 200ml BPX substratum that contains 10 μ g/ml kantlex 37 ℃ in the Erlenmeyer flask of 500ml band baffle plate as the MB1306 of acquisition as described in the embodiment 3,300rpm cultivated 5 days, obtained the 4550ml cultured solution of broth.

Embodiment 5

The pectate lyase gene of clone Bacillus subtilus DSM 14979

With the PCR primer sets that following oligonucleotide is formed the dna sequence dna from Bacillus subtilus DSM 14979 of coding pectate lyase is done pcr amplification:

SEQ ID NO:16 (primer L)

5?′-CAT?TCT?GCA?GCC?GCG?GCA?GCT?GAT?TTA?GGC?CAC?CAG?ACG-3’

SEQ ID NO:17 (primer M)

5’-GTA?CCT?CGC?GAG?CGG?CCG?CTT?CTT?AAT?TTA?ATT?TAC?CCG?CAC?CCG?C-3’

Restriction site Sac II and Not I are added with underscore.

Each 50pmol of oligonucleotide is used for the PCR reaction, and this reacts on and contains dNTP each 200 μ M, 3.5mM MgCl ₂, 2.5 units AmpliTaq Gold ^TM(Perkin-Elmer) carry out in the PCR damping fluid (10mM Tris-HCl, pH8.3,50mM KCl), about 100 to 200ng by the isolating genomic dna of method as mentioned above as the template of pcr amplification reaction.Cumulative volume is 50 μ l.This PCR is reflected in the Perkin-Elmer GeneAmp PCR system 2400 and carries out.

The recycle scheme of this PCR reaction is:

94 ℃-10 minutes; 1 circulation

94 ℃-1 minute, 55 ℃-30 seconds, 72 ℃-1 minute 30 seconds; 25 circulations

72 ℃-7 minutes; 1 circulation.

(Agarose SIGMA) goes up electrophoretic analysis 5 μ l amplified productions at 1.0% sepharose.Size shows that for the appearance of the dna fragmentation of 1.2kb gene fragment is correctly increased.The sequencing fragment result has disclosed the dna sequence dna (be shown in SEQ ID NO:18) of coding from the ripe pectate lyase (being shown among the SEQ IDNO:19) of Bacillus subtilus DSM 14979.

Embodiment 6

Measure the stability of pectate lyase in liquid washing agent

The washing composition stability of pectate lyase variant of the present invention is to determine by method is as described below measured variant behind incubation enzyme and detergent mixture activity.

Residual activity is measured

In two sample hoses, be respectively charged into the mixture of 30 microlitre enzyme solution (culture supernatants or pure enzyme) and the potent type liquid washing agent in the typical Europe of 1ml or the U.S..Wherein a pipe is housed on ice, and another pipe was in 40 ℃ of incubations 90 minutes.The washing composition of the water of 30 microlitres and 1ml mixed being incorporated in incubation on ice, as object of reference.

Behind the incubation, the icy water of 9ml adds in the sample, acutely mixes and is housed in and treat further analysis on ice.

For the mensuration of enzymic activity, at first mix 50 microlitre enzyme-detergent mixtures and 5ml and detect damping fluid (100mM Tris-HCl, 0.68mM CaCl ₂, pH8.0), the substrate solution (the detection damping fluid that contains 1% polygalacturonic acid) that secondly takes out 75 microlitres and 75 microlitre prepared fresh from this solution mixes and is incorporated in 40 ℃ of incubations 10 minutes.Again, the incubation mixture of 100 microlitres is added in 100 microlitre stop buffer (50mM H in the transparent microtiter plate of UV- ₃PO ₄) in, and with the light absorption value at spectrophotometer measurement 235nm place.Water-washing composition sample is used for regulating spectrophotometric zero point.

At this moment, by calculating activity (A235 absorption value) and the active relative value that is housed in sample on ice, obtain the residual activity value at 90 minutes sample of 40 ℃ of incubations:

Residual activity (RA)=absorption value [incubation 40 ℃ sample]/absorption value [incubation 0 ℃ sample]

Therefore, residual activity is equivalent to the washing composition stability of enzyme.Fig. 1 has listed the residual activity of raising of some alternative variations of the MB331 pectate lyase of incubation in typical European potent type liquid washing agent.With respect to parent's pectate lyase, the washing composition stability of most of alternative variations has improved more than 50%.Be equal to ground, the alternative variations of the MB331 pectate lyase of listing in Fig. 2 has also improved the stability of enzyme significantly when incubation is in the potent type liquid washing agent of the typical U.S..

Fig. 1. cause in the potent type liquid washing agent in Europe substituting in residual activity increases behind the incubation the SEQ ID NO:2 pectate lyase.

Sudden change	Residual activity	Residual activity % with respect to parent enzyme
Sudden change	Residual activity	Residual activity % with respect to parent enzyme	SEQ ID NO:2 pectate lyase (parent enzyme)	????23	????100
?Q40E	????28	????121.7	SEQ ID NO:2 pectate lyase (parent enzyme)	????23	????100
?Q40E	????28	????121.7	?F251I	????30	????130.4
?A332P	????31	????134.8	?F251I	????30	????130.4
?A332P	????31	????134.8	?L106Q	????31	????134.8
?R272H	????31	????134.8	?L106Q	????31	????134.8
?R272H	????31	????134.8	?R272Y	????31	????134.8
?A91E	????33	????143.5	?R272Y	????31	????134.8
?A91E	????33	????143.5	?K115I+K213E	????33	????143.5
?K139I+K213N	????33	????143.5	?K115I+K213E	????33	????143.5
?K139I+K213N	????33	????143.5	?H5R+K257N+S302A	????34	????147.8
?K139M	????34	????147.8	?H5R+K257N+S302A	????34	????147.8
?K139M	????34	????147.8	?K87A	????34	????147.8
?D48P	????35	????152.2	?K87A	????34	????147.8

?K99I+I196V	????35	????152.2
?K99I+I196V	????35	????152.2	?T105P	????35	????152.2
?K115A+K118A	????36	????156.5	?T105P	????35	????152.2
?K115A+K118A	????36	????156.5	?K115A+K118A+M122N	????36	????156.5
?K115Q	????36	????156.5	?K115A+K118A+M122N	????36	????156.5
?K115Q	????36	????156.5	?K213T	????36	????156.5
?V141E+C199S+K213E	????36	????156.5	?K213T	????36	????156.5
?V141E+C199S+K213E	????36	????156.5	?K115I+Q146H	????37	????160.9
?K257N	????37	????160.9	?K115I+Q146H	????37	????160.9
?K257N	????37	????160.9	?K71E+K118E	????38	????165.2
?S331P	????38	????165.2	?K71E+K118E	????38	????165.2
?S331P	????38	????165.2	?T49P+N156S	????38	????165.2
?K314N+S340P	????39	????169.6	?T49P+N156S	????38	????165.2
?K314N+S340P	????39	????169.6	?V141E+I235V	????39	????169.6
?G46D+K257N	????40	????173.9	?V141E+I235V	????39	????169.6
?G46D+K257N	????40	????173.9	?Q146H	????40	????173.9
?A305P	????41	????178.3	?Q146H	????40	????173.9
?A305P	????41	????178.3	?K218P	????41	????178.3
?S28T+S30F+K334E+N363S	????41	????178.3	?K218P	????41	????178.3
?S28T+S30F+K334E+N363S	????41	????178.3	?D48E+L106Q+I140V+F215Y+K218E	????42	????182.6
?H193Y+S256C+V389I+A393V	????42	????182.6	?D48E+L106Q+I140V+F215Y+K218E	????42	????182.6
?H193Y+S256C+V389I+A393V	????42	????182.6	?R272C	????42	????182.6
?E9G+H31N+N50D+L106Q+A111E+T136S+V14 ?1L+F201L+N202K+ ?F215Y+G286A+A381D+H384N	????43	????187.0	?R272C	????42	????182.6
	????43	????187.0	?K213N+T258I	????43	????187.0
?E9G+H31N+L106Q+D303S+A305P+T335S+H3 ?84N+S391N	????44	????191.3	?K213N+T258I	????43	????187.0
?E9G+H31N+L106Q+D303S+A305P+T335S+H3 ?84N+S391N	????44	????191.3	?E9G+H31N+D48E+L106Q+A111E+S301Y+D30 ?3S+A305P+T378S+H384N+S391N	????45	????195.7
?L45V+N50Y+N185H	????46	????200.0		????45	????195.7
?L45V+N50Y+N185H	????46	????200.0	?N11Y+K87E+K99N	????46	????200.0
?E9G+D48E+L106Q+S316F+A381D	????48	????208.7	?N11Y+K87E+K99N	????46	????200.0

?S30P+K115I+K139I+Q146H+S337C	????49	????213.0
?S30P+K115I+K139I+Q146H+S337C	????49	????213.0	?E9G+H31N+D48E+L106Q+I140V+F215Y+D303 ?S+A305P+T378S+H384N+S391N	????51	????221.7
?H31N+T105A+L106Q+A111E+V141L+K218E+ ?D303S+A305P+D326N+T335S+H384N+S391N	????51	????221.7		????51	????221.7
	????51	????221.7	?K26Q+K47N+L106Q+I140V+F215Y+D303S+A ?305P+T378S+H384N+S391N	????53	????230.4
?D48E+L106Q+I140V+F215Y+D303S+A305P+T ?378S+H384N+S391N	????56	????243.5		????53	????230.4
?D48E+L106Q+I140V+F215Y+D303S+A305P+T ?378S+H384N+S391N	????56	????243.5	?K213N	????60	????260.9
?K213T+K218L+A305P	????64	????278.3	?K213N	????60	????260.9
?K213T+K218L+A305P	????64	????278.3	?S337C	????65	????282.6
?M64F+K213T+K218L+A305P	????70	????304.3	?S337C	????65	????282.6
?M64F+K213T+K218L+A305P	????70	????304.3	?M64F+M122K+K118E+K213T+K218L+A305P	????71	????308?7
?K139I+Q146H+S337C	????74	????321.7	?M64F+M122K+K118E+K213T+K218L+A305P	????71	????308?7
?K139I+Q146H+S337C	????74	????321.7	?K139I+Q146H+K257N+S337C	????75	????326.1
?M64F+K139I+Q146H+S337C	????90	????391.3	?K139I+Q146H+K257N+S337C	????75	????326.1

Fig. 2. cause in the potent type liquid washing agent of the U.S. substituting in residual activity increases behind the incubation the SEQ ID NO:2 pectate lyase.

Mutant	Residual activity	Parent enzyme residual activity %
Mutant	Residual activity	Parent enzyme residual activity %	SEQ ID NO:2 pectate lyase (parent enzyme)	????45	????100
?R272Y	????50	????111.1	SEQ ID NO:2 pectate lyase (parent enzyme)	????45	????100
?R272Y	????50	????111.1	?M237I	????51	????113.3
?C199N	????51	????113.3	?M237I	????51	????113.3
?C199N	????51	????113.3	?F215Y	????52	????115.6
?K213T	????52	????115.6	?F215Y	????52	????115.6
?K213T	????52	????115.6	?A228I+F251I	????53	????117.8
?K115I+Q146H	????57	????126.7	?A228I+F251I	????53	????117.8

?K257N	????59	????131.1
?K257N	????59	????131.1	?K213N+T258I	????60	????133.3
?M122Q	????61	????135.6	?K213N+T258I	????60	????133.3
?M122Q	????61	????135.6	?K218P	????61	????135.6
?K386P	????61	????135.6	?K218P	????61	????135.6
?K386P	????61	????135.6	?A332P	????62	????137.8
?F251I	????64	????142.2	?A332P	????62	????137.8
?F251I	????64	????142.2	?A305P	????64	????142.2
?D48P	????64	????142.2	?A305P	????64	????142.2
?D48P	????64	????142.2	?S134L+K257E	????66	????146.7
?S331P	????75	????166.7	?S134L+K257E	????66	????146.7
?S331P	????75	????166.7	?K139I+Q146H+S337C	????100	????222.2

Scourability embodiment

The scourability embodiment A

With full scale (fullscale) test pectate lyase of the present invention.

In the potent type liquid washing agent in Europe, use a series of different specimen in full scale is washed, to go evaluation from the pectate lyase (SEQ ID NO:2) (following table is shown the MB331 enzyme) of clone MB331.Employed enzyme level is 0.05mg MB331 enzyme/every liter of washing water.On many different fruit or vegetables base spot, find very high scourability, shown in seeing the following form with this dosage level.

Sample	Δ reflectivity (remission)
Sample	Δ reflectivity (remission)	Blueberry sauce	????15.0
Avocado	????14.9	Blueberry sauce	????15.0
Avocado	????14.9	The tomato enriched material	????13.3
Tomato ketchup	????10.6	The tomato enriched material	????13.3
Tomato ketchup	????10.6	Banana	????8.4
Tomato puree	????7.3	Banana	????8.4
Tomato puree	????7.3	Pears	????7.2

Apple	????5.6
Apple	????5.6	Plum	????5.5
Blackberry, blueberry	????4.8	Plum	????5.5
Blackberry, blueberry	????4.8	Radix Dauci Sativae juice	????3.3

Method is described

Use following equipment and wash conditions.

Washer: AEG  kolavamat 86820 (latest type)

Washing procedure: 40 ℃ short washings

The potent type liquid washing agent in washing composition: 5g/l Europe

The water hardness: 15 ° of dH (4: 1 Ca/Mg; 2.14mM CaCl ₂With 0.54mM MgCl ₂)

Enzyme: every liter of washing water 0.05mg MB331 pectate lyase albumen

Sample: from the pre-preparation food stains of Equest

Assessment: measure reflectivity at the 460nm place with Elrepho 2000 reflectivity spectrophotometers.

The Δ reflectivity is according to following calculating:

R _Enzyme-R _{No enzyme}, wherein R is the reflectivity at the 460nm place.

The scourability Embodiment B

Polygalacturonase is to the scourability of banana sample in washing on a small scale.

Using laundrometer (Laundermeter) and banana sample in the small-scale washing test, in the potent type liquid washing agent in Europe, will make comparisons with other pectate lyase from the pectate lyase (SEQID NO:2) of clone MB331.The results are shown in following table, wherein the scourability effect of MB331 is transferred to 100%.

Pectate lyase (SEQ ID NO:2 from Bacillus subtilus DSM14218, from clone MB331) and from pectate lyase (the SEQ ID NO:7 of Bacillus subtilus A168, from clone MB1306) observe and have best scourability, be lower than 50% scourability yet demonstrate from the pectate lyase of Bacillus licheniformis (SP958) and B.agaradherens (SP956).

Enzyme is other	Enzyme	Scourability
Enzyme is other	Enzyme	Scourability	Pectate lyase	The MB331 Bacillus subtilus	????100％
Pectate lyase	A168, the MB1306 Bacillus subtilus	????97％	Pectate lyase	The MB331 Bacillus subtilus	????100％
Pectate lyase	A168, the MB1306 Bacillus subtilus	????97％	Pectate lyase	Bacillus licheniformis (the SEQ ID NO:4 among the WO99/27084)	????43％
Pectate lyase	B.agaradherens (the SEQ ID NO:2 among the WO99/27084)	????36％	Pectate lyase		????43％

Method is described

Preparation banana sample: three bananas are smashed to pieces and in stirrer, added 70ml deionized water homogenate 3-4 minute.Suspension is introduced in the dish and placed about 2 hours.The cotton sample (from Testfabrics Inc., model 400) of cleaning is immersed in this suspension, extruding and dried overnight between two rolls.

Scourability is measured: banana sample usefulness laundrometer (Laundermeter) is potent type liquid washing agent and 15 ° of dH water (4: washing 1Ca/Mg) in Europe.Water hardness is by adding CaCl ₂(2.14mM) and MgCl ₂(0.54mM) regulate.Each beaker (500ml) adds 20 steel balls, the 200ml detergent solution, 0.05mg/l polygalacturonase and 3 banana samples (5cm * 5cm).Washing procedure is to heat 10 minutes from 25 ℃ to 40 ℃, subsequently 40 ℃ of washings 20 minutes.Sample is rinsing and dried overnight at room temperature in tap water.

Assessment: the reflectivity of sample is measured at the 440nm place with MacBeth ColorEye 7000 reflectivity spectrophotometers.

The scourability Embodiment C

The MB331 variant that in liquid washing agent, has the stability of raising

Produced and in potent type liquid washing agent, had the MB331 pectate lyase variant that improves stability.Measure its stability over 7 days by 35 ℃ of following storage pectate lyases in liquid washing agent, use the banana sample in laundrometer (Laundermeter) test, to assess scourability subsequently.Following table shows that 4 kinds of different MB331 variants have strong improvement on storage stability.MB331-varl47 and 168 scourability do not change, yet the scourability of MB331-varl 35 and 137 is more lower slightly than the scourability of MB331.

The MB331 variant	Storage stability	Scourability
The MB331 variant	Storage stability	Scourability	Wild-type	????0％	????100％
M64F+K139I+Q146H+S337C	????74％	????68％	Wild-type	????0％	????100％
M64F+K139I+Q146H+S337C	????74％	????68％	M64F+V123I+K139I+Q146H+S337C	????74％	????79％
K139N+E158N+Q146H+S337C	????57％	????101％	M64F+V123I+K139I+Q146H+S337C	????74％	????79％
K139N+E158N+Q146H+S337C	????57％	????101％	M64F+M237I+K139I+Q146H+S337C	????59％	????94％

Method is described

Polygalacturonase in the potent type liquid washing agent in Europe in 35 ℃ of water-baths incubation 7 days.The sample of storage and fresh enzyme are with buying from the banana spot sample of Equest and 0.01mg/l polygalacturonase at laundrometer (Laundermeter) test in the scourability analysis (seeing described in the scourability Embodiment B).The scourability of fresh MB331 is adjusted to 100%.Store after seven days, the storage stability of enzyme is used and is compared residual scourability % with fresh enzyme and calculate.

Washing composition embodiment

The washing composition example I

Particle fabric detergent composition of the present invention can be prepared as follows:

A???????????B????????????C

LAS?????????????????6.5?????????8.0??????????8.0

Sodium sulfate 15.0 20.0 20.0

Zeolite A 26.0 20.0 25.0

Sodium nitrilo triacetate 5.0-2.0

Enzyme 0.01 0.001 0.05 of the present invention

PVP?????????????????0.5?????????-????????????0.5

TAED????????????????3.0?????????-????????????2.0

Boric acid 4.0--

PB1?????????????????18.0????????15.00????????15.00

NACA-OBS????????????-???????????-????????????1.0

Metal catalyst 0.02--

Sulfocarbolate 0.1--

Micro substance (other enzyme, whitening agent, spices, dyestuff ...) reaches 100

The washing composition example II

Dense granule fabric detergent composition of the present invention (density 800g/l) can be prepared as follows:

A???????????????B

45AS??????????????8.0?????????????8.0

25E3S?????????????2.0?????????????2.0

25E5??????????????3.0?????????????3.0

25E3??????????????3.0?????????????3.0

TFAA??????????????2.5?????????????2.5

Zeolite A 17.0 17.0

NaSKS-6???????????12.0????????????12.0

Citric acid 3.0 3.0

Carbonate 7.0 7.0

MA/AA?????????????5.0?????????????5.0

CMC???????????????0.4?????????????0.4

Enzyme 0.05 0.05

Enzyme 0.01 0.001 of the present invention

TAED??????????????6.0?????????????6.0

Percarbonate 22.0 22.0

EDDS??????????????0.3?????????????0.3

Particle froth suppressor 3.5 3.5

Water/micro substance (whitening agent, spices, dyestuff ...) reaches 100%

The washing composition EXAMPLE III

Especially can be used for the particle fabric detergent composition of the present invention of washing colored fabric, be prepared as follows:

A?????????????B

LAS??????????10.7??????????-

TAS??????????2.4???????????-

TFAA?????????-?????????????4.0

45AS???????????????3.1????????????10.0

45E7???????????????4.0????????????-

25E3S??????????????-??????????????3.0

68E11??????????????1.8????????????-

25E5???????????????-??????????????8.0

Citrate trianion 15.0 7.0

Carbonate-10

Citric acid 2.5 3.0

Zeolite A 32.1 25.0

Na-SKS-6???????????-??????????????9.0

MA/AA??????????????5.0????????????5.0

DETPMP?????????????0.2????????????0.8

Enzyme 0.10 0.05 of the present invention

Silicate 2.5-

Vitriol 5.2 3.0

PVP????????????????0.5????????????-

PVPVI??????????????-??????????????0.2

PB1????????????????1.0????????????-

Sulfocarbolate 0.2-

Water/micro substance reaches 100%

The washing composition EXAMPLE IV

Can in washing process, provide the particle fabric detergent composition of the present invention of softening power to be prepared as follows:

A??????????????B

45AS????????????-??????????????10.0

LAS?????????????7.6????????????-

68AS????????????1.3????????????-

45E7????????????4.0????????????-

25E3??????????????????????-?????????????????5.0

Cocounut oil alkyl-dimethyl hydroxyethyl 1.4 1.0

The ammonium muriate

Citrate trianion 5.0 3.0

Na-SKS-6??????????????????-?????????????????11.0

Zeolite A 15.0 15.0

MA/AA?????????????????????4.0???????????????4.0

DETPMP????????????????????0.4???????????????0.4

PB1???????????????????????15.0??????????????-

Percarbonate-15.0

TAED??????????????????????5.0???????????????5.0

Smectic clays 10.0 10.0

HMWPEO????????????????????-?????????????????0.1

Enzyme 0.10 0.05 of the present invention

Silicate 3.0 5.0

Carbonate 10.0 10.0

Particle froth suppressor 1.0 4.0

CMC???????????????????????0.2???????????????0.1

Water/micro substance reaches 100%

The washing composition EXAMPLE V

Following laundry composition can be particulate state or sheet, and it is produced according to the present invention.

A??????B??????C??????D??????E

Based product

C45AS/TAS?????8.0????5.0????3.0????3.0????3.0

LAS???????????8.0????-??????8.0????-??????7.0

C25AE3S???????0.5????2.0????1.0????-??????-

C25AE5/AF3????2.0????-??????5.0????2.0????2.0

QAS??????????????????-????????-??????-???????1.0????1.0

Zeolite A 20.0 18.0 11.0-10.0

SKS-6 (I) (the dry adding)--9.0--

MA/AA????????????????2.0??????2.0????2.0?????-??????4.0

Citrate trianion-2.0---

Citric acid 2.0-1.5 2.0-

DTPA?????????????????0.2??????0.2????-???????-??????-

EDDS?????????????????-????????-??????0.5?????0.1????-

HEDP?????????????????-????????-??????0.2?????0.1????-

PB1??????????????????3.0??????5.0????10.0????-??????4.0

Percarbonate---18.0-

NOBS?????????????????3.0??????4.0?????-??????-??????4.0

NACAOBS??????????????-????????-???????2.0????-??????-

TAED?????????????????-????????-???????2.0????5.0????-

Metal catalyst-0.02---

Carbonate 15.0 18.0 8.0 15.0 15.0

Vitriol 5.0 12.0 2.0 17.0 3.0

Silicate-1.0--8.0

Enzyme 0.001 0.002 0.02 0.05 0.005 of the present invention

Moisture and impurity reach 100%

The washing composition example VI

Following granulated detergent is produced according to the present invention:

A???????B??????C??????D??????E??????F???????G??????H

Base particle

STPP??????-???????22.0???-??????15.0???-??????22.0????-??????15.0

Zeolite A 30.0-24.0 5.0 30.0-24.0 5.0

Vitriol 5.5 5.0 7.0 7.0 5.5 5.0 7.0 7.0

MA/AA????3.0?????12?????-??????6.0-???3.0????12.0????2.0????6.0

LAS??????14.0????10.0???9.0????20.0???14.0???10.0????9.0????20.0

C45AS??????????????8.0????7.0????9.0????7.0????8.0????7.0????9.0????7.0

C45AE11S???????????-??????1.0????-??????1.0????-??????1.0????-??????1.0

MES????????????????0.5????4.0????6.0????-??????0.5????4.0????6.0????-

SADS???????????????2.5????-??????-??????1.0????2.5????-??????-??????1.0

Silicate-1.0 0.5 10.0-1.0 0.5 10.0

Soap-2.0---2.0--

Whitening agent 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2

Carbonate 6.0 9.0 8.0 10.0 6.0 9.0 8.0 10.0

PEG4000????????????-??????1.0????1.5????-??????-??????1.0????1.5????-

DTPA???????????????-??????0.4????-??????-??????-??????0.4????-??????-

Spray to:

C25E9??????????????-??????-??????-??????5.0????-??????-??????-??????5.0

C45E7??????????????1.0????1.0????-??????-??????1.0????1.0????-??????-

C23E9??????????????-??????1.0????2.5????-??????-??????1.0????2.5????-

Spices 0.2 0.3 0.3-0.2 0.3 0.3-

Dry additive

Carbonate 5.0 10.0 13.0 8.0 5.0 10.0 13.0 8.0

PVPVI/PVO??????????0.5????-??????0.3????-??????0.5????-??????0.3????-

Enzyme 0.04 0.03 0.03 .04 0.04 0.03 0.03 0.01

Enzyme 0.001 0.02 0.03 0.015 0.001 0.02 0.03 0.015 of the present invention

DTPA???????????????0.5????0.3????0.5????1.0????0.5????0.3????0.5????1.0

PB1????????????????5??????3.0????10?????4.0????5??????3.0????10?????4.0

NOBS/TAED??????????0.5????0.3????0.5????0.6????0.5????0.3????0.5????0.6

Vitriol 4.0 5.0-5.0 4.0 5.0-5.0

SRP????????????????-??????0.4????-??????-??????-??????0.4????-??????-

Particle froth suppressor-0.5---0.5--

Specking (speckle) 0.9-2.7 1.2 0.9-2.7 1.2

Moisture and impurity reach 100%

The washing composition example VII A

Following liquid detergent preparation is produced according to the present invention:

A???????B??????C???????D??????E

LAS??????????????????11.5????9.0????-???????4.0????-

C25E2.5S?????????????-???????3.0????18.0????-??????16.0

C45E2.25S????????????11.5????3.0????-???????16.0???-

C23E9????????????????-???????3.0????2.0?????2.0????1.0

C23E7????????????????3.2?????-??????-???????-??????-

CFAA?????????????????-???????-??????5.0?????-??????3.0

TopPalmKernel lipid acid 2.0-2.0 0.5 2.0

Citric acid (50%) 6.5 1.0 2.5 4.0 2.5

Ca and/or Ca formate 0.6 0.7 0.2 0.05 0.05

SCS??????????????????4.0?????1.0????3.0?????1.2????-

Borate 0.6-3.0 2.0 3.0

Sodium hydroxide 6.0 2.0 3.5 4.0 3.0

Ethanol 2.0 1.0 4.0 4.0 3.0

1,2-propylene glycol 3.0 2.0 8.0 8.0 5.0

Monoethanolamine MEA BASF 3.0 1.5 1.0 2.5 1.0

TEPAE????????????????2.0?????-??????1.0?????1.0????1.0

Enzyme 0.001 0.002 0.01 0.01 0.005 of the present invention

Enzyme 0.03 0.01 0.03 0.02 0.02

SRP??????????????????0.2?????-??????0.1?????-??????-

DTPA?????????????????-???????-??????0.3?????-??????-

PVNO?????????????????-???????-??????0.3?????-??????0.2

Whitening agent 0.2 0.07 0.1--

Froth suppressor 0.04 0.02 0.1 0.1 0.1

Impurity and water

Washing composition example VII A I

The potent liquid fabric detergent composition of the present invention can be prepared as follows:

A??????????????B

The LAS-25.0 of acid form

Citric acid 5.0 2.0

The 25AS 8.0 of acid form-

The 25AE2S 3.0 of acid form-

25AE7???????????????????????????8.0????????????-

CFAA????????????????????????????5??????????????-

DETPMP??????????????????????????1.0????????????1.0

Lipid acid 8-

Oleic acid-1.0

Ethanol 4.0 6.0

Propylene glycol 2.0 6.0

Enzyme 0.10 0.05 of the present invention

Cocounut oil alkyl dimethyl hydroxyethyl ammonium muriate-3.0

Smectic clays-5.0

PVP?????????????????????????????2.0????????????-

Water/micro substance reaches 100%

Washing composition example I X

A??????????B???????????C

C25AES????????18.0???????15.0????????14.0

LAS???????????5.8????????5.0?????????4.0

C810 amine 1.4 2.0-

NI24-7????????2.8????????2.0?????????3.0

Citric acid 2.5 3.0 3.0

Lipid acid 8.5 3.0 3.0

Enzyme 0.02 0.02 0.006

Boric acid 2.0 2.0 2.0

Ethoxylation four inferior second

Base five amine 0.9 1.0 1.0

The polymine ethoxy

Baseization thing 0.7-1.0

DETPMP????????????0.3?????????-???????????-

HEDP??????????????0.35????????-???????????-

Ethanol 1.0 3.0 3.0

1,2-propylene glycol 8.0 4.0 5.0

MEA???????????????9.8?????????2.0?????????2.0

NaCS??????????????2.0?????????-???????????-

Froth suppressor 0.25 0.01 0.01

Micro substance (spices, whitening agent etc.) and water reach 100%

Washing composition embodiment X

Below illustrate the detergent tablet of the present invention that is suitable for dishwasher.

A??????B???????C???????D???????E??????F

First phase

STPP??????5.0????9.6?????10.0????7.10????6.0????11.5

Silicate 1.7 0.67 1.6 1.0 1.0 2.4

SKS-6?????2.5????1.5?????-???????2.3?????2.25

Carbonate 5.00 2.74 3.5 3.59 4.10 5.25

HEDP??????0.25???0.18????0.18????0.28????0.28???0.28

PB1???????3.5????2.45????2.45????3.68????3.68???3.68

PAAC??????0.002??0.002???0.002???0.003???0.004??0.004

Citric acid-0.5-0.2--

Mierocrystalline cellulose--0.65 0.8--

Enzyme 0.01 0.008 0.008 0.02 0.01 0.01

Nonionogenic tenside 0.90 0.80 0.80 1.20 1.20 1.20

PEG4000??????????0.4?????0.36????0.26???0.38????0.39????0.39

BTA??????????????0.01????0.04????0.04???-???????0.06????0.06

Paraffin 0.16 0.17 0.10 0.15 0.15 0.15

Spices 0.02 0.02 0.02 0.013 0.013 0.013

Vitriol---0.502 0.05 2.838

Total amount19.65g 19.7g 19.77g 21.54g 19.43g 28.0g

Second phase

Enzyme 0.03 0.03 0.03 0.03 0.03 0.03

Enzyme 0.01 0.02 0.005 0.001 0.003 0.005 of the present invention

Citric acid and/or

Citrate trianion 0.3 0.20 0.3 0.3 0.20 0.3

Thionamic acid-0.30--0.30-

Two carbonate 0.92 0.25 0.45 1.09 0.30 0.45

Supercarbonate-0.55--0.55-

Silicate--0.64--0.64

CaCl ₂???????????-???????0.07????-??????-???????0.07????-

PEG400???????????0.15????-???????-??????-???????-???????-

PEG4000??????????0.08????0.06????0.06???0.06????0.06????0.06

Total amount2.0g 2.0g 2.0g 2.0g 2.0g 2.0g

Washing composition embodiment XI

Below illustrate the liquid detergent composition of the present invention that is suitable for dishwasher:

Embodiment	????A	????B	????C	????D	????E
Embodiment	????A	????B	????C	????D	????E	?KOH	????14.3	????14.3	????14.3	????11.4	????4.7
?H ₂SO ₄	????11.3	????11.3	????11.3	????9.00	????-	?KOH	????14.3	????14.3	????14.3	????11.4	????4.7
?H ₂SO ₄	????11.3	????11.3	????11.3	????9.00	????-	?STPP	????16.00	????16.00	????16.00	????20.00	????-
?SKTP	????-	????-	????-	????-	????30.00	?STPP	????16.00	????16.00	????16.00	????20.00	????-
?SKTP	????-	????-	????-	????-	????30.00	1, the 2-propylene glycol	????0.50	????0.50	????0.50	????0.5	????6.00
Boric acid	????3.00	????3.00	????3.00	????3.0	????4.00	1, the 2-propylene glycol	????0.50	????0.50	????0.50	????0.5	????6.00
Boric acid	????3.00	????3.00	????3.00	????3.0	????4.00	The coagel pre-composition	????24.40	????24.40	????24.40	????24.00	????24.40
?PVPVI	????0.02	????-	????-	????-	????-	The coagel pre-composition	????24.40	????24.40	????24.40	????24.00	????24.40
?PVPVI	????0.02	????-	????-	????-	????-	?SLF18	????1.0	????-	????1.0	????1.00	????-
?C ₁₆Amine oxide	????0.6	????0.6	????-	????2.00	????2.00	?SLF18	????1.0	????-	????1.0	????1.00	????-
?C ₁₆Amine oxide	????0.6	????0.6	????-	????2.00	????2.00	?ACNI	????0.3	????0.3	????-	????-	????3.00
?CaCl ₂	????0.04	????0.04	????0.04	????0.4	????0.4	?ACNI	????0.3	????0.3	????-	????-	????3.00
?CaCl ₂	????0.04	????0.04	????0.04	????0.4	????0.4	Sodium Benzoate	????0.6	????0.6	????0.6	????0.6	????0.6
Sanitas	????0.05	????0.05	????0.05	????0.05	????0.05	Sodium Benzoate	????0.6	????0.6	????0.6	????0.6	????0.6
Sanitas	????0.05	????0.05	????0.05	????0.05	????0.05	Enzyme	????0.06	????0.03	????0.03	????0.03	????0.03
Enzyme of the present invention	????0.03	????0.015	????0.02	????0.02	????0.01	Enzyme	????0.06	????0.03	????0.03	????0.03	????0.03
Enzyme of the present invention	????0.03	????0.015	????0.02	????0.02	????0.01	Water and micro substance	To 100%

????0-1 ? ? ????0-1-1	Utilize during the relevant statement of the microorganism of form-PCT/RO/134 (EASY) and preservation or other biomaterial (PCT detailed rules and regulations the 13rd 2) preparation	PCT-EASY version 2 .92 (01.01.2002 renewal)
????0-1 ? ? ????0-1-1		PCT-EASY version 2 .92 (01.01.2002 renewal)	????0-2	International application no	PCT/DK01/00574
????0-3	Applicant or procuratorial with reference to reel number	10171-WO	????0-2	International application no	PCT/DK01/00574

????1 ? ????1-1 ????1-2	List below with specification sheets in the microorganism or the relevant statement of other biomaterial of the preservation mentioned: the page or leaf row	? ? 3 23-27
????1 ? ????1-1 ????1-2		? ? 3 23-27	????1-3 ????1-3-1 ????1-3-2 ? ? ????1-3-3 ????1-3-4	Preservation organization names preservation mechanism address preservation date preserving number is identified in preservation	The microbial preservation center Mascheroder weg 1b of DSMZ-Germany, D-38124 Braunschweig, Germany (05.04.2001) DSMZ on April 5 calendar year 2001 14218
????1-4	Other statement	Do not have	????1-3 ????1-3-1 ????1-3-2 ? ? ????1-3-3 ????1-3-4
????1-4	Other statement	Do not have	????1-5	The designated state that statement is suitable for	All designated states
????1-6	These statements that provide separately of statement will be submitted to international office afterwards	Do not have	????1-5	The designated state that statement is suitable for	All designated states

????2 ? ????2-1 ????2-2	List below with specification sheets in the microorganism or the relevant statement of other biomaterial of the preservation mentioned: the page or leaf row	? ? 57 14-15
????2 ? ????2-1 ????2-2		? ? 57 14-15	????1-3 ????1-3-1 ????1-3-2 ? ? ????1-3-3 ????1-3-4	Preservation organization names preservation mechanism address preservation date preserving number is identified in preservation	The microbial preservation center Mascheroder weg 1b of DSMZ-Germany, D-38124 Braunschweig, Germany (03.05.2002) DSMZ on May 3rd, 2,002 14979
????1-4	Other statement	Do not have	????1-3 ????1-3-1 ????1-3-2 ? ? ????1-3-3 ????1-3-4
????1-4	Other statement	Do not have	????1-5	The designated state that statement is suitable for	All designated states
????1-6	These statements that provide separately of statement will be submitted to international office afterwards	Do not have	????1-5	The designated state that statement is suitable for	All designated states

Sequence table

＜110〉Novozymes A/S

＜120〉comprise the detergent composition of Bacillus subtilus pectate lyase

<130>10171.204-WO

<160>19

<170>PatentIn?version?3.1

<210>1

<211>1200

<212>DNA

＜213〉Bacillus subtilus (Bacillus subtilis)

<220>

<221>CDS

<222>(1)..(1200)

<223>

<400>1

gct?gat?tta?ggc?cac?cag?acg?tta?gaa?tca?aat?gat?ggc?tgg?ggc?gcg?????????48

Ala?Asp?Leu?Gly?His?Gln?Thr?Leu?Glu?Ser?Asn?Asp?Gly?Trp?Gly?Ala

1???????????????5???????????????????10??????????????????15

tac?tcg?acc?ggc?aca?aca?ggc?gga?tca?aaa?gct?tcg?tca?tcc?cac?gtg?????????96

Tyr?Ser?Thr?Gly?Thr?Thr?Gly?Gly?Ser?Lys?Ala?Ser?Ser?Ser?His?Val

20??????????????????25??????????????????30

tat?acc?gtc?agc?aac?aga?aac?cag?ctt?gtc?tcg?gca?tta?ggc?aag?gac????????144

Tyr?Thr?Val?Ser?Asn?Arg?Asn?Gln?Leu?Val?Ser?Ala?Leu?Gly?Lys?Asp

35??????????????????40??????????????????45

acc?aac?aca?acg?cca?aaa?atc?att?tat?att?aag?gga?acg?att?gac?atg????????192

Thr?Asn?Thr?Thr?Pro?Lys?Ile?Ile?Tyr?Ile?Lys?Gly?Thr?Ile?Asp?Met

50??????????????????55??????????????????60

aac?gtc?gat?gac?aat?ctg?aag?ccg?ctt?ggt?cta?aat?gat?tat?aaa?gat????????240

Asn?Val?Asp?Asp?Asn?Leu?Lys?Pro?Leu?Gly?Leu?Asn?Asp?Tyr?Lys?Asp

65??????????????????70??????????????????75??????????????????80

cca?gag?tac?gat?ttg?gac?aaa?tat?ttg?aaa?gcc?tat?gac?cct?agc?aca????????288

Pro?Glu?Tyr?Asp?Leu?Asp?Lys?Tyr?Leu?Lys?Ala?Tyr?Asp?Pro?Ser?Thr

85??????????????????90??????????????????95

tgg?ggc?aaa?aag?gag?ccg?tcg?ggg?aca?cta?gaa?gag?gcg?aga?gca?cga????????336

Trp?Gly?Lys?Lys?Glu?Pro?Ser?Gly?Thr?Leu?Glu?Glu?Ala?Arg?Ala?Arg

100?????????????????105?????????????????110

tct?cag?aaa?aat?caa?aaa?gca?cga?gtc?atg?gtg?gat?att?ccg?gca?aac????????384

Ser?Gln?Lys?Asn?Gln?Lys?Ala?Arg?Val?Met?Val?Asp?Ile?Pro?Ala?Asn

115?????????????????120?????????????????125

acg?acg?atc?gtc?ggt?tca?ggg?aca?aat?gcc?aaa?atc?gtg?ggc?gga?aat????????432

Thr?Thr?Ile?Val?Gly?Ser?Gly?Thr?Asn?Ala?Lys?Ile?Val?Gly?Gly?Asn

130?????????????????135?????????????????140

ttc?cag?atc?aag?agt?gat?aat?gtc?atc?atc?cgc?aac?atc?gaa?ttc?cag????????480

Phe?Gln?Ile?Lys?Ser?Asp?Asn?Val?Ile?Ile?Arg?Asn?Ile?Glu?Phe?Gln

145?????????????????150?????????????????155?????????????????160

gat?gct?tat?gat?tat?ttt?ccg?caa?tgg?gat?ccg?act?gac?ggc?agc?tca????????528

Asp?Ala?Tyr?Asp?Tyr?Phe?Pro?Gln?Trp?Asp?Pro?Thr?Asp?Gly?Ser?Ser

165?????????????????170?????????????????175

gga?aac?tgg?aac?tca?caa?tac?gac?aac?atc?aca?ata?aac?ggc?ggc?acg????????576

Gly?Asn?Trp?Asn?Ser?Gln?Tyr?Asp?Asn?Ile?Thr?Ile?Asn?Gly?Gly?Thr

180?????????????????185?????????????????190

cat?ata?tgg?att?gat?cat?tgt?aca?ttt?aat?gac?ggt?tcc?cgt?ccg?gac????????624

His?Ile?Trp?Ile?Asp?His?Cys?Thr?Phe?Asn?Asp?Gly?Ser?Arg?Pro?Asp

195?????????????????200?????????????????205

agc?aca?tcg?cca?aag?tat?ttc?ggc?aga?aaa?tat?cag?cac?cat?gac?ggc????????672

Ser?Thr?Ser?Pro?Lys?Tyr?Phe?Gly?Arg?Lys?Tyr?Gln?His?His?Asp?Gly

210?????????????????215?????????????????220

caa?acc?gat?gct?tct?aac?ggc?gct?aac?tat?atc?acg?atg?tct?tac?aac????????720

Gln?Thr?Asp?Ala?Ser?Asn?Gly?Ala?Asn?Tyr?Ile?Thr?Met?Ser?Tyr?Asn

225?????????????????230?????????????????235?????????????????240

tat?tat?cac?gat?cat?gat?aaa?agc?tcc?att?ttc?gga?tca?agc?gac?agc????????768

Tyr?Tyr?His?Asp?His?Asp?Lys?Ser?Ser?Ile?Phe?Gly?Ser?Ser?Asp?Ser

245?????????????????250?????????????????255

aaa?aca?tct?gat?gac?ggc?aaa?tta?aaa?atc?acg?ctc?cat?cat?aac?cgc????????816

Lys?Thr?Ser?Asp?Asp?Gly?Lys?Leu?Lys?Ile?Thr?Leu?His?His?Asn?Arg

260?????????????????265?????????????????270

tat?aaa?aat?atc?gtc?cag?cgc?gca?ccg?aga?gtc?cgc?ttc?ggg?cag?gtg????????864

Tyr?Lys?Asn?Ile?Val?Gln?Arg?Ala?Pro?Arg?Val?Arg?Phe?Gly?Gln?Val

275?????????????????280?????????????????285

cac?gtt?tac?aac?aac?tat?tat?gaa?ggc?agc?aca?agc?tcc?tcg?gat?tat????????912

His?Val?Tyr?Asn?Asn?Tyr?Tyr?Glu?Gly?Ser?Thr?Ser?Ser?Ser?Asp?Tyr

290?????????????????295?????????????????300

gcc?ttc?agc?tat?gcg?tgg?gga?atc?gga?aaa?tca?tct?aaa?atc?tac?gct????????960

Ala?Phe?Ser?Tyr?Ala?Trp?Gly?Ile?Gly?Lys?Ser?Ser?Lys?Ile?Tyr?Ala

305?????????????????310?????????????????315?????????????????320

caa?aac?aat?gtc?att?gac?gtg?cct?gga?ctg?tca?gcc?gct?aaa?acg?atc???????1008

Gln?Asn?Asn?Val?Ile?Asp?Val?Pro?Gly?Leu?Ser?Ala?Ala?Lys?Thr?Ile

325?????????????????330?????????????????335

agc?gta?ttc?agc?ggg?gga?acg?gct?tta?tat?gac?tca?ggc?aca?ttg?ctg???????1056

Ser?Val?Phe?Ser?Gly?Gly?Thr?Ala?Leu?Tyr?Asp?Ser?Gly?Thr?Leu?Leu

340?????????????????345?????????????????350

aat?ggc?acg?cag?atc?aac?gca?tcg?gct?gca?aac?ggg?ctg?agt?tct?tct???????1104

Asn?Gly?Thr?Gln?Ile?Asn?Ala?Ser?Ala?Ala?Asn?Gly?Leu?Ser?Ser?Ser

355?????????????????360?????????????????365

gtc?ggc?tgg?aca?ccg?tct?ctg?cac?ggc?aca?atc?gat?gct?tcc?gcg?cat???????1152

Val?Gly?Trp?Thr?Pro?Ser?Leu?His?Gly?Thr?Ile?Asp?Ala?Ser?Ala?His

370?????????????????375?????????????????380

gta?aaa?tcg?aat?gtt?ata?tct?caa?gcg?ggt?gcg?ggt?aaa?tta?aat?taa??????1200

Val?Lys?Ser?Asn?Val?Ile?Ser?Gln?Ala?Gly?Ala?Gly?Lys?Leu?Asn

385?????????????????390?????????????????395

<210>2

<211>399

<212>PRT

＜213〉Bacillus subtilus

<400>2

Ala?Asp?Leu?Gly?His?Gln?Thr?Leu?Glu?Ser?Asn?Asp?Gly?Trp?Gly?Ala

1???????????????5???????????????????10??????????????????15

Tyr?Ser?Thr?Gly?Thr?Thr?Gly?Gly?Ser?Lys?Ala?Ser?Ser?Ser?His?Val

20??????????????????25??????????????????30

Tyr?Thr?Val?Ser?Asn?Arg?Asn?Gln?Leu?Val?Ser?Ala?Leu?Gly?Lys?Asp

35??????????????????40??????????????????45

Thr?Asn?Thr?Thr?Pro?Lys?Ile?Ile?Tyr?Ile?Lys?Gly?Thr?Ile?Asp?Met

50??????????????????55??????????????????60

Asn?Val?Asp?Asp?Asn?Leu?Lys?Pro?Leu?Gly?Leu?Asn?Asp?Tyr?Lys?Asp

65??????????????????70??????????????????75??????????????????80

Pro?Glu?Tyr?Asp?Leu?Asp?Lys?Tyr?Leu?Lys?Ala?Tyr?Asp?Pro?Ser?Thr

85??????????????????90??????????????????95

Trp?Gly?Lys?Lys?Glu?Pro?Ser?Gly?Thr?Leu?Glu?Glu?Ala?Arg?Ala?Arg

100?????????????????105?????????????????110

Ser?Gln?Lys?Asn?Gln?Lys?Ala?Arg?Val?Met?Val?Asp?Ile?Pro?Ala?Asn

115?????????????????120?????????????????125

Thr?Thr?Ile?Val?Gly?Ser?Gly?Thr?Asn?Ala?Lys?Ile?Val?Gly?Gly?Asn

130?????????????????135?????????????????140

Phe?Gln?Ile?Lys?Ser?Asp?Asn?Val?Ile?Ile?Arg?Asn?Ile?Glu?Phe?Gln

145?????????????????150?????????????????155?????????????????160

Asp?Ala?Tyr?Asp?Tyr?Phe?Pro?Gln?Trp?Asp?Pro?Thr?Asp?Gly?Ser?Ser

165?????????????????170?????????????????175

Gly?Asn?Trp?Asn?Ser?Gln?Tyr?Asp?Asn?Ile?Thr?Ile?Asn?Gly?Gly?Thr

180?????????????????185?????????????????????????190

His?Ile?Trp?Ile?Asp?His?Cys?Thr?Phe?Asn?Asp?Gly?Ser?Arg?Pro?Asp

195?????????????????200?????????????????205

Ser?Thr?Ser?Pro?Lys?Tyr?Phe?Gly?Arg?Lys?Tyr?Gln?His?His?Asp?Gly

210?????????????????215?????????????????220

Gln?Thr?Asp?Ala?Ser?Asn?Gly?Ala?Asn?Tyr?Ile?Thr?Met?Ser?Tyr?Asn

225?????????????????230?????????????????235?????????????????240

Tyr?Tyr?His?Asp?His?Asp?Lys?Ser?Ser?Ile?Phe?Gly?Ser?Ser?Asp?Ser

245?????????????????250?????????????????255

Lys?Thr?Ser?Asp?Asp?Gly?Lys?Leu?Lys?Ile?Thr?Leu?His?His?Asn?Arg

260?????????????????265?????????????????270

Tyr?Lys?Asn?Ile?Val?Gln?Arg?Ala?Pro?Arg?Val?Arg?Phe?Gly?Gln?Val

275?????????????????280?????????????????285

His?Val?Tyr?Asn?Asn?Tyr?Tyr?Glu?Gly?Ser?Thr?Ser?Ser?Ser?Asp?Tyr

290?????????????????295?????????????????300

Ala?Phe?Ser?Tyr?Ala?Trp?Gly?Ile?Gly?Lys?Ser?Ser?Lys?Ile?Tyr?Ala

305?????????????????310?????????????????315?????????????????320

Gln?Asn?Asn?Val?Ile?Asp?Val?Pro?Gly?Leu?Ser?Ala?Ala?Lys?Thr?Ile

325?????????????????330?????????????????335

Ser?Val?Phe?Ser?Gly?Gly?Thr?Ala?Leu?Tyr?Asp?Ser?Gly?Thr?Leu?Leu

340?????????????????345?????????????????350

Asn?Gly?Thr?Gln?Ile?Asn?Ala?Ser?Ala?Ala?Asn?Gly?Leu?Ser?Ser?Ser

355?????????????????360?????????????????365

Val?Gly?Trp?Thr?Pro?Ser?Leu?His?Gly?Thr?Ile?Asp?Ala?Ser?Ala?His

370?????????????????375?????????????????380

Val?Lys?Ser?Asn?Val?Ile?Ser?Gln?Ala?Gly?Ala?Gly?Lys?Leu?Asn

385?????????????????390?????????????????395

<210>3

<211>6661

<212>DNA

＜213〉artificial sequence

<220>

＜223〉plasmid pMOL995

<400>3

gaattcctta?gtgctttcat?agattaaact?cacatcacgc?tttaaatcgc?ttattttaga?????60

ctttaaagac?ttgttttctt?caagcaactc?attataatca?tttacatttt?cattaaatcg?????120

ctctacaaga?ccactatatt?tttctttaac?ttgcccatgt?tctttactta?attttttata?????180

ttctctcgcc?atatcagtac?tcatgagatt?tctaacatgc?tgttttaacc?tatcgttatc?????240

tctcgcagca?gtcactaagt?ttttataatc?acgctccgat?ataacaacat?ttttggttgg?????300

tttcttttct?gttttcatta?tttcttttcc?caaaccaaac?atggactttt?cacccgttgg?????360

cacttcaaca?cttttcatgt?gtcgtttcgc?tggtacttct?aaatctgatt?taactttatc?????420

gctataagca?gtccattcat?cttttttaac?tgctaaattt?ttttctagaa?aatcaatctc?????480

tttttccaaa?gtttgttttt?taaatttagc?tgtctcaata?tgtttacggt?cagagccacg?????540

ttcaccacgc?ttcaactcaa?aaccctgttt?tttcatatgc?tcggggaatt?tatcttgtag?????600

ccataacagt?tcttgacgat?taaacacatt?ttttccttgc?agttttccat?cacgcatagg?????660

cacaacacct?aaatgcatgt?gaggggtttg?ctcatcatta?tgaactgttg?cataagcaat?????720

attttgcttg?ccatatcgtt?cggaaaataa?tttataactt?tcctcaaaaa?atcgtttttg?????780

ttctcctgga?tccagttgct?caaaaaaatc?tcggtcagat?gttactagca?actcatttac?????840

aagaacagca?tctttcctcg?tttttcttgt?acctgttttt?tgtgattcaa?taatttcttt?????900

gacacgttcg?ttgtaatcaa?tatttttatc?atttttcaaa?tcataatttt?cacgtgttcg?????960

ctcatggtca?atatcatcat?tcgttctact?ttttcgctct?ctttgattat?gaaattgcat????1020

gccttttagt?ccagctgatt?tcactttttg?cattctacaa?actgcataac?tcatatgtaa????1080

atcgctcctt?tttaggtggc?acaaatgtga?ggcattttcg?ctctttccgg?caaccacttc????1140

caagtaaagt?ataacacact?atactttata?ttcataaagt?gtgtgctctg?cgaggctgtc????1200

ggcagtgccg?accaaaacca?taaaaccttt?aagacctttc?ttttttttac?gagaaaaaag????1260

aaacaaaaaa?acctgccctc?tgccacctca?gcaaaggggg?gttttgctct?cgtgctcgtt????1320

taaaaatcag?caagggacag?gtagtatttt?ttgagaagat?cactcaaaaa?atctccacct????1380

ttaaaccctt?gccaattttt?attttgtccg?ttttgtctag?cttaccgaaa?gccagactca????1440

gcaagaataa?aatttttatt?gtctttcggt?tttctagtgt?aacggacaaa?accactcaaa????1500

ataaaaaaga?tacaagagag?gtctctcgta?tcttttattc?agcaatcgcg?cccgattgct????1560

gaacagatta?ataatagatt?ttagcttttt?atttgttgaa?aaaagctaat?caaattgttg????1620

tcgggatcaa?ttactgcaaa?gtctcgttca?tcccaccact?gatcttttaa?tgatgtattg????1680

gggtgcaaaa?tgcccaaagg?cttaatatgt?tgatataatt?catcaattcc?ctctacttca????1740

atgcggcaac?tagcagtacc?agcaataaac?gactccgcac?ctgtacaaac?cggtgaatca????1800

ttactacgag?agcgccagcc?ttcatcactt?gcctcccata?gatgaatccg?aacctcatta????1860

cacattagaa?ctgcgaatcc?atcttcatgg?tgaaccaaag?tgaaacctag?tttatcgcaa????1920

taaaaaccta?tactcttttt?aatatccccg?actggcaatg?ccggggcggc?cgacatacat????1980

tcgctttgcc?ccaccgggtc?cgtctgttat?taatgccgcc?aaacctgaat?ttgcaaccga????2040

gctgtcgcct?tcccttgtcc?agccgacaat?gtcatggtgg?tcgaaataat?catgctgtgc????2100

tccgtacgca?tactgttttc?tcgcttttaa?gatcggttca?attttgtgtt?tcaaggcagg????2160

aatttcgcgc?tgggagtctc?ctttcgtccc?gtacatatcc?ccgtagaaaa?cctgagggta????2220

tccagattcc?cttgtgagaa?taaaagcgta?agcaagcggc?ttaaaccatg?tttggacagt????2280

cgaccccttc?cctcttatcc?aacctttatt?gcttcaccca?aaccgaaacg?gaccctccat????2340

taacagagaa?attaccccat?ccgtctgcat?taattgtgac?ggtgcctgtc?ctatttccgg????2400

taatatctct?ccaaacttgt?cccgctttat?ttttccccac?atacatccat?ttgttaccac????2460

ctggaccatc?tgacataatg?gtggcaaggc?ctgaatttgg?atgggagcta?tttccctctc????2520

ttgtccaacc?gataatatca?tgatgatcaa?agtaatcatg?ctgcgtacca?taggcaaaag????2580

tttgacgtgc?ctgcagaaga?gggtctattt?tagatttcat?agccggaaca?ccatgggttg????2640

ggataccgta?gtaatcccca?taaaatacgg?aaggataacc?ttgttccctt?gtcagaacca????2700

atgcatatgc?aagtggttta?aaccattgtt?gaacaaagga?ttccaatgct?tccccgggct????2760

gagaatcatg?gttatcaaca?aaagtaacgg?catgtgttgg?atgtttttgc?accacagaac????2820

catttaaaat?atttctcata?tcataataac?caccgctatt?agatgcattg?tacaaattat????2880

agtggagagg?aacatcaaac?accgagtgat?tccaacttgt?tttattcaaa?tagttttcaa????2940

ttgcaccaag?gtcatttttc?caaaactcag?ccactgcaaa?cattggttta?cctgtggtgt????3000

tacgcacatg?tgtaagccaa?tctctcgtaa?agctatattt?tatatgtttc?actgcatcta????3060

ttctaaatcc?atcaaggttc?agtgtattcg?tataccacac?tccccagttt?ctaagttcat????3120

gtattacttc?tgggtgatcc?atatccacgt?ctgcatacat?aagatagtca?tagttgccat????3180

tctctgtatc?gacttcccag?tcccaggcct?tgcctgttcc?cctgaattta?tatattttgt????3240

tttgaagctg?gcgtgactga?tcccaatctg?tcccatcaaa?atgataccag?cgccacttaa????3300

agctggaatg?gttatttcct?cttccaggaa?aatcaaactt?tgtccacgct?tctattgcat????3360

actctcctga?ggtttcctgg?tttcggttgc?tccgattcac?ttctaccgca?tttacaattt????3420

ccgtaccatc?tgctccacct?ttatgattca?tgacgacatc?accatatacc?tgaatgccgt????3480

tattttttaa?agaggtcacc?gcagcctgta?gctggttgcg?tgttccatat?tttgtacgaa????3540

ccgtcccctt?ctggttaaac?tctccaagat?catataaatc?ataggctcca?taacctacat????3600

cattctggga?agtccccttc?catgcaggtg?ggatccatac?agctgttatc?cctttactct????3660

ttaagttagc?tgcgtcatcc?ctcaacctgt?tccaatgatt?cccgtcattt?ggcaaatacc????3720

attcgaaata?ttgcatcata?gtaccatttg?ttccattatg?atgggccgcg?gatgtttttg????3780

taatcggcaa?actgacaaat?aacagcgtgc?acataagcac?aagtctgaac?gaaactgtcc????3840

gctttcgttt?ttgaatcatg?tttcctctcc?ctctcatttt?cttatacaaa?ttatatttta????3900

catatcagta?aaataataac?aacccccctt?tattccttat?ttttacacag?cggacagtct????3960

ggacagcata?aaaaataccc?tgtctgatga?cagacaaggt?atttttatgg?tcttcttctt????4020

ttctcaaaca?atcgatccac?ttcttcagcc?aaatcatcag?tcatcaagag?ctcccgggat????4080

agactgtaac?attctcacgc?ataaaatccc?ctttcatttt?ctaatgtaaa?tctattacct????4140

tattattaat?tcaattcgct?cataattaat?cctttttctt?attacgcaaa?atggcccgat????4200

ttaagcacac?cctttattcc?gttaatgcgc?catgacagcc?atgataatta?ctaatactag????4260

gagaagttaa?taaatacgta?accaacatga?ttaacaatta?ttagaggtca?tcgttcaaaa????4320

tggtatgcgt?tttgacacat?ccactatata?tccgtgtcgt?tctgtccact?cctgaatccc????4380

attccagaaa?ttctctagcg?attccagaag?tttctcagag?tcggaaagtt?gaccagacat????4440

tacgaactgg?cacagatggt?cataacctga?aggaagatct?gattgcttaa?ctgcttcagt????4500

taagaccgaa?gcgctcgtcg?tataacagat?gcgatgatgc?agaccaatca?acatggcacc????4560

tgccattgct?acctgtacag?tcaaggatgg?tagaaatgtt?gtcggtcctt?gcacacgaat????4620

attacgccat?ttgcctgcat?attcaaacag?ctcttctacg?ataagggcac?aaatcgcatc????4680

gtggaacgtt?tgggcttcta?ccgatttagc?agtttgatac?actttctcta?agtatccacc????4740

tgaatcataa?atcggcaaaa?tagagaaaaa?ttgaccatgt?gtaagcggcc?aatctgattc????4800

cacctgagat?gcataatcta?gtagaatctc?ttcgctatca?aaattcactt?ccaccttcca????4860

ctcaccggtt?gtccattcat?ggctgaactc?tgcttcctct?gttgacatga?cacacatcat????4920

ctcaatatcc?gaatagggcc?catcagtctg?acgaccaaga?gagccataaa?caccaatagc????4980

cttaacatca?tccccatatt?tatccaatat?tcgttcctta?atttcatgaa?caatcttcat????5040

tctttcttct?ctagtcatta?ttattggtcc?attcactatt?ctcattccct?tttcagataa????5100

ttttagattt?gcttttctaa?ataagaatat?ttggagagca?ccgttcttat?tcagctatta????5160

ataactcgtc?ttcctaagca?tccttcaatc?cttttaataa?caattatagc?atctaatctt????5220

caacaaactg?gcccgtttgt?tgaactactc?tttaataaaa?taatttttcc?gttcccaatt????5280

ccacattgca?ataatagaaa?atccatcttc?atcggctttt?tcgtcatcat?ctgtatgaat????5340

caaatcgcct?tcttctgtgt?catcaaggtt?taatttttta?tgtatttctt?ttaacaaacc????5400

accataggag?attaaccttt?tacggtgtaa?accttcctcc?aaatcagaca?aacgtttcaa????5460

attcttttct?tcatcatcgg?tcataaaatc?cgtatccttt?acaggatatt?ttgcagtttc????5520

gtcaattgcc?gattgtatat?ccgatttata?tttatttttc?ggtcgaatca?tttgaacttt????5580

tacatttgga?tcatagtcta?atttcattgc?ctttttccaa?aattgaatcc?attgtttttg????5640

attcacgtag?ttttctgtat?tcttaaaata?agttggttcc?acacatacca?atacatgcat????5700

gtgctgatta?taagaattat?ctttattatt?tattgtcact?tccgttgcac?gcataaaacc????5760

aacaagattt?ttattaattt?ttttatattg?catcattcgg?cgaaatcctt?gagccatatc????5820

tgacaaactc?ttatttaatt?cttcgccatc?ataaacattt?ttaactgtta?atgtgagaaa????5880

caaccaacga?actgttggct?tttgtttaat?aacttcagca?acaacctttt?gtgactgaat????5940

gccatgtttc?attgctctcc?tccagttgca?cattggacaa?agcctggatt?tacaaaacca????6000

cactcgatac?aactttcttt?cgcctgtttc?acgattttgt?ttatactcta?atatttcagc????6060

acaatctttt?actctttcag?cctttttaaa?ttcaagaata?tgcagaagtt?caaagtaatc????6120

aacattagcg?attttctttt?ctctccatgg?tctcactttt?ccactttttg?tcttgtccac????6180

taaaaccctt?gatttttcat?ctgaataaat?gctactatta?ggacacataa?tattaaaaga????6240

aacccccatc?tatttagtta?tttgtttagt?cacttataac?tttaacagat?ggggtttttc????6300

tgtgcaacca?attttaaggg?ttttcaatac?tttaaaacac?atacatacca?acacttcaac????6360

gcacctttca?gcaactaaaa?taaaaatgac?gttatttcta?tatgtatcaa?gataagaaag????6420

aacaagttca?aaaccatcaa?aaaaagacac?cttttcaggt?gcttttttta?ttttataaac????6480

tcattccctg?atctcgactt?cgttcttttt?ttacctctcg?gttatgagtt?agttcaaatt????6540

cgttcttttt?aggttctaaa?tcgtgttttt?cttggaattg?tgctgtttta?tcctttacct????6600

tgtctacaaa?ccccttaaaa?acgtttttaa?aggcttttaa?gccgtctgta?cgttccttaa????6660

g????????????????????????????????????????????????????????????????????6661

<210>4

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer A

<400>4

cattctgcag?ccgcggcagc?tgatttaggc?caccagacg??????????????????????39

<210>5

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer B

<400>5

gtacctcgcg?agtcgacttc?ttaatttaat?ttacccgcac?ccgc?????????????????44

<210>6

<211>1200

<212>DNA

＜213〉Bacillus subtilus

<220>

<221>CDS

<222>(1)..(1200)

<223>

<400>6

gct?gat?tta?ggc?cac?cag?acg?ttg?gga?tcc?aat?gat?ggc?tgg?ggc?gcg?????????48

Ala?Asp?Leu?Gly?His?Gln?Thr?Leu?Gly?Ser?Asn?Asp?Gly?Trp?Gly?Ala

1???????????????5???????????????????10??????????????????15

tac?tcg?acc?ggc?acg?aca?ggc?gga?tca?aaa?gca?tcc?tcc?tca?aat?gtg????????96

Tyr?Ser?Thr?Gly?Thr?Thr?Gly?Gly?Ser?Lys?Ala?Ser?Ser?Ser?Asn?Val

20??????????????????25??????????????????30

tat?acc?gtc?agc?aac?aga?aac?cag?ctt?gtc?tcg?gca?tta?ggg?aag?gaa????????144

Tyr?Thr?Val?Ser?Asn?Arg?Asn?Gln?Leu?Val?Ser?Ala?Leu?Gly?Lys?Glu

35??????????????????40??????????????????45

acg?aac?aca?acg?cca?aaa?atc?att?tat?atc?aag?gga?acg?att?gac?atg????????192

Thr?Asn?Thr?Thr?Pro?Lys?Ile?Ile?Tyr?Ile?Lys?Gly?Thr?Ile?Asp?Met

50??????????????????55??????????????????60

aac?gtg?gat?gac?aat?ctg?aag?ccg?ctt?ggc?cta?aat?gac?tat?aaa?gat????????240

Asn?Val?Asp?Asp?Asn?Leu?Lys?Pro?Leu?Gly?Leu?Asn?Asp?Tyr?Lys?Asp

65??????????????????70??????????????????75??????????????????80

ccg?gag?tat?gat?ttg?gac?aaa?tat?ttg?aaa?gcc?tat?gat?cct?agc?aca????????288

Pro?Glu?Tyr?Asp?Leu?Asp?Lys?Tyr?Leu?Lys?Ala?Tyr?Asp?Pro?Ser?Thr

85??????????????????90??????????????????95

tgg?ggc?aaa?aaa?gag?ccg?tcg?gga?aca?caa?gaa?gaa?gcg?aga?gca?cgc????????336

Trp?Gly?Lys?Lys?Glu?Pro?Ser?Gly?Thr?Gln?Glu?Glu?Ala?Arg?Ala?Arg

100?????????????????105?????????????????110

tct?cag?aaa?aac?caa?aaa?gca?cgg?gtc?atg?gtg?gat?atc?cct?gca?aac????????384

Ser?Gln?Lys?Asn?Gln?Lys?Ala?Arg?Val?Met?Val?Asp?Ile?Pro?Ala?Asn

115?????????????????120?????????????????125

acg?acg?atc?gtc?ggt?tca?ggg?act?aac?gct?aaa?gtc?gtg?gga?gga?aac????????432

Thr?Thr?Ile?Val?Gly?Ser?Gly?Thr?Asn?Ala?Lys?Val?Val?Gly?Gly?Asn

130?????????????????135?????????????????140

ttc?caa?atc?aag?agt?gat?aac?gtc?att?att?cgc?aac?att?gaa?ttc?cag????????480

Phe?Gln?Ile?Lys?Ser?Asp?Asn?Val?Ile?Ile?Arg?Asn?Ile?Glu?Phe?Gln

145?????????????????150?????????????????155?????????????????160

gat?gcc?tat?gac?tat?ttt?ccg?caa?tgg?gat?ccg?act?gac?gga?agc?tca????????528

Asp?Ala?Tyr?Asp?Tyr?Phe?Pro?Gln?Trp?Asp?Pro?Thr?Asp?Gly?Ser?Ser

165?????????????????170?????????????????175

ggg?aac?tgg?aac?tca?caa?tac?gac?aac?atc?acg?ata?aac?ggc?ggc?aca????????576

Gly?Asn?Trp?Asn?Ser?Gln?Tyr?Asp?Asn?Ile?Thr?Ile?Asn?Gly?Gly?Thr

180?????????????????185?????????????????190

cac?atc?tgg?att?gat?cac?tgt?aca?ttt?aat?gac?ggt?tcg?cgt?ccg?gac????????624

His?Ile?Trp?Ile?Asp?His?Cys?Thr?Phe?Asn?Asp?Gly?Ser?Arg?Pro?Asp

195?????????????????200?????????????????205

agc?aca?tca?ccg?aaa?tat?tat?gga?aga?aaa?tat?cag?cac?cat?gac?ggc????????672

Ser?Thr?Ser?Pro?Lys?Tyr?Tyr?Gly?Arg?Lys?Tyr?Gln?His?His?Asp?Gly

210?????????????????215?????????????????220

caa?acg?gat?gct?tcc?aac?ggt?gct?aac?tat?atc?acg?atg?tcc?tac?aac????????720

Gln?Thr?Asp?Ala?Ser?Asn?Gly?Ala?Asn?Tyr?Ile?Thr?Met?Ser?Tyr?Asn

225?????????????????230?????????????????235?????????????????240

tat?tat?cac?gat?cat?gat?aaa?agc?tcc?att?ttc?gga?tca?agt?gac?agc????????768

Tyr?Tyr?His?Asp?His?Asp?Lys?Ser?Ser?Ile?Phe?Gly?Ser?Ser?Asp?Ser

245?????????????????250?????????????????255

aaa?acc?tcc?gat?gac?ggc?aaa?tta?aaa?att?acg?ctg?cat?cat?aac?cgc????????816

Lys?Thr?Ser?Asp?Asp?Gly?Lys?Leu?Lys?Ile?Thr?Leu?His?His?Asn?Arg

260?????????????????265?????????????????270

tat?aaa?aat?att?gtc?cag?cgc?gcg?ccg?aga?gtc?cgc?ttc?ggg?caa?gtg????????864

Tyr?Lys?Asn?Ile?Val?Gln?Arg?Ala?Pro?Arg?Val?Arg?Phe?Gly?Gln?Val

275?????????????????280?????????????????285

cac?gta?tac?aac?aac?tat?tat?gaa?gga?agc?aca?agc?tct?tca?agt?tat????????912

His?Val?Tyr?Asn?Asn?Tyr?Tyr?Glu?Gly?Ser?Thr?Ser?Ser?Ser?Ser?Tyr

290?????????????????295?????????????????300

cct?ttt?agc?tat?gca?tgg?gga?atc?gga?aag?tca?tct?aaa?atc?tat?gcc????????960

Pro?Phe?Ser?Tyr?Ala?Trp?Gly?Ile?Gly?Lys?Ser?Ser?Lys?Ile?Tyr?Ala

305?????????????????310?????????????????315?????????????????320

caa?aac?aat?gtc?att?gac?gta?ccg?gga?ctg?tca?gct?gct?aaa?acg?atc???????1008

Gln?Asn?Asn?Val?Ile?Asp?Val?Pro?Gly?Leu?Ser?Ala?Ala?Lys?Thr?Ile

325?????????????????330?????????????????335

agc?gta?ttc?agc?ggg?gga?acg?gct?tta?tat?gac?tcc?ggc?acg?ttg?ctg???????1056

Ser?Val?Phe?Ser?Gly?Gly?Thr?Ala?Leu?Tyr?Asp?Ser?Gly?Thr?Leu?Leu

340?????????????????345?????????????????350

aac?ggc?aca?cag?atc?aac?gca?tcg?gct?gca?aac?ggg?ctg?agc?tct?tct??????1104

Asn?Gly?Thr?Gln?Ile?Asn?Ala?Ser?Ala?Ala?Asn?Gly?Leu?Ser?Ser?Ser

355?????????????????360?????????????????365

gtc?ggc?tgg?acg?ccg?tct?ctg?cat?gga?tcg?att?gat?gct?tct?gct?aat???????1152

Val?Gly?Trp?Thr?Pro?Ser?Leu?His?Gly?Ser?Ile?Asp?Ala?Ser?Ala?Asn

370?????????????????375?????????????????380

gtg?aaa?tca?aat?gtt?ata?aat?caa?gcg?ggt?gcg?ggt?aaa?tta?aat?taa??????1200

Val?Lys?Ser?Asn?Val?Ile?Asn?Gln?Ala?Gly?Ala?Gly?Lys?Leu?Asn

385?????????????????390?????????????????395

<210>7

<211>399

<212>PRT

＜213〉Bacillus subtilus

<400>7

Ala?Asp?Leu?Gly?His?Gln?Thr?Leu?Gly?Ser?Asn?Asp?Gly?Trp?Gly?Ala

1???????????????5???????????????????10??????????????????15

Tyr?Ser?Thr?Gly?Thr?Thr?Gly?Gly?Ser?Lys?Ala?Ser?Ser?Ser?Asn?Val

20??????????????????25??????????????????30

Tyr?Thr?Val?Ser?Asn?Arg?Asn?Gln?Leu?Val?Ser?Ala?Leu?Gly?Lys?Glu

35??????????????????40??????????????????45

Thr?Asn?Thr?Thr?Pro?Lys?Ile?Ile?Tyr?Ile?Lys?Gly?Thr?Ile?Asp?Met

50??????????????????55??????????????????60

Asn?Val?Asp?Asp?Asn?Leu?Lys?Pro?Leu?Gly?Leu?Asn?Asp?Tyr?Lys?Asp

65??????????????????70??????????????????75??????????????????80

Pro?Glu?Tyr?Asp?Leu?Asp?Lys?Tyr?Leu?Lys?Ala?Tyr?Asp?Pro?Ser?Thr

85??????????????????90??????????????????95

Trp?Gly?Lys?Lys?Glu?Pro?Ser?Gly?Thr?Gln?Glu?Glu?Ala?Arg?Ala?Arg

100?????????????????105?????????????????110

Ser?Gln?Lys?Asn?Gln?Lys?Ala?Arg?Val?Met?Val?Asp?Ile?Pro?Ala?Asn

115?????????????????120?????????????????125

Thr?Thr?Ile?Val?Gly?Ser?Gly?Thr?Asn?Ala?Lys?Val?Val?Gly?Gly?Asn

130?????????????????135?????????????????140

Phe?Gln?Ile?Lys?Ser?Asp?Asn?Val?Ile?Ile?Arg?Asn?Ile?Glu?Phe?Gln

145?????????????????150?????????????????155?????????????????160

Asp?Ala?Tyr?Asp?Tyr?Phe?Pro?Gln?Trp?Asp?Pro?Thr?Asp?Gly?Ser?Ser

165?????????????????170?????????????????175

Gly?Asn?Trp?Asn?Ser?Gln?Tyr?Asp?Asn?Ile?Thr?Ile?Asn?Gly?Gly?Thr

180?????????????????185?????????????????190

His?Ile?Trp?Ile?Asp?His?Cys?Thr?Phe?Asn?Asp?Gly?Ser?Arg?Pro?Asp

195?????????????????200?????????????????205

Ser?Thr?Ser?Pro?Lys?Tyr?Tyr?Gly?Arg?Lys?Tyr?Gln?His?His?Asp?Gly

210?????????????????215?????????????????220

Gln?Thr?Asp?Ala?Ser?Asn?Gly?Ala?Asn?Tyr?Ile?Thr?Met?Ser?Tyr?Asn

225?????????????????230?????????????????235?????????????????240

Tyr?Tyr?His?Asp?His?Asp?Lys?Ser?Ser?Ile?Phe?Gly?Ser?Ser?Asp?Ser

245?????????????????250?????????????????255

Lys?Thr?Ser?Asp?Asp?Gly?Lys?Leu?Lys?Ile?Thr?Leu?His?His?Asn?Arg

260?????????????????265?????????????????270

Tyr?Lys?Asn?Ile?Val?Gln?Arg?Ala?Pro?Arg?Val?Arg?Phe?Gly?Gln?Val

275?????????????????280?????????????????285

His?Val?Tyr?Asn?Asn?Tyr?Tyr?Glu?Gly?Ser?Thr?Ser?Ser?Ser?Ser?Tyr

290?????????????????295?????????????????300

Pro?Phe?Ser?Tyr?Ala?Trp?Gly?Ile?Gly?Lys?Ser?Ser?Lys?Ile?Tyr?Ala

305?????????????????310?????????????????315?????????????????320

Gln?Asn?Asn?Val?Ile?Asp?Val?Pro?Gly?Leu?Ser?Ala?Ala?Lys?Thr?Ile

325?????????????????330?????????????????335

Ser?Val?Phe?Ser?Gly?Gly?Thr?Ala?Leu?Tyr?Asp?Ser?Gly?Thr?Leu?Leu

340?????????????????345?????????????????350

Asn?Gly?Thr?Gln?Ile?Asn?Ala?Ser?Ala?Ala?Asn?Gly?Leu?Ser?Ser?Ser

355?????????????????360?????????????????365

Val?Gly?Trp?Thr?Pro?Ser?Leu?His?Gly?Ser?Ile?Asp?Ala?Ser?Ala?Asn

370?????????????????375?????????????????380

Val?Lys?Ser?Asn?Val?Ile?Asn?Gln?Ala?Gly?Ala?Gly?Lys?Leu?Asn

385?????????????????390?????????????????395

<210>8

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer C

<400>8

gtcgccgggg?cggccgctat?caattggtaa?ctgtatctca??gc?????????????????????????42

<210>9

<211>64

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer D

<400>9

gtcgcccggg?agctctgatc?aggtaccaag?cttgtcgacc?tgcagaatga?ggcagcaaga???????60

agat????????????????????????????????????????????????????????????????????64

<210>10

<211>61

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer E

<400>10

gtcggcggcc?gctgatcacg?taccaagctt?gtcgacctgc?agaatgaggc?agcaagaaga????60

t????????????????????????????????????????????????????????????????????61

<210>11

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primers F

<400>11

gtcggagctc?tatcaattgg?taactgtatc?tcagc????????????????????????????????35

<210>12

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer G

<400>12

aacagctgat?cacgactgat?cttttagctt?ggcac???????????????????????????????35

<210>13

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer H

<400>13

aactgcagcc?gcggcacatc?ataatgggac?aaatggg??????????????????????????????37

<210>14

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer J

<400>14

cattctgcag?ccgcggcagc?tgatttaggc?caccagacg????????????????????????????39

<210>15

<211>46

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer K

<400>15

gtacctcgcg?agcggccgct?tcttaattta?atttacccgc?acccge????????????????????46

<210>16

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer L

<400>16

cattctgcag?ccgcggcagc?tgatttaggc?caccagacg????????????????????????????39

<210>17

<211>46

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer M

<400>17

gtacctcgcg?agcggccgct?tcttaattta?atttacccgc?acccgc????????????????????46

<210>18

<211>1200

<212>DNA

＜213〉Bacillus subtilus

<220>

<221>CDS

<222>(1)..(1200)

<223>

<400>18

gct?gat?tta?ggc?cac?cag?acg?tta?ggg?tca?aat?gat?ggc?tgg?ggc?gcg?????????48

Ala?Asp?Leu?Gly?His?Gln?Thr?Leu?Gly?Ser?Asn?Asp?Gly?Trp?Gly?Ala

1???????????????5???????????????????10??????????????????15

tac?tcg?acc?ggc?acg?aca?ggc?gga?tca?aaa?gca?tcg?tcc?tcg?aac?gtg????????96

Tyr?Ser?Thr?Gly?Thr?Thr?Gly?Gly?Ser?Lys?Ala?Ser?Ser?Ser?Asn?Val

20??????????????????25??????????????????30

tat?acc?gtc?agc?aac?aga?aac?caa?ctt?gtc?tct?gca?tta?ggc?aag?aaa????????144

Tyr?Thr?Val?Ser?Asn?Arg?Asn?Gln?Leu?Val?Ser?Ala?Leu?Gly?Lys?Lys

35??????????????????40??????????????????45

acg?gat?acg?acg?cct?aaa?atc?att?tac?atc?aag?ggt?gcg?att?gac?atg????????192

Thr?Asp?Thr?Thr?Pro?Lys?Ile?Ile?Tyr?Ile?Lys?Gly?Ala?Ile?Asp?Met

50??????????????????55??????????????????60

aat?gtt?gat?gac?aat?ctg?aag?ccg?ctt?gac?gca?gat?gat?tat?gct?gac????????240

Asn?Val?Asp?Asp?Asn?Leu?Lys?Pro?Leu?Asp?Ala?Asp?Asp?Tyr?Ala?Asp

65??????????????????70??????????????????75??????????????????80

cca?gag?tac?gat?ttg?aac?aaa?tat?tta?aaa?gct?tat?gat?cca?agc?aca????????288

Pro?Glu?Tyr?Asp?Leu?Asn?Lys?Tyr?Leu?Lys?Ala?Tyr?Asp?Pro?Ser?Thr

85??????????????????90??????????????????95

tgg?ggc?aaa?aaa?gag?cct?tcc?ggc?aca?cag?gaa?gaa?gcc?aga?gaa?cgt????????336

Trp?Gly?Lys?Lys?Glu?Pro?Ser?Gly?Thr?Gln?Glu?Glu?Ala?Arg?Glu?Arg

100?????????????????105?????????????????110

tct?caa?aaa?aac?caa?aaa?gca?aga?gtg?atg?gtt?gac?att?cct?gcg?aat????????384

Ser?Gln?Lys?Asn?Gln?Lys?Ala?Arg?Val?Met?Val?Asp?Ile?Pro?Ala?Asn

115?????????????????120?????????????????125

aca?acg?att?gtc?ggt?tca?ggg?aca?aat?gcc?aaa?atc?gtg?ggc?gga?aac???????432

Thr?Thr?Ile?Val?Gly?Ser?Gly?Thr?Asn?Ala?Lys?Ile?Val?Gly?Gly?Asn

130?????????????????135?????????????????140

ttc?caa?atc?aag?agt?gat?aat?gtc?atc?atc?cgc?aac?atc?gaa?ttc?caa???????480

Phe?Gln?Ile?Lys?Ser?Asp?Asn?Val?Ile?Ile?Arg?Asn?Ile?Glu?Phe?Gln

145?????????????????150?????????????????155?????????????????160

gat?gct?tat?gac?tat?ttt?ccg?caa?tgg?gac?ccg?act?gac?ggt?agc?tca????????528

Asp?Ala?Tyr?Asp?Tyr?Phe?Pro?Gln?Trp?Asp?Pro?Thr?Asp?Gly?Ser?Ser

165?????????????????170?????????????????175

gga?aac?tgg?aac?tca?caa?tat?gac?aac?atc?aca?ata?aac?ggc?ggc?acg???????576

Gly?Asn?Trp?Asn?Ser?Gln?Tyr?Asp?Asn?Ile?Thr?Ile?Asn?Gly?Gly?Thr

180?????????????????185?????????????????190

cat?ata?tgg?att?gac?cat?tgc?aca?ttt?aat?gac?gga?tcc?cgt?cct?gac????????624

His?Ile?Trp?Ile?Asp?His?Cys?Thr?Phe?Asn?Asp?Gly?Ser?Arg?Pro?Asp

195?????????????????200?????????????????205

agt?aca?tca?cca?aag?tat?tac?gga?aga?gaa?tat?cag?cat?cat?gac?ggc????????672

Ser?Thr?Ser?Pro?Lys?Tyr?Tyr?Gly?Arg?Glu?Tyr?Gln?His?His?Asp?Gly

210?????????????????215?????????????????220

caa?aca?gat?gct?tct?aac?ggc?gcc?aac?tat?atc?acg?atg?tct?tac?aac????????720

Gln?Thr?Asp?Ala?Ser?Asn?Gly?Ala?Asn?Tyr?Ile?Thr?Met?Ser?Tyr?Asn

225?????????????????230?????????????????235?????????????????240

tat?tat?cac?gat?cat?gat?aaa?agc?tcc?att?ttc?gga?tca?agc?gac?agc????????768

Tyr?Tyr?His?Asp?His?Asp?Lys?Ser?Ser?Ile?Phe?Gly?Ser?Ser?Asp?Ser

245?????????????????250?????????????????255

aaa?acc?tcc?gat?gat?ggc?aaa?tta?aaa?att?acg?ctt?cac?cat?aac?cgc????????816

Lys?Thr?Ser?Asp?Asp?Gly?Lys?Leu?Lys?Ile?Thr?Leu?His?His?Asn?Arg

260?????????????????265?????????????????270

tac?aaa?aat?atc?gtt?cag?cgc?gca?ccg?aga?gtc?cgc?ttc?ggg?caa?gtg????????864

Tyr?Lys?Asn?Ile?Val?Gln?Arg?Ala?Pro?Arg?Val?Arg?Phe?Gly?Gln?Val

275?????????????????280?????????????????285

cac?gta?tac?aac?aac?tat?tat?gaa?ggc?agc?aaa?agc?tca?tcc?gga?tat????????912

His?Val?Tyr?Asn?Asn?Tyr?Tyr?Glu?Gly?Ser?Lys?Ser?Ser?Ser?Gly?Tyr

290?????????????????295?????????????????300

gct?ttc?agt?tat?gca?tgg?gga?atc?ggc?aag?tca?tct?aaa?atc?tat?gct????????960

Ala?Phe?Ser?Tyr?Ala?Trp?Gly?Ile?Gly?Lys?Ser?Ser?Lys?Ile?Tyr?Ala

305?????????????????310?????????????????315?????????????????320

caa?aac?aat?gtc?att?gac?gta?ccg?gga?ctg?tca?gct?gag?aaa?aca?atc???????1008

Gln?Asn?Asn?Val?Ile?Asp?Val?Pro?Gly?Leu?Ser?Ala?Glu?Lys?Thr?Ile

325?????????????????330?????????????????335

agc?gtg?ttt?aaa?ggt?gga?acg?gct?tta?tat?gac?tca?ggc?aca?ttg?ctg???????1056

Ser?Val?Phe?Lys?Gly?Gly?Thr?Ala?Leu?Tyr?Asp?Ser?Gly?Thr?Leu?Leu

340?????????????????345?????????????????350

aat?ggc?acg?cgg?atc?agc?gca?tca?gct?gca?aac?ggg?ctg?agc?tct?tct??????1104

Asn?Gly?Thr?Arg?Ile?Ser?Ala?Ser?Ala?Ala?Asn?Gly?Leu?Ser?Ser?Ser

355?????????????????360?????????????????365

gtc?ggc?tgg?aca?cca?tct?ctc?cac?ggc?aca?atc?gat?gat?tcc?gcg?aat???????1152

Val?Gly?Trp?Thr?Pro?Ser?Leu?His?Gly?Thr?Ile?Asp?Asp?Ser?Ala?Asn

370?????????????????375?????????????????380

gtg?aaa?tcg?aat?gtt?ata?tcc?caa?gcg?ggt?gcg?ggt?aaa?tta?aat?taa???????1200

Val?Lys?Ser?Asn?Val?Ile?Ser?Gln?Ala?Gly?Ala?Gly?Lys?Leu?Asn

385?????????????????390?????????????????395

<210>19

<211>399

<212>PRT

＜213〉Bacillus subtilus

<400>19

Ala?Asp?Leu?Gly?His?Gln?Thr?Leu?Gly?Ser?Asn?Asp?Gly?Trp?Gly?Ala

1???????????????5???????????????????10??????????????????15

Tyr?Ser?Thr?Gly?Thr?Thr?Gly?Gly?Ser?Lys?Ala?Ser?Ser?Ser?Asn?Val

20??????????????????25??????????????????30

Tyr?Thr?Val?Ser?Asn?Arg?Asn?Gln?Leu?Val?Ser?Ala?Leu?Gly?Lys?Lys

35??????????????????40??????????????????45

Thr?Asp?Thr?Thr?Pro?Lys?Ile?Ile?Tyr?Ile?Lys?Gly?Ala?Ile?Asp?Met

50??????????????????55??????????????????60

Asn?Val?Asp?Asp?Asn?Leu?Lys?Pro?Leu?Asp?Ala?Asp?Asp?Tyr?Ala?Asp

65??????????????????70??????????????????75??????????????????80

Pro?Glu?Tyr?Asp?Leu?Asn?Lys?Tyr?Leu?Lys?Ala?Tyr?Asp?Pro?Ser?Thr

85??????????????????90??????????????????95

Trp?Gly?Lys?Lys?Glu?Pro?Ser?Gly?Thr?Gln?Glu?Glu?Ala?Arg?Glu?Arg

100?????????????????105?????????????????110

Ser?Gln?Lys?Asn?Gln?Lys?Ala?Arg?Val?Met?Val?Asp?Ile?Pro?Ala?Asn

115?????????????????120?????????????????125

Thr?Thr?Ile?Val?Gly?Ser?Gly?Thr?Asn?Ala?Lys?Ile?Val?Gly?Gly?Asn

130?????????????????135?????????????????140

Phe?Gln?Ile?Lys?Ser?Asp?Asn?Val?Ile?Ile?Arg?Asn?Ile?Glu?Phe?Gln

145?????????????????150?????????????????155?????????????????160

Asp?Ala?Tyr?Asp?Tyr?Phe?Pro?Gln?Trp?Asp?Pro?Thr?Asp?Gly?Ser?Ser

165?????????????????170?????????????????175

Gly?Asn?Trp?Asn?Ser?Gln?Tyr?Asp?Asn?Ile?Thr?Ile?Asn?Gly?Gly?Thr

180?????????????????185?????????????????190

His?Ile?Trp?Ile?Asp?His?Cys?Thr?Phe?Asn?Asp?Gly?Ser?Arg?Pro?Asp

195?????????????????200?????????????????205

Ser?Thr?Ser?Pro?Lys?Tyr?Tyr?Gly?Arg?Glu?Tyr?Gln?His?His?Asp?Gly

210?????????????????215?????????????????220

Gln?Thr?Asp?Ala?Ser?Asn?Gly?Ala?Asn?Tyr?Ile?Thr?Met?Ser?Tyr?Asn

225?????????????????230?????????????????235?????????????????240

Tyr?Tyr?His?Asp?His?Asp?Lys?Ser?Ser?Ile?Phe?Gly?Ser?Ser?Asp?Ser

245?????????????????250?????????????????255

Lys?Thr?Ser?Asp?Asp?Gly?Lys?Leu?Lys?Ile?Thr?Leu?His?His?Asn?Arg

260?????????????????265?????????????????270

Tyr?Lys?Asn?Ile?Val?Gln?Arg?Ala?Pro?Arg?Val?Arg?Phe?Gly?Gln?Val

275?????????????????280?????????????????285

His?Val?Tyr?Asn?Asn?Tyr?Tyr?Glu?Gly?Ser?Lys?Ser?Ser?Ser?Gly?Tyr

290?????????????????295?????????????????300

Ala?Phe?Ser?Tyr?Ala?Trp?Gly?Ile?Gly?Lys?Ser?Ser?Lys?Ile?Tyr?Ala

305?????????????????310?????????????????315?????????????????320

Gln?Asn?Asn?Val?Ile?Asp?Val?Pro?Gly?Leu?Ser?Ala?Glu?Lys?Thr?Ile

325?????????????????330?????????????????335

Ser?Val?Phe?Lys?Gly?Gly?Thr?Ala?Leu?Tyr?Asp?Ser?Gly?Thr?Leu?Leu

340?????????????????345?????????????????350

Asn?Gly?Thr?Arg?Ile?Ser?Ala?Ser?Ala?Ala?Asn?Gly?Leu?Ser?Ser?Ser

355?????????????????360?????????????????365

Val?Gly?Trp?Thr?Pro?Ser?Leu?His?Gly?Thr?Ile?Asp?Asp?Ser?Ala?Asn

370?????????????????375?????????????????380

Val?Lys?Ser?Asn?Val?Ile?Ser?Gln?Ala?Gly?Ala?Gly?Lys?Leu?Asn

385?????????????????390?????????????????395

Claims

1. detergent composition, it comprises tensio-active agent and pectate lyase (EC 4.2.2.2) or its stabilized variants, and wherein said pectate lyase is by the endogenous dna sequence encoding of Bacillus subtilus bacterial strain.

2. detergent composition as claimed in claim 1, wherein said polypeptide is by the dna sequence encoding that obtains from Bacillus subtilus DSM 14218.

3. detergent composition as claimed in claim 1, wherein said polypeptide is by the dna sequence encoding of the 1-1197 position that comprises SEQ IDNO:1.

4. detergent composition as claimed in claim 1, wherein said polypeptide are the stabilized variants of pectate lyase of dna sequence encoding that comprises the 1-1197 position of SEQ IDNO:1.

5. any one detergent composition of claim as described above, wherein pectate lyase calculates by the weight percentage that pure enzyme accounts for whole composition, and preferred 0.0005% to 0.5% with 0.0001% to 2%, more preferably 0.001% to 0.02% level exists.

6. any one detergent composition of claim as described above, it also comprises one or more and is selected from following enzyme: proteolytic enzyme, cellulase (endoglucanase), beta-glucanase, hemicellulase, lipase, peroxidase, laccase, α-Dian Fenmei, glucoamylase, at, polygalacturonase, reductase enzyme, oxydase, phenol oxidase, lignoenzyme, Starch debranching enzyme, pectate lyase, xyloglucanase enzymes, zytase, the pectin acetylase, polygalacturonase, the phammogalacturonane enzyme, pectin lyase, other mannase, pectin methylesterase, cellobiohydrolase, trans-glutaminases, with their mixture.

7. any one detergent composition of claim as described above; it also comprises SYNTHETIC OPTICAL WHITNER, transition metal complex, hydrogen peroxide releasing agent and the amino hexylyloxy of 4-[N-(nonanoyl) of the preferred rigid ligand of ring mostly]-combination of benzene sulfonic acid sodium salt and/or their mixture.

8. any one detergent composition of claim as described above, wherein tensio-active agent is an anion surfactant, its content is no more than 30% of detergent composition gross weight, and preferred 10% to 25%.

With any one detergent composition clean textile, dish or the crust of claim as described above to obtain the method for good clean-up performance.

As described above any one detergent composition of claim clean fabric and/or fabric decontaminating and/or fabric whiteness maintenance and/or fabric-softening and/or fabric color manifests and/or the dye for fabrics metastasis inhibition in purposes.

11. detergent composition the purposes in cleaning of hard surfaces such as floor, wall, bathroom tile and analogue any as claim 1-8.

12. detergent composition the purposes in hand washing and machine washing dish any as claim 1-8.

13. detergent composition the purposes in oral cavity and/or tooth application any as claim 1-8.

14. have the active polypeptide of pectate lyase (EC 4.2.2.2), it is one of following that this polypeptide is selected from:

(a) by the polypeptide of the dna sequence encoding of the 1-1197 position of SEQ ID NO:1;

(b) cell of the dna sequence dna of the 1-1197 position by will containing SEQ ID NO:1 is cultivated the polypeptide that produces under the condition that can express this dna sequence dna;

(c) (a) or the fixed point variant of polypeptide (b), this variant by change one, two, three, four or the five amino acid residue obtained stabilization for other amino-acid residue.

15. as the polypeptide of claim 14, this polypeptide obtains from maybe obtaining preferably to belong to the bacterial strain of Bacillus subtilus kind from bacterium preferred Bacillus subtilus DSM 14218.

16. as the polypeptide of claim 14, this polypeptide is isolating and does not contain homology impurity.

17. an enzyme preparation, it comprises enzyme and conventional weighting agent or the stablizer any one as claim 14-16.

18. as the goods of claim 17, it also comprises one or more and is selected from following enzyme: proteolytic enzyme, cellulase (endoglucanase), beta-glucanase, hemicellulase, lipase, peroxidase, laccase, α-Dian Fenmei, glucoamylase, at, polygalacturonase, reductase enzyme, oxydase, phenol oxidase, lignoenzyme, Starch debranching enzyme, pectate lyase, xyloglucanase enzymes, zytase, the pectin acetylase, polygalacturonase, the phammogalacturonane enzyme, pectin lyase, other mannase, pectin methylesterase, cellobiohydrolase, trans-glutaminases, with their mixture.

19. coding has the isolating polynucleotide molecule of the active polypeptide of pectate lyase, this polynucleotide molecule is hybridized with the double chain DNA probe of sex change under the moderate stringent condition, wherein this probe is selected from: comprise the dna probe of sequence shown in the 1-1197 position of SEQ ID NO:1 and comprise the dna probe of subsequence of the 1-1197 position of SEQ IDNO:1, this subsequence has the length at least about 100 base pairs.

20. expression vector, it comprises the following element that is operably connected: transcripting promoter; Be selected from coding that (a) comprise the nucleotide sequence shown in the Nucleotide of SEQ ID NO:1 1-1197 position and have the polynucleotide molecule of the active polypeptide of pectate lyase and (b) dna fragmentation of the degenerate core nucleotide sequence of (a); And transcription terminator.

21. imported the culturing cell of the expression vector of claim 20, this dna fragmentation encoded polypeptides of wherein said cell expressing.

Have the method for the active polypeptide of pectate lyase 22. produce, this method comprises that cultivation has imported the cell of the expression vector of claim 20, this dna fragmentation encoded polypeptides of described thus cell expressing; With this polypeptide of recovery.