CN1233286A - Alpha-amylase fused to cellulose binding domain, for starch degradation - Google Patents

Abstract

The invention relates to a starch conversion method wherein the starch substrate is treated in aqueous medium with an CBD/enzyme hybrid. Further, the invention also relates to an isolated DNA sequence encoding a stable CBD/enzyme hybrid, a DNA construct comprising said DNA sequence of the invention, an expression vector comprising the DNA sequence of the invention, and a CBD/enzyme hybrid.

Description

α-Dian Fenmei and cellulose binding domain merge to be used for starch degradation

Invention field

In particular, the present invention relates to carbohydrate-binding domain (" CBD ") and be used for industrial starch processing (it should be noted that and be used for sweeting agent, particularly contain the starch processing of the syrupy production (vide infra) of glucose and/or fructose) the application of this zymoid heterozygote, described class of enzymes specifically is an amylolytic enzyme, the α-Dian Fenmei that for example is used for so-called " starch liquefacation effect " processing (vide infra), wherein starch degradation (often being called " gelatinization ") is arrived less oligomerization and/or polysaccharide fragment, or process relevant being used for so-called " saccharification " amylopectin deutero-starch fragment is taken off the debranching factor (for example isoamylase or Starch debranching enzyme) that props up, " saccharification " course of processing (vide infra) is normally carried out after liquefaction stage.The present invention also relates to the heterozygote enzyme formed by CDB-joint-enzyme.

Background of invention

As mentioned above, the present invention is valuable especially in starch processing (starch transformation) field.The conventional starch converting process and the condition of liquefaction and/or saccharification technology are described in for example United States Patent (USP) 3,912,590 and EP252730 and EP063909.From the Starch Production sweeting agent

Syrupy " tradition " technology that contains glucose and/or fructose from Starch Production is made up of three successive enzymic process usually, and promptly liquefaction process is followed by saccharification process and (when being used to produce the syrup that contains fructose) isomerization process.During liquefaction process, usually between the pH5.5-6.2 and under 95-160 ℃ temperature, by α-Dian Fenmei [EC3.2.1.1; Termanyl for example ^TM(bacillus licheniformis alpha-amylase) can be from Novo Nordisk A/S, and Bagsvaerd, Denmark obtains] about 2 hours of effect and starch (the starch suspension form from water-bearing media begins) is degraded to dextrin (oligomerization of starch and polysaccharide fragment).In order to ensure best enzyme stability is arranged under these conditions, calcium (free calcium ions of about 40ppm) that usually will about 1mM joins in the starch suspension.

After liquefaction process, by adding glucoamylase (amyloglucosidase, EC3.2.1.3; AMG for example ^TM, obtain from Novo Nordisk A/S), and add debranching factor usually, for example isoamylase (EC3.2.1.68) or Starch debranching enzyme (EC3.2.1.41; Promozyme for example ^TM, obtain from Novo Nordisk A/S) and change dextrin into dextrose (D-glucose).Before this step, common pH with medium is reduced to and is lower than 4.5 (for example pH4.3), and keeps high temperature (more than 95 ℃), thereby makes the alpha-amylase activity sex change of liquification.Usually temperature is turned down 60 ℃ then, and added glucoamylase and debranching factor.Liquefaction process was carried out 24-72 hour.After finishing in the saccharification stage, the pH of medium is elevated in the scope of 6-8, preferably pH 7.5, and remove calcium ion by ion exchange chromatography.Can utilize immobilized " glucose isomerase " (xylose isomerase, EC5.3.1.5 then; Sweetzyme for example ^TM, obtain from NovoNordisk A/S) and change the syrup (dextrose syrup) that obtains into high fructose syrups.

To carry out some improvement may be necessary to being used for characteristic that starch transforms the enzyme of processing at present.With regard to starch liquefacation, can carry out at least 3 kinds of improvement to the α-Dian Fenmei of liquification, be summarized in hereinafter; Various improvement are regarded as unilaterally useful, might obtain better effect though they are used in combination (for example 1+2,1+3,2+3, or 1+2+3).Improve 1 and reduce the liquescency α-Dian Fenmei Ca ²⁺Dependency

Have suitable high stability in order to ensure the α-Dian Fenmei that is used for starch liquefacation at present, need to add free ca (calcium ion), but exist calcium ion can cause the activity of use therein glucose isomerase is produced strongly inhibited in the medium in isomery stage.Therefore be necessary the calcium ion content in the medium to be reduced to the free ca level that is lower than about 3-5ppm by means of the device operation (for example ion-exchange) of costliness, perhaps the mode with other is reduced to minimum with the restraining effect of calcium, and for example the magnesium ion of the bonded amount by will being enough to suitably " competitive cancellation " calcium ion and glucose isomerase after the saccharification stage joins in the medium.Can not carry out liquefaction process if do not add calcium ion yet, not remove calcium ion or its restraining effect is reduced to minimum, then can economize on the use of funds significantly thereby do not need subsequently costliness to remedy the device operation.

For this reason, need be at the free ca of lower concentration (＜stable and highly active Alpha-starch lytic enzyme in 40ppm).Preferably, the optimum pH of such enzyme is more preferably in the pH4.5-5.5 scope in the pH4.5-6.5 scope.Improving 2 reduces the formation of useless Maillard product

The formation degree of useless Maillard product depends on pH during liquefaction process.Low pH helps reducing the formation of Maillard product.Therefore wish and the pH of this process can be reduced to about pH4.5 from about pH6.0; Unfortunately, known all the heat-staple liquescency α-Dian Fenmei of the public are less stable when low pH value (being pH＜6.0), and its specific activity is lower usually.

For achieving the above object, need to obtain in the pH4.5-6.5 scope, relatively to stablize and preferably keep the Alpha-starch lytic enzyme of high specific acitivity.Improve of the influence reduction of 3 liquescency α-Dian Fenmei to the saccharification process

The someone reported (United States Patent (USP) 5234823) in the past, when carrying out saccharification with aspergillus niger glucoamylase and B.acidopullulyticus Starch debranching enzyme, if α-Dian Fenmei is not by inactivation before the saccharification stage, the remaining alpha-amylase activity that keeps after liquefaction process can cause the dextrose productive rate lower so.(referring to above) as mentioned above, usually by being reduced to 60 ℃ in temperature carrying out before the saccharification, 95 ℃ with pH regulator below pH4.5 and carry out this inactivation.

Reason to this negative impact of dextrose output is not made clear at present fully, and this is (Termanyl for example because employed liquescency alpha Amylase preparation but hypothesis is arranged ^TMProduct, for example Termanyl ^TM120L) by 1 of the hydrolysis amylopectin tapping point and both sides near, 4-α-glucoside bond and produce " limit dextrin " (it is the relatively poor substrate of B.acidopullulyticus Starch debranching enzyme) and cause.Glucoamylase causes producing the trisaccharide panose to the hydrolysis of these limit dextrins, and this sugar is only lentamente by the glucose starch enzymic hydrolysis.

It will be an important process modification that exploitation no longer is subjected to the heat-staple Alpha-starch lytic enzyme of this shortcoming puzzlement, because will no longer need independent deactivation step like this.

One of purpose of the present invention is to obtain the Alpha-starch lytic enzyme relevant with the starch liquefacation process to improve performance-for example by obtaining one or more improvement of summarizing above-pass through the affinity of this enzyme of change and starch substrates, makes modified enzyme contact tightr with this substrate.

Summary of the invention

One aspect of the present invention relates to the improved enzymatic means that is used for liquefying starch of the liquescency α-Dian Fenmei of using modified forms, and wherein said α-Dian Fenmei is connected on the aminoacid sequence that comprises carbohydrate-binding domain and (vide infra).

The present invention also relates to be used for the improved enzymatic means of liquefying starch, wherein, also handle with debranching factor except with the modified α-Dian Fenmei.Can modify this debranching factor by being connected on the aminoacid sequence that comprises carbohydrate-binding domain.

Similarly, also within the scope of the present invention be, the debranching factor (for example isoamylase or Starch debranching enzyme) through similar modification (being the CBD deutero-) form is used in estimation in saccharifying, amylopectin deutero-starch fragment (for example relevant with the saccharification step of the starch transition process of above-outlined) is taken off, the Zhi Xingneng reinforcement will be caused taking off, thereby the output of dextrose can be increased.Detailed Description Of The Invention

Therefore first aspect of the present invention relates to the method that is used for liquefying starch, wherein starch substrates is handled with modified enzyme (enzyme heterozygote) in water-bearing media, described modified enzyme comprises the α-Dian Fenmei aminoacid sequence that is connected (promptly covalently bound) with the aminoacid sequence that contains carbohydrate-binding domain (CBD).

The present invention also relates to be used for the improved enzymatic means of liquefying starch, wherein, also handle with debranching factor except with the modified α-Dian Fenmei.This debranching factor can obtain on the aminoacid sequence that comprise carbohydrate-binding domain modifying by being connected to.

Another aspect of the present invention relates to and is used for method that the starch that carried out liquefaction processing is carried out saccharification, wherein the reaction mixture after the liquefaction is handled with modified enzyme (enzyme heterozygote), and described modified enzyme comprises the aminoacid sequence of the amylopectin debranching factor (as isoamylase or Starch debranching enzyme) that is connected (promptly covalently bound) with the aminoacid sequence that contains carbohydrate-binding domain (CBD).

Should understand in the present invention the starch liquefacation process of describing and not comprise such as the destarch of textiles and handling, in described processing, from fabric or textiles, remove the starch (" slurry ") that is present in fabric or the textiles (be commonly referred to cellulosic or contain cellulosic fiber or textiles) by enzymic process.Carbohydrate-binding domain

Carbohydrate-binding domain (CBD) is preferential and many or oligosaccharides (carbohydrate) bonded polypeptid acid sequence, and it usually but be not definitely can only combine with the water-insoluble form (comprising crystal) of many or oligosaccharides.

Though described the CBD of many types in patent and scientific literature, wherein most of--many Mierocrystalline cellulose lyase (cellulase) that derive from them--are commonly called " cellulose binding domain "; Therefore typical cellulose binding domain is the CBD that appears in the cellulase.Similarly, other subclass of CBD should comprise for example chitin binding domains (appearing at the CBD in the chitinase usually), xylan structural domain (appearing at the CBD in the zytase usually), mannosans binding domains (appearing at the CBD in the mannase usually), starch binding domains (some amylolytic enzymes are some glucoamylase for example, or the CBD that for example may occur in the cyclodextrin glucanotrasferase enzyme (" CGT enzyme ")) and other.

Big polypeptide or proteinic integrated part that CBD is made up of 2 or a plurality of polypeptid acid sequences district, especially be present in the lytic enzyme, described lytic enzyme comprises a catalyst structure domain that contains the avtive spot that is useful on substrate hydrolysis and one usually and is used for and purpose carbohydrate substrate bonded carbohydrate-binding domain (CBD).Such enzyme can comprise more than one catalyst structure domain and one, two or three CBD, also optionally comprise one or more polypeptid acid sequence districts that CBD and described catalyst structure domain are coupled together, the zone of back one type is commonly called " joint ".The example that comprises the lytic enzyme of CBD has: cellulase, and zytase, mannase, arabinofuranosidase, acetylase and chitinase, some of them are mentioned hereinbefore.Also in algae, the CBD of non-water-disintegrable polysaccharide conjugated protein form that has for example found to derive from cellulase and zytase in red algae Porphyra purpurea is (referring to people such as P.Tomme, cellulose binding domain-classification and the characteristic in the enzymatic degradation of insolubility carbohydrate, John N.Saddler and Michael H.Penner (Eds), ACS can be serial, No.618 (1996)).But most of known CBD (by people such as P.Tomme (source is the same), classification and called after " cellulose binding domain ") derive from cellulase and zytase.

In the present invention, term " cellulose binding domain " is identical with implication in back one reference people's (source is the same) such as () P.Tomme, and therefore used herein writing a Chinese character in simplified form " CBD " often can be understood as broad sense (carbohydrate-binding domain) or understand to narrow sense (cellulose binding domain) in principle.People's such as P.Tomme reference is divided into 10 families (I-X) with " cellulose binding domain " more than 120 kinds, and they may have different functions or effect aspect the substrate bonding mechanism.Yet, estimate also to occur new family's representative and other CBD family in the future.

For the protein/polypeptide that wherein has CBD (for example enzyme, normally lytic enzyme), CBD may be positioned at N or C-terminal or be positioned at interior location.

That part that itself constitutes the polypeptide of CBD or protein (for example lytic enzyme) usually by about more than 30 and about amino-acid residue below 250 form.For example: describe according to people such as P.Tomme (source is the same), those CBD that list in family's I and classify are made up of 33-37 amino-acid residue, the CBD that lists in the II a of family and classify is made up of 95-108 amino-acid residue, the CBD that lists in family's VI and classify is made up of 85-92 amino-acid residue, and lists in family's VII and a kind of CBD (deriving from thermal fiber end spore bacterium) of classifying is made up of 240 amino-acid residues.Therefore, the molecular weight of aminoacid sequence that itself constitutes CBD and is usually less than about 35kD normally in about 4kD arrives the scope of about 40kD.The enzyme heterozygote

The recommendation (1992) of enzyme classification that relates in specification sheets of the present invention and claims number (EC number) and international biological chemistry and NK of molecular biology federation, academic press, 1992 unanimities.

Point out (referring to above) to a certain extent, modified herein enzyme (also being referred to as the enzyme heterozygote hereinafter) comprises those kinds of the aminoacid sequence that contains the amylolytic enzyme that is connected (promptly covalently bound) with the aminoacid sequence that comprises CBD [for example can be in the present invention α-Dian Fenmei (EC3.2.1.1), isoamylase (EC3.2.1.68) or Starch debranching enzyme (EC3.2.1.41)].

The purpose enzyme heterozygote that other relevant with starch degradation contains CBD comprises, for example contain dextran 1,4-α-maltose lytic enzyme (EC3.2.1.133), p-amylase (EC3.2.1.2), glucoamylase (EC3.2.1.3), or the heterozygote of the aminoacid sequence of new Starch debranching enzyme (EC3.2.1.135).

The enzyme heterozygote that contains CBD with and the detailed description of preparation and purifying be those skilled in that art known (referring to, WO90/00609 for example, WO94/24158 and WO95/16782, and people such as Greenwood, biotechnology and biotechnology 44 (1994), 1295-1305).They can be for example by DNA construct is transformed in the host cell (described DNA construct comprise by or not by joint and at least one fragment of DNA of the coding cellulose binding domain that is connected with the dna sequence dna of coding purpose enzyme), cultivate transformed host cells with the expression fusion gene.The recombinant products that is obtained (enzyme heterozygote) often is called " fused protein " in this area, can be described by following general formula: A-CBD-MR-X

In this general formula, A-CBD is N-end or the C-stub area that comprises an aminoacid sequence of carbohydrate-binding domain (CBD) itself at least.MR is region intermediate (" joint "), and X is the amino acid residue sequence by the coded polypeptide of dna sequence dna of the enzyme (or other protein) that is connected with CBD of coding.

A part or do not have (A-CBD is exactly CBD itself like this, does not promptly comprise other amino-acid residue except the amino-acid residue that constitutes CBD) perhaps can be the sequence (its function is the end extension of CBD itself) of one or more amino-acid residues.Joint (MR) can be a key, or comprises about 2 linking groups to the weak point of about 100 carbon atom, particularly 2-40 carbon atoms.But MR preferably has about 2 to about 100 amino-acid residues, more preferably 2-40 amino-acid residue, for example sequence of 2-15 amino-acid residue.

The X part can constitute the N-end or the C-stub area of whole enzyme heterozygote.

Therefore according to top described, the CBD in the enzyme heterozygote of described type obviously can be positioned at C-end, the N-end or inner of this enzyme heterozygote.Can be used for preparing the cellulase (cellulose enzyme gene) of CBD

The technology that is applicable to the plain enzyme gene of separated fiber is that those skilled in that art know.In the present invention, term " cellulase " but be meant that catalyse cellulose is degraded to glucose, cellobiose, the enzyme of procellose and/or other cell-oligosaccharide.

The preferred cellulase of the present invention (cellulase that promptly comprises preferred CBD) is a microbial cellulase, particularly bacterium or fungal cellulase.Endoglucanase (EC3.2.1.4), particularly single composition (reorganization) endoglucanase is preferred cellulase type.

The example of useful bacteria cellulose enzyme be derive from following group bacterium or can be by the cellulase that they produced: pseudomonas, genus bacillus, cellulomonas cartae, clostridium, Microspora, the thermobacillus of dwelling, Caldocellum and actinomycetes are streptomycete for example, Thermomonospora and Acidothemus, particularly come from following group: Pseudomonas cellulolyticus, bacillus lautus, Bacillus agaradherens, the muck cellulomonas cartae, the thermal fiber Zymomonas mobilis, Clostridium stercorarium, Microspora bispora, Termomonospora fusca, Termomonosporacellulolyticum and Acidothemus cellulolyticus.

Cellulase can be acidity, neutrality or alkali cellulose enzyme, promptly shows the enzyme of maximum decomposition fiber-reactive respectively in acidity, neutrality or alkaline range.

Useful cellulase is an acidic cellulase, fungi acidic cellulase preferably, it be derive from following each genus group fungi or can be by the cellulase that they produced: Trichoderma, Myrothecium, Aspergillus, Phanaerochaete, the mould genus of arteries and veins spore, Neocallimastix and Staphlosporonites.

Preferred useful acidic cellulase is to derive from following various fungi or can be by the acidic cellulase that they produced: viride, Trichoderma reesei, Trichodermalongibrachiatum, myrothecium verrucaria, aspergillus niger, aspergillus oryzae, Phanaerochaetechrysosporium, coarse arteries and veins spore is mould, Neocallimastix partriciarum and Botrytis cinerea.

Another kind of useful cellulase is neutrality or alkali cellulose enzyme, preferably neutrality of fungi or alkali cellulose enzyme, it derive from each of following group belong in fungi or can produce by them: Aspergillus, Penicillium, myceliophthora, Humicola, the rake Pseudomonas, fusarium, grape fringe enzyme belongs to, and the broom enzyme belongs to, Chaetomium, the mould genus of wart spore, Verticillium, Myrothecium, Papulospora, Gliocladium, Cephalosporium and Acremonium.

Preferred alkali cellulose enzyme be derive from following group various fungies or can be by the alkali cellulose enzyme that they produced: Humicola insolens, point sickle spore, thermophilic ruin the silk mould, little purple mould and cephalo are mould, preferably come from following group or the alkali cellulose enzyme that can produce by their: Humicola insolens DSM1800, point sickle spore DSM2672, thermophilic mould CBS117.65 of silk and the mould RYM-202 of cephalo of ruining.

Preferred cellulase is a kind of alkaline endo glucanase, it with at highly purified, derive from Humicola insolens DSM1800～43kD endoglucanase and the antibody that produces has immunoreactivity, perhaps it be this～derivative and the plain enzymic activity of display fibers of 43kD endoglucanase.

Other example of useful cellulase is the variant of the plain enzyme of the precursor fiber of fungi or bacterial origin (for example deriving from fungi Humicola, the plain enzyme of the precursor fiber of the bacterial strain of the kind of one of Trichoderma or fusarium).Can be used for preparing other protein (protein gene) of CBD

As already mentioned, other type lytic enzyme that comprises CBD for example has zytase, mannase, arabinofuranosidase, acetylase and chitinase.Also mentioned in the past, for example in some algae (as red algae Porphyra purpurea), also found the CBD of non-water-disintegrable polysaccharide binding proteins matter form.Can be with reference to people's such as P.Tomme (source is the same) the further detail knowledge of document detail about the source (organism belongs to and plants) of this CBD.Other CBD related to the present invention comprises the CBD that derives from glucoamylase (EC3.2.1.3) or derive from CGT enzyme (EC2.4.1.19).

CBD derived from this source also is applicable to the present invention usually.At this on the one hand, be applicable to and for example separate that the technology of xylanase gene, mannase gene, arabinofuranosidase gene, 0-acetyl esterase gene, chitinase gene (with other genes involved) is that those skilled in that art know.The separation of CBD

In order to separate for example cellulose binding domain of cellulase, can use several genetically engineered approach.A kind of method is to use Restriction Enzyme to remove the part of gene, then the gene one carrier segments frame that stays is as one man merged, to obtain coding for the special genes fragment proteinic mutator gene of brachymemma to some extent.Another kind method relates to the use exonuclease, and Bal31 for example is from 5 ' and the 3 ' end of DNA restricted breach systematicness disappearance Nucleotide internally externally or in the gene.These genetically deficient methods cause producing the mutator gene of the gene molecule that coding shortens, and the substrate of expression product that can estimate this mutator gene then is in conjunction with (for example cellulase in conjunction with) ability.The suitable substrates that is used to estimate binding ability comprises cellulosic material, for example Avicel ^TMAnd cotton fabric.Other method comprises that use can be from selectivity or the specific protease of the remainder cracking CBD (for example terminal CBD) of the polypeptide chain of target protein matter.

(referring to above) as already explained, in case identified the nucleotide sequence of coding substrate in conjunction with (carbohydrate combination) property zone, no matter be cDNA or chromosomal DNA, can operate so that its dna sequence dna with coding purpose enzyme is merged it by various modes so.The DNA of the dna fragmentation of the carbohydrate associativity of will encoding then aminoacid sequence and coding purpose enzyme by or do not couple together by joint.Can adopt the whole bag of tricks that the connection dna sequence dna that obtains is operated to obtain expression then.Preferred microbial expression host comprises some aspergillus (for example aspergillus niger or aspergillus oryzae), genus bacillus and the organism such as intestinal bacteria or cereuisiae fermentum.Amylolytic enzyme

The amylase (particularly α-Dian Fenmei) that is applicable to the basis of the CBD/ amylase heterozygote type of using as the present invention comprises the enzyme of bacterium and originated from fungus.In this, also comprise this diastatic mutant through chemistry or genetically engineered modified.Relevant α-Dian Fenmei comprises the α-Dian Fenmei that for example can derive from genus bacillus, particularly can derive from the α-Dian Fenmei of a special bacterial strain of Bacillus licheniformis of describing in detail among the GB1296839.Relevant commercially available amylase comprises Duramyl ^TM, Termamyl ^TM, Fungamyl ^TMAnd BAN ^TM(all can derive from NovoNordisk A/S, Bagsvaerd, Denmark), and Rapidase ^TMWith Maxamyl P ^TM(can derive from Gist-Brocades, Holland), Optitherm ^TM(can obtain) from Solvay, Spezym AA ^TMWith Spezyme Delta AA (can obtain) from Genencor, and Keistase ^TM(can obtain) from Daiwa.

Other amylase (particularly α-Dian Fenmei) that is suitable as the basis of the CBD/ amylase heterozygote type of using among the present invention comprises by BAN 1-35 the-terminal amino acid of (can from Novo Nordisk obtain) and have one or more following sudden changes: H156Y, A181T, N190F, A209V, the Termanyl of Q264S

The heterozygote α-Dian Fenmei that terminal 36-483 the C-end amino acid of C-of (can obtain from Novo Nordisk) formed; Or have one or more following sudden changes: I201E, D207H, E211Q, the Termamyl of H205S Or have one or more following sudden changes: H133Y, the Maxamyl of N188P, S ^TM(can obtain) from Gist-Brocades/Genencor.Starch or starch fragment debranching factor

Isoamylase: the isoamylase (EC3.2.1.68) that is suitable as the basis of the CBD/ isoamylase heterozygote type that the present invention uses comprises those of bacterial origin.The mutant that so also comprises this isoamylase through chemistry or genetically engineered modification.Relevant isoamylase comprises for example can be from pseudomonas (for example pseudomonas SMP1 or starch skin pseudomonas SB15), genus bacillus (for example bacillus amyloliquefaciens), the isoamylase that Flavobacterium or Cytophaga (molten bacillus) obtain.

Starch debranching enzyme: the Starch debranching enzyme (EC3.2.1.41) that is suitable as the basis of the CBD/ Starch debranching enzyme heterozygote type that the present invention uses comprises those of bacterial origin.The mutant that so also comprises this side chain powder enzyme through chemistry or genetically engineered modification.Relevant Starch debranching enzyme for example comprises can be from genus bacillus (B.acidopullulyticus for example; As Promozyme ^TM, can obtain from Novo Nordisk A/S) and the Starch debranching enzyme that obtains.Plasmid

The preparation of plasmid that can expressed fusion protein matter be those skilled in that art know (referring to, for example WO90/00609 and WO95/16782), described fused protein has the fragment deutero-aminoacid sequence from an above polypeptide.Expression cassette can be included in and be used for keeping episomal duplicating in the system at suitable cell host, perhaps can provide the expression cassette that does not have dubbing system, and it will be incorporated in the host genome this moment.Can be according to known technology, for example conversion, micro-injection or the like import to this DNA among the host.

In case this fusion gene is imported in the appropriate host, then can cultivate the host so that express this fusion gene.Usually, add in addition that fusion gene excretory signal sequence can be provided is more gratifying.The exemplary of useful fusion gene is: signal sequence--(former peptide)--, and carbohydrate-binding domain-joint-purpose enzyme, or signal sequence--(former peptide)-purpose enzyme-joint-carbohydrate-binding domain, its Central Plains peptide sequence contains 5-25 amino-acid residue usually.

Recombinant products can be glycosylated or nonglycosylated.The mensuration of Alpha-starch lytic enzyme activity (KNU)

Utilize yam starch as substrate, can measure the Alpha-starch degrading activity of enzyme or enzyme heterozygote.This method is based on the cracking (hydrolysis) of the yam starch of modification, and the sample with starch/enzyme or starch/heterozygote enzyme solution after the reaction mixes with iodine solution.Originally, form black slightly blue color, but between the burst times of starch, a little less than blue color becomes more and more, and change into red slightly brown gradually.The glass calibration standard of gained color with the band look compared.

1,000 Novo α-Dian Fenmei units (KNU) are defined as under standard conditions that (promptly at 37 ± 0.05 ℃, the 0.0003M calcium ion makes the amount of the enzyme (enzyme heterozygote) of 5.26 gram starch dry matter (MerckAmylum of solubility) dextrinizations under pH5.6).Be suitable for assessing the test condition of the usefulness of enzyme heterozygote in starch processing that contains CBD

The test condition (for example pH, temperature, calcium concn or the like condition) that is suitable for testing aforesaid CBD/ αDian Fenmei, CBD/ isoamylase or CBD/ Starch debranching enzyme heterozygote can be toply to transform processing and those conditions of describing with regard to industrial starch.Be suitable for measuring polytype enzyme (pH for example under various conditions, temperature, calcium concn or the like, the measuring method of the enzymic activity characteristic that depends on the enzyme heterozygote) is known in the art, described enzyme is suitable for being connected with CBD as herein described, and those of ordinary skill in the art can easily select to be suitable for being evaluated at the testing method of the enzyme usefulness of the enzyme heterozygote of application herein.

The present invention also relates to the encode DNA isolation sequence of heterozygote enzyme with amylolytic activity, this sequence comprises:

(a) dna sequence dna of coding amylolytic activity;

(b) dna sequence dna of coding CBD; With

(c) dna sequence dna or its fragment of joint sequence shown in the coding SEQ ID NO:21.

A FAQs that contains the heterozygote enzyme of the enzyme that connects by joint and CBD is that they are not very stable owing to there is joint.The inventor has been found that when utilizing joint shown in the SEQ IDNO:21 or its essential part, heterozygote is very stable.

DNA isolation sequence of the present invention coding usually has amylolytic activity such as alpha-amylase activity, especially genus bacillus alpha-amylase activity, particularly Termamyl

Active or its mutant, or the enzyme of one of the top amylolytic activity of mentioning in " amylolytic enzyme " part.CBD can be any CBD, for example at the described CBD of above-mentioned " carbohydrate-binding domain " part.In a preferred embodiment, CBD is the CBD of Bacillus agaradherens NCIMBNo.40482 alkali cellulose enzyme Cel5A or the CBD dimer of Clostridium stercorarium (NCIMB11754) XynA.

In specific embodiments of the present invention, this separated DNA sequence is the Termamyl that is encoded by plasmid pMB492 shown in the SEQ ID NO:19 -joint-Cel5A-CBD.

The present invention relates to the DNA construct that contains the DNA isolation sequence of the present invention that is operably connected with one or more control sequences on the other hand, and described control sequence can instruct the expression of this dna sequence dna in suitable expressive host.

Promotor can be any dna sequence dna that shows transcriptional activity in selected host cell, and it can get own coding and host cell homology or allogenic proteinic gene.The suitable promotor that can instruct the DNA of code book invention cellulolytic enzyme to transcribe in bacterial host cell for example has the promotor of bacstearothermophilus Fructus Hordei Germinatus ative starch enzyme gene, bacillus licheniformis alpha amylase gene, bacillus amyloliquefaciens BAN amylase gene, bacillus subtilis alkali proteinase gene or bacillus pumilus zytase or xylosidase gene etc., lambda particles phage P _ROr P _LPromotor, or intestinal bacteria lac, trp or tac promotor.

The example that is applicable to the promotor of yeast host cell comprises from Yeast sugar glycolysis gene (people (1980) journal of biological chemistry such as Hitzeman, 255:12073-12080; Alber and Kawasaki (1982) molecular application genetics magazine 1:419-434) or alcohol dehydrogenase gene [people (1982) such as Young, produce chemical preparations people such as (compile) Hollaender at microbiological genetic engineering, Plenum press, New York] promotor, or TPI1 (U.S. 4599311) or ADH2-4C (people (1983) such as Russell, nature, 304:652-654) promotor.

For CBD/ enzyme heterozygote being imported the Secretory Pathway of host cell, can in expression vector, provide a secretory signal sequence (also being known as leader sequence, preceding former sequence or presequence).Secretory signal sequence is that the dna sequence dna with correct reading frame and codase heterozygote links together.Secretory signal sequence is usually located at 5 ' end of the dna sequence dna of coding amylolytic enzyme.Secretory signal sequence can be usually the secretory signal sequence relevant with amylolytic enzyme, maybe can come the gene of the another kind of secretory protein of own coding.

In a preferred embodiment, expression vector of the present invention can contain and (the substantially the same secretory signal sequence of secretion signal encoding sequence of amylase gene of Bacillus licheniformis described in the WO86/05812.

And, can for example increase expression by series connection amplification technique (relating to list or double exchange) or by multiple copied technology (described in the U.S. 4959316 or WO91/09129).Perhaps, expression vector can comprise a temperature sensitive replication origin, for example described in the EP283075.

Be used for connecting respectively the encode dna sequence dna, promotor of cellulolytic enzyme and selectively terminator and/or secretory signal sequence, and the method that their insertions are contained in the suitable carrier that duplicates information needed is (referring to people (1989) such as for example Sambrook, ibid) well known to those skilled in the art.

The present invention also relates to contain DNA construct of the present invention, promotor and transcribe recombinant expression vector with the translation termination signal.

Another object of the present invention provides the host cell that contains DNA construct of the present invention.

The host cell of the present invention that desire imports DNA construct of the present invention or recombinant expression vector therein can be any cell that can produce amylolytic enzyme, comprises bacterium, yeast, fungi and higher eucaryotic cells.

The example that can produce the bacterial host cell of cellulolytic enzyme of the present invention when cultivating is a gram positive bacterium, as Bacillus strain, subtilis particularly, Bacillus licheniformis, bacillus lentus, bacillus brevis, bacstearothermophilus, Alkaliphilic bacillus, bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, bacillus lautus, bacillus megaterium, bacillus pumilus, the bacterial strain of bacillus thuringiensis or B.agaradherens, or streptomycete bacterial strain, the bacterial strain of particularly shallow Streptomyces glaucoviolaceus or mouse ash streptomycete, or gram negative bacterium such as intestinal bacteria.The conversion of bacterium can be by protoplast transformation or is utilized competent cell to carry out (for example, people such as Sambrook (1989), ibid) by known methods.

When at bacterium such as expression in escherichia coli CBD/ enzyme heterozygote, enzyme can be retained in the tenuigenin, normally as insoluble particle (being known as endosome), perhaps can be directed in the periplasmic space by the bacterium secretion sequence.In the previous case, lysing cell also reclaims particle and sex change, after this, and the folded fibre lytic enzyme again by dilution sex change reagent.Under one situation of back, with the content in the release periplasmic space and reclaim the heterozygote enzyme, promptly can reclaim the heterozygote enzyme from periplasmic space by smudge cells (for example ultrasonic wave or wait to ooze shock).

Then under the condition that allows cellulolytic enzyme to express, in suitable nutritional medium, cultivate aforesaid the conversion or the host cell of transfection, after this, promptly from culture, reclaim the cellulolytic enzyme of gained.

The substratum that is used for culturing cell can be any conventional substratum that is suitable for cultivating host cell, as minimum medium or contain the complex medium of suitable fill-in.Can obtain suitable substratum or can prepare from commercial supplier according to disclosed prescription (for example catalogue of American type culture collection).Then can be according to the type of purpose cellulolytic enzyme, reclaim the cellulolytic enzyme that cell produces by ordinary method from culture, described method comprises by centrifugal or filtration separates host cell from substratum, method precipitation supernatant liquor by salt (for example ammonium sulfate) or the protein component in the filtered liquid, and carry out purifying through multiple chromatography method such as ion exchange chromatography, gel permeation chromatography, affinity chromatography etc.

The invention still further relates to the method that is used to produce CBD/ enzyme heterozygote of the present invention, described method comprises that (a) cultivates Bacillus strain contains described polypeptide with generation supernatant liquor; (b) reclaim this polypeptide.

The invention still further relates to the method that is used to produce heterozygote enzyme of the present invention, described method comprises that (a) cultivates host cell helping to express under the condition of described polypeptide; (b) reclaim this polypeptide.

In two kinds of methods, all utilize methods known in the art culturing cell in being suitable for producing the nutritional medium of heterozygote enzyme.For example; can and allow under expression of polypeptides and/or the isolating condition in suitable substratum; by shake-flask culture, in laboratory or industrial fermentation jar, carry out small-scale or large scale fermentation (comprise successive, in batches, batch feeding or solid state fermentation) and culturing cell.In containing the proper nutrition substratum of carbon and nitrogenous source and inorganic salt, utilize methods known in the art cultivate (referring to, bacterium and zymic reference; Bennett, J.W. and LaSure, L., editor (1991), " the more polygene operation in the fungi ", the academic press, CA).Can obtain suitable substratum from commercial supplier, or can prepare according to disclosed composition (for example, in the catalogue of American type culture collection).If polypeptide is secreted in the nutritional medium, just can from substratum, directly reclaim polypeptide.If polypeptide is not secreted, then can from cell lysate, reclaim polypeptide.

Can utilize the method that is specific to the heterozygote enzyme known in the art to detect the heterozygote enzyme.These detection methods comprise the application of specific antibody, the formation of enzyme product, or the disappearance of enzyme substrates.For example, can utilize the enzyme test to determine the activity of enzyme.The method of determining amylolytic activity is known in the art, will be described below.

Can reclaim the heterozygote enzyme that obtains by methods known in the art.For example, can pass through ordinary method, include but not limited to centrifugal, filtration, extraction, spraying drying, evaporation or precipitation etc. and from nutritional medium, reclaim the heterozygote enzyme.Then, can pass through various chromatography methods, for example ion exchange chromatography, gel permeation chromatography, affinity chromatography etc. are further purified the heterozygote enzyme that has reclaimed.

Can pass through the whole bag of tricks known in the art, include but not limited to chromatography (for example ion-exchange, affine, hydrophobic, chromatofocusing and size exclusion chromatography), electrophoresis method (for example preparation property isoelectrofocusing (IEF), difference solvability (for example ammonium sulfate precipitation), or extraction (referring to, for example, protein purification (Janson and Ryden edit), VCH press, New York, 1989) purifying heterozygote enzyme of the present invention.

Relate to by the separation of separated DNA sequence encoding of the present invention and the CBD/ enzyme heterozygote, the particularly heterozygote shown in the SEQ ID NO:20 of purifying aspect last of the present invention.

Material and method

Material:

Enzyme and enzyme heterozygote:

Termamvl

-joint-CBD _EGV: Termamyl With fungi CBD from Humicola insolensEGV _EGVHeterozygote.In embodiment 9, narrated the building process of this heterozygote.

CBD _CenA-Termamyl CBD from muck cellulomonas cartae endoglucanase A (CenA) _CenAAnd Tcrmamyl Heterozygote via the joint connection.In embodiment 8, narrated the building process of this heterozygote.

Termamyl (obtaining) from Novo Nordisk A/S

Plasmid:

PDN1528 (S.J

People such as rgensen (1991), the bacteriology magazine, 173 volumes, p559-567)

pBluescriptKSII-(Stratagene,USA)

PDNl981 (P.L.J

Rgensen, C.K.Hansen, G.B.Poulsen and B.Diderichsen (1990), the vivo gene engineering: as the homologous recombination of the instrument of plasmid construction, gene, 96, p37-41)

PSJl678: in WO94/19454, narrate; PDN1981: by J People such as rgensen (1990), gene, 96:37-41) record.

Bacterial strain:

The genus bacillus ACl3 NCIMB 40482 (identical) of the dna sequence dna of the SEQ ID NO:1 of the endoglucanase of coding described in the EXAMPLE l below expressing with Bacillus agaradherensDSM8721

Coli strain: preparation can be used for intestinal bacteria SJ2 cell electroporation, coding for alpha acetolactate decarboxylase (a kind of excision enzyme from bacillus brevis) (people (1990) such as Diderichsen, the bacteriology magazine, 172:4315-4321), and be used to GenePulser from BIO-RAD ^TMElectroporation apparatus transforms according to the described electroporation that carries out of manufacturer.

Utilize the host strain of subtilis PL2306 as conversion reaction.This is a kind of cellulase negative strain, and it is to destroy (Diderichsen, B., the Wedsted that produces by importing one in the transcription unit of known subtilis cellulose enzyme gene in bacillus subtilis strain DN1885, U., Hedegaard, L., Jensen, B.R., Sj

Holm, the clone of the aldB of C. (1990) coding for alpha acetolactate decarboxylase (a kind of excision enzyme) from bacillus brevis.The bacteriology magazine, 172:4315-4321).Not only the cellulose enzyme gene of DN1885 is destroyed, and two protease-encoding genes are also destroyed, and these two proteinase genes are aprE (Stahl, M.L. and E.Ferrari, 1984, utilize the deletion mutantion in external source to substitute subtilis subtilisin structure gene.The bacteriology magazine, 158:411-418) and nprE gene (Yang, people such as M.Y., 1984, the clone of subtilis neutral protease gene and utilize cloned genes to produce the deletion mutantion in external source.The bacteriology magazine, 160:16-21).

Basically as carry out described in subtilis and other gram positive bacterium gene disruption (A.LSonenshein, J.A.Hoch and Richard Losick compile, U.S. microbiology association, 1993, p618).

Subtilis: utilize ToC46 (Diderichsen, B., Wedsted, U., Hedegaard, L., Jensen, B.R., Sj

Holm, the clone of the aldB of C. (1990) coding for alpha acetolactate decarboxylase (a kind of excision enzyme) from bacillus brevis.The bacteriology magazine, 172,4315-4321) as secondary expressive host, carry out competent cell preparation and conversion as mentioned above.

Solution/substratum/reagent

Waxy corn from Cerestar

W-Gum Cerestar (89%DS) GL 03406 lot number 624362

TY and LB agar (as Ausubel, " the contemporary molecular biology method " that people such as F.M. edit.John Wiley and Sohs are described in 1995).

SB:32 restrains Tryptones, 20 gram yeast extracts, and 5 gram NaCl and 5 milliliters of 1NNaOH are mixed to final volume in sterilized water be 1 liter.At 121 ℃ by high pressure steam sterilization 20 minutes and with solution sterilization.

10%Avicel: it is 1 liter that 100 Avicel (FLUKA, Switzerland) that restrain are mixed to final volume with sterilized water, by 121 ℃ of autoclaved 20 minutes 10%Avicel being sterilized.

Damping fluid: 0.05M potassiumphosphate, pH7.5

Method

DE measures

DE (dextrose equivalent) is defined as in the sample amount (being measured as dextrose equivalent) of the reductibility carbohydrate that the w/w% with the dissolved dry-matter represents.It is by neocuproine thermometrically (bioid academic year comments for Dygert, LiFloridana (1965), 368).The neocuproine testing principle is to add CuSO in sample ₄, reducing sugar reduction Cu ⁺⁺, and the neocuproine mixture that forms at 450 nano measurements.

General molecular biology method:

Utilize molecular biological standard method to carry out DNA operation and transform (people (1989) such as Sambrook, molecular cloning: laboratory manual, cold spring harbor laboratory, cold spring port, New York; Ausubel, people such as F.M. edit " contemporary molecular biology method ", John Wiley and Sons, 1995; Harwood, C.R., and Cutting, S.M. edit " molecular biology method that is used for genus bacillus ", John Wiley and Sons, 1990).

According to the enzyme in supplier's the specification sheets use DNA operation.

Cellulytic activity

Cellulytic activity can be expressed as utilizes carboxymethyl cellulose (CMC) as substrate, in the cellulase viscosity unit (CEVU) that pH9.0 determines.

(＜1% water remains on-20 ℃ of N with respect to the enzyme standard ₂In the gas; Major criterion is kept at-80 ℃) determine cellulase viscosity unit.The standard 17-1187 that uses is pH9.0 in the standard incubation conditions, Tris damping fluid 0.1M, and CMC Hercules 7 LFD substrates 33.3 grams per liters, 40.0 ℃ was 4400 CEVU/ grams under 30 minutes.

α-Dian Fenmei-Termamyl

Active

Referring to the Novo Nordisk analytical procedure AF 9/6 that can ask for.

Embodiment

The purpose that provides following embodiment is to those of ordinary skills' full disclosure and describes how to produce and use various constructs, carries out the whole bag of tricks of the present invention, does not plan to be used for limiting the inventor and is considered as their scope of invention.Unless point out in addition, each several part is all with weight fraction metering, and temperature is degree centigrade, and pressure is or near normal atmosphere.Guaranteed the accuracy (for example length of dna sequence dna, molecular weight, quantity, special component or the like) of used numeral as far as possible, but should consider to have some deviations.

Embodiment 1

The clone of Bacillus agaradherens endo glucanase gene

The preparation of genomic dna

Described in WO94/01532, propagation bacterial strain NCIMB40482 (identical) in the liquid medium within Bacillus agaradherens DSM 8721.After 30 ℃ and 300rpm cultivate 16 hours, harvested cell, by people such as Pitcher (1989), applied microbiology communication, the method isolation of genomic DNA of 8:151-156 narration.

Make up genomic library

Utilize restriction enzyme Sau3A part digested genomic dna, electrophoresis carries out the size fractionation separation on 0.7% sepharose.(people (1981) such as Dretzen comments, 112:295-298) bioid academic year to separate the fragment of size between 2-7kb to the DEAE cellulose paper by electrophoresis.The separated DNA fragment is connected on the pSJl678 plasmid DNA of BamH I digestion.

Pcr amplification

In order to obtain being connected to the endo glucanase gene on the pSJl678 carrier, will connect mixture and in PCR reaction, be used as template, contain in the described PCR reaction each 200mM various Nucleotide (dATP, dCTP, dGTP, dTTP), 2.5mM MgCl ₂, amplification high frequency high fidelity damping fluid, the amplification high frequency high fidelity PCR system enzyme mixture of 2.0 units and the following primer of each 300nM:

Primer 1 (#9555): 5 '-TCACAGATCCTC-GCGAATTGGTGCGGCCGCGTNGTNG-ARGARCAYGGNC-3 ' (SEQID No.3).

Primer 1 be designed to WO94/01532 in the degenerated primer of aminoacid sequence (Val-Val-Glu-Glu-His-Gly-Gln) (SEQ ID NO:4) coupling of the N-terminal aminoacid sequence that provides.Last amino acid is only provided by first Nucleotide of this codon C.C is a 3 ' Nucleotide of this primer.

In addition, comprise a Not I site to be used to clone purpose at 5 ' end, these Nucleotide are represented by underscoring.Primer 2 (#9029);

5′-CAGAGCAAGAGATTACGCGC-3′(SEQ?ID?NO:5).

Primer 2 is corresponding to the one section sequence that exists in the pSJ1678 carrier.

In Hans Landgraf THERMOCYCLER (HansLandgraf, Germany), carry out the PCR circulation according to following proposal:

1 * (94 ℃ 120 seconds);

10 * (94 ℃ 10 seconds, 55 ℃ 30 seconds, 72 ℃ 240 seconds);

30 * (94 ℃ 10 seconds; 55 ℃ 30 seconds; 72 ℃ 180 seconds; Each newly circulates in 72 ℃ increases by 20 second hold-time); With

1 * (72 ℃ 300 seconds).

In 0.7% sepharose, the PCR product is carried out gel-purified, cuts out relevant fragment (approximately 1.7kb) from gel by gel electrophoresis, and utilize QIAquick Gel extract test kit (Qiagen, USA), according to manufacturer's explanation purifying.At 50 μ l 10mMTris-HCl, the wash-out DNA of purifying among the pH8.5.

This DNA as the template that PCR increases again, is wherein still used primer same as described above, mixture and recycle scheme.

Gel electrophoresis in 0.7% sepharose and the PCR product is carried out gel-purified cuts out relevant fragment from gel, and utilizes QIAquick Gel to extract the test kit purifying.At 50 μ l 10mM Tris-HCl, the wash-out DNA of purifying among the pH8.5.

Utilize Not I and Hind III to digest the DNA of purifying, carry out gel-purified as mentioned above, and be connected to also carrier pBluescript II KS-through Not I and the digestion of Hind III (Stratagene, USA) in, utilize to connect mixture transformed into escherichia coli SJ2.

Contain penbritin (200 μ g/ml) and adding pair cell bed board on the LB agar plate of X-gal (5-bromo-4-chloro-3-indoles-α-D-galactopyranoside, 50 mcg/ml).The evaluation of positive colony and sign

Be coated with cell transformed containing penbritin (200 mcg/ml) and added on the LB agar plate of X-gal (5-bromo-4-chloro-3-indoles-α-D-galactopyranoside, 50 mcg/ml), and 37 ℃ of overnight incubation.Second day, breed by on fresh LB-penbritin agar plate, white colony being rule again, 37 ℃ of overnight incubation.After one day, with single colony lift of each clone in the liquid LB substratum that contains penbritin (200 mcg/ml), and at 37 ℃ in 250rpm jolting overnight incubation.

Utilize the little test kit of QIAgen plasmid purification from liquid culture, to extract plasmid.Utilize Not I and Hind III to digest 5 microlitre plasmid samples.By (NuSieve FMC) goes up gel electrophoresis and checks digest at 0.7% sepharose.The dna fragmentation that about 1.0kb occurs represents that this is a positive colony.The segmental nucleotide sequencing of cloned DNA

Utilize the terminal cycle sequencing test kit of Taq deoxidation (Perkin Elmer, USA) and following " oppositely " primer or " forward " primer the plasmid DNA through the Qiagen purifying is checked order:

Oppositely: 5 '-GTTTTCC-CAGTCACGAC-3 ' (SEQ ID No.6),

Forward: 5 '-GCGGATAACAATTTCACACAGG-3 ' (SEQ ID No.7).

Utilize APPlied Biosystems 373A automatic sequencer, according to manufacturer's explanation to dna sequencing.According to people such as Devereux (1984), nucleic acids research, the method for 12:387-395 is carried out the analysis of sequence data.

From this sequence, can design new primer be used to carry out inverse PCR [referring to people such as McPherson (volume), " PCR-practical approach "; 1991, IRL press].Genomic dna to bacterial strain NCIMB 40482 carries out inverse PCR

Isolation of genomic DNA as mentioned above.Utilize 2 milligrams of pure genomic dnas of EcoR I digestion.Heated 20 minutes and heat inactivation EcoR I at 65 ℃, carry out the phenol of DNA then: the chloroform extracting.Last ethanol sedimentation DNA, resuspending is in 20 milliliters of TE.

In the reaction mixture of 100 milliliters of T4-DNA ligase enzymes (Boehringer Mannheim, Germany) that contain T4 ligase enzyme damping fluid and 1 unit, utilize the T4 ligase enzyme to connect 1 milliliter of DNA through the digestion of EcoR I.After 18 hours, heat 10 fens Zhongli's heat inactivation ligase enzymes 14 ℃ of connections at 68 ℃.In order before inverse PCR, to make the genomic DNA fragment linearizing of cyclisation, in connecting mixture, add 10U BstE II (inside of the dna sequence dna that BstE II site obtains above being present in).

50 milliliters of connection mixtures through the digestion of BstE II are used as template in the PCR reaction, contain each Nucleotide (dATP, dCTP, dGTP and dTTP) of each 200mM in this PCR reaction, 2.5mM MgCl ₂, amplification high frequency high fidelity damping fluid, following each primer of 2.0 units amplification high frequency high fidelity PCR system enzyme mixture and each 300nM:

Primer 3 (#19719): 5 '-TGACCCGTACGGTCCGTGGG-3 ' (SEQ ID No.8) and

Primer 4 (#19720): 5 '-GGCTCTTGATTTTGTGTCCACC-3 ' (SEQ ID No.9).

In Hans Landgraf THERMOCYCLER (Hans Landgraf, Germany), carry out the PCR circulation according to following scheme:

1 * (94 ℃ 120 seconds)

10 * (94 ℃ 10 seconds; 55 ℃ 30 seconds; 72 ℃ 240 seconds);

30 * (94 ℃ 10 seconds; 55 ℃ 30 seconds; 72 ℃ 180 seconds, each new circulation all increased retention time 20 seconds at 72 ℃); With

1 * (72 ℃ 300 seconds).

Gel-purified PCR product in 0.7% sepharose cuts out relevant fragment (about 4-5kb) from gel, and utilizes QIAquick Gel to extract the test kit purifying.At 50 μ l 10mMTris-HCl, the wash-out DNA of purifying among the pH8.5.The nucleotide sequencing of inverse PCR dna fragmentation

Utilize terminal cycle sequencing test kit (Perkin Elmer, the U.S.) of Taq deoxidation and aforesaid primer 1,3 and 4, utilize the 373A of applying biological system automatic sequencer, according to manufacturer's explanation, to dna sequencing through the Qiagen purifying.Method according to people such as Devereux (1984, ibid) is carried out the analysis of sequence data.In order to clone the alkaline endo glucanase gene shown in SEQ ID NO:12, according to two new primers of the sequences Design that has obtained.They are #20887 (SEQ ID NO:10) and #100084 (SEQ ID NO:14), and are as described below.

Embodiment 2

In subtilis, express alkaline endo glucanase

By the nucleotide sequence among the PCR clone SEQ 1D NO:12 to be used for importing to expression plasmid pDN1981.

As described below, utilize following two primers that contain Nde I and Kpn I (Kpn I site is positioned at extension increasing sequence easily) restriction site that the 500ng genomic dna is carried out PCR, wherein restriction site can be used for the dna sequence dna of coding endoglucanase is imported among the pDN1981 so that express:

Primer 5 (#20887): 5 '-GTA GGC TCA GTC ATA TGT TAC ACA TTG AAA GGG GAG GAG AAT CATGAA AAA GAT AAC TAC TAT TTT TGT CG-3 ' (SEQ ID No.10) and

Primer 7 (#100084): 5 '-CCT CGC GAG GTA CCA GCG GCC GCG TAC CAC CAA TTA AGT ATG GTAC-3 ' (SEQID No.14)

Underscore Nucleotide correspondence in the primer 5 is in Nde I site, and the underscore Nucleotide in the primer 7 is the part in the Kpn I site that exists in the sequence.

Use Expand ^TMLength template PCR system (can be from Boehringer Mannheim, Germany obtains), utilization contains the damping fluid 1 of 10 times of dilutions and the various dNTP of each 200 μ M, and the mixture of the various primers of 2.5 unit enzyme mixtures (Boehringer Mannheim, Germany) and each 500pmole increases.

Utilize DNA thermo cycler (can be from Landgraf, Germany obtains) to carry out the PCR reaction.Earlier 94 ℃ of incubations one time 2 minutes, the Recycle design of following was 94 ℃ of sex change 10 seconds, 55 ℃ of annealing 30 seconds, and 68 ℃ were extended 4 minutes, and carried out 10 round-robin PCR.Carrying out Recycle design subsequently is 94 ℃ of sex change 10 seconds, 55 ℃ of 25 circulations of PCR (each round-robin time of extending all prolongs 20 seconds in 25 circulations) of annealing and extending in 30 seconds and 68 ℃ 3 minutes.

By 0.7% sepharose (NuSieve, FMC) in electrophoresis, utilize ReadyLoad100bp dna ladder scale standard (GibcoBRL, Denmark) as molecular weight marker, 10 μ l aliquots containigs of analysing amplified product.

After the PCR circulation, utilize QIAquick PCR post test kit (Qiagen, the U.S.), according to manufacturer's explanation purifying PCR fragment.At 50 μ l 10mM Tris-HCl, the wash-out DNA of purifying among the pH8.5 utilizes the digestion of Nde I and Kpn I and is connected among the pDN1981 that has digested.Utilize and connect mixture conversion subtilis PL2304.

Preparation competent cell and as [Yasbin R E, Wilson G A and Young F E as described in the people such as Yasbin; Conversion in the lysogenic strain of subtilis and transfection: the evidence of in competent cell, optionally inducing prophage; The bacteriology magazine, 1975,121 296-304] transform.Separate and test subtilis transformant

With the transformant bed board to containing 10 mg/ml kantlex, 0.4% glucose, 10mM KH ₂PO ₄On the LB agar plate of 0.1%AZCL HE-Mierocrystalline cellulose (Megazyme, Australia), and cultivated 18 hours at 37 ℃.There is the bacterium colony of blue circle to be accredited as the positive bacterium colony of endoglucanase on every side.

Cultivate each positive transformant at 10 milliliters of TY inoculation of mediums that contain the 10mg/ml kantlex.After cultivating 1 day under 37 ℃ and the 250rpm vibration, take out 50 milliliters of supernatant liquors.Thereby add 50 milliliters of supernatant liquors in the hole by puncture generation in the agar that contains the cellulosic LB agar plate of 0.1%AZCL HE-and identify endoglucanase activities.

After 16 hours, the indigo plant circle around the hole is represented the expression of endoglucanase in the subtilis 37 ℃ of cultivations.

Embodiment 3

Analyze the sequence of having cloned

The protein sequence that derives from the endo glucanase gene of having cloned shows that this is a kind of endoglucanase with following composition:

Amino-acid residue 1 to 26 corresponding to signal peptide; Amino-acid residue 27 to 326 constitutes real endoglucanase (with other glycosyl hydrolase homology of family 5); Amino-acid residue 327 to 354 corresponding to joint; Amino-acid residue 355 to 400 is corresponding to cellulose binding domain (as described in example 3 above); Amino-acid residue 401 to 416 corresponding to joint; And amino-acid residue 417 to 462 constitutes second cellulose binding domain [with first (at amino-acid residue 355 to 400) height homology].

It is 146.370 that molar extinction coefficient is defined as.Molecular weight is about 52 kilodaltons.

For the protein that does not have signal sequence, its molar extinction coefficient is defined as 146.370.Molecular weight is about 49 kilodaltons.

This enzyme does not have halfcystine, calculates the pI value by charged amino acid and is about 4.

Embodiment 4

Part Termamyl The subclone of sequence

The αDian Fenmei gene of pDN1528 being gone up coding carries out pcr amplification, so that import a BamH I site at 3 ' end of coding region.Followingly carry out PCR and clone.In the every kind of dNTP that has added each 200 μ M, 2.6 unit high frequency high fidelity

The high frequency high fidelity of following every kind of primer of amplification enzyme mixture and each 300pmol

Pcr amplification about 10 to 20ng plasmid pDN1528 in the PCR damping fluid (Boehringer Mannheim, Germany):

Primer 8, #5289

5′-GCT?TTA?CGC?CCG?ATT?GCT?GAC?GCT?G-3′(SEQ?ID?No.15)

Primer 9, #26748

5′-GCG?ATG?AGA?CGC?GCG?GCC?GCC?TAT?CTT?TGA?ACA?TAA?ATT?GAA?ACG

GAT?CCG-3′(SEQ?ID?No.16)

Restriction site BamH I underscoring.

Utilize DNA thermal cycler (Landgraf, Germany) to carry out the PCR reaction.Earlier 94 ℃ 2 minutes, 60 ℃ of 30 seconds and 72 ℃ of 45 seconds incubations are once, then according to following Recycle design: 94 ℃ of sex change 30 seconds, annealed 30 seconds for 60 ℃, extend 10 circulations carrying out PCR in 45 seconds at 72 ℃, be 94 ℃ of sex change 30 seconds then, 60 ℃ of 30 seconds and 72 ℃ carried out 20 circulations (extend step at each round-robin and all increase by 20 seconds) in 45 seconds.By (NuSieve, FMC) middle electrophoresis utilizes the 10 μ l aliquots containigs of ReadyLoad 100bp dna ladder scale standard (GibcoBRL, Denmark) as the analysing amplified product of molecular weight marker at 1.0% sepharose.

40 mul aliquots samples of the PCR product that produces are as mentioned above utilized QIAquickPCR purification kit (Qiagen, the U.S.), carry out purifying according to manufacturer's explanation.At 50 microlitre 10mM Tris-HCl, the wash-out DNA of purifying among the pH8.5.Utilize BamH I and Pst I to digest the 25 microlitres PCR fragment of purifying, and at agarose (the SeaPlaque GTG of 1.0% low gelling temperature, FMC) electrophoresis in the gel, from gel, cut out relevant fragment, utilize QIAquick Gel to extract test kit (Qiagen, the U.S.) and carry out purifying according to manufacturer's explanation.Then the separated DNA fragment is connected on the pBluescript II KS-of BamH I-Pst I digestion, utilizes to connect mixture transformed into escherichia coli SJ2.

The cell bed board is being contained penbritin (200 mcg/ml) and adding on the LB agar plate of X-gal (5-bromo-4-chloro-3-indoles-α-D-galactopyranoside, 50 mcg/ml), and 37 ℃ of overnight incubation.Second day, with white colony line again on fresh LB penbritin agar plate, and 37 ℃ of overnight incubation.Second day, with single colony lift in the liquid LB substratum that contains penbritin (200 mcg/ml) and at 37 ℃ in 250rpm jolting overnight incubation.

Utilize the little test kit of QIAgen plasmid purification (Qiagen, the U.S.), from liquid culture, extract plasmid according to manufacturer's explanation.Utilize Pst I and BamH I to digest 5 microlitre plasmid samples.(NuSieve FMC) goes up the electrophoretic examinations digest at 1.0% sepharose.To contain the segmental positive colony called after pMB335 of the Pst I-BamH I that comprises αDian Fenmei gene-part.Then, this plasmid is used to make up αDian Fenmei-CBD heterozygote.The amplification in vitro of the C-terminal CBD of joint and Bacillus agaradherens NCIMB 40482

In the every kind of dNTP that has added each 200 μ M, 2.6 unit high frequency high fidelity The high frequency high fidelity of following every kind of primer of amplification enzyme mixture and each 300pmol

About 100-200ng chromosomal DNA (as described in above-mentioned embodiment 1 to 3) that pcr amplification obtains from Bacillus agaradherens NCIMB40482 in the PCR damping fluid (BoeringerMannheim, Germany):

Primer 10, #110150A5 '-GCT GCA GGA TCC GTT TCA ATT TAT GTT CAA AGA TCT GAT CCA GATTCA GGA G-3 ' (SEQ ID No.17)

Primer 11, #1000845 '-CCT CGC GAG GTA CCA GCG GCC GCG TAC CAC CAA TTA AGT ATG GTAC-3 ' (SEQ ID NO.18)

Restriction site BamH I and Not I are marked by underscore.

Design of primers become the to increase C-terminal CBD of endoglucanase encoding gene of the Bacillusagaradherens NCIMB 40482 that narrates among joint and the top embodiment.

Utilize DNA thermal cycler (Landgraf. Germany) to carry out the PCR reaction.Earlier 94 ℃ 2 minutes, 60 ℃ of 30 seconds and 72 ℃ carried out one time incubation in 45 seconds, then carrying out Recycle design is 94 ℃ of sex change 30 minutes, annealed 30 seconds for 60 ℃, 72 ℃ of PCR10 circulations of extending 45 seconds, then 94 ℃ 30 seconds, 60 ℃ of 30 seconds and 72 ℃ carried out 20 circulations (extend step at each round-robin and increase by 20 seconds) in 45 seconds.By (NuSieve, FMC) middle electrophoresis utilizes Ready Load 100bp dna ladder scale standard (GibcoBRL, Denmark) as molecular weight marker, 10 μ l aliquots containigs of analysing amplified product at 1.5% sepharose.(PCR) clones by the polymerase chain reaction: the segmental subclone of PCR

Utilize QIAquick PCR purification kit (Qiagen, the U.S.),, the PCR product 40 mul aliquots samples that produce are as mentioned above carried out purifying according to manufacturer's explanation.At 50 microlitre 10mM Tris-HCl, the pH8.5 wash-out is the DNA of purifying.Utilize the Not I to digest the 25 microlitres PCR fragment of purifying, utilize the BamH I partly to digest again, agarose (SeaPlaque GTG at 1.5% low gelling temperature, FMC) electrophoresis in the gel, from gel, cut out relevant fragment, utilize QIAquick Gel to extract test kit (Qiagen, the U.S.), carry out purifying according to manufacturer's explanation.The separated DNA fragment is connected with the pMB335 that digests through BamH I-Not I then, with connecting mixture transformed into escherichia coli SJ2.The evaluation of positive colony and sign

On the LB agar plate that contains z (200 mcg/ml), be coated with cell, and 37 ℃ of overnight incubation.Second day, on fresh LB penbritin agar plate, bacterium colony is rule again, and cultivate incubation at 37 ℃.Second day, with single colony lift in the liquid LB substratum that contains (200 mcg/ml) and at 37 ℃ in 250rpm jolting overnight incubation.

Utilize the little test kit of QIAgen plasmid purification (Qiagen, the U.S.), from liquid culture, extract plasmid according to manufacturer's explanation.Utilize BamH I and Not I to digest 5 microlitre plasmid samples.By (NuSieve FMC) goes up the electrophoretic examinations digest at 1.5% sepharose.The onesize dna fragmentation of seeing in appearance and the pcr amplification shows that this is a positive colony.

The positive colony called after MBamyC5ANewlink of fusion constructs that will contain the CBD of αDian Fenmei gene and Bacillus agaradherens NCIMB No.40482 alkali cellulose enzyme Cel5A.Fusion constructs is cloned in the expression vector based on genus bacillus

The pDN1528 carrier contains the amyL gene of Bacillus licheniformis, and this gene is expressed in subtilis actively, causes producing the active α-Dian Fenmei in the present supernatant liquor.In order to express, the DNA of the encoding fusion protein that as above makes up is imported among the pDN1528.

By utilizing Sal I-Not I digestion pMBamyC5ANewlink and pDN1528, the purifying fragment, and 4.7kb pDN1528 Sal I-Not I fragment and 0.5kbpMBamyC5ANewlink Sal I-Not I fragment coupled together promptly reached top purpose.This has produced heterozygosis construct and the consistent syzygy of the frame of Termamyl gene.Sequence (SEQ ID NO:19) referring to pMB492.

The competent cell that mixture is used to transform PL2306 will be connected.Cell is coated on the LB agar plate that contains paraxin (6 mcg/ml), 0.4% glucose and 10mM potassium hydrogen phosphate, and 37 ℃ of overnight incubation.Second day, on fresh LBPG chloramphenicol agar flat board, bacterium colony is rule again, and 37 ℃ of overnight incubation.After one day, with each clone's single colony lift in the liquid LB substratum that contains paraxin (6 mcg/ml), and at 37 ℃ in 250rpm jolting overnight incubation.

Utilize the little test kit of QIAgen plasmid purification (Qiagen, the U.S.), according to manufacturer's explanation, extract plasmid from liquid culture, different is, before 37 ℃ of lysing cell 15 minutes, added 1 mg/ml egg albumen N,O-Diacetylmuramidase (Sigma, the U.S.) in the resuspension damping fluid.Utilize BamH I and Not I to digest 5 microlitre plasmid samples.By (NuSieve FMC) goes up the electrophoretic examinations digestion product at 1.5% sepharose.Occur showing that as the onesize dna fragmentation of seeing in the pcr amplification this is a positive colony.With a positive colony called after MB492.The expression of fused protein, secretion and functional analysis

To clone the MB492 (Termamyl that the Cel5A-joint of expression and Bacillus agaradherens-CBD merges ) under 37 ℃ and 250rpm, in the SB substratum, cultivated 20 hours.1 milliliter of acellular supernatant liquor is mixed with 200 microlitre 10%Avicel.Mixture was kept incubation 1 hour at 0 ℃.After this of CBD and Avicel combines, the Avicel that will contain CBD centrifugal 5 minutes in 5000g.Pellet resuspended in the SDS-PAGE of 100 microlitres damping fluid, was boiled 5 minutes at 95 ℃, centrifugal 5 minutes of 5000g, again with sample on the 25 μ l to 4-20% Laemmli Tris-Glycine, on the SDS-PAGE NOVEX gel (Novex, the U.S.).According to manufacturer's explanation, at Xcell ^TMElectrophoresis sample among the Mini-Cell (NOVEX, the U.S.).To all operations of gel, comprise and utilize Coomassie blue stain that decolouring and dry is all carried out according to manufacturer's explanation subsequently.

The appearance of a protein band of about 60 kilodaltons is illustrated in the Termamyl that has expressed coding among the plasmid pMB492 (SEQ ID NO:19) in the subtilis

-joint-CBD syzygy.SEQ ID NO:20 has provided the marking protein sequence of the fusion constructs of pMB492.

The purpose connector area of narrating in this embodiment is following particular sequence:

SDPDSGEPDPTPPSDPG(SEQ?ID?No.21)

Embodiment 5

Isolation of genomic DNA from muck clostridium NCIMB11754

In specific culture medium, cultivate muck clostridium NCIMB11754 in 60 ℃ of anaerobism as state-run industry and marine bacteria preservation company limited (Scotland) recommendation.Pass through centrifugal collecting cell.As people such as Pitcher (Pitcher, D.G., Saunders, N.A., Owen, R.J. (1989) utilizes guanidine thiocyanate rapid extraction bacterial genomes DNA.Applied microbiology is used communication, 8,151-156) described isolation of genomic DNA.The dimeric amplification in vitro of CBD of muck clostridium (NCIMB 11754) XynA is in the every kind of dNTP that has added each 200 μ M, 2.6 unit high frequency high fidelity

Extend the high frequency high fidelity of enzyme mixture and the following every kind of primer of each 300pmol

The genomic dna of the about 100-200ng of pcr amplification in the PCR damping fluid (BoehringerMannheim, Germany) (separating as mentioned above):

Primer 12, #1141355 '-GCT GCA GGA TCC GTT TCA ATT TAT GTT CAA AGA TCT CCA ACT CCTGCC CCA TCT CAA AGC-3 ' (SEQ ID NO.22)

Primer 13, #1101515 '-GCG ATG AGA CGC GCG GCC GCT ACT ACC AGT CAA CAT TAA CAG GACCTG AG-3 ' (SEQ ID NO.23)

Restriction site BamH I and Not I are marked by underscore.

The design primer is so that the DNA of the cellulose binding domain of amplification coding muck clostridium (NCIMB 11754) XynA according to registration number D13325, obtains this dna sequence dna from database GenBank.

Utilize the DNA thermal cycler to carry out PCR reaction (Landgraf. Germany).Earlier 94 ℃ 2 minutes, 60 ℃ 30 seconds, 72 ℃ were carried out one time incubation in 45 seconds, then utilize recycle scheme: 94 ℃ of sex change 30 seconds, annealed 30 seconds for 60 ℃, 72 ℃ are extended 10 circulations carrying out PCR in 45 seconds, and then 94 ℃ of sex change 30 seconds, 60 ℃ of 30 seconds and 72 ℃ carried out circulate for 20 times (each circulation all increases by 20 seconds in this extension step) in 45 seconds.By (NuSieve, FMC) middle electrophoresis utilizes the 10 mul aliquots samples of ReadyLoad 100bp dna ladder scale standard (GibcoBRL, Denmark) as the analysing amplified product of molecular weight marker at 1.0% sepharose.(PCR) clones by the polymerase chain reaction: the segmental subclone of PCR

Utilize QIAquick PCR purification kit (Qiagen, the U.S.),, the PCR product 40 mul aliquots samples that produce are as mentioned above carried out purifying according to manufacturer's explanation.At 50 microlitre 10mM Tris-HCl, the wash-out DNA of purifying among the pH8.5.Utilize BamH I and Eag I to digest the 25 microlitres PCR fragment of purifying, agarose (SeaPlaque GTG at 1.0% low gelling temperature, FMC) electrophoresis in the gel, from gel, cut out relevant fragment, utilize QIAquick Gel to extract test kit (Qiagen, the U.S.), carry out purifying according to manufacturer's explanation.Then the separated DNA fragment is connected with the pMB335 that digests through BamH I-Not I, with connecting mixture transformed into escherichia coli SJ2.

Then, carry out following step as mentioned above:

-identify and the sign positive colony.

-fusion constructs is cloned in the expression vector based on genus bacillus.

-expression, secretion and functional analysis fused protein.

A protein belt that occurs about 87 kilodaltons on the painted SDS-PAGE of coomassie is presented at that the expression of heterozygote is positive in the subtilis.

So, in subtilis clone MBXynCBD2, expressed heterozygote, this heterozygote is by dna sequence dna SEQ ID NO:24 coding, and this dna sequence dna can be translated into the protein sequence shown in the SEQ ID NO:25.

Embodiment 6

CBD _Cel5A-joint-Termamyl

Starch is processed us and has been studied CBD _Cel5A-joint-Termamyl

(be joint and the Termamyl shown in the SEQ ID NO:21 that pass through that makes up described in the embodiment 4

The Bacillusagaradherens NCIMB 40482 endoglucanase C-terminal CBD that connect) and Termamyl Compare, at pH6.0 and 40ppm Ca ²⁺In, whether its every microgram zymoprotein/gram dry-matter increases to the liquification of starch.

The oil bath of jolting is heated to 105 ℃.Prepare two kinds of starch suspensions and (contain 40ppm Ca ⁺⁺30%DS), utilize NaOH with pH regulator to 6.0.Respectively with CBD _Cel5A-joint-Termamyl

And Termamyl

With the suspension mixing.

From every kind of suspension, in respectively get 4 parts, every part 10 the gram.Place the Erlenmeyer flask that has screw cap with every part.Flask is placed 105 ℃ oil bath 8 minutes, placed 90 minutes at 95 ℃ then.

In oil bath, handle after 7 minutes 45 seconds, the thermostatted of oil bath is adjusted to 95.4 ℃, in oil bath, add 2 liters of oil under the room temperature.Start clock, sampling (every kind of suspension is got 1 flask) in 20,40,60 and 90 minutes.Add 2 1N HCl with inactivation amylase to each flask.

Then, determine the DE value, so that compare CBD as the function of time _Cel5A-joint-Termamyl

With Termamyl

The starch liquefacation ability of every microgram enzyme/gram DS.

Embodiment 7

Make up CBD _CenAExpression vector pCBDT001

To encode from the 103 residue CBD of muck cellulomonas cartae endoglucanase A (CenA) _CenAGene fragment clone in high-expression vector pTugEO7K3.By PCR at CBD _CenAThe suitable restriction site of 5 ' and 3 ' terminal importing of gene.Every kind of PCR mixture (50 milliliters of cumulative volumes) contains 25ng template DNA (pTZ18R-1.6cenA; Damude 1995, Ph D dissertation, University of British Columbia, Canada), 25-50pmol primer (5 ' SAENH and 3 ' SAENH), 10% dimethyl sulfoxide (DMSO), 0.4mM 2 '-deoxynucleoside-5 ' triphosphoric acid and 1 Vent of unit archaeal dna polymerase (New England's biology laboratory) in " Thermopol " damping fluid.94 ℃ of sex change 30 seconds then 55 ℃ of annealing 30 seconds and 72 ℃ of primer extensions 54 seconds, are so carried out 20 circulations continuously.Utilize oligonucleotide (5 ' SAENH) as primer, at CBD _CenA5 ' the terminal Spe I site (underscoring) that imports of gene fragment:

Primer 14

5′-AGGTCTACTAGTCCCGGCTGCCGCGTCGAC-3′(SEQ?ID?No.27)

Utilize oligonucleotide (3 ' SAENH) as primer, at CBD _CenA3 ' the terminal EcoR I (underscoring) that imports of sequence, Nhe I (black matrix) and Hind III (italic) restriction site:

Primer 15

5′-CCGATTAAAGCTTATTAGCTAGCACGGAATTCCGTGGGGCTGGTCGTCGGCAC-3′(SEQ?ID?No.28)

Utilize the 0.38kb PCR fragment that obtains of Spe I and Hind III digestion, and meet frame ground and be connected in the Nhe of pTugE07K3 I-Hind III site with the Cex leading peptide, wherein pTugEO7K3 digests with Nhe I and Hind III in advance and has removed CBD _CexGene fragment.Confirm last construct pCBDT001 by restricted cutting and pcr analysis.Make up CBD-Termamyl Heterozygosis expression vector pNAMK1.0.Contain Termamyl by standard method from the genus bacillus separation

The plasmid pSJ3368 of gene, it is a kind of derivative (S.J of pDN1528

People such as rgensen (1991), the bacteriology magazine, 173 volumes, p559-567).Import suitable restriction site to be used for cloning Termamyl again by PCR at escherichia coli vector pCBDT001 Gene fragment and structure heterozygote.Every kind of PCR reaction mixture (50 milliliters of cumulative volumes) contains 15ng template DNA (pSJ3368), 3pmol primer (PAM1 and PAM2), 2mM MgSO ₄, 10% dimethyl sulfoxide (DMSO), 0.4mM 2 '-deoxynucleoside-5 '-triphosphoric acid and 1 Vent of unit archaeal dna polymerase (New England's biology laboratory) in " Thermopol " damping fluid.Followingly carry out the circulation of 30 successive: 95 ℃ of sex change 1 minute, 55 ℃ of annealing 1 minute and 72 ℃ of primer extensions 1.54 minutes.

With oligonucleotide (PAM1) as primer, in a 5 ' terminal Nhe I (underscoring) and Nco I site of importing of gene:

Primer 16

5′-TCATGAGCCATGGCTAGCGCAAATCTTAATGGGACGCTGATG-3′(SEQ?ID?NO.29)

Utilize oligonucleotide (PAM2) as primer, in a 3 ' terminal Spe I (black matrix) and the Hind III site (underscoring) of importing of Termamyl gene:

Primer 17

5′-ATGACTAAGCTTAC?TTACTTAGTGATGGTGATGGTGATGACTAGTTCTTTGAACATAAATTGAAACCGA-3′(SEQ?ID?NO.30)

So also imported a His ₆Mark (italic) can be convenient to by immobilized metal affinity chromatography (IMAC) purifying heterozygote protein, and is close to before Hind III restriction sequence and has connect a terminator codon.The 1.5kb fragment of utilizing the digestion of Nhe I and Hind III to obtain, and and CBD _CenABe cloned into Nhe I-Hind III site of pCBDT001 with meeting frame, obtain pNAMK1.0.By utilizing Nhe I and Hind III restriction digestion and automatic sequencing to confirm construct.CBD _CenA-PTPTTP-Termamyl

Generation and purifying

Adding 1.25mM CaCl ₂Nutrient broth (TB with the 100mg/ml kantlex; Contain 12 gram peptones in 1 liter, 24 gram yeast extracts, 9.8 gram K ₂HPO ₄, 2.2 gram KH ₂PO ₄With 8 gram (10 milliliters) glycerine) (people such as Sambrook, 1989) (reference: Sambrook J., Fritsch, E.F.Maniatis, T. (1989) molecular cloning: laboratory manual, second edition, cold spring harbor laboratory publishes, cold spring port, New York) will contain 500 times of the intestinal bacteria JM101 overnight culture dilutions of plasmid pNAM1.0 in, and cultivate A at 30 ℃ ₆₀₀Be 3.0-5.0.By adding and propyl group-β-D-thiogalactoside (IPTG) is 0.1mM and induced protein generation to final concentration.Cultivated culture again 18 hours at 30 ℃, during this period of time CBD-Termamyl Heterozygote will leak in the substratum.By removing cell in centrifugal 10 minutes in 13000 * g at 4 ℃.Utilize 70% (NH ₄) ₂SO ₄And spend the night and from clarifying supernatant liquor precipitating proteins 4 ℃ of stirrings.By at the centrifugal recovery protein of 11000 * g, with resolution of precipitate at 20mM Tris-HCl, among the pH8.0 (binding buffer liquid).After 15000 * g is further centrifugal, with sample on the clarifying supernatant liquor to Ni ²⁺Agarose column (Novagen, Markham, ON) in.Utilize the 40mM imidazoles, 200mM NaCl, 20mM Tris-HCl, pH8.0 (lavation buffer solution) washing column.Imidazoles gradient (0-500mM) wash-out of utilization in containing the 20mM Tris-HCl damping fluid of 500mM NaCl be bonded protein.In each fraction, add CaCl immediately ₂To final concentration be 1mM, with stabilizing protein.Analyze each fraction last the reaching of SDS-PAGE (12%) by activity measurement.

SEQ ID NO:31 has shown the nucleotide sequence of NAM1.0, can translate into the aminoacid sequence shown in the SEQID NO:32.

Embodiment 8Termamyl-joint-from the fungi of Humicola insolens EGV.pNAMK6.1

CBD (Termamyl

Joint-CBD _EGV) the Termamyl carrier NAM2.0 of CBD C-terminal: every kind of PCR reaction mixture (50 milliliters of cumulative volumes) contains 15ng template DNA (pSJ3368), 3pmol primer (5Term2 and 3Term2), 2mM MgSO ₄, 10% dimethyl sulfoxide (DMSO), 0.4mM 2 '-deoxynucleoside-5 '-triphosphoric acid and 1 Vent of unit archaeal dna polymerase (New England's biology laboratory) in " Thermopol " damping fluid.Followingly carry out 30 continuously circulations: 95 ℃ of sex change 1 minute, 55 ℃ of annealing 1 minute and 72 ℃ of primer extensions 1.54 minutes.

With oligonucleotide (5Term2) as primer, 5 ' terminal Nhe I (underscoring) and EcoR I (black matrix) site of importing at the Termamyl gene: primer 185 '-CATATGGCTAGCGAATTCGCAAATCTTAATGGGACGCTG-3 ' (SEQ ID NO.33) utilizes oligonucleotide (3Term2) as primer, at Termamyl 3 ' the terminal Stu I (underscoring) that imports of gene, Spe I (black matrix) and Hind III (italic) site:

Primer 195 '-AAGCTTACTAGTAGGCCTTCTTTGAACATAAATT GAAA-3 ' (SEQ ID NO.34)

Confirm construct by restriction digestion and automatic sequencing.

Fungi CBD carrier:

By will be from the coding CBD of Humicola insolens EGV (WO91/17243) _EGVGene fragment clone in pTugEO7K3, obtain pCBDT006.By PCR at CBD _EGVThe suitable restriction site of 5 ' and 3 ' terminal importing of gene.Every kind of PCR mixture (50 milliliters of cumulative volumes) contains the 25ng template DNA, 25-50pmol primer (N137 and NIPTcs), 10% dimethyl sulfoxide (DMSO), 0.4mM 2 '-deoxynucleoside-5 '-triphosphoric acid and 1 Vent of unit archaeal dna polymerase (New England's biology laboratory) in " Thermopol " damping fluid.Carry out the circulation of 20 successive by following: 96 ℃ of sex change 45 seconds, 50 ℃ of annealing 60 seconds and 72 ℃ of primer extensions 35 seconds.Extended 90 seconds at 72 ℃ last circulation back.

Utilize oligonucleotide (5CBDT6) as primer, 3 ' end in the CBDEGV sequence, at manual splice (lowercase, italic) imports Nhe I (underscoring), EcoR I (black matrix before, underscoring), Stu I (black matrix) restriction site, import Spe I (italic in the joint back, underscoring) and Eco47 III (small letter, black matrix) site: primer 2 05 '-CCATGGGCTAGCCCTGAATTCAGGCCTccaacccccACTAGTCCGagcgctCCCAG CGGCTGCACTGCTG-3 ' (SEQ ID No.35)

Utilize oligonucleotide (3CBDT6) as primer, at CBD _EGV3 ' terminal Hind III (underscoring) restriction site that imports of sequence.Primer 21

5′-AGCCTAAGCTTACAGGCACTGATGGTACCAGT-3′(SEQ?ID?No.36)

Utilize the 0.18kb PCR fragment that obtains of Nhe I and Hind III digestion, and it and Cex leading peptide frame as one man are connected to Nhe I-Hind III site of pTugEO7K3, wherein pTugEO7K3 digests to remove CBD with Nhe I and Hind III in advance _CexGene fragment.Confirm last construct pCBDT006 by restrictive diges-tion and pcr analysis.Make up heterozygote NAMK6.1 (Termamyl

Joint-CBD _EGV)

With Nhe I and Stu I digestion Termamyl

Carrier NAM2.0 utilizes the resulting 1.48kb fragment of GeneClean (Bio101) test kit gel-purified, again with it and coding CBD _EGVThe fragment frame as one man be connected in advance among the pCBDT006 with Nhe I and the digestion of Stu I, obtain pNAMK6.1.

This product has following feature: MW60863; 537 amino-acid residues altogether; At first be Termamyl

Catalytic amylase is the joint that single-letter is represented: RPPTPTSPSAPS (SEQ ID NO:37) then, is 38 residues from fungi CBD at last.SEQ ID NO:26 has shown that the complete nucleotide sequence of pNAMK6.1 (contains Termamyl

CBD _EGVInsert segmental pTugK).

Embodiment 9

Termamyl Joint-CBD _EGVStarch processing

We have studied at pH6.0 and 40ppm Ca ²⁺In, with Termamyl

Compare Termamyl Joint-CBD _EGV(as the Termamyl of structure as described in the top embodiment 9

Whether every microgram zymoprotein/gram dry-matter joint-from the fungi CBD of Humicola insolens EGV) can obtain better starch liquefacation effect.

The oil bath of jolting is heated to 105 ℃.Prepare three kinds of starch suspensions and (contain 40ppm Ca ⁺⁺30%DS), utilize NaOH to regulate pH to 6.0.According to following pattern with enzyme and all suspension mixings:

Suspension 1:Termamyl

Joint-CBD _EGV10.9 microgram/gram DS starch

Suspension 2:Termamyl

Joint-CBD _EGV8.72 microgram/gram DS starch

Suspension 3:Termamyl 10.9 microgram/gram DS starch

Get four parts from every kind of suspension, every part 10 gram.Place the Erlenmeyer flask that has screw cap with every part.Flask is placed 105 ℃ oil bath 8 minutes, placed 90 minutes at 95 ℃ then.

In oil bath, place after 7 minutes 45 seconds, the thermostatted of oil bath is adjusted to 95.4 ℃, in oil bath, add 2 liters of oil of room temperature.Start clock, sampling (each suspension is got 1 flask) in 20,40,60 and 90 minutes.In each flask, add 2 1N HCl, with inactivation amylase.

Be defined as the DE value of the function of time:

As can be seen from the above table, with Termamyl

Compare Termamyl

Joint-CBD _EGVThe liquefaction effect of every microgram enzyme/gram DS increase.

Embodiment 10

CBD _CenA-Termamyl Starch processing

Also studied at pH6.0 and 40ppm Ca ²⁺Down, with Termamyl

Compare CBD _CenA-Termamyl

(muck cellulomonas cartae endoglucanase ACBD and the Termamyl described in the top embodiment 8 via the joint connection The starch liquefacation effect of every activity unit/gram dry-matter whether improve.The jolting oil bath is heated to 105 ℃.Prepare two parts of starch suspensions and (contain 40ppm Ca ⁺⁺30%DS), utilize NaOH to be adjusted to pH6.0.According to following pattern with enzyme and suspension mixing:

Suspension 1:CBD _CenA-Termamyl

75NU/ gram DS starch

Suspension 2:Termamyl

75NU/ gram DS starch

From each suspension, respectively get four parts, every part 10 gram.Place the Erlenmeyer flask that has screw cap with every part.Flask is placed 105 ℃ of oil baths 8 minutes, placed 90 minutes at 95 ℃ then.

In oil bath, place after 7 minutes 45 seconds, the thermostatted of oil bath is adjusted to 95.4 ℃, and in oil bath, add 2 liters of the oil of room temperature.Start clock, sampling (each suspension is got a flask) in 20,40,60 and 90 minutes.In each flask, add 2 1N HCl, with inactivation amylase.

Be defined as the DE value of the function of time:

Passable from last table, with Termamyl Compare CBD _CenA-Termamyl

The liquification better effects if of every activity unit/gram DS.

Sequence table (1) general information: (I) applicant:

(A) title: Novo Nordisk A/S

(B) street: Novo Alle

(C) city: Bagsvaerd

(E) country: Denmark

(F) postcode (ZIP): DK-2880

(G) phone :+45 4,444 8888

(H) fax :+45 4,449 3256 (ⅱ) denomination of invention: heterozygote enzyme/starch processing (ⅲ) sequence number: 37 (ⅳ) computer-reader form:

(A) media type: floppy disk

(B) computer: IBM PC compatible

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

(D) software: the data of PatentIn Release#1.0.#1.30 version (EPO) (2) SEQ ID NO:1: (I) sequence signature:

(A) length: 1203 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: cDNA (ⅳ) source:

(A) organism: Bacillus agaradherens

(B) bacterial strain: AC13 (ⅸ) feature:

(A) title/keyword: CDS

, (B) position: 1..1203, (ⅹ ⅰ) sequence description: SEQ ID NO:1ATG AAA AAG ATA ACT ACT ATT TTT GTC GTA TTG CTT ATG ACA GTG GCG 48Met Lys Lys Ile Thr Thr Ile Phe Val Val Leu Leu Met Thr Val Ala 15 10 15TTG TTC AGT ATA GGA AAC ACG ACT GCT GCT GAT AAT GAT TCA GTT GTA 96Leu Phe Ser Ile Gly Asn Thr Thr Ala Ala Asp Asn Asp Ser Val Val

20??????????????????25??????????????????30GAA?GAA?CAT?GGG?CAA?TTA?AGT?ATT?AGT?AAC?GGT?GAA?TTA?GTC?AAT?GAA?144Glu?Glu?His?Gly?Gln?Leu?Ser?Ile?Ser?Asn?Gly?Glu?Leu?Val?Asn?Glu

35??????????????????40??????????????????45CGA?GGC?GAA?CAA?GTT?CAG?TTA?AAA?GGG?ATG?AGT?TCC?CAT?GGT?TTG?CAA?192Arg?Gly?Glu?Gln?Val?Gln?Leu?Lys?Gly?Met?Ser?Ser?His?Gly?Leu?Gln

50??????????????????55??????????????????60TGG?TAC?GGT?CAA?TTT?GTA?AAC?TAT?GAA?AGT?ATG?AAA?TGG?CTA?AGA?GAT?240Trp?Tyr?Gly?Gln?Phe?Val?Asn?Tyr?Glu?Ser?Met?Lys?Trp?Leu?Arg?Asp?65??????????????????70??????????????????75??????????????????80GAT?TGG?GGA?ATA?AAT?GTA?TTC?CGA?GCA?GCA?ATG?TAT?ACC?TCT?TCA?GGA?288Asp?Trp?Gly?Ile?Asn?Val?Phe?Arg?Ala?Ala?Met?Tyr?Thr?Ser?Ser?Gly

85??????????????????90??????????????????95GGA?TAT?ATT?GAT?GAT?CCA?TCA?GTA?AAG?GAA?AAA?GTA?AAA?GAG?GCT?GTT?336Gly?Tyr?Ile?Asp?Asp?Pro?Ser?Val?Lys?Glu?Lys?Val?Lys?Glu?Ala?Val

100?????????????????105?????????????????110GAA?GCT?GCG?ATA?GAC?CTT?GAT?ATA?TAT?GTG?ATC?ATT?GAT?TGG?CAT?ATC?384Glu?Ala?Ala?Ile?Asp?Leu?Asp?Ile?Tyr?Val?Ile?Ile?Asp?Trp?His?Ile

115?????????????????120?????????????????125CTT?TCA?GAC?AAT?GAC?CCA?AAT?ATA?TAT?AAA?GAA?GAA?GCG?AAG?GAT?TTC?432Leu?Ser?Asp?Asn?Asp?Pro?Asn?Ile?Tyr?Lys?Glu?Glu?Ala?Lys?Asp?Phe

130?????????????????135?????????????????140TTT?GAT?GAA?ATG?TCA?GAG?TTG?TAT?GGA?GAC?TAT?CCG?AAT?GTG?ATA?TAC?480Phe?Asp?Glu?Met?Ser?Glu?Leu?Tyr?Gly?Asp?Tyr?Pro?Asn?Val?Ile?Tyr145?????????????????150?????????????????155?????????????????160GAA?ATT?GCA?AAT?GAA?CCG?AAT?GGT?AGT?GAT?GTT?ACG?TGG?GGC?AAT?CAA?528Glu?Ile?Ala?Asn?Glu?Pro?Asn?Gly?Ser?Asp?Val?Thr?Trp?Gly?Asn?Gln

165?????????????????170?????????????????175ATA?AAA?CCG?TAT?GCA?GAG?GAA?GTC?ATT?CCG?ATT?ATT?CGT?AAC?AAT?GAC?576Ile?Lys?Pro?Tyr?Ala?Glu?Glu?Val?Ile?Pro?Ile?Ile?Arg?Asn?Asn?Asp

180?????????????????185?????????????????190CCT?AAT?AAC?ATT?ATT?ATT?GTA?GGT?ACA?GGT?ACA?TGG?AGT?CAG?GAT?GTC?624Pro?Asn?Asn?Ile?Ile?Ile?Val?Gly?Thr?Gly?Thr?Trp?Ser?Gln?Asp?Val

195?????????????????200?????????????????205CAT?CAT?GCA?GCT?GAT?AAT?CAG?CTT?GCA?GAT?CCT?AAC?GTC?ATG?TAT?GCA?672His?His?Ala?Ala?Asp?Asn?Gln?Leu?Ala?Asp?Pro?Asn?Val?Met?Tyr?Ala

210?????????????????215?????????????????220TTT?CAT?TTT?TAT?GCA?GGG?ACA?CAT?GGT?CAA?AAT?TTA?CGA?GAC?CAA?GTA?720Phe?His?Phe?Tyr?Ala?Gly?Thr?His?Gly?Gln?Asn?Leu?Arg?Asp?Gln?Val225?????????????????230?????????????????235?????????????????240GAT?TAT?GCA?TTA?GAT?CAA?GGA?GCA?GCG?ATA?TTT?GTT?AGT?GAA?TGG?GGA?768Asp?Tyr?Ala?Leu?Asp?Gln?Gly?Ala?Ala?Ile?Phe?Val?Ser?Glu?Trp?Gly

245?????????????????250?????????????????255ACA?AGT?GCA?GCT?ACA?GGT?GAT?GGT?GGC?GTG?TTT?TTA?GAT?GAA?GCA?CAA??816Thr?Ser?Ala?Ala?Thr?Gly?Asp?Gly?Gly?Val?Phe?Leu?Asp?Glu?Ala?Gln

260?????????????????265?????????????????270GTG?TGG?ATT?GAC?TTT?ATG?GAT?GAA?AGA?AAT?TTA?AGC?TGG?GCC?AAC?TGG??864Val?Trp?Ile?Asp?Phe?Met?Asp?Glu?Arg?Asn?Leu?Ser?Trp?Ala?Asn?Trp

275?????????????????280?????????????????285TCT?CTA?ACG?CAT?AAA?GAT?GAG?TCA?TCT?GCA?GCG?TTA?ATG?CCA?GGT?GCA??912Ser?Leu?Thr?His?Lys?Asp?Glu?Ser?Ser?Ala?Ala?Leu?Met?Pro?Gly?Ala

290?????????????????295?????????????????300AAT?CCA?ACT?GGT?GGT?TGG?ACA?GAG?GCT?GAA?CTA?TCT?CCA?TCT?GGT?ACA??960Ash?Pro?Thr?Gly?Gly?Trp?Thr?Glu?Ala?Glu?Leu?Ser?Pro?Ser?Gly?Thr305?????????????????310?????????????????315?????????????????320TTT?GTG?AGG?GAA?AAA?ATA?AGA?GAA?TCA?GCA?TCT?ATT?CCG?CCA?AGC?GAT?1008Phe?Val?Arg?Glu?Lys?Ile?Arg?Glu?Ser?Ala?Ser?Ile?Pro?Pro?Ser?Asp

325?????????????????330?????????????????335CCA?ACA?CCG?CCA?TCT?GAT?CCA?GGA?GAA?CCG?GAT?CCA?ACG?CCC?CCA?AGT?1056Pro?Thr?Pro?Pro?Ser?Asp?Pro?Gly?Glu?Pro?Asp?Pro?Thr?Pro?Pro?Ser

340?????????????????345?????????????????350GAT?CCA?GGA?GAG?TAT?CCA?GCA?TGG?GAT?CCA?AAT?CAA?ATT?TAC?ACA?AAT?1104Asp?Pro?Gly?Glu?Tyr?Pro?Ala?Trp?Asp?Pro?Ash?Gln?Ile?Tyr?Thr?Asn

355?????????????????360?????????????????365GAA?ATT?GTG?TAC?CAT?AAC?GGC?CAG?CTA?TGG?CAA?GCA?AAA?TGG?TGG?ACA?1152Glu?Ile?Val?Tyr?His?Asn?Gly?Gln?Leu?Trp?Gln?Ala?Lys?Trp?Trp?Thr

The data of 370 375 380CAA AAT CAA GAG CCA GGT GAC CCG TAC GGT CCG TGG GAA CCA CTC AAT 1200Gln Ash Gln Glu Pro Gly Asp Pro Tyr Gly Pro Trp Glu Pro Leu Asn385 390 395 400TAA 1203 (2) SEQ ID NO:2:

(ⅰ) sequence signature:

(A) length: 400 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: protein

(ⅹ ⅰ) sequence description: SEQ ID NO:2Met Lys Lys Ile Thr Thr Ile Phe Val Val Leu Leu Met Thr Val Ala 15 10 15Leu Phe Ser Ile Gly Asn Thr Thr Ala Ala Asp Asn Asp Ser Val Val

20??????????????????25??????????????????30Glu?Glu?His?Gly?Gln?Leu?Ser?Ile?Ser?Asn?Gly?Glu?Leu?Val?Asn?Glu

35??????????????????40??????????????????45Arg?Gly?Glu?Gln?Val?Gln?Leu?Lys?Gly?Met?Ser?Ser?His?Gly?Leu?Gln

50??????????????????55??????????????????60Trp?Tyr?Gly?Gln?Phe?Val?Asn?Tyr?Glu?Ser?Met?Lys?Trp?Leu?Arg?Asp?65??????????????????70??????????????????75??????????????????80Asp?Trp?Gly?Ile?Asn?Val?Phe?Arg?Ala?Ala?Met?Tyr?Thr?Ser?Ser?Gly

85??????????????????90??????????????????95Gly?Tyr?Ile?Asp?Asp?Pro?Ser?Val?Lys?Glu?Lys?Val?Lys?Glu?Ala?Val

100?????????????????105?????????????????110Glu?Ala?Ala?Ile?Asp?Leu?Asp?Ile?Tyr?Val?Ile?Ile?Asp?Trp?His?Ile

115?????????????????120?????????????????125Leu?Ser?Asp?Asn?Asp?Pro?Asn?Ile?Tyr?Lys?Glu?Glu?Ala?Lys?Asp?Phe

130?????????????????135?????????????????140Phe?Asp?Glu?Met?Ser?Glu?Leu?Tyr?Gly?Asp?Tyr?Pro?Asn?Val?Ile?Tyr145?????????????????150?????????????????155?????????????????160Glu?Ile?Ala?Asn?Glu?Pro?Asn?Gly?Ser?Asp?Val?Thr?Trp?Gly?Asn?Gln

165?????????????????170?????????????????175Ile?Lys?Pro?Tyr?Ala?Glu?Glu?Val?Ile?Pro?Ile?Ile?Arg?Asn?Asn?Asp

180?????????????????185?????????????????190Pro?Asn?Asn?Ile?Ile?Ile?Val?Gly?Thr?Gly?Thr?Trp?Ser?Gln?Asp?Val

195?????????????????200?????????????????205His?His?Ala?Ala?Asp?Asn?Gln?Leu?Ala?Asp?Pro?Asn?Val?Met?Tyr?Ala

210?????????????????215?????????????????220Phe?His?Phe?Tyr?Ala?Gly?Thr?His?Gly?Gln?Asn?Leu?Arg?Asp?Gln?Val225?????????????????230?????????????????235?????????????????240Asp?Tyr?Ala?Leu?Asp?Gln?Gly?Ala?Ala?Ile?Phe?Val?Ser?Glu?Trp?Gly

245?????????????????250?????????????????255Thr?Ser?Ala?Ala?Thr?Gly?Asp?Gly?Gly?Val?Phe?Leu?Asp?Glu?Ala?Gln

260?????????????????265?????????????????270Val?Trp?Ile?Asp?Phe?Met?Asp?Glu?Arg?Asn?Leu?Ser?Trp?Ala?Asn?Trp

275?????????????????280?????????????????285Ser?Leu?Thr?His?Lys?Asp?Glu?Ser?Ser?Ala?Ala?Leu?Met?Pro?Gly?Ala

290?????????????????295?????????????????300Asn?Pro?Thr?Gly?Gly?Trp?Thr?Glu?Ala?Glu?Leu?Ser?Pro?Ser?Gly?Thr305?????????????????310?????????????????315?????????????????320Phe?Val?Arg?Glu?Lys?Ile?Arg?Glu?Ser?Ala?Ser?Ile?Pro?Pro?Ser?Asp

325?????????????????330?????????????????335Pro?Thr?Pro?Pro?Ser?Asp?Pro?Gly?Glu?Pro?Asp?Pro?Thr?Pro?Pro?Ser

340?????????????????345?????????????????350Asp?Pro?Gly?Glu?Tyr?Pro?Ala?Trp?Asp?Pro?Asn?Gln?Ile?Tyr?Thr?Asn

355?????????????????360?????????????????365Glu?Ile?Val?Tyr?His?Asn?Gly?Gln?Leu?Trp?Gln?Ala?Lys?Trp?Trp?Thr

The data of 370 375 380Gln Asn Gln Glu Pro Gly Asp Pro Tyr Gly Pro Trp Glu Pro Leu Asn385,390 395 400 (2) SEQ ID N0:3:

(ⅰ) sequence signature:

(A) length: 49 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 1 (#9555) "

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 33,36,39,42,45,48

(D) out of Memory :/note N=A, G, C or T

R=G or A

Y=C or T

(ⅹ ⅰ) sequence description: SEQ ID NO:3

The data of TCACAGATCC TCGCGAATTG GTGCGGCCGC GTNGTNGARG ARCAYGGNC 49 (2) SEQ ID NO:4:

(ⅰ) sequence signature:

(A) length: 7 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: protein

(ⅹ ⅰ) sequence description: SEQ ID NO:4Val Val Glu Glu His Gly Gln

The data of 5 (2) SEQ ID NO:5:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 2 "

(ⅹ ⅰ) sequence description: SEQ ID NO:5

The data of CAGAGCAAGAG ATTACGCGC 19 (2) SEQ ID NO:6:

(ⅰ) sequence signature:

(A) length: 17 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" reverse primer "

(ⅹ ⅰ) sequence description: SEQ ID NO:6

The data of GTTTTCCCAG TCACGAC 17 (2) SEQ ID NO:7:

(ⅰ) sequence signature:

(A) length: 22 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" forward primer "

(ⅹ ⅰ) sequence description: SEQ ID NO:7

The data of GCGGATAACA ATTTCACACA GG 22 (2) SEQ ID NO:8:

(ⅰ) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 3, #19719 "

(ⅹ ⅰ) sequence description: SEQ ID NO:8

The data of TGACCCGTAC GGTCCGTGGG 20 (2) SEQ ID NO:9:

(ⅰ) sequence signature:

(A) length: 22 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 4, #19720 "

(ⅹ ⅰ) sequence description: the data of SEQ ID NO:9GGCTCTTGAT TTTGTGTCCA CC 22 (2) SEQ ID NO:10:

(ⅰ) sequence signature:

(A) length: 71 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 5, #20887 "

(ⅹ ⅰ) sequence description: the data of SEQ ID NO:10GTAGGCTCAG TCATATGTTA CACATTGAAA GGGGAGGAGA ATCATGAAAA AGATAACTAC 60TATTTTTGTC G 71 (2) SEQ ID NO:11:

(ⅰ) sequence signature:

(A) length: 51 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 6 "

(ⅹ ⅰ) sequence description: the data of SEQ ID NO:11GTACCTCGCG GGTACCAAGC GGCCGCTTAA TTGAGTGGTT CCCACGGACC G 51 (2) SEQ ID NO:12:

(ⅰ) sequence signature:

(A) length: 1386 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: DNA (genome)

(ⅵ) primary source:

(A) organism: Bacillus agaradherens

(B) bacterial classification: AC13

(ⅸ) feature:

(A) title/keyword: CDS

(B) position: 1..1386

(ⅹ ⅰ) sequence description: SEQ ID NO:12:ATG AAA AAG ATA ACT ACT ATT TTT GTC GTA TTG CTT ATG ACA GTG GCG 48Met Lys Lys Ile Thr Thr Ile Phe Val Val Leu Leu Met Thr Val Ala 15 10 15TTG TTC AGT ATA GGA AAC ACG ACT GCT GCT GAT AAT GAT TCA GTT GTA 96Leu Phe Ser Ile Gly Asn Thr Thr Ala Ala Asp Asn Asp Ser Val Val

20??????????????????25??????????????????30GAA?GAA?CAT?GGG?CAA?TTA?AGT?ATT?AGT?AAC?GGT?GAA?TTA?GTC?AAT?GAA??144Glu?Glu?His?Gly?Gln?Leu?Ser?Ile?Ser?Asn?Gly?Glu?Leu?Val?Asn?Glu

35??????????????????40??????????????????45CGA?GGC?GAA?CAA?GTT?CAG?TTA?AAA?GGG?ATG?AGT?TCC?CAT?GGT?TTG?CAA?192Arg?Gly?Glu?Gln?Val?Gln?Leu?Lys?Gly?Met?Ser?Ser?His?Gly?Leu?Gln

50??????????????????55??????????????????60TGG?TAC?GGT?CAA?TTT?GTA?AAC?TAT?GAA?AGT?ATG?AAA?TGG?CTA?AGA?GAT?240Trp?Tyr?Gly?Gln?Phe?Val?Asn?Tyr?Glu?Ser?Met?Lys?Trp?Leu?Arg?Asp?65??????????????????70??????????????????75??????????????????80GAT?TGG?GGA?ATA?AAT?GTA?TTC?CGA?GCA?GCA?ATG?TAT?ACC?TCT?TCA?GGA?288Asp?Trp?Gly?Ile?Asn?Val?Phe?Arg?Ala?Ala?Met?Tyr?Thr?Ser?Ser?Gly

85??????????????????90??????????????????95GGA?TAT?ATT?GAT?GAT?CCA?TCA?GTA?AAG?GAA?AAA?GTA?AAA?GAG?GCT?GTT?336Gly?Tyr?Ile?Asp?Asp?Pro?Ser?Val?Lys?Glu?Lys?Val?Lys?Glu?Ala?Val

100?????????????105?????????????????110GAA?GCT?GCG?ATA?GAC?CTT?GAT?ATA?TAT?GTG?ATC?ATT?GAT?TGG?CAT?ATC?384Glu?Ala?Ala?Ile?Asp?Leu?Asp?Ile?Tyr?Val?Ile?Ile?Asp?Trp?His?Ile

115?????????????????120?????????????????125CTT?TCA?GAC?AAT?GAC?CCA?AAT?ATA?TAT?AAA?GAA?GAA?GCG?AAG?GAT?TTC?432Leu?Ser?Asp?Asn?Asp?Pro?Asn?Ile?Tyr?Lys?Glu?Glu?Ala?Lys?Asp?Phe

130?????????????????135?????????????????140TTT?GAT?GAA?ATG?TCA?GAG?TTG?TAT?GGA?GAC?TAT?CCG?AAT?GTG?ATA?TAC?480Phe?Asp?Glu?Met?Ser?Glu?Leu?Tyr?Gly?Asp?Tyr?Pro?Asn?Val?Ile?Tyr145?????????????????150?????????????????155?????????????????160GAA?ATT?GCA?AAT?GAA?CCG?AAT?GGT?AGT?GAT?GTT?ACG?TGG?GGC?AAT?CAA?528Glu?Ile?Ala?Asn?Glu?Pro?Asn?Gly?Ser?Asp?Val?Thr?Trp?Gly?Asn?Gln

165?????????????????170?????????????????175ATA?AAA?CCG?TAT?GCA?GAG?GAA?GTC?ATT?CCG?ATT?ATT?CGT?AAC?AAT?GAC?576Ile?Lys?Pro?Tyr?Ala?Glu?Glu?Val?Ile?Pro?Ile?Ile?Arg?Asn?Asn?Asp

180?????????????????185?????????????????190CCT?AAT?AAC?ATT?ATT?ATT?GTA?GGT?ACA?GGT?ACA?TGG?AGT?CAG?GAT?GTC?624Pro?Asn?Asn?Ile?Ile?Ile?Val?Gly?Thr?Gly?Thr?Trp?Ser?Gln?Asp?Val

195?????????????????200?????????????????205CAT?CAT?GCA?GCT?GAT?AAT?CAG?CTT?GCA?GAT?CCT?AAC?GTC?ATG?TAT?GCA?672His?His?Ala?Ala?Asp?Asn?Gln?Leu?Ala?Asp?Pro?Asn?Val?Met?Tyr?Ala

210?????????????????215?????????????????220TTT?CAT?TTT?TAT?GCA?GGG?ACA?CAT?GGT?CAA?AAT?TTA?CGA?GAC?CAA?GTA?720Phe?His?Phe?Tyr?Ala?Gly?Thr?His?Gly?Gln?Asn?Leu?Arg?Asp?Gln?Val225?????????????????230?????????????????235?????????????????240GAT?TAT?GCA?TTA?GAT?CAA?GGA?GCA?GCG?ATA?TTT?GTT?AGT?GAA?TGG?GGA?768Asp?Tyr?Ala?Leu?Asp?Gln?Gly?Ala?Ala?Ile?Phe?Val?Ser?Glu?Trp?Gly

245?????????????????250?????????????????255ACA?AGT?GCA?GCT?ACA?GGT?GAT?GGT?GGC?GTG?TTT?TTA?GAT?GAA?GCA?CAA?816Thr?Ser?Ala?Ala?Thr?Gly?Asp?Gly?Gly?Val?Phe?Leu?Asp?Glu?Ala?Gln

260?????????????????265?????????????????270GTG?TGG?ATT?GAC?TTT?ATG?GAT?GAA?AGA?AAT?TTA?AGC?TGG?GCC?AAC?TGG?864Val?Trp?Ile?Asp?Phe?Met?Asp?Glu?Arg?Asn?Leu?Ser?Trp?Ala?Asn?Trp

275?????????????????280?????????????????285TCT?CTA?ACG?CAT?AAA?GAT?GAG?TCA?TCT?GCA?GCG?TTA?ATG?CCA?GGT?GCA?912Ser?Leu?Thr?His?Lys?Asp?Glu?Ser?Ser?Ala?Ala?Leu?Met?Pro?Gly?Ala

290?????????????????295?????????????????300AAT?CCA?ACT?GGT?GGT?TGG?ACA?GAG?GCT?GAA?CTA?TCT?CCA?TCT?GGT?ACA?960Asn?Pro?Thr?Gly?Gly?Trp?Thr?Glu?Ala?Glu?Leu?Ser?Pro?Ser?Gly?Thr305?????????????????310?????????????????315?????????????????320TTT?GTG?AGG?GAA?AAA?ATA?AGA?GAA?TCA?GCA?TCT?ATT?CCG?CCA?AGC?GAT?1008Phe?Val?Arg?Glu?Lys?Ile?Arg?Glu?Ser?Ala?Ser?Ile?Pro?Pro?Ser?Asp

325?????????????????330?????????????????335CCA?ACA?CCG?CCA?TCT?GAT?CCA?GGA?GAA?CCG?GAT?CCA?ACG?CCC?CCA?AGT?1056Pro?Thr?Pro?Pro?Ser?Asp?Pro?Gly?Glu?Pro?Asp?Pro?Thr?Pro?Pro?Ser

340?????????????????345?????????????????350GAT?CCA?GGA?AAG?TAT?CCA?GCA?TGG?GAT?CCA?AAT?CAA?ATT?TAC?ACA?AAT?1104Asp?Pro?Gly?Lys?Tyr?Pro?Ala?Trp?Asp?Pro?Asn?Gln?Ile?Tyr?Thr?Asn

355?????????????????360?????????????????365GAA?ATT?GTG?TAC?CAT?AAC?GGC?CAG?CTA?TGG?CAA?GCA?AAA?TGG?TGG?ACA?1152Glu?Ile?Val?Tyr?His?Asn?Gly?Gln?Leu?Trp?Gln?Ala?Lys?Trp?Trp?Thr

370?????????????????375?????????????????380CAA?AAT?CAA?GAG?CCA?GGT?GAC?CCG?TAC?GGT?CCG?TGG?GAA?CCA?CTC?AAA?1200Gln?Asn?Gln?Glu?Pro?Gly?Asp?Pro?Tyr?Gly?Pro?Trp?Glu?Pro?Leu?Lys385?????????????????390?????????????????395?????????????????400TCT?GAT?CCA?GAT?TCA?GGA?GAA?CCG?GAT?CCA?ACG?CCC?CCA?AGT?GAT?CCA?1248Ser?Asp?Pro?Asp?Ser?Gly?Glu?Pro?Asp?Pro?Thr?Pro?Pro?Ser?Asp?Pro

405?????????????????410?????????????????415GGA?GAA?TAT?CCA?GCA?TGG?GAC?CCA?ACG?CAA?ATT?TAC?ACA?GAT?GAA?ATT?1296Gly?Glu?Tyr?Pro?Ala?Trp?Asp?Pro?Thr?Gln?Ile?Tyr?Thr?Asp?Glu?Ile

420?????????????????425?????????????????430GTG?TAC?CAT?AAC?GGC?CAG?CTA?TGG?CAA?GCC?AAA?TGG?TGG?ACA?CAA?AAT?1344Val?Tyr?His?Asn?Gly?Gln?Leu?Trp?Gln?Ala?Lys?Trp?Trp?Thr?Gln?Asn

435?????????????????440?????????????????445CAA?GAG?CCA?GGT?GAC?CCA?TAC?GGT?CCG?TGG?GAA?CCA?CTC?AAT?????????1386Gln?Glu?Pro?Gly?Asp?Pro?Tyr?Gly?Pro?Trp?Glu?Pro?Leu?Asn

The data of 450 455 460 (2) SEQ ID NO:13:

(ⅰ) sequence signature:

(A) length: 462 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: protein

(ⅹ ⅰ) sequence description: SEQ ID NO:13Met Lys Lys Ile Thr Thr Ile Phe Val Val Leu Leu Met Thr Val Ala 15 10 15Leu Phe Ser Ile Gly Asn Thr Thr Ala Ala Asp Asn Asp Ser Val Val

20??????????????????25??????????????????30Glu?Glu?His?Gly?Gln?Leu?Ser?Ile?Ser?Asn?Gly?Glu?Leu?Val?Asn?Glu

35??????????????????40??????????????????45Arg?Gly?Glu?Gln?Val?Gln?Leu?Lys?Gly?Met?Ser?Ser?His?Gly?Leu?Gln

50??????????????????55??????????????????60Trp?Tyr?Gly?Gln?Phe?Val?Asn?Tyr?Glu?Ser?Met?Lys?Trp?Leu?Arg?Asp?65??????????????????70??????????????????75??????????????????80Asp?Trp?Gly?Ile?Asn?Val?Phe?Arg?Ala?Ala?Met?Tyr?Thr?Ser?Ser?Gly

85??????????????????90??????????????????95Gly?Tyr?Ile?Asp?Asp?Pro?Ser?Val?Lys?Glu?Lys?Val?Lys?Glu?Ala?Val

100?????????????????105?????????????????110Glu?Ala?Ala?Ile?Asp?Leu?Asp?Ile?Tyr?Val?Ile?Ile?Asp?Trp?His?Ile

115?????????????????120?????????????????125Leu?Ser?Asp?Asn?Asp?Pro?Asn?Ile?Tyr?Lys?Glu?Glu?Ala?Lys?Asp?Phe

130?????????????????135?????????????????140Phe?Asp?Glu?Met?Ser?Glu?Leu?Tyr?Gly?Asp?Tyr?Pro?Asn?Val?Ile?Tyr145?????????????????150?????????????????155?????????????????160Glu?Ile?Ala?Asn?Glu?Pro?Asn?Gly?Ser?Asp?Val?Thr?Trp?Gly?Asn?Gln

165?????????????????170?????????????????175Ile?Lys?Pro?Tyr?Ala?Glu?Glu?Val?Ile?Pro?Ile?Ile?Arg?Asn?Asn?Asp

180?????????????????185?????????????????190Pro?Asn?Asn?Ile?Ile?Ile?Val?Gly?Thr?Gly?Thr?Trp?Ser?Gln?Asp?Val

195?????????????????200?????????????????205His?His?Ala?Ala?Asp?Asn?Gln?Leu?Ala?Asp?Pro?Asn?Val?Met?Tyr?Ala

210?????????????????215?????????????????220Phe?His?Phe?Tyr?Ala?Gly?Thr?His?Gly?Gln?Asn?Leu?Arg?Asp?Gln?Val225?????????????????230?????????????????235?????????????????240Asp?Tyr?Ala?Leu?Asp?Gln?Gly?Ala?Ala?Ile?Phe?Val?Ser?Glu?Trp?Gly

245?????????????????250?????????????????255Thr?Ser?Ala?Ala?Thr?Gly?Asp?Gly?Gly?Val?Phe?Leu?Asp?Glu?Ala?Gln

260?????????????????265?????????????????270Val?Trp?Ile?Asp?Phe?Met?Asp?Clu?Arg?Asn?Leu?Ser?Trp?Ala?Asn?Trp

275?????????????????280?????????????????285Ser?Leu?Thr?His?Lys?Asp?Glu?Ser?Ser?Ala?Ala?Leu?Met?Pro?Gly?Ala

290?????????????????295?????????????????300Asn?Pro?Thr?Gly?Gly?Trp?Thr?Glu?Ala?Glu?Leu?Ser?Pro?Ser?Gly?Thr305?????????????????310?????????????????315?????????????????320Phe?Val?Arg?Glu?Lys?Ile?Arg?Glu?Ser?Ala?Ser?Ile?Pro?Pro?Ser?Asp

325?????????????????330?????????????????335Pro?Thr?Pro?Pro?Ser?Asp?Pro?Gly?Glu?Pro?Asp?Pro?Thr?Pro?Pro?Ser

340?????????????????345?????????????????350Asp?Pro?Gly?Lys?Tyr?Pro?Ala?Trp?Asp?Pro?Asn?Gln?Ile?Tyr?Thr?Asn

355?????????????????360?????????????????365Glu?Ile?Val?Tyr?His?Asn?Gly?Gln?Leu?Trp?Gln?Ala?Lys?Trp?Trp?Thr

370?????????????????375?????????????????380Gln?Asn?Gln?Glu?Pro?Gly?Asp?Pro?Tyr?Gly?Pro?Trp?Glu?Pro?Leu?Lys385?????????????????390?????????????????395?????????????????400Ser?Asp?Pro?Asp?Ser?Gly?Glu?Pro?Asp?Pro?Thr?Pro?Pro?Ser?Asp?Pro

405?????????????????410?????????????????415Gly?Glu?Tyr?Pro?Ala?Trp?Asp?Pro?Thr?Gln?Ile?Tyr?Thr?Asp?Glu?Ile

420?????????????????425?????????????????430Val?Tyr?His?Asn?Gly?Gln?Leu?Trp?Gln?Ala?Lys?Trp?Trp?Thr?Gln?Asn

435?????????????????440?????????????????445Gln?Glu?Pro?Gly?Asp?Pro?Tyr?Gly?Pro?Trp?Glu?Pro?Leu?Asn

The data of 450 455 460 (2) SEQ ID NO:14:

(ⅰ) sequence signature:

(A) length: 46 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 7, #100084 "

(ⅹ ⅰ) sequence description: the data of SEQ ID NO:14CCTCGCGAGG TACCAGCGGC CGCGTACCAC CAATTAAGTA TGGTAC 46 (2) SEQ ID NO:15:

(ⅰ) sequence signature:

(A) length: 35 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/dese=" primer 8, #5289 "

(ⅹ ⅰ) sequence description: the data of SEQ ID NO:15GCTTTACGCC CGATTGCTGA CGCTG 35 (2) SEQ ID NO:16:

(ⅰ) sequence signature:

(A) length: 51 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 9, #26748 "

(ⅹ ⅰ) sequence description: the data of SEQ ID NO:16GCGATGAGAC GCGCGGCCGC CTATCTTTGA ACATAAATTG AAACGGATCC G 51 (2) SEQ ID NO:17:

(ⅰ) sequence signature:

(A) length: 52 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 10, #110150A "

(ⅹ ⅰ) sequence description: SEQ ID NO:17GCTGCAGGAT CCGTTTCAAT TTATGTTCAA AGATCTGATC CAGATTCAGG AG 52

(2) data of SEQ ID NO:18:

(ⅰ) sequence signature:

(A) length: 46 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 11, #100084 "

(ⅹ ⅰ) sequence description: SEQ ID NO:18CCTCGCGAGG TACCAGCGGC CGCGTACCAC CAATTAAGTA TGGTAC 46

(2) data of SEQ ID NO:19:

(ⅰ) sequence signature:

(A) length: 1725 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(A) describe :/desc=" heterozygote "

(ⅸ) feature:

(A) title/keyword: CDS

(B) position: 1..1725

(ⅹ ⅰ) sequence description: SEQ ID NO:19ATG AAA CAA CAA AAA CGG CTT TAC GCC CGA TTG CTG ACG CTG TTA TTT 48Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu Leu Phe 15 10 15 GCG CTC ATC TTC TTG CTG CCT CAT TCT GCA GCA GCG GCG GCA AAT CTT 96Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ala Asn Leu

20??????????????????25??????????????????30?AAT?GGG?ACG?CTG?ATG?CAG?TAT?TTT?GAA?TGG?TAC?ATG?CCC?AAT?GAC?GGC???????????144Asn?Gly?Thr?Leu?Met?Gln?Tyr?Phe?Glu?Trp?Tyr?Met?Pro?Asn?Asp?Gly

35??????????????????40??????????????????45CAA?CAT?TGG?AAG?CGT?TTG?CAA?AAC?GAC?TCG?GCA?TAT?TTG?GCT?GAA?CAC???????????192Gln?His?Trp?Lys?Arg?Leu?Gln?Asn?Asp?Ser?Ala?Tyr?Leu?Ala?Glu?His

50??????????????????55??????????????????60GGT?ATT?ACT?GCC?GTC?TGG?ATT?CCC?CCG?GCA?TAT?AAG?GGA?ACG?AGC?CAA???????????240Gly?Ile?Thr?Ala?Val?Trp?Ile?Pro?Pro?Ala?Tyr?Lys?Gly?Thr?Ser?Gln?65??????????????????70??????????????????75??????????????????80GCG?GAT?GTG?GGC?TAC?GGT?GCT?TAC?GAC?CTT?TAT?GAT?TTA?GGG?GAG?TTT???????????288Ala?Asp?Val?Gly?Tyr?Gly?Ala?Tyr?Asp?Leu?Tyr?Asp?Leu?Gly?Glu?Phe

85??????????????????90??????????????????95CAT?CAA?AAA?GGG?ACG?GTT?CGG?ACA?AAG?TAC?GGC?ACA?AAA?GGA?GAG?CTG???????????336His?Gln?Lys?Gly?Thr?Val?Arg?Thr?Lys?Tyr?Gly?Thr?Lys?Gly?Glu?Leu

100?????????????????105?????????????????110CAA?TCT?GCG?ATC?AAA?AGT?CTT?CAT?TCC?CGC?GAC?ATT?AAC?GTT?TAC?GGG???????????384Gln?Ser?Ala?Ile?Lys?Ser?Leu?His?Ser?Arg?Asp?Ile?Asn?Val?Tyr?Gly

115?????????????????120?????????????????125GAT?GTG?GTC?ATC?AAC?CAC?AAA?GGC?GGC?GCT?GAT?GCG?ACC?GAA?GAT?GTA???????????432Asp?Val?Val?Ile?Asn?His?Lys?Gly?Gly?Ala?Asp?Ala?Thr?Glu?Asp?Val

130?????????????????135?????????????????140ACC?GCG?GTT?GAA?GTC?GAT?CCC?GCT?GAC?CGC?AAC?CGC?GTA?ATC?TCA?GGA???????????480Thr?Ala?Val?Glu?Val?Asp?Pro?Ala?Asp?Arg?Asn?Arg?Val?Ile?Ser?Gly145?????????????????150?????????????????155?????????????????160GAA?CAC?CTA?ATT?AAA?GCC?TGG?ACA?CAT?TTT?CAT?TTT?CCG?GGG?GCC?GGC???????????528Glu?His?Leu?Ile?Lys?Ala?Trp?Thr?His?Phe?His?Phe?Pro?Gly?Ala?Gly

165?????????????????170?????????????????175AGC?ACA?TAC?AGC?GAT?TTT?AAA?TGG?CAT?TGG?TAC?CAT?TTT?GAC?GGA?ACC???????????576Ser?Thr?Tyr?Ser?Asp?Phe?Lys?Trp?His?Trp?Tyr?His?Phe?Asp?Gly?Thr

180?????????????????185?????????????????190GAT?TGG?GAC?GAG?TCC?CGA?AAG?CTG?AAC?CGC?ATC?TAT?AAG?TTT?CAA?GGA???????????624Asp?Trp?Asp?Glu?Ser?Arg?Lys?Leu?Asn?Arg?Ile?Tyr?Lys?Phe?Gln?Gly

195?????????????????200?????????????????205AAG?GCT?TGG?GAT?TGG?GAA?GTT?TCC?AAT?GAA?AAC?GGC?AAC?TAT?GAT?TAT???????????672Lys?Ala?Trp?Asp?Trp?Glu?Val?Ser?Asn?Glu?Asn?Gly?Asn?Tyr?Asp?Tyr

210?????????????????215?????????????????220TTG?ATG?TAT?GCC?GAC?ATC?GAT?TAT?GAC?CAT?CCT?GAT?GTC?GCA?GCA?GAA???????????720Leu?Met?Tyr?Ala?Asp?Ile?Asp?Tyr?Asp?His?Pro?Asp?Val?Ala?Ala?Glu225?????????????????230?????????????????235?????????????????240ATT?AAG?AGA?TGG?GGC?ACT?TGG?TAT?GCC?AAT?GAA?CTG?CAA?TTG?GAC?GGA???????????768Ile?Lys?Arg?Trp?Gly?Thr?Trp?Tyr?Ala?Asn?Glu?Leu?Gln?Leu?Asp?Gly

245?????????????????250?????????????????255AAC?CGT?CTT?GAT?GCT?GTC?AAA?CAC?ATT?AAA?TTT?TCT?TTT?TTG?CGG?GAT???????????816Asn?Arg?Leu?Asp?Ala?Val?Lys?His?Ile?Lys?Phe?Ser?Phe?Leu?Arg?Asp

260?????????????????265?????????????????270TGG?GTT?AAT?CAT?GTC?AGG?GAA?AAA?ACG?GGG?AAG?GAA?ATG?TTT?ACG?GTA???????????864Trp?Val?Asn?His?Val?Arg?Glu?Lys?Thr?Gly?Lys?Glu?Met?Phe?Thr?Val

275?????????????????280?????????????????285GCT?GAA?TAT?TGG?CAG?AAT?GAC?TTG?GGC?GCG?CTG?GAA?AAC?TAT?TTG?AAC??912Ala?Glu?Tyr?Trp?Gln?Asn?Asp?Leu?Gly?Ala?Leu?Glu?Asn?Tyr?Leu?Asn

290?????????????????295?????????????????300AAA?ACA?AAT?TTT?AAT?CAT?TCA?GTG?TTT?GAC?GTG?CCG?CTT?CAT?TAT?CAG??960Lys?Thr?Asn?Phe?Asn?His?Ser?Val?Phe?Asp?Val?Pro?Leu?His?Tyr?Gln305?????????????????310?????????????????315?????????????????320TTC?CAT?GCT?GCA?TCG?ACA?CAG?GGA?GGC?GGC?TAT?GAT?ATG?AGG?AAA?TTG?1008Phe?His?Ala?Ala?Ser?Thr?Gln?Gly?Gly?Gly?Tyr?Asp?Met?Arg?Lys?Leu

325?????????????????330?????????????????335CTG?AAC?GGT?ACG?GTC?GTT?TCC?AAG?CAT?CCG?TTG?AAA?TCG?GTT?ACA?TTT?1056Leu?Asn?Gly?Thr?Val?Val?Ser?Lys?His?Pro?Leu?Lys?Ser?Val?Thr?Phe

340?????????????????345?????????????????350GTC?GAT?AAC?CAT?GAT?ACA?CAG?CCG?GGG?CAA?TCG?CTT?GAG?TCG?ACT?GTC?1104Val?Asp?Asn?His?Asp?Thr?Gln?Pro?Gly?Gln?Ser?Leu?Glu?Ser?Thr?Val

355?????????????????360?????????????????365CAA?ACA?TGG?TTT?AAG?CCG?CTT?GCT?TAC?GCT?TTT?ATT?CTC?ACA?AGG?GAA?1152Gln?Thr?Trp?Phe?Lys?Pro?Leu?Ala?Tyr?Ala?Phe?Ile?Leu?Thr?Arg?Glu

370?????????????????375?????????????????380TCT?GGA?TAC?CCT?CAG?GTT?TTC?TAC?GGG?GAT?ATG?TAC?GGG?ACG?AAA?GGA?1200Ser?Gly?Tyr?Pro?Gln?Val?Phe?Tyr?Gly?Asp?Met?Tyr?Gly?Thr?Lys?Gly385?????????????????390?????????????????395?????????????????400GAC?TCC?CAG?CGC?GAA?ATT?CCT?GCC?TTG?AAA?CAC?AAA?ATT?GAA?CCG?ATC?1248Asp?Ser?Gln?Arg?Glu?Ile?Pro?Ala?Leu?Lys?His?Lys?Ile?Glu?Pro?Ile

405?????????????????410?????????????????415TTA?AAA?GCG?AGA?AAA?CAG?TAT?GCG?TAC?GGA?GCA?CAG?CAT?GAT?TAT?TTC?1296Leu?Lys?Ala?Arg?Lys?Gln?Tyr?Ala?Tyr?Gly?Ala?Gln?His?Asp?Tyr?Phe

420?????????????????425?????????????????430GAC?CAC?CAT?GAC?ATT?GTC?GGC?TGG?ACA?AGG?GAA?GGC?GAC?AGC?TCG?GTT?1344Asp?His?His?Asp?Ile?Val?Gly?Trp?Thr?Arg?Glu?Gly?Asp?Ser?Ser?Val

435?????????????????440?????????????????445GCA?AAT?TCA?GGT?TTG?GCG?GCA?TTA?ATA?ACA?GAC?GGA?CCC?GGT?GGG?GCA?1392Ala?Asn?Ser?Gly?Leu?Ala?Ala?Leu?Ile?Thr?Asp?Gly?Pro?Gly?Gly?Ala

450?????????????????455?????????????????460AAG?CGA?ATG?TAT?GTC?GGC?CGG?CAA?AAC?GCC?GGT?GAG?ACA?TGG?CAT?GAC?1440Lys?Arg?Met?Tyr?Val?Gly?Arg?Gln?Asn?Ala?Gly?Glu?Thr?Trp?His?Asp465?????????????????470?????????????????475?????????????????480ATT?ACC?GGA?AAC?CGT?TCG?GAG?CCG?GTT?GTC?ATC?AAT?TCG?GAA?GGC?TGG?1488Ile?Thr?Gly?Asn?Arg?Ser?Glu?Pro?Val?Val?Ile?Asn?Ser?Glu?Gly?Trp

485?????????????????490?????????????????495GGA?GAG?TTT?CAC?GTA?AAC?GGC?GGA?TCC?GTT?TCA?ATT?TAT?GTT?CAA?AGA?1536Gly?Glu?Phe?His?Val?Asn?Gly?Gly?Ser?Val?Ser?Ile?Tyr?Val?Gln?Arg

500?????????????????????????505?????????510TCT?GAT?CCA?GAT?TCA?GGA?GAA?CCG?GAT?CCA?ACG?CCC?CCA?AGT?GAT?CCA?1584Ser?Asp?Pro?Asp?Ser?Gly?Glu?Pro?Asp?Pro?Thr?Pro?Pro?Ser?Asp?Pro

515?????????????????520?????????????????525GGA?GAA?TAT?CCA?GCA?TGG?GAC?CCA?ACG?CAA?ATT?TAC?ACA?GAT?GAA?ATT?1632Gly?Glu?Tyr?Pro?Ala?Trp?Asp?Pro?Thr?Gln?Ile?Tyr?Thr?Asp?Glu?Ile

The data of 530 535 540GTG TAC CAT AAC GGC CAG CTA TGG CAA GCC AAA TGG TGG ACA CAA AAT 1680Val Tyr His Asn Gly Gln Leu Trp Gln Ala Lys Trp Trp Thr Gln Asn545,550 555 560 (2) SEQ ID NO:20:

(ⅰ) sequence signature:

(A) length: 575 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: protein

(ⅹ ⅰ) sequence description: SEQ ID NO:20Met Lys Gln G1n Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu Leu Phe 15 10 15 Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ala Asn Leu

20??????????????????25??????????????????30Asn?Gly?Thr?Leu?Met?Gln?Tyr?Phe?Glu?Trp?Tyr?Met?Pro?Asn?Asp?Gly

35??????????????????40??????????????????45Gln?His?Trp?Lys?Arg?Leu?Gln?Asn?Asp?Ser?Ala?Tyr?Leu?Ala?Glu?His

50??????????????????55??????????????????60Gly?Ile?Thr?Ala?Val?Trp?Ile?Pro?Pro?Ala?Tyr?Lys?Gly?Thr?Ser?Gln?65??????????????????70??????????????????75??????????????????80Ala?Asp?Val?Gly?Tyr?Gly?Ala?Tyr?Asp?Leu?Tyr?Asp?Leu?Gly?Glu?Phe

85??????????????????90??????????????????95His?Gln?Lys?Gly?Thr?Val?Arg?Thr?Lys?Tyr?Gly?Thr?Lys?Gly?Glu?Leu

100?????????????????105?????????????????110Gln?Ser?Ala?Ile?Lys?Ser?Leu?His?Ser?Arg?Asp?Ile?Asn?Val?Tyr?Gly

115?????????????????120?????????????????125Asp?Val?Val?Ile?Asn?His?Lys?Gly?Gly?Ala?Asp?Ala?Thr?Glu?Asp?Val

130?????????????????135?????????????????140Thr?Ala?Val?Glu?Val?Asp?Pro?Ala?Asp?Arg?Asn?Arg?Val?Ile?Ser?Gly145?????????????????150?????????????????155?????????????????160Glu?His?Leu?Ile?Lys?Ala?Trp?Thr?His?Phe?His?Phe?Pro?Gly?Ala?Gly

165?????????????????170?????????????????175Ser?Thr?Tyr?Ser?Asp?Phe?Lys?Trp?His?Trp?Tyr?His?Phe?Asp?Gly?Thr

180?????????????????185?????????????????190Asp?Trp?Asp?Glu?Ser?Arg?Lys?Leu?Asn?Arg?Ile?Tyr?Lys?Phe?Gln?Gly

195?????????????????200?????????????????205Lys?Ala?Trp?Asp?Trp?Glu?Val?Ser?Asn?Glu?Asn?Gly?Asn?Tyr?Asp?Tyr

210?????????????????215?????????????????220Leu?Met?Tyr?Ala?Asp?Ile?Asp?Tyr?Asp?His?Pro?Asp?Val?Ala?Ala?Glu225?????????????????230?????????????????235?????????????????240Ile?Lys?Arg?Trp?Gly?Thr?Trp?Tyr?Ala?Asn?Glu?Leu?Gln?Leu?Asp?Gly

245?????????????????250?????????????????255Asn?Arg?Leu?Asp?Ala?Val?Lys?His?Ile?Lys?Phe?Ser?Phe?Leu?Arg?Asp

260?????????????????265?????????????????270Trp?Val?Asn?His?Val?Arg?Glu?Lys?Thr?Gly?Lys?Glu?Met?Phe?Thr?Val

275?????????????????280?????????????????285Ala?Glu?Tyr?Trp?Gln?Asn?Asp?Leu?Gly?Ala?Leu?Glu?Asn?Tyr?Leu?Asn

290?????????????????295?????????????????300Lys?Thr?ASn?Phe?Asn?His?Ser?Val?Phe?Asp?Val?Pro?Leu?His?Tyr?Gln305?????????????????310?????????????????315?????????????????320Phe?His?Ala?Ala?Ser?Thr?Gln?Gly?Gly?Gly?Tyr?Asp?Met?Arg?Lys?Leu

325?????????????????330?????????????????335Leu?Asn?Gly?Thr?Val?Val?Ser?Lys?His?Pro?Leu?Lys?Ser?Val?Thr?Phe

340?????????????????345?????????????????350Val?Asp?Asn?His?Asp?Thr?Gln?Pro?Gly?Gln?Ser?Leu?Glu?Ser?Thr?Val

355?????????????????360?????????????????365Gln?Thr?Trp?Phe?Lys?Pro?Leu?Ala?Tyr?Ala?Phe?Ile?Leu?Thr?Arg?Glu

370?????????????????375?????????????????380Ser?Gly?Tyr?Pro?Gln?Val?Phe?Tyr?Gly?Asp?Met?Tyr?Gly?Thr?Lys?Gly385?????????????????390?????????????????395?????????????????400Asp?Ser?Gln?Arg?Glu?Ile?Pro?Ala?Leu?Lys?His?Lys?Ile?Glu?Pro?Ile

405?????????????????410?????????????????415Leu?Lys?Ala?Arg?Lys?Gln?Tyr?Ala?Tyr?Gly?Ala?Gln?His?Asp?Tyr?Phe

420?????????????????425?????????????????430Asp?His?His?Asp?Ile?Val?Gly?Trp?Thr?Arg?Glu?Gly?Asp?Ser?Ser?Val

435?????????????????440?????????????????445Ala?Asn?Ser?Gly?Leu?Ala?Ala?Leu?Ile?Thr?Asp?Gly?Pro?Gly?Gly?Ala

450?????????????????455?????????????????460Lys?Arg?Met?Tyr?Val?Gly?Arg?Gln?Asn?Ala?Gly?Glu?Thr?Trp?His?Asp465?????????????????470?????????????????475?????????????????480I1e?Thr?Gly?Asn?Arg?Ser?Glu?Pro?Val?Val?Ile?Asn?Ser?Glu?Gly?Trp

485?????????????????490?????????????????495Gly?Glu?Phe?His?Val?Asn?Gly?Gly?Ser?Val?Ser?Ile?Tyr?Val?Gln?Arg

500?????????????????505?????????????????510Ser?Asp?Pro?Asp?Ser?Gly?Glu?Pro?Asp?Pro?Thr?Pro?Pro?Ser?Asp?Pro

515?????????????????520?????????????????525Gly?Glu?Tyr?Pro?Ala?Trp?Asp?Pro?Thr?Gln?Ile?Tyr?Thr?Asp?Glu?Ile

530?????????????????535?????????????????540Val?Tyr?His?Asn?Gly?Gln?Leu?Trp?Gln?Ala?Lys?Trp?Trp?Thr?Gln?Asn545?????????????????550?????????????????555?????????????????560Gln?Glu?Pro?Gly?Asp?Pro?Tyr?Gly?Pro?Trp?Glu?Pro?Leu?Asn???*

The data of 565 570 575 (2) SEQ ID NO:21:

(ⅰ) sequence signature:

(A) length: 17 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: protein

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" joint "

(ⅹ ⅰ) sequence description: SEQ ID NO:21Ser Asp Pro Asp Ser Gly Glu Pro Asp Pro Thr Pro Pro Ser Asp Pro Gly

The data of 5 10 15 (2) SEQ ID NO:22:

(ⅰ) sequence signature:

(A) length: 60 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 12, #114135 "

(ⅹ ⅰ) sequence description: the data of SEQ ID NO:22GCTGCAGGAT CCGTTTCAAT TTATGTTCAA AGATCTCCAA CTCCTGCCCC ATCTCAAAGC 60 (2) SEQ ID NO:23:

(ⅰ) sequence signature:

(A) length: 50 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 13, #110151 "

(ⅹ ⅰ) sequence description: the data of SEQ ID NO:23GCGATGAGAC GCGCGGCCGC TACTACCAGT CAACATTAAC AGGACCTGAG 50 (2) SEQ ID NO:24:

(ⅰ) sequence signature:

(A) length: 2346 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(A) describe :/desc=" heterozygote "

(ⅸ) feature:

(A) title/keyword: CDS

(B) position: 1..2346

(ⅹ ⅰ) sequence description: SEQ ID NO:24ATG AAA CAA CAA AAA CGG CTT TAC GCC CGA TTG CTG ACG CTG TTA TTT 48Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu Leu Phe 15 10 15GCG CTC ATC TTC TTG CTG CCT CAT TCT GCA GCA GCG GCG GCA AAT CTT 96Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ala Asn Leu

20??????????????????25??????????????????30AAT?GGG?ACG?CTG?ATG?CAG?TAT?TTT?GAA?TGG?TAC?ATG?CCC?AAT?GAC?GGC?144Asn?Gly?Thr?Leu?Met?Gln?Tyr?Phe?Glu?Trp?Tyr?Met?Pro?Asn?Asp?Gly

35??????????????????40??????????????????45CAA?CAT?TGG?AAG?CGT?TTG?CAA?AAC?GAC?TCG?GCA?TAT?TTG?GCT?GAA?CAC?192Gln?His?Trp?Lys?Arg?Leu?Gln?Asn?Asp?Ser?Ala?Tyr?Leu?Ala?Glu?His

50??????????????????55??????????????????60GGT?ATT?ACT?GCC?GTC?TGG?ATT?CCC?CCG?GCA?TAT?AAG?GGA?ACG?AGC?CAA??240Gly?Ile?Thr?Ala?Val?Trp?Ile?Pro?Pro?Ala?Tyr?Lys?Gly?Thr?Ser?Gln?65??????????????????70??????????????????75??????????????????80GCG?GAT?GTG?GGC?TAC?GGT?GCT?TAC?GAC?CTT?TAT?GAT?TTA?GGG?GAG?TTT??288Ala?Asp?Val?Gly?Tyr?Gly?Ala?Tyr?Asp?Leu?Tyr?Asp?Leu?Gly?Glu?Phe

85??????????????????90??????????????????95CAT?CAA?AAA?GGG?ACG?GTT?CGG?ACA?AAG?TAC?GGC?ACA?AAA?GGA?GAG?CTG??336His?Gln?Lys?Gly?Thr?Val?Arg?Thr?Lys?Tyr?Gly?Thr?Lys?Gly?Glu?Leu

100?????????????????105?????????????????110CAA?TCT?GCG?ATC?AAA?AGT?CTT?CAT?TCC?CGC?GAC?ATT?AAC?GTT?TAC?GGG??384Gln?Ser?Ala?Ile?Lys?Ser?Leu?His?Ser?Arg?Asp?Ile?Asn?Val?Tyr?Gly

115?????????????????120?????????????????125GAT?GTG?GTC?ATC?AAC?CAC?AAA?GGC?GGC?GCT?GAT?GCG?ACC?GAA?GAT?GTA??432Asp?Val?Val?Ile?Asn?His?Lys?Gly?Gly?Ala?Asp?Ala?Thr?Glu?Asp?Val

130?????????????????135?????????????????140ACC?GCG?GTT?GAA?GTC?GAT?CCC?GCT?GAC?CGC?AAC?CGC?GTA?ATC?TCA?GGA??480Thr?Ala?Val?Glu?Val?Asp?Pro?Ala?Asp?Arg?Asn?Arg?Val?Ile?Ser?Gly145?????????????????150?????????????????155?????????????????160GAA?CAC?CTA?ATT?AAA?GCC?TGG?ACA?CAT?TTT?CAT?TTT?CCG?GGG?GCC?GGC??528Glu?His?Leu?Ile?Lys?Ala?Trp?Thr?His?Phe?His?Phe?Pro?Gly?Ala?Gly

165?????????????????170?????????????????175AGC?ACA?TAC?AGC?GAT?TTT?AAA?TGG?CAT?TGG?TAC?CAT?TTT?GAC?GGA?ACC??576Ser?Thr?Tyr?Ser?Asp?Phe?Lys?Trp?His?Trp?Tyr?His?Phe?Asp?Gly?Thr

180?????????????????185?????????????????190GAT?TGG?GAC?GAG?TCC?CGA?AAG?CTG?AAC?CGC?ATC?TAT?AAG?TTT?CAA?GGA??624Asp?Trp?Asp?Glu?Ser?Arg?Lys?Leu?Asn?Arg?Ile?Tyr?Lys?Phe?Gln?Gly

195?????????????????200?????????????????205AAG?GCT?TGG?GAT?TGG?GAA?GTT?TCC?AAT?GAA?AAC?GGC?AAC?TAT?GAT?TAT??672Lys?Ala?Trp?Asp?Trp?Glu?Val?Ser?Asn?Glu?Asn?Gly?Asn?Tyr?Asp?Tyr

210?????????????????215?????????????????220TTG?ATG?TAT?GCC?GAC?ATC?GAT?TAT?GAC?CAT?CCT?GAT?GTC?GCA?GCA?GAA??720Leu?Met?Tyr?Ala?Asp?Ile?Asp?Tyr?Asp?His?Pro?Asp?Val?Ala?Ala?Glu225?????????????????230?????????????????235?????????????????240ATT?AAG?AGA?TGG?GGC?ACT?TGG?TAT?GCC?AAT?GAA?CTG?CAA?TTG?GAC?GGA??768Ile?Lys?Arg?Trp?Gly?Thr?Trp?Tyr?Ala?Asn?Glu?Leu?Gln?Leu?Asp?Gly

245?????????????????250?????????????????255AAC?CGT?CTT?GAT?GCT?GTC?AAA?CAC?ATT?AAA?TTT?TCT?TTT?TTG?CGG?GAT??816Asn?Arg?Leu?Asp?Ala?Val?Lys?His?Ile?Lys?Phe?Ser?Phe?Leu?Arg?Asp

260?????????????????265?????????????????270TGG?GTT?AAT?CAT?GTC?AGG?GAA?AAA?ACG?GGG?AAG?GAA?ATG?TTT?ACG?GTA??864Trp?Val?Asn?His?Val?Arg?Glu?Lys?Thr?Gly?Lys?Glu?Met?Phe?Thr?Val

275?????????????????280?????????????????285GCT?GAA?TAT?TGG?CAG?AAT?GAC?TTG?GGC?GCG?CTG?GAA?AAC?TAT?TTG?AAC??912Ala?Glu?Tyr?Trp?Gln?Asn?Asp?Leu?Gly?Ala?Leu?Glu?Asn?Tyr?Leu?Asn

290?????????????????295?????????????????300AAA?ACA?AAT?TTT?AAT?CAT?TCA?GTG?TTT?GAC?GTG?CCG?CTT?CAT?TAT?CAG??960Lys?Thr?Asn?Phe?Asn?His?Ser?Val?Phe?Asp?Val?Pro?Leu?His?Tyr?Gln305?????????????????310?????????????????315?????????????????320TTC?CAT?GCT?GCA?TCG?ACA?CAG?GGA?GGC?GGC?TAT?GAT?ATG?AGG?AAA?TTG?1008Phe?His?Ala?Ala?Ser?Thr?Gln?Gly?Gly?Gly?Tyr?Asp?Met?Arg?Lys?Leu

325?????????????????330?????????????????335CTG?AAC?GGT?ACG?GTC?GTT?TCC?AAG?CAT?CCG?TTG?AAA?TCG?GTT?ACA?TTT?1056Leu?Asn?Gly?Thr?Val?Val?Ser?Lys?His?Pro?Leu?Lys?Ser?Val?Thr?Phe

340?????????????????345?????????????????350GTC?GAT?AAC?CAT?GAT?ACA?CAG?CCG?GGG?CAA?TCG?CTT?GAG?TCG?ACT?GTC?1104Val?Asp?Asn?His?Asp?Thr?Gln?Pro?Gly?Gln?Ser?Leu?Glu?Ser?Thr?Val

355?????????????????360?????????????????365CAA?ACA?TGG?TTT?AAG?CCG?CTT?GCT?TAC?GCT?TTT?ATT?CTC?ACA?AGG?GAA?1152Gln?Thr?Trp?Phe?Lys?Pro?Leu?Ala?Tyr?Ala?Phe?Ile?Leu?Thr?Arg?Glu

370?????????????????375?????????????????380TCT?GGA?TAC?CCT?CAG?GTT?TTC?TAC?GGG?GAT?ATG?TAC?GGG?ACG?AAA?GGA?1200Ser?Gly?Tyr?Pro?Gln?Val?Phe?Tyr?Gly?Asp?Met?Tyr?Gly?Thr?Lys?Gly385?????????????????390?????????????????395?????????????????400GAC?TCC?CAG?CGC?GAA?ATT?CCT?GCC?TTG?AAA?CAC?AAA?ATT?GAA?CCG?ATC?1248Asp?Ser?Gln?Arg?Glu?Ile?Pro?Ala?Leu?Lys?His?Lys?Ile?Glu?Pro?Ile

405?????????????????410?????????????????415TTA?AAA?GCG?AGA?AAA?CAG?TAT?GCG?TAC?GGA?GCA?CAG?CAT?GAT?TAT?TTC?1296Leu?Lys?Ala?Arg?Lys?Gln?Tyr?Ala?Tyr?Gly?Ala?Gln?His?Asp?Tyr?Phe

420?????????????????425?????????????????430GAC?CAC?CAT?GAC?ATT?GTC?GGC?TGG?ACA?AGG?GAA?GGC?GAC?AGC?TCG?GTT?1344Asp?His?His?Asp?Ile?Val?Gly?Trp?Thr?Arg?Glu?Gly?Asp?Ser?Ser?Val

435?????????????????440?????????????????445GCA?AAT?TCA?GGT?TTG?GCG?GCA?TTA?ATA?ACA?GAC?GGA?CCC?GGT?GGG?GCA?1392Ala?Asn?Ser?Gly?Leu?Ala?Ala?Leu?Ile?Thr?Asp?Gly?Pro?Gly?Gly?Ala

450?????????????????455?????????????????460AAG?CGA?ATG?TAT?GTC?GGC?CGG?CAA?AAC?GCC?GGT?GAG?ACA?TGG?CAT?GAC?1440Lys?Arg?Met?Tyr?Val?Gly?Arg?Gln?Asn?Ala?Gly?Glu?Thr?Trp?His?Asp465?????????????????470?????????????????475?????????????????480ATT?ACC?GGA?AAC?CGT?TCG?GAG?CCG?GTT?GTC?ATC?AAT?TCG?GAA?GGC?TGG?1488Ile?Thr?Gly?Asn?Arg?Ser?Glu?Pro?Val?Val?Ile?Asn?Ser?Glu?Gly?Trp

485?????????????????490?????????????????495GGA?GAG?TTT?CAC?GTA?AAC?GGC?GGA?TCC?GTT?TCA?ATT?TAT?GTT?CAA?AGA?1536Gly?Glu?Phe?His?Val?Asn?Gly?Gly?Ser?Val?Ser?Ile?Tyr?Val?Gln?Arg

500?????????????????505?????????????????510TCT?CCA?ACT?CCT?GCC?CCA?TCT?CAA?AGC?CCA?ATT?AGA?AGA?GAT?GCA?TTT?1584Ser?Pro?Thr?Pro?Ala?Pro?Ser?Gln?Ser?Pro?Ile?Arg?Arg?Asp?Ala?Phe

515?????????????????520?????????????525TCA?ATA?ATC?GAA?GCG?GAA?GAA?TAT?AAC?AGC?ACA?AAT?TCC?TCC?ACT?TTA?1632Ser?Ile?Ile?Glu?Ala?Glu?Glu?Tyr?Asn?Ser?Thr?Asn?Ser?Ser?Thr?Leu

530?????????????????535?????????????????540CAA?GTG?ATT?GGA?ACG?CCA?AAT?AAT?GGC?AGA?GGA?ATT?GGT?TAT?ATT?GAA?1680Gln?Val?Ile?Gly?Thr?Pro?Asn?Asn?Gly?Arg?Gly?Ile?Gly?Tyr?Ile?Glu545?????????????????550?????????????????555?????????????????560AAT?GGT?AAT?ACC?GTA?ACT?TAC?AGC?AAT?ATA?GAT?TTT?GGT?AGT?GGT?GCA?1728Asn?Gly?Asn?Thr?Val?Thr?Tyr?Ser?Asn?Ile?Asp?Phe?Gly?Ser?Gly?Ala

565?????????????????570?????????????????575ACA?GGG?TTC?TCT?GCA?ACT?GTT?GCA?ACG?GAG?GTT?AAT?ACC?TCA?ATT?CAA?1776Thr?Gly?Phe?Ser?Ala?Thr?Val?Ala?Thr?Glu?Val?Asn?Thr?Ser?Ile?Gln

580?????????????????585?????????????????590ATC?CGT?TCT?GAC?AGT?CCT?ACC?GGA?ACT?CTA?CTT?GGT?ACC?TTA?TAT?GTA?1824Ile?Arg?Ser?Asp?Ser?Pro?Thr?Gly?Thr?Leu?Leu?Gly?Thr?Leu?Tyr?Val

595?????????????????600?????????????????605AGT?TCT?ACC?GGC?AGC?TGG?AAT?ACA?TAT?CAA?ACC?GTA?TCT?ACA?AAC?ATC?1872Ser?Ser?Thr?Gly?Ser?Trp?Asn?Thr?Tyr?Gln?Thr?Val?Ser?Thr?Asn?Ile

610?????????????????615?????????????????620AGC?AAA?ATT?ACC?GGC?GTT?CAT?GAT?ATT?GTA?TTG?GTA?TTC?TCA?GGT?CCA?1920Ser?Lys?Ile?Thr?Gly?Val?His?Asp?Ile?Val?Leu?Val?Phe?Ser?Gly?Pro625?????????????????630?????????????????635?????????????????640GTC?AAT?GTG?GAC?AAC?TTC?ATA?TTT?AGC?AGA?AGT?TCA?CCA?GTG?CCT?GCA?1968Val?Asn?Val?Asp?Asn?Phe?Ile?Phe?Ser?Arg?Ser?Ser?Pro?Val?Pro?Ala

645?????????????????650?????????????????655CCT?GGT?GAT?AAC?ACA?AGA?GAC?GCA?TAT?TCT?ATC?ATT?CAG?GCC?GAG?GAT?2016Pro?Gly?Asp?Asn?Thr?Arg?Asp?Ala?Tyr?Ser?Ile?Ile?Gln?Ala?Glu?Asp

660?????????????????665?????????????????670TAT?GAC?AGC?AGT?TAT?GGT?CCC?AAC?CTT?CAA?ATC?TTT?AGC?TTA?CCA?GGT?2064Tyr?Asp?Ser?Ser?Tyr?Gly?Pro?Asn?Leu?Gln?Ile?Phe?Ser?Leu?Pro?Gly

675?????????????????680?????????????????685GGT?GGC?AGC?GCC?ATT?GGC?TAT?ATT?GAA?AAT?GGT?TAT?TCC?ACT?ACC?TAT?2112Gly?Gly?Ser?Ala?Ile?Gly?Tyr?Ile?Glu?Asn?Gly?Tyr?Ser?Thr?Thr?Tyr

690?????????????????695?????????????????700AAA?AAT?ATT?GAT?TTT?GGT?GAC?GGC?GCA?ACG?TCC?GTA?ACA?GCA?AGA?GTA?2160Lys?Asn?Ile?Asp?Phe?Gly?Asp?Gly?Ala?Thr?Ser?Val?Thr?Ala?Arg?Val705?????????????????710?????????????????715?????????????????720GCT?ACC?CAG?AAT?GCT?ACT?ACC?ATT?CAG?GTA?AGA?TTG?GGA?AGT?CCA?TCG?2208Ala?Thr?Gln?Asn?Ala?Thr?Thr?Ile?Gln?Val?Arg?Leu?Gly?Ser?Pro?Ser

725?????????????????730?????????????????735GGT?ACA?TTA?CTT?GGA?ACA?ATT?TAC?GTG?GGG?TCC?ACA?GGA?AGC?TTT?GAT?2256Gly?Thr?Leu?Leu?Gly?Thr?Ile?Tyr?Val?Gly?Ser?Thr?Gly?Ser?Phe?Asp

740?????????????????????745?????????????????750ACT?TAT?AGG?GAT?GTA?TCC?GCT?ACC?ATT?AGT?AAT?ACT?GCG?GGT?GTA?AAA?2304Thr?Tyr?Arg?Asp?Val?Ser?Ala?Thr?Ile?Ser?Asn?Thr?Ala?Gly?Val?Lys

755?????????????????760?????????????????765GAT?ATT?GTT?CTT?GTA?TTC?TCA?GGT?CCT?GTT?AAT?GTT?GAC?TGG?????????2346Asp?Ile?Val?Leu?Val?Phe?Ser?Gly?Pro?Val?Asn?Val?Asp?Trp

The data of 770 775 780 (2) SEQ ID NO:25:

(ⅰ) sequence signature:

(A) length: 782 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: protein

(ⅹ ⅰ) sequence description: SEQ ID NO:25Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu Leu Phe 15 10 15 Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ala Asn Leu

20??????????????????25??????????????????30??Asn?Gly?Thr?Leu?Met?Gln?Tyr?Phe?Glu?Trp?Tyr?Met?Pro?Asn?Asp?Gly

35??????????????????40??????????????????45Gln?His?Trp?Lys?Arg?Leu?Gln?Asn?Asp?Ser?Ala?Tyr?Leu?Ala?Glu?His

50??????????????????55??????????????????60Gly?Ile?Thr?Ala?Val?Trp?Ile?Pro?Pro?Ala?Tyr?Lys?Gly?Thr?Ser?Gln?65??????????????????70??????????????????75??????????????????80Ala?Asp?Val?Gly?Tyr?Gly?Ala?Tyr?Asp?Leu?Tyr?Asp?Leu?Gly?Glu?Phe

85??????????????????90??????????????????95His?Gln?Lys?Gly?Thr?Val?Arg?Thr?Lys?Tyr?Gly?Thr?Lys?Gly?Glu?Leu

100?????????????????105?????????????????110Gln?Ser?Ala?Ile?Lys?Ser?Leu?His?Ser?Arg?Asp?Ile?Asn?Val?Tyr?Gly

115?????????????????120?????????????????125Asp?Val?Val?Ile?Asn?His?Lys?Gly?Gly?Ala?Asp?Ala?Thr?Glu?Asp?Val

130?????????????????135?????????????????140Thr?Ala?Val?Glu?Val?Asp?Pro?Ala?Asp?Arg?Asn?Arg?Val?Ile?Ser?Gly145?????????????????150?????????????????155?????????????????160Glu?His?Leu?Ile?Lys?Ala?Trp?Thr?His?Phe?His?Phe?Pro?Gly?Ala?Gly

165?????????????????170?????????????????175Ser?Thr?Tyr?Ser?Asp?Phe?Lys?Trp?His?Trp?Tyr?His?Phe?Asp?Gly?Thr

180?????????????????185?????????????????190Asp?Trp?Asp?Glu?Ser?Arg?Lys?Leu?Asn?Arg?Ile?Tyr?Lys?Phe?Gln?Gly

195?????????????????200?????????????????205Lys?Ala?Trp?Asp?Trp?Glu?Val?Ser?Asn?Glu?Asn?Gly?Asn?Tyr?Asp?Tyr

210?????????????????215?????????????????220Leu?Met?Tyr?Ala?Asp?Ile?Asp?Tyr?Asp?His?Pro?Asp?Val?Ala?Ala?Glu225?????????????????230?????????????????235?????????????????240Ile?Lys?Arg?Trp?Gly?Thr?Trp?Tyr?Ala?Asn?Glu?Leu?Gln?Leu?Asp?Gly

245?????????????????250?????????????????255Asn?Arg?Leu?Asp?Ala?Val?Lys?His?Ile?Lys?Phe?Ser?Phe?Leu?Arg?Asp

260?????????????????265?????????????????270Trp?Val?Asn?His?Val?Arg?Glu?Lys?Thr?Gly?Lys?Glu?Met?Phe?Thr?Val

275?????????????????280?????????????????285Ala?Glu?Tyr?Trp?Gln?Asn?Asp?Leu?Gly?Ala?Leu?Glu?Asn?Tyr?Leu?Asn

290?????????????????295?????????????????300Lys?Thr?Asn?Phe?Asn?His?Ser?Val?Phe?Asp?Val?Pro?Leu?His?Tyr?Gln305?????????????????310?????????????????315?????????????????320Phe?His?Ala?Ala?Ser?Thr?Gln?Gly?Gly?Gly?Tyr?Asp?Met?Arg?Lys?Leu

325?????????????????330?????????????????335Leu?Asn?Gly?Thr?Val?Val?Ser?Lys?His?Pro?Leu?Lys?Ser?Val?Thr?Phe

340?????????????????345?????????????????350Val?Asp?Asn?His?Asp?Thr?Gln?Pro?Gly?Gln?Ser?Leu?Glu?Ser?Thr?Val

355?????????????????360?????????????????365Gln?Thr?Trp?Phe?Lys?Pro?Leu?Ala?Tyr?Ala?Phe?Ile?Leu?Thr?Arg?Glu

370?????????????????375?????????????????380Ser?Gly?Tyr?Pro?Gln?Val?Phe?Tyr?Gly?Asp?Met?Tyr?Gly?Thr?Lys?Gly385?????????????????390?????????????????395?????????????????400Asp?Ser?Gln?Arg?Glu?Ile?Pro?Ala?Leu?Lys?His?Lys?Ile?Glu?Pro?Ile

405?????????????????410?????????????????415Leu?Lys?Ala?Arg?Lys?Gln?Tyr?Ala?Tyr?Gly?Ala?Gln?His?Asp?Tyr?Phe

420?????????????????425?????????????????430Asp?His?His?Asp?Ile?Val?Gly?Trp?Thr?Arg?Glu?Gly?Asp?Ser?Ser?Val

435?????????????????440?????????????????445Ala?Asn?Ser?Gly?Leu?Ala?Ala?Leu?Ile?Thr?Asp?Gly?Pro?Gly?Gly?Ala

450?????????????????455?????????????????460Lys?Arg?Met?Tyr?Val?Gly?Arg?Gln?Asn?Ala?Gly?Glu?Thr?Trp?His?Asp465?????????????????470?????????????????475?????????????????480Ile?Thr?Gly?Asn?Arg?Ser?Glu?Pro?Val?Val?Ile?Asn?Ser?Glu?Gly?Trp

485?????????????????490?????????????????495Gly?Glu?Phe?His?Val?Asn?Gly?Gly?Ser?Val?Ser?Ile?Tyr?Val?Gln?Arg

500?????????????????505?????????????????510Ser?Pro?Thr?Pro?Ala?Pro?Ser?Gln?Ser?Pro?Ile?Arg?Arg?Asp?Ala?Phe

515?????????????????520?????????????????525Ser?Ile?Ile?Glu?Ala?Glu?Glu?Tyr?Asn?Ser?Thr?Asn?Ser?Ser?Thr?Leu

530?????????????????535?????????????????540Gln?Val?Ile?Gly?Thr?Pro?Asn?Asn?Gly?Arg?Gly?Ile?Gly?Tyr?Ile?Glu545?????????????????550?????????????????555?????????????????560Asn?Gly?Asn?Thr?Val?Thr?Tyr?Ser?Asn?Ile?Asp?Phe?Gly?Ser?Gly?Ala

565?????????????????570?????????????????575Thr?Gly?Phe?Ser?Ala?Thr?Val?Ala?Thr?Glu?Val?Asn?Thr?Ser?Ile?Gln

580?????????????????585?????????????????590Ile?Arg?Ser?Asp?Ser?Pro?Thr?Gly?Thr?Leu?Leu?Gly?Thr?Leu?Tyr?Val

595?????????????????600?????????????????605Ser?Ser?Thr?Gly?Ser?Trp?Asn?Thr?Tyr?Gln?Thr?Val?Ser?Thr?Asn?Ile

610?????????????????615?????????????????620Ser?Lys?Ile?Thr?Gly?Val?His?Asp?Ile?Val?Leu?Val?Phe?Ser?Gly?Pro625?????????????????630?????????????????635?????????????????640Val?Asn?Val?Asp?Asn?Phe?Ile?Phe?Ser?Arg?Ser?Ser?Pro?Val?Pro?Ala

645?????????????????650?????????????????655Pro?Gly?Asp?Asn?Thr?Arg?Asp?Ala?Tyr?Ser?Ile?Ile?Gln?Ala?Glu?Asp

660?????????????????665?????????????????670Tyr?Asp?Ser?Ser?Tyr?Gly?Pro?Asn?Leu?Gln?Ile?Phe?Ser?Leu?Pro?Gly

675?????????????????680?????????????????685Gly?Gly?Ser?Ala?Ile?Gly?Tyr?Ile?Glu?Asn?Gly?Tyr?Ser?Thr?Thr?Tyr

690?????????????????695?????????????????700Lys?Asn?Ile?Asp?Phe?Gly?Asp?Gly?Ala?Thr?Ser?Val?Thr?Ala?Arg?Val705?????????????????710?????????????????715?????????????????720Ala?Thr?Gln?Asn?Ala?Thr?Thr?Ile?Gln?Val?Arg?Leu?Gly?Ser?Pro?Ser

725?????????????????730?????????????????735Gly?Thr?Leu?Leu?Gly?Thr?Ile?Tyr?Val?Gly?Ser?Thr?Gly?Ser?Phe?Asp

740?????????????????745?????????????????750Thr?Tyr?Arg?Asp?Val?Ser?Ala?Thr?Ile?Ser?Asn?Thr?Ala?Gly?Val?Lys

755?????????????????760?????????????????765Asp?Ile?Val?Leu?Val?Phe?ser?Gly?ProVal?A5n?Val?Asp?Trp

The data of 770 775 780 (2) SEQ ID NO:26:

(ⅰ) sequence signature:

(A) length: 6136 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: cDNA

(Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO: 26 TTTGACAGCT TATCATCGAC TGCACGGTGC ACCAATGCTT CTGGCGTCAG GCAGCCATCG 60 GAAGCTGTGG TATGGCTGTG CAGGTCGTAA ATCACTGCAT AATTCGTGTC GCTCAAGGCG 120 CACTCCCGTT CTGGATAATG TTTTTTGCGC CGACATCATA ACGGTTCTGG CAAATATTCT 180 GAAATGAGCT GTTGACAATT AATCATCGGC TCGTATAATG TGTGGAATTG TGAGCGGATA 240 ACAATTTCAC ACAGGAAACA GAATTGATCC ATAACTAACT AATCTAGTAA TAATTTTGTT 300 TAACTTTAAG AAGGAGATAT ATCCATGGAT CCTAGGACCA CGCCCGCACC CGGCCACCCG 360 GCCCGCGGCG CCCGCACCGC TCTGCGCACG ACGCTCGCCG CCGCGGCGGC GACGCTCGTC 420 GTCGGCGCCA CGGTCGTGCT GCCCGCCCAG GCCGCTAGCG AATTCGCAAA TCTTAATGGG 480 ACGCTGATGC AGTATTTTGA ATGGTACATG CCCAATGACG GCCAACATTG GAGGCGTTTG 540 CAAAACGACT CGGCATATTT GGCTGAACAC GGTATTACTG CCGTCTGGAT TCCCCCGGCA 600 TATAAGGGAA CGAGCCAAGC GGATGTGGGC TACGGTGCTT ACGACCTTTA TGATTTAGGG 660 GAGTTTCATC AAAAAGGGAC GGTTCGGACA AAGTACGGCA CAAAAGGAGA GCTGCAATCT 720 GCGATCAAAA GTCTTCATTC CCGCGACATT AACGTTTACG GGGATGTGGT CATCAACCAC 780 AAAGGCGGCG CTGATGCGAC CGAAGATGTA ACCGCGGTTG AAGTCGATCC CGCTGACCGC 840 AACCGCGTAA TCTCAGGAGA ACACCTAATT AAAGCCTGGA CACATTTTCA TTTTCCGGGG 900 CGCGGCAGCA CATACAGCGA TTTTAAATGG CATTGGTACC ATTTTGACGG AACCGATTGG 960 GACGAGTCCC GAAAGCTGAA CCGCATCTAT AAGTTTCAAG GAAAGGCTTG GGATTGGGAA 1020 GTTTCCAATG AAAACGGCAA CTATGATTAT TTGATGTATG CCGACATCGA TTATGACCAT 1080 CCTGATGTCG CAGCAGAAAT TAAGAGATGG GGCACTTGGT ATGCCAATGA ACTGCAATTG 1140 GACGGTTTCC GTCTTGATGC TGTCAAACAC ATTAAATTTT CTTTTTTGCG GGATTGGGTT 1200 AATCATGTCA GGGAAAAAAC GGGGAAGGAA ATGTTTACGG TAGCTGAATA TTGGCAGAAT 1260 GACTTGGGCG CGCTGGAAAA CTATTTGAAC AAAACAAATT TTAATCATTC AGTGTTTGAC 1320 GTGCCGCTTC ATTATCAGTT CCATGCTGCA TCGACACAGG GAGGCGGCTA TGATATGAGG 1380 AAATTGCTGA ACGGTACGGT CGTTTCCAAG CATCCGTTGA AATCGGTTAC ATTTGTCGAT 1440 AACCATGATA CACAGCCGGG GCAATCGCTT GAGTCGACTG TCCAAACATG GTTTAAGCCG 1500 CTTGCTTACG CTTTTATTCT CACAAGGGAA TCTGGATACC CTCAGGTTTT CTACGGGGAT 1560 ATGTACGGGA CGAAAGGAGA CTCCCAGCGC GAAATTCCTG CCTTGAAACA CAAAATTGAA 1620 CCGATCTTAA AAGCGAGAAA ACAGTATGCG TACGGAGCAC AGCATGATTA TTTCGACCAC 1680 CATGACATTG TCGGCTGGAC AAGGGAAGGC GACAGCTCGG TTGCAAATTC AGGTTTGGCG 1740 GCATTAATAA CAGACGGACC CGGTGGGGCA AAGCGAATGT ATGTCGGCCG GCAAAACGCC 1800 GGTGAGACAT GGCATGACAT TACCGGAAAC CGTTCGGAGC CGGTTGTCAT CAATTCGGAA 1860 GGCTGGGGAG AGTTTCACGT AAACGGCGGG TCGGTTTCAA TTTATGTTCA AAGAAGGCCT 1920 CCAACCCCCA CTAGTCCGAG CGCTCCCAGC GGCTGCACTG CTGAGAGGTG GGCTCAGTGC 1980 GGCGGCAATG GCTGGAGCGG CTGCACCACC TGCGTCGCTG GCAGCACTTG CACGAAGATT 2040 AATGACTGGT ACCATCAGTG CCTGTAAGCT TATTATATTA CTAATTAATT GGGGACCCTA 2100 GAGGTCCCCT TTTTTATTTT AGCTTCACGC TGCCGCAAGC ACTCAGGGCG CAAGGGCTGC 2160 TAAAGGAAGC GGAACACGTA GAAAGCCAGT CCGCAGAAAC GGTGCTGACC CCGGATGAAT 2220 GTCAGCTACT GGGCTATCTG GACAAGGGAA AACGCAAGCG CAAAGAGAAA GCAGGTAGCT 2280 TGCAGTGGGC TTACATGGCG ATAGCTAGAC TGGGCGGTTT TATGGACAGC AAGCGAACCG 2340 GAATTGCCAG CTGGGGCGCC CTCTGGTAAG GTTGGGAAGC CCTGCAAAGT AAACTGGATG 2400 GCTTTCTTGC CGCCAAGGAT CTGATGGCGC AGGGGATCAA GATCTGATCA AGAGACAGGA 2460 TGAGGATCGT TTCGCATGAT TGAACAAGAT GGATTGCACG CAGGTTCTCC GGCCGCTTGG 2520 GTGGAGAGGC TATTCGGCTA TGACTGGGCA CAACAGACAA TCGGCTGCTC TGATGCCGCC 2580 GTGTTCCGGC TGTCAGCGCA GGGGCGCCCG GTTCTTTTTG TCAAGACCGA CCTGTCCGGT 2640 GCCCTGAATG AACTGCAGGA CGAGGCAGCG CGGCTATCGT GGCTGGCCAC GACGGGCGTT 2700 CCTTGCGCAG CTGTGCTCGA CGTTGTCACT GAAGCGGGAA GGGACTGGCT GCTATTGGGC 2760 GAAGTGCCGG GGCAGGATCT CCTGTCATCT CACCTTGCTC CTGCCGAGAA AGTATCCATC 2820 ATGGCTGATG CAATGCGGCG GCTGCATACG CTTGATCCGG CTACCTGCCC ATTCGACCAC 2880 CAAGCGAAAC ATCGCATCGA GCGAGCACGT ACTCGGATGG AAGCCGGTCT TGTCGATCAG 2940 GATGATCTGG ACGAAGAGCA TCAGGGGCTC GCGCCAGCCG AACTGTTCGC CAGGCTCAAG 3000 GCGCGCATGC CCGACGGCGA GGATCTCGTC GTGACACATG GCGATGCCTG CTTGCCGAAT 3060 ATCATGGTGG AAAATGGCCG CTTTTCTGGA TTCATCGACT GTGGCCGGCT GGGTGTGGCG 3120 GACCGCTATC AGGACATAGC GTTGGCTACC CGTGATATTG CTGAAGAGCT TGGCGGCGAA 3180 TGGGCTGACC GCTTCCTCGT GCTTTACGGT ATCGCCGCTC CCGATTCGCA GCGCATCGCC 3240 TTCTATCGCC TTCTTGACGA GTTCTTCTGA GCGGGACTCT GGGGTTCGAA ATGACCGACC 3300 AAGCGACGCC CAACCTGCCA TCACGAGATT TCGATTCCAC CGCCGCCTTC TATGAAAGGT 3360 TGGGCTTCGG AATCGTTTTC CGGGACGCCG GCTGGATGAT CCTCCAGCGC GGGGATCTCA 3420 TGCTGGAGTT CTTCGCCCAC CCCAAAAGGA TCTAGGTGAA GATCCTTTTT GATAATCTCA 3480 TGACCAAAAT CCCTTAACGT GAGTTTTCGT TCCACTGAGC GTCAGACCCC GTAGAAAAGA 3540 TCAAAGGATC TTCTTGAGAT CCTTTTTTTC TGCGCGTAAT CTGCTGCTTG CAAACAAAAA 3600 AACCACCGCT ACCAGCGGTG GTTTGTTTGC CGGATCAAGA GCTACCAACT CTTTTTCCGA 3660 AGGTAACTGG CTTCAGCAGA GCGCAGATAC CAAATACTGT CCTTCTAGTG TAGCCGTAGT 3720 TAGGCCACCA CTTCAAGAAC TCTGTAGCAC CGCCTACATA CCTCGCTCTG CTAATCCTGT 3780 TACCAGTGGC TGCTGCCAGT GGCGATAAGT CGTGTCTTAC CGGGTTGGAC TCAAGACGAT 3840 AGTTACCGGA TAAGGCGCAG CGGTCGGGCT GAACGGGGGG TTCGTGCACA CAGCCCAGCT 3900 TGGAGCGAAC GACCTACACC GAACTGAGAT ACCTACAGCG TGAGCTATGA GAAAGCGCCA 3960 CGCTTCCCGA AGGGAGAAAG GCGGACAGGT ATCCGGTAAG CGGCAGGGTC GGAACAGGAG 4020 AGCGCACGAG GGAGCTTCCA GGGGGAAACG CCTGGTATCT TTATAGTCCT GTCGGGTTTC 4080 GCCACCTCTG ACTTGAGCGT CGATTTTTGT GATGCTCGTC AGGGGGGCGG AGCCTATGGA 4140 AAAACGCCAG CAACGCGGCC TTTTTACGGT TCCTGGCCTT TTGCTGGCCT TTTGCTCACA 4200 TGTTCTTTCC TGCGTTATCC CCTGATTCTG TGGATAACCG TATTACCGCC TTTGAGTGAG 4260 CTGATACCGC TCGCCGCAGC CGAACGACCG AGCGCAGCGA GTCAGTGAGC GAGGAAGCGG 4320 AAGAGCGCCT GATGCGGTAT TTTCTCCTTA CGCATCTGTG CGGTATTTCA CACCGCATAT 4380 GCAGATATTT TGTTAAAATT CGCGTTAAAT TTTTGTTAAA TCAGCTCATT TTTTAACCAA 4440 TAGGCCGAAA TCGGCAAAAT CCCTTATAAA TCAAAAGAAT AGACCGAGAT AGGGTTGAGT 4500 GTTGTTCCAG TTTGGAACAA GAGTCCACTA TTAAAGAACG TGGACTCCAA CGTCAAAGGG 4560 CGAAAAACCG TCTATCAGGG CGATGGCCCA CTACGTGAAC CATCACCCTA ATCAAGTTTT 4620 TTGGGGTCGA GGTGCCGTAA AGCACTAAAT CGGAACCCTA AAGGGAGCCC CCGATTTAGA 4680 GCTTGACGGG GAAAGCCGGC GAACGTGGCG AGAAAGGAAG GGAAGAAAGC GAAAGGAGCG 4740 GGCGCTAGGG CGCTGGCAAG TGTAGCGGTC ACGCTGCGCG TAACCACCAC ACCCGCCGCG 4800 CTTAATGCGC CGCTACAGGG CGCGTCAGGT GGCACTTTTC GGGGAAATGT GCGCGGAACC 4860 CCTATTTGTT TATTTTTCTA AATACATTCA AATATGTATC CGCTCATGAG ACAATAACCC 4920 TGCTGCATTT ACGTTGACAC CATCGAATGG TGCAAAACCT TTCGCGGTAT GGCATGATAG 4980 CGCCCGGAAG AGAGTCAATT CAGGGTGGTG AATGTGAAAC CAGTAACGTT ATACGATGTC 5040 GCAGAGTATG CCGGTGTCTC TTATCAGACC GTTTCCCGCG TGGTGAACCA GGCCAGCCAC 5100 GTTTCTGCGA AAACGCGGGA AAAAGTGGAA GCGGCGATGG CGGAGCTGAA TTACATTCCC 5160 AACCGCGTGG CACAACAACT GGCGGGCAAA CAGTCGTTGC TGATTGGCGT TGCCACCTCC 5220 AGTCTGGCCC TGCACGCGCC GTCGCAAATT GTCGCGGCGA TTAAATCTCG CGCCGATCAA 5280 CTGGGTGCCA GCGTGGTGGT GTCGATGGTA GAACGAAGCG GCGTCGAAGC CTGTAAAGCG 5340 GCGGTGCACA ATCTTCTCGC GCAACGCGTC AGTGGGCTGA TCATTAACTA TCCGCTGGAT 5400 GACCAGGATG CCATTGCTGT GGAAGCTGCC TGCACTAATG TTCCGGCGTT ATTTCTTGAT 5460 GTCTCTGACC AGACACCCAT CAACAGTATT ATTTTCTCCC ATGAAGACGG TACGCGACTG 5520 GGCGTGGAGC ATCTGGTCGC ATTGGGTCAC CAGCAAATCG CGCTGTTAGC GGGCCCATTA 5580 AGTTCTGTCT CGGCGCGTCT GCGTCTGGCT GGCTGGCATA AATATCTCAC TCGCAATCAA 5640 ATTCAGCCGA TAGCGGAACG GGAAGGCGAC TGGAGTGCCA TGTCCGGTTT TCAACAAACC 5700 ATGCAAATGC TGAATGAGGG CATCGTTCCC ACTGCGATGC TGGTTGCCAA CGATCAGATG 5760 GCGCTGGGCG CAATGCGCGC CATTACCGAG TCCGGGCTGC GCGTTGGTGC GGATATCTCG 5820 GTAGTGGGAT ACGACGATAC CGAAGACAGC TCATGTTATA TCCCGCCGTT AACCACCATC 5880 AAACAGGATT TTCGCCTGCT GGGGCAAACC AGCGTGGACC GCTTGCTGCA ACTCTCTCAG 5940 GGCCAGGCGG TGAAGGGCAA TCAGCTGTTG CCCGTCTCAC TGGTGAAAAG AAAAACCACC 6000 CTGGCGCCCA ATACGCAAAC CGCCTCTCCC CGCGCGTTGG CCGATTCATT AATGCAGCTG 6060 GCACGACAGG TTTCCCGACT GGAAAGCGGG CAGTGAGCGC AACGCAATTA ATGTGAGTTA 6120 GCGCGAATTG ATCTGG 6136 (2) SEQ ID NO: 27 of the data: ...

(ⅰ) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 14 "

(ⅹ ⅰ) sequence description: the data of SEQ ID NO:27AGGTCTACTA GTCCCGGCTG CCGCGTCGAC 30 (2) SEQ ID NO:28:

(ⅰ) sequence signature:

(A) length: 53 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 15 "

(ⅹ ⅰ) sequence description: the data of SEQ ID NO:28CCGATTAAAG CTTATTAGCT AGCACGGAAT TCCGTGGGGC TGGTCGTCGG CAC 53 (2) SEQ ID NO:29:

(ⅰ) sequence signature:

(A) length: 42 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 16 "

(ⅹ ⅰ) sequence description: the data of SEQ ID NO:29TCATGAGCCA TGGCTAGCGC AAATCTTAAT GGGACGCTGA TG 42 (2) SEQ ID NO:30:

(ⅰ) sequence signature:

(A) length: 69 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 17 "

(ⅹ ⅰ) sequence description: SEQ ID NO:30ATGACTAAGC TTACTTACTT AGTGATGGTG ATGGTGATGA CTAGTTCTTT GAACATAAAT TGAAACCGA

The data of 69 (2) SEQ ID NO:31:

(ⅰ) sequence signature:

(A) length: 1959 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: cDNA

(ⅸ) feature:

(A) title/keyword: CDS

(B) position: 1..1959

(ⅹ ⅰ) sequence description: SEQ IDNO:31ATG GAT CCT AGG ACC ACG CCC GCA CCC GGC CAC CCG GCC CGC GGC GCC 48Met Asp Pro Arg Thr Thr Pro Ala Pro Gly His Pro Ala Arg Gly Ala 15 10 15CGC ACC GCT CTG CGC ACG ACG CTC GCC GCC GCG GCG GCG ACG CTC GTC 96Arg Thr Ala Leu Arg Thr Thr Leu Ala Ala Ala Ala Ala Thr Leu Val

20??????????????????25??????????????????30GTC?GGC?GCC?ACG?GTC?GTG?CTG?CCC?GCC?CAG?GCC?GCT?AGT?CCC?GGC?TGC?144Val?Gly?Ala?Thr?Val?Val?Leu?Pro?Ala?Gln?Ala?Ala?Ser?Pro?Gly?Cys

35??????????????????40??????????????????45CGC?GTC?GAC?TAC?GCC?GTC?ACC?AAC?CAG?TGG?CCC?GGC?GGC?TTC?GGC?GCC?192Arg?Val?Asp?Tyr?Ala?Val?Thr?Asn?Gln?Trp?Pro?Gly?Gly?Phe?Gly?Ala

50??????????????????55??????????????????60AAC?GTC?ACG?ATC?ACC?AAC?CTC?GGC?GAC?CCC?GTC?TCG?TCG?TGG?AAG?CTC??240Asn?Val?Thr?Ile?Thr?Asn?Leu?Gly?Asp?Pro?Val?Ser?Ser?Trp?Lys?Leu?65??????????????????70??????????????????75??????????????????80GAC?TGG?ACC?TAC?ACC?GCA?GGC?CAG?CGG?ATC?CAG?CAG?CTG?TGG?AAC?GGC??288Asp?Trp?Thr?Tyr?Thr?Ala?Gly?Gln?Arg?Ile?Gln?Gln?Leu?Trp?Asn?Gly

85??????????????????90??????????????????95ACC?GCG?TCG?ACC?AAC?GGC?GGC?CAG?GTC?TCC?GTC?ACC?AGC?CTG?CCC?TGG??336Thr?Ala?Ser?Thr?Asn?Gly?Gly?Gln?Val?Ser?Val?Thr?Ser?Leu?Pro?Trp

100?????????????????105?????????????????110AAC?GGC?AGC?ATC?CCG?ACC?GGC?GGC?ACG?GCG?TCG?TTC?GGG?TTC?AAC?GGC??384Aan?Gly?Ser?Ile?Pro?Thr?Gly?Gly?Thr?Ala?Ser?Phe?Gly?Phe?Asn?Gly

115?????????????????120?????????????????125TCG?TGG?GCC?GGG?TCC?AAC?CCG?ACG?CCG?GCG?TCG?TTC?TCG?CTC?AAC?GGC??432Ser?Trp?Ala?Gly?Ser?Asn?Pro?Thr?Pro?Ala?Ser?Phe?Ser?Leu?Asn?Gly

130?????????????????135?????????????????140ACC?ACC?TGC?ACG?GGC?ACC?GTG?CCG?ACG?ACC?AGC?CCC?ACG?GAA?TTC?CGT??480Thr?Thr?Cys?Thr?Gly?Thr?Val?Pro?Thr?Thr?Ser?Pro?Thr?Glu?Phe?Arg145?????????????????150?????????????????155?????????????????160GCT?AGC?GCA?AAT?CTT?AAT?GGG?ACG?CTG?ATG?CAG?TAT?TTT?GAA?TGG?TAC??528Ala?Ser?Ala?Asn?Leu?Asn?Gly?Thr?Leu?Met?Gln?Tyr?Phe?Glu?Trp?Tyr

165?????????????????170?????????????????175ATG?CCC?AAT?GAC?GGC?CAA?CAT?TGG?AAG?CGC?TTG?CAA?AAC?GAC?TCG?GCA??576Met?Pro?Asn?Asp?Gly?Gln?His?Trp?Lys?Arg?Leu?Gln?Asn?Asp?Ser?Ala

180?????????????????185?????????????????190TAT?TTG?GCT?GAA?CAC?GGT?ATT?ACT?GCC?GTC?TGG?ATT?CCC?CCG?GCA?TAT??624Tyr?Leu?Ala?Glu?His?Gly?Ile?Thr?Ala?Val?Trp?Ile?Pro?Pro?Ala?Tyr

195?????????????????200?????????????????205AAG?GGA?ACG?AGC?CAA?GCG?GAT?GTG?GGC?TAC?GGT?GCT?TAC?GAC?CTT?TAT??672Lys?Gly?Thr?Ser?Gln?Ala?Asp?Val?Gly?Tyr?Gly?Ala?Tyr?Asp?Leu?Tyr

210?????????????????215?????????????????220GAT?TTA?GGG?GAG?TTT?CAT?CAA?AAA?GGG?ACG?GTT?CGG?ACA?AAG?TAC?GGC??720Asp?Leu?Gly?Glu?Phe?His?Gln?Lys?Gly?Thr?Val?Arg?Thr?Lys?Tyr?Gly225?????????????????230?????????????????235?????????????????240ACA?AAA?GGA?GAG?CTG?CAA?TCT?GCG?ATC?AAA?AGT?CTT?CAT?TCC?CGC?GAC??768Thr?Lys?Gly?Glu?Leu?Gln?Ser?Ala?Ile?Lys?Ser?Leu?His?Ser?Arg?Asp

245?????????????????250?????????????????255ATT?AAC?GTT?TAC?GGG?GAT?GTG?GTC?ATC?AAC?CAC?AAA?GGC?GGC?GCT?GAT??816Ile?Asn?Val?Tyr?Gly?Asp?Val?Val?Ile?Asn?His?Lys?Gly?Gly?Ala?Asp

260?????????????????265?????????????????270GCG?ACC?GAA?GAT?GTA?ACC?GCG?GTT?GAA?GTC?GAT?CCC?GCT?GAC?CGC?AAC??864Ala?Thr?Glu?Asp?Val?Thr?Ala?Val?Glu?Val?Asp?Pro?Ala?Asp?Arg?Asn

275?????????????????280?????????????????285CGC?GTA?ATT?TCA?GGA?GAA?CAC?TTA?ATT?AAA?GCC?TGG?ACA?CAT?TTT?CAT??912Arg?Val?Ile?Ser?Gly?Glu?His?Leu?Ile?Lys?Ala?Trp?Thr?His?Phe?His

290?????????????????295?????????????????300TTT?CCG?GGG?CGC?GGC?AGC?ACA?TAC?AGC?GAT?TTT?AAA?TGG?CAT?TGG?TAC??960Phe?Pro?Gly?Arg?Gly?Ser?Thr?Tyr?Ser?Asp?Phe?Lys?Trp?His?Trp?Tyr305?????????????????310?????????????????315?????????????????320CAT?TTT?GAC?GGA?ACC?GAT?TGG?GAC?GAG?TCC?CGA?AAG?CTG?AAC?CGC?ATC?1008His?Phe?Asp?Gly?Thr?Asp?Trp?Asp?Glu?Ser?Arg?Lys?Leu?Asn?Arg?Ile

325?????????????????330?????????????????335TAT?AAG?TTT?CAA?GGA?AAG?GCT?TGG?GAT?TGG?GAA?GTT?TCC?AAT?GAA?AAC?1056Tyr?Lys?Phe?Gln?Gly?Lys?Ala?Trp?Asp?Trp?Glu?Val?Ser?Asn?Glu?Asn

340?????????????????345?????????????????350GGC?AAC?TAT?GAT?TAT?TTG?ATG?TAT?GCC?GAC?ATC?GAT?TAT?GAT?CAT?CCT?1104Gly?Asn?Tyr?Asp?Tyr?Leu?Met?Tyr?Ala?Asp?Ile?Asp?Tyr?Asp?His?Pro

355?????????????????360?????????????????365GAT?GTC?GCA?GCA?GAA?ATT?AAG?AGA?TGG?GGC?ACT?TGG?TAT?GCC?AAT?GAA?1152Asp?Val?Ala?Ala?Glu?Ile?Lys?Arg?Trp?Gly?Thr?Trp?Tyr?Ala?Asn?Glu

370?????????????????375?????????????????380CTG?CAA?TTG?GAC?GGT?TTC?CGT?CTT?GAT?GCT?GTC?AAA?CAC?ATT?AAA?TTT?1200Leu?Gin?Leu?Asp?Gly?Phe?Arg?Leu?Asp?Ala?Val?Lys?His?Ile?Lys?Phe385?????????????????390?????????????????395?????????????????400TCT?TTT?TTG?CGG?GAT?TGG?GTT?AAT?CAT?GTC?AGG?GAA?AAA?ACG?GGG?AAG?1248Ser?Phe?Leu?Arg?Asp?Trp?Val?Asn?His?Val?Arg?Glu?Lys?Thr?Gly?Lys

405??????????????????410????????????????415GAA?ATG?TTT?ACG?GTA?GCT?GAA?TAT?TGG?CAG?AAT?GAC?TTG?GGC?GCG?CTG?1296Glu?Met?Phe?Thr?Val?Ala?Glu?Tyr?Trp?Gln?Asn?Asp?Leu?Gly?Ala?Leu

420?????????????????425?????????????????430GAA?AAC?TAT?TTG?AAC?AAA?ACA?AAT?TTT?AAT?CAT?TCA?GTG?TTT?GAC?GTG?1344Glu?Asn?Tyr?Leu?Asn?Lys?Thr?Asn?Phe?Asn?His?Ser?Val?Phe?Asp?Val

435?????????????????440?????????????????445CCG?CTT?CAT?TAT?CAG?TTC?CAT?GCT?GCA?TCG?ACA?CAG?GGA?GGC?GGC?TAT?1392Pro?Leu?His?Tyr?Gln?Phe?His?Ala?Ala?Ser?Thr?Gln?Gly?Gly?Gly?Tyr

450?????????????????455?????????????????460GAT?ATG?AGG?AAA?TTG?CTG?AAC?GGT?ACG?GTC?GTT?TCC?AAG?CAT?CCG?TTG?1440Asp?Met?Arg?Lys?Leu?Leu?Asn?Gly?Thr?Val?Val?Ser?Lys?His?Pro?Leu465?????????????????470??????????????????475????????????????480AAA?GCG?GTT?ACA?TTT?GTC?GAT?AAC?CAT?GAT?ACA?CAG?CCG?GGG?CAA?TCG?1488Lys?Ala?Val?Thr?Phe?Val?Asp?Asn?His?Asp?Thr?Gln?Pro?Gly?Gln?Ser

485?????????????????490?????????????????495CTT?GAG?TCG?ACT?GTC?CAA?ACA?TGG?TTT?AAG?CCG?CTT?GCT?TAC?GCT?TTT?1536Leu?Glu?Ser?Thr?Val?Gln?Thr?Trp?Phe?Lys?Pro?Leu?Ala?Tyr?Ala?Phe

500?????????????????505?????????????????510ATT?CTC?ACA?AGG?GAA?TCT?GGA?TAC?CCT?CAG?GTT?TTC?TAC?GGG?GAT?ATG?1584Ile?Leu?Thr?Arg?Glu?Ser?Gly?Tyr?Pro?Gln?Val?Phe?Tyr?Gly?Asp?Met

515?????????????????520?????????????????525TAC?GGG?ACG?AAA?GGA?GAC?TCC?CAG?CGC?GAA?ATT?CCT?GCC?TTG?AAA?CAC?1632Tyr?Gly?Thr?Lys?Gly?Asp?Ser?Gln?Arg?Glu?Ile?Pro?Ala?Leu?Lys?His

530?????????????????535?????????????????540AAA?ATT?GAA?CCG?ATC?TTA?AAA?GCG?AGA?AAA?CAG?TAT?GCG?TAC?GGA?GCA?1680Lys?Ile?Glu?Pro?Ile?Leu?Lys?Ala?Arg?Lys?Gln?Tyr?Ala?Tyr?Gly?Ala545?????????????????550?????????????????555?????????????????560CAG?CAT?GAT?TAT?TTC?GAC?CAC?CAT?GAC?ATT?GTC?GGC?TGG?ACA?AGG?GAA?1728Gln?His?Asp?Tyr?Phe?Asp?His?His?Asp?Ile?Val?Gly?Trp?Thr?Arg?Glu

565?????????????????570?????????????????575GGC?GAC?AGC?TCG?GTT?GCA?AAT?TCA?GGT?TTG?GCG?GCA?TTA?ATA?ACA?GAC?1776Gly?Asp?Ser?Ser?Val?Ala?Asn?Ser?Gly?Leu?Ala?Ala?Leu?Ile?Thr?Asp

580?????????????????585?????????????????590GGA?CCC?GGT?GGG?GCA?AAG?CGA?ATG?TAT?GTC?GGC?CGG?CAA?AAC?GCC?GGT?1824Gly?Pro?Gly?Gly?Ala?Lys?Arg?Met?Tyr?Val?Gly?Arg?Gln?Asn?Ala?Gly

595?????????????????600?????????????????605GAG?ACA?TGG?CAT?GAC?ATT?ACC?GGA?AAC?CGT?TCG?GAG?CCG?GTT?GTC?ATC?1872Glu?Thr?Trp?His?Asp?Ile?Thr?Gly?Asn?Arg?Ser?Glu?Pro?Val?Val?Ile

610?????????????????615?????????????????620AAT?TCG?GAA?GGC?TGG?GGA?GAG?TTT?CAC?GTA?AAC?GGC?GGG?TCG?GTT?TCA?1920Asn?Ser?Glu?Gly?Trp?Gly?Glu?Phe?His?Val?Asn?Gly?Gly?Ser?Val?Ser625?????????????????630????????????????635??????????????????640ATT?TAT?GTT?CAA?AGA?ACT?AGT?CAT?CAC?CAT?CAC?CAT?CACIle?Tyr?Val?Gln?Arg?Thr?Ser?His?His?His?His?His?His

The data of 645 650 (2) SEQ ID NO:32:

(ⅰ) sequence signature:

(A) length: 653 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: protein

(ⅹ ⅰ) sequence description: SEQ ID NO:32Met Asp Pro Arg Thr Thr Pro Ala Pro Gly His Pro Ala Arg Gly Ala 15 10 15Arg Thr Ala Leu Arg Thr Thr Leu Ala Ala Ala Ala Ala Thr Leu Val

20??????????????????25??????????????????30Val?Gly?Ala?Thr?Val?Val?Leu?Pro?Ala?Gln?Ala?Ala?Ser?Pro?Gly?Cys

35??????????????????40??????????????????45Arg?Val?Asp?Tyr?Ala?Val?Thr?Asn?Gln?Trp?Pro?Gly?Gly?Phe?Gly?Ala

50??????????????????55??????????????????60Asn?Val?Thr?Ile?Thr?Asn?Leu?Gly?Asp?Pro?Val?Ser?Ser?Trp?Lys?Leu?65??????????????????70??????????????????75??????????????????80Asp?Trp?Thr?Tyr?Thr?Ala?Gly?Gln?Arg?Ile?Gln?Gln?Leu?Trp?Asn?Gly

85??????????????????90??????????????????95Thr?Ala?Ser?Thr?Asn?Gly?Gly?Gln?Val?Ser?Val?Thr?Ser?Leu?Pro?Trp

100?????????????????105?????????????????110Asn?Gly?Ser?Ile?Pro?Thr?Gly?Gly?Thr?Ala?Ser?Phe?Gly?Phe?Asn?Gly

115?????????????????120?????????????????125Ser?Trp?Ala?Gly?Ser?Asn?Pro?Thr?Pro?Ala?Ser?Phe?Ser?Leu?Asn?Gly

130?????????????????135?????????????????140Thr?Thr?Cys?Thr?Gly?Thr?Val?Pro?Thr?Thr?Ser?Pro?Thr?Glu?Phe?Arg145?????????????????150?????????????????155?????????????????160Ala?Ser?Ala?Asn?Leu?Asn?Gly?Thr?Leu?Met?Gln?Tyr?Phe?Glu?Trp?Tyr

165?????????????????170?????????????????175Met?Pro?Asn?Asp?Gly?Gln?His?Trp?Lys?Arg?Leu?Gln?Asn?Asp?Ser?Ala

180?????????????????185?????????????????190Tyr?Leu?Ala?Glu?His?Gly?Ile?Thr?Ala?Val?Trp?Ile?Pro?Pro?Ala?Tyr

195?????????????????200?????????????????205Lys?Gly?Thr?Ser?Gln?Ala?Asp?Val?Gly?Tyr?Gly?Ala?Tyr?Asp?Leu?Tyr

210?????????????????215?????????????????220Asp?Leu?Gly?Glu?Phe?His?Gln?Lys?Gly?Thr?Val?Arg?Thr?Lys?Tyr?Gly225?????????????????230?????????????????235?????????????????240Thr?Lys?Gly?Glu?Leu?Gln?Ser?Ala?Ile?Lys?Ser?Leu?His?Ser?Arg?Asp

245?????????????????250?????????????????255Ile?Asn?Val?Tyr?Gly?Asp?Val?Val?Ile?Asn?His?Lys?Gly?Gly?Ala?Asp

260?????????????????265?????????????????270Ala?Thr?Glu?Asp?Val?Thr?Ala?Val?Glu?Val?Asp?Pro?Ala?Asp?Arg?Asn

275?????????????????280?????????????????285Arg?Val?Ile?Ser?Gly?Glu?His?Leu?Ile?Lys?Ala?Trp?Thr?His?Phe?His

290?????????????????295?????????????????300Phe?Pro?Gly?Arg?Gly?Ser?Thr?Tyr?Ser?Asp?Phe?Lys?Trp?His?Trp?Tyr305?????????????????310?????????????????315?????????????????320His?Phe?Asp?Gly?Thr?Asp?Trp?Asp?Glu?Ser?Arg?Lys?Leu?Asn?Arg?Ile

325?????????????????330?????????????????335Tyr?Lys?Phe?Gln?Gly?Lys?Ala?Trp?Asp?Trp?Glu?Val?Ser?Asn?Glu?Asn

340?????????????????345?????????????????350Gly?Asn?Tyr?Asp?Tyr?Leu?Met?Tyr?Ala?Asp?Ile?Asp?Tyr?Asp?His?Pro

355?????????????????360?????????????????365Asp?Val?Ala?Ala?Glu?Ile?Lys?Arg?Trp?Gly?Thr?Trp?Tyr?Ala?Asn?Glu

370?????????????????375?????????????????380Leu?Gln?Leu?Asp?Gly?Phe?Arg?Leu?Asp?Ala?Val?Lys?His?Ile?Lys?Phe385?????????????????390?????????????????395?????????????????400Ser?Phe?Leu?Arg?Asp?Trp?Val?Asn?His?Val?Arg?Glu?Lys?Thr?Gly?Lys

405?????????????????410?????????????????415Glu?Met?Phe?Thr?Val?Ala?Glu?Tyr?Trp?Gln?Asn?Asp?Leu?Gly?Ala?Leu

420?????????????????425?????????????????430Glu?Asn?Tyr?Leu?Asn?Lys?Thr?Asn?Phe?Asn?His?Ser?Val?Phe?Asp?Val

435?????????????????440?????????????????445Pro?Leu?His?Tyr?Gln?Phe?His?Ala?Ala?Ser?Thr?Gln?Gly?Gly?Gly?Tyr

450?????????????????455?????????????????460Aep?Met?Arg?Lys?Leu?Leu?Asn?Gly?Thr?Val?Val?Ser?Lys?His?Pro?Leu465?????????????????470?????????????????475?????????????????480Lys?Ala?Val?Thr?Phe?Val?Asp?Asn?His?Asp?Thr?Gln?Pro?Gly?Gln?Ser

485?????????????????490?????????????????495Leu?Glu?Ser?Thr?Val?Gln?Thr?Trp?Phe?Lys?Pro?Leu?Ala?Tyr?Ala?Phe

500?????????????????505?????????????????510Ile?Leu?Thr?Arg?Glu?Ser?Gly?Tyr?Pro?Gln?Val?Phe?Tyr?Gly?Asp?Met

515?????????????????520?????????????????525Tyr?Gly?Thr?Lys?Gly?Asp?Ser?Gln?Arg?Glu?Ile?Pro?Ala?Leu?Lys?His

530?????????????????535?????????????????540Lys?Ile?Glu?Pro?Ile?Leu?Lys?Ala?Arg?Lys?Gln?Tyr?Ala?Tyr?Gly?Ala545?????????????????550?????????????????555?????????????????560Gln?His?Asp?Tyr?Phe?Asp?His?His?Asp?Ile?Val?Gly?Trp?Thr?Arg?Glu

565?????????????????570?????????????????575Gly?Asp?Ser?Ser?Val?Ala?Asn?Ser?Gly?Leu?Ala?Ala?Leu?Ile?Thr?Asp

580?????????????????585?????????????????590Gly?Pro?Gly?Gly?Ala?Lys?Arg?Met?Tyr?Val?Gly?Arg?Gln?Asn?Ala?Gly

595?????????????????600?????????????????605Glu?Thr?Trp?His?Asp?Ile?Thr?Gly?Asn?Arg?Ser?Glu?Pro?Val?Val?Ile

610?????????????????615?????????????????620Asn?Ser?Glu?Gly?Trp?Gly?Glu?Phe?His?Val?Asn?Gly?Gly?Ser?Val?Ser625?????????????????630?????????????????635?????????????????640Ile?Tyr?Val?Gln?Arg?Thr?Ser?His?His?His?His?His?His

The data of 645 650 (2) SEQ ID NO:33:

(ⅰ) sequence signature:

(A) length: 29 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 18 "

(ⅹ ⅰ) sequence description: the data of SEQ ID NO:33CATATGGCTA GCGAATTCGC AAATCTTAAT GGGACGCTG 29 (2) SEQ ID NO:34:

(ⅰ) sequence signature:

(A) length: 28 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 19 "

(ⅹ ⅰ) sequence description: the data of SEQ ID NO:34AAGCTTACTA GTAGGCCTTC TTTGAACATA AATTGAAA 28 (2) SEQ ID NO:35:

(ⅰ) sequence signature:

(A) length: 70 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 20 "

(ⅹ ⅰ) sequence description: the data of SEQ ID NO:35CCATGGGCTA GCCCTGAATT CAGGCCTCCA ACCCCCACTA GTCCGAGCGC TCCCAGCGGCTGCACTGCTG 70 (2) SEQ ID NO:36:

(ⅰ) sequence signature:

(A) length: 32 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: other nucleic acid

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" primer 21 "

(ⅹ ⅰ) sequence description: the data of SEQ ID NO:36AGCCTAAGCT TACAGGCACT GATGGTACCA GT 32 (2) SEQ ID NO:37:

(ⅰ) sequence signature:

(A) length: 12 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: protein

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) out of Memory :/desc=" joint "

(ⅹ ⅰ) sequence description: SEQ ID NO:37Arg Pro Pro Thr Pro Thr Ser Pro Ser Ala Pro Ser1 5 10

Claims (23)

1. method that is used for liquefying starch, wherein starch substrates is handled with modified enzyme (enzyme heterozygote) in water-bearing media, this modified enzyme comprises the α-Dian Fenmei aminoacid sequence that has connected the aminoacid sequence that contains carbohydrate-binding domain (CBD).

2. the method for liquefying starch according to claim 1 wherein also comprises debranching factor.

3. method according to claim 2, wherein debranching factor is the modified debranching factor (enzyme heterozygote) that has connected the aminoacid sequence that contains carbohydrate-binding domain (CBD).

4. the method that the starch that is used for having processed having carried out liquefying carries out saccharification, reaction mixture after wherein will liquefying is handled with modified enzyme (enzyme heterozygote), and described modified enzyme comprises the debranching factor aminoacid sequence that has connected on the aminoacid sequence that contains carbohydrate-binding domain (CBD).

5. according to the described method of claim 2,3 or 4, wherein said debranching factor is isoamylase or Starch debranching enzyme.

6. the method that the starch that is used for having processed having carried out liquefying carries out saccharification, reaction mixture after wherein will liquefying is handled with a kind of modified enzyme (enzyme heterozygote), and described modified enzyme comprises the glucose starch enzyme amino acid sequence that has connected the aminoacid sequence that contains carbohydrate-binding domain (CBD).

7. according to the described method of each claim of front, wherein said CBD is the CBD that comes from cellulase, zytase, mannase, arabinofuranosidase, acetylase, chitinase, glucoamylase or CGT enzyme.

8. the purposes of modified enzyme (enzyme heterozygote) in liquefying starch that comprises the α-Dian Fenmei aminoacid sequence that has connected the aminoacid sequence that contains carbohydrate-binding domain (CBD).

9. the purposes of modified enzyme (enzyme heterozygote) in the saccharification processing of the starch that has carried out liquefaction processing that comprises the debranching factor aminoacid sequence that has connected the aminoacid sequence that contains carbohydrate-binding domain (CBD).

10. the purposes of modified enzyme (enzyme heterozygote) in the saccharification processing of the starch that has carried out liquefaction processing that comprises the glucose starch enzyme amino acid sequence that has connected the aminoacid sequence that contains carbohydrate-binding domain (CBD).

11. coding has the separated DNA sequence of the heterozygote enzyme of amylolytic activity, wherein comprises:

(a) dna sequence dna of coding amylolytic activity;

(b) dna sequence dna of coding CBD; With

(c) dna sequence dna or its fragment of joint sequence shown in the coding SEQ ID NO:21.

12. separated DNA sequence according to claim 11, wherein amylolytic activity is an alpha-amylase activity, especially genus bacillus α-Dian Fenmei, particularly Termamyl Or the activity of its variant.

13. according to claim 11 or 12 described separated DNA sequences, wherein said CBD is the CBD of Bacillus agaradherens NCIMB No.40482 alkali cellulose enzyme Cel5A.

14. separated DNA sequence according to claim 13, the Termamyl shown in its coding SEQ ID NO:19 by plasmid pMB492 coding

-joint-Cel5A-CBD.

15. according to claim 11 or 12 described separated DNA sequences, wherein said CBD is the CBD dimer of Clostridium stercorarium (NCIMB 11754) XynA.

16. a DNA construct wherein comprises each described dna sequence dna among the claim 11-15 of one or more control sequences that have been operably connected, described control sequence can instruct this dna sequence dna to express in suitable expressive host.

17. DNA construct according to claim 16, wherein comprise the nucleotide sequence that coding is selected from following group promotor: the promotor of bacstearothermophilus Fructus Hordei Germinatus ative starch enzyme gene, the promotor of bacillus licheniformis alpha amylase gene, bacillus amyloliquefaciens BAN

The promotor of amylase gene, the promotor of bacillus subtilis alkali proteinase gene, or the promotor of bacillus pumilus cellulase or xylosidase gene.

18. a recombinant expression vector wherein comprises claim 16 or 17 described DNA construct, promotor and transcribing and the translation termination signal.

19. comprise the host cell of claim 16 or 17 described DNA construct.

20. cell according to claim 19, wherein cell is a kind of bacillus cell that comes from following group bacterial strain: bacillus subtilis strain, Bacillus licheniformis, bacillus lentus, bacillus brevis, bacstearothermophilus, Alkaliphilic bacillus, bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, bacillus lautus, bacillus megaterium, bacillus pumilus, bacillus thuringiensis or B.agaradherens.

21. a method of producing CBD/ heterozygote enzyme is included under the condition that allows this enzyme to produce and cultivates claim 19 or 20 described cells, and reclaim this enzyme from culture.

22. CBD/ enzyme heterozygote by the isolating and purifying of each described dna sequence encoding among the claim 11-15.

23. CBD/ enzyme heterozygote according to claim 22, it is the heterozygote enzyme shown in the SEQ ID NO:20.