CN101962633A - Alpha-amylase, encoding gene and expression thereof - Google Patents

Alpha-amylase, encoding gene and expression thereof Download PDF

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CN101962633A
CN101962633A CN2009100552044A CN200910055204A CN101962633A CN 101962633 A CN101962633 A CN 101962633A CN 2009100552044 A CN2009100552044 A CN 2009100552044A CN 200910055204 A CN200910055204 A CN 200910055204A CN 101962633 A CN101962633 A CN 101962633A
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gly
asp
ala
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tyr
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CN101962633B (en
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叶秀云
靳伟刚
张洋
罗鋆琳
赖庆安
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Fujian Fuda Biotech Co Ltd
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Fujian Fuda Biotech Co Ltd
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Abstract

The invention relates to alpha-amylase, an encoding gene and expression thereof. The invention disclose alpha-amylase with a general formula sequence disclosed by SEQ ID NO.11and also discloses the encoding gene of the alpha-amylase, an expression vector and cells with the encoding gene, a preparation method and the application of the alpha-amylase.

Description

α-Dian Fenmei, its encoding gene and expression thereof
Technical field
The invention belongs to bioengineering field, more specifically, the present invention relates to α-Dian Fenmei, its encoding gene contains the recombinant plasmid and the bacterial strain of this gene and expression method.
Background technology
Starch (Starch) is a kind of carbohydrate, almost completely is that unit is polymerized with the alpha-D-glucose, and the chain mean length is a 500-1000 glucosyl residue.The hot water soluble part of energy is amylose starch (Amylase) in the starch, the undissolvable amylopectin (Amylopectin) that cries, once be called alpha-amylose or red amylose starch (Erythroamylose), these two kinds of main components that composition is a starch granules, content can reach 75%-80%.
The amylose molecule amount forms with the condensation of α-1,4 glucosides of bonding between 10-2000kD, is curled into volution, meets iodine and is hyacinthine, and each sugar chain contains several thousand glucosyl residues, and amylose starch is maximum photoabsorption at 620-680nm.The amylopectin molecular weight is between 50-400000kD, mainly with α-1,4 glucosides of bonding condensations form, and are formed by the condensation of α-1,6 glucosides of bonding on a small quantity, the α-1 of average per 24 1 terminal glucose residues of having an appointment, 6 side chains occur, and amylopectin divides sub-branch a lot, and swelling becomes starch paste in hot water, meet iodine and be red-purple, be maximum light absorption at 530-550nm.
Starch almost is present in the most tissues of all green plantss, is a kind of storage form of plant nutrient substance, is present in the plant organism with particle shape at microscopically.Can mainly corn as the crop of starch material as things go, next is potato class, paddy and wheat, China's total output of grain reached 46947.2 ten thousand tons in 2004, and these four kinds of food crop annual production account for China's food crop ultimate production more than 90%, so be quite abundant at China's starch resource.
Amylase is the common name of hydrolyzed starch and glycogen enzyme, extensively is present in animals and plants and the microorganism.Because therefore the important value of amylase in industrial or agricultural have the people that amylase is studied very early.Payen in 1833 and Persoz are separated to amylase with alcohol precipitation first from the water extract of Fructus Hordei Germinatus.Japanese peak in 1894 allows Ji cultivate aspergillus oryzae (Aspergillus oryzae) with wheat bran and uses water extraction, again with alcohol precipitation, obtain the enzyme as digestive pharmaceutical, i.e. taka-diastase.French Boidin in 1919 and Effront produce amylase with Bacillus subtilus first, for the industrial production of microbial enzyme lays the foundation.1949, the submerged fermentation of the α-Dian Fenmei of the carrying out of Japanese's success, thus promoted the development of the scale operation of enzyme.Amylase is broadly divided into four big classes according to the difference of hydrolyzed starch mode: (1) α-Dian Fenmei (EC 3.2.1.1), it is a substrate with glycogen or starch, cut α-1,4 glycosidic link and make substrate hydrolysis become dextrin and a spot of glucose and maltose from intramolecule.(2) beta-amylase (EC 3.2.1.2): from the non-reduced end of substrate at interval incision α-1 successively, 4 glycosidic links, therefore the product of gained is a maltose when acting on amylose starch, and can only obtain 50%-65% maltose, residual about 40% limit dextrin down when acting on side chain.(3) glucoamylase (EC 3.2.1.3): be called for short saccharifying enzyme traditionally,, generate glucose from substrate non reducing end hydrolyzing alpha successively-1,4 glycosidic link and tapping point α-1,6 glycosidic link.(4) isoamylase (EC 3.2.1.9): a hydrolysis glycogen or amylopectin tapping point α-1,6 glycosidic link, downcut whole side shoot.
On the catalysis specificity, α-Dian Fenmei is classified as the 13rd family of glycoside hydrolysis enzyme (glycosyl hydrases), belong to alpha-glucosaccharase lytic enzyme (α-Glycosyl hydrases, EC 3.2.1), (α-amylase) is called liquefying amylase again to α-Dian Fenmei, its systematic name is α-1,4-dextran-4-glucan hydrolase (α-1,4-glucan-glucanhydrolase EC 3.2.1.1), the common name α-Dian Fenmei, have another name called α-1,4 dextrinases are a kind of restriction endonucleases, the α-1 in the energy hydrolyzed starch molecule, 4 glycosidic links, it is cut into short chain dextrin different in size and a spot of low relative molecular mass carbohydrate arbitrarily, and amylose starch and amylopectin all decompose with random form, thereby the viscosity of starch paste is descended rapidly, promptly " liquefaction " work, so α-Dian Fenmei is called Ye Huamei again.
α-Dian Fenmeishuixie starch mechanism is elaborated after the fifties gradually in eighties of last century: α-Dian Fenmei is that amylose starch at random promptly is degraded into small molecules dextrin, maltose and trisaccharide maltose for the effect the first step of amylose starch, and second step was hydrolyzed to oligose glucose and maltose lentamente.The hydrolysis end product has α-limit dextrin, part oligose, maltose and glucose.In α-1,4 glycosidic link hydrolysis ground process, rupture between the C1-O-C4 key ground C1-O key between glucose unit.
α-Dian Fenmeiyehua starch causes the depolymerization of starch part, loses the character of viscosity and chance iodine colour generation, and the reducing power of hydrolyzate increases, and speed of response reduces with the reducing power increase in the time of near arriving achromic point, reaches the hydrolysis limit gradually.But the α-Dian Fenmei of different sources, factors such as the starch of different sources, the pretreatment mode to starch, treatment temp and pH value of solution all can influence this hydrolytic process, cause α-Dian Fenmei to act on the hydrolysis limit difference of starch, generate low oligosaccharides composition and there are differences the limit dextrin different with structure.
The α-Dian Fenmei of different sources, amylatic limit difference.When α-Dian Fenmei acts on starch, decline along with viscosity, Iod R is by the blue stain purple, become redness again, until colourless, the reinforcement gradually of the reducing power of reaction solution, near the increase speed of response with reducing power in the time of the arrival achromic point reduces gradually, the percent hydrolysis of this moment is different because of the source of enzyme, and the α-Dian Fenmei of bacterium, mould, Fructus Hordei Germinatus is 10-20%; Pancreatic juice, saliva and bacterium saccharification type α-Dian Fenmei are about 40%.
α-Dian Fenmei extensively is present in the various biologies such as comprising people, animal, plant, fungi and bacterium, separate α-Dian Fenmei since reported first in 1956, separated at present and identifies above 120 kinds of α-Dian Fenmei, microbe-derived in quantity α-Dian Fenmei accounts for major part.The genus that can produce α-Dian Fenmei in the microorganism has: Bacillus, Thermomonospor, Acinetobacter, Pseudomonas, Streptomyces, Aspergillus, Penicillus etc.α-Dian Fenmei in industrial a large amount of uses is mainly derived from subtilis (Bacillus subtilis) at present, Bacillus licheniformis (Bacillus licheniformis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), aspergillus niger (Aspergillus niger) and aspergillus oryzae (Aspergillus oryzae).
α-Dian Fenmei has sizable commercial value, is widely used in starch deep processing industries, alcohol industry, brewing industry, citric acid industry, monosodium glutamate and mashing industry, pharmaceutical industry and textile industry.At present in the tame zymin of China more than 40 factory, amylase is the main enzyme preparation product of quite a few producer.Domestic production wherein be applicable to the α-Dian Fenmei of different purposes have 10 surplus kind of formulation, but in the majority with adsorptive type, wrap less by type or high purity formulation.Mainly producing bacterial strain in China's α-Dian Fenmei is subtilis and aspergillus niger.But these produce bacterial strains nearly all obtained or passed through induced mutations from the nature screening bacterial strain, and the character of enzyme is very ungood, and the aftertreatment technology of enzyme falls behind in addition, and product profit is generally not high.The engineering strain that has an independent intellectual property right in China realizes that the report of α-Dian Fenmei of suitability for industrialized production is few.
Therefore, at present this area also is necessary to find the encoding gene of the new α-Dian Fenmei of superior performance, develop high performance amylase, upgrade and improve diastatic kind, for the deep processing of starch material provides better condition, start new enzyme method technique, improve recovery rate, reduce and consume, improve the quality of products, increase benefit, thereby can further promote the development of starch material deep processing industry, significantly improve economic benefit and social benefit.
Summary of the invention
The object of the present invention is to provide α-Dian Fenmei, its encoding gene contains the recombinant plasmid and the bacterial strain of this gene and expression method.
In a first aspect of the present invention, a kind of isolating α-Dian Fenmei is provided, the aminoacid sequence of described α-Dian Fenmei shown in SEQ ID NO:11, wherein,
The 14th amino acids is selected from Tyr or His; The 124th amino acids is selected from Asp or Asn;
The 133rd amino acids is selected from His or Arg; The 134th amino acids is selected from Arg or Gln;
The 164th amino acids is selected from Asp or Gly; The 180th amino acids is selected from Glu or Lys;
The 211st amino acids is selected from Glu or Lys; The 264th amino acids is selected from Gln or Pro;
The 272nd amino acids is selected from Asn or Thr; The 310th amino acids is selected from Gly or Ser;
The 373rd amino acids is selected from Pro or Ser; Or the 396th amino acids be selected from Tyr or His.
In a preference, the aminoacid sequence of described α-Dian Fenmei such as SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 is shown in SEQ ID NO:9 or the SEQ ID NO:10.
In another aspect of this invention, provide a kind of isolating polynucleotide, described polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is selected from down group:
(a) polynucleotide of the described α-Dian Fenmei of coding; Or
(b) with polynucleotide (a) complementary polynucleotide.
In a preference, the nucleotide sequence of described polynucleotide such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 is shown in SEQ ID NO:4 or the SEQ ID NO:5.
In another aspect of this invention, provide a kind of carrier, described carrier comprises described polynucleotide.
In a preference, described carrier is an escherichia coli plasmid.
In another preference, described carrier is selected from pTrcHis2-BSAmy, pTrcHis2-BCAmy, pTrcHis2-BL16Amy, pTrcHis2-BLAmy or pTrcHis2-BPAmy.
In another aspect of this invention, provide a kind of cell, it comprises described carrier, or is integrated with described polynucleotide in its genome.
In a preference, described cell is a Bacillus coli cells.
In another preference, described cell is the e. coli bl21 that comprises described nucleic acid molecule or transform with described carrier.
In another aspect of this invention, provide a kind of method of producing described α-Dian Fenmei, comprising: cultivate described cell, from culture, isolate expression product.
In another aspect of this invention, provide a kind of composition, contain described α-Dian Fenmei in the described composition; And acceptable carrier on the bromatology.
In another aspect of this invention, provide the purposes of described α-Dian Fenmei, be used for hydrolysis glycogen or starch.
In another aspect of this invention, provide the method for a kind of hydrolysis glycogen or starch, described method comprises: handle glycogen or starch with described α-Dian Fenmei.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1: from coding for alpha-diastatic nucleotide sequence BS-Amy (SEQ ID NO:1) and the amino acid sequence coded (SEQ ID NO:6) thereof of subtilis.
Fig. 2: from the coding for alpha-diastatic nucleotide sequence BC-Amy (SEQ ID NO:2) and the amino acid sequence coded (SEQ ID NO:7) thereof of bacillus cereus.
Fig. 3: from coding for alpha-diastatic nucleotide sequence BL16-Amy (SEQ ID NO:3) and the amino acid sequence coded (SEQ ID NO:8) thereof of Bacillus licheniformis.
Fig. 4: from coding for alpha-diastatic nucleotide sequence BL-Amy (SEQ ID NO:4) and the amino acid sequence coded (SEQ ID NO:9) thereof of Bacillus licheniformis.
Fig. 5: from coding for alpha-diastatic nucleotide sequence BP-Amy (SEQ ID NO:5) and the amino acid sequence coded (SEQ ID NO:10) thereof of bacillus pumilus.
Fig. 6: the comparison of the α-Dian Fenmei aminoacid sequence in five kinds of sources.
Fig. 7: the optimal reaction pH of escherichia coli expression BS-Amy, BC-Amy, BL16-Amy, BL-Amy and the coded α-Dian Fenmei of BP-Amy sequence.
Fig. 8: the optimal reactive temperature of escherichia coli expression BS-Amy, BC-Amy, BL16-Amy, BL-Amy and the coded α-Dian Fenmei of BP-Amy sequence.
Fig. 9: the pH stability of escherichia coli expression BS-Amy, BC-Amy, BL16-Amy, BL-Amy and the coded α-Dian Fenmei of BP-Amy sequence.
Figure 10: the thermostability of escherichia coli expression BS-Amy, BC-Amy, BL16-Amy, BL-Amy and the coded α-Dian Fenmei of BP-Amy sequence.
Figure 11: the SDS-PAGE of sample in the coded α-Dian Fenmei fermenting process of escherichia coli expression BS-Amy, BC-Amy, BL16-Amy, BL-Amy and BP-Amy sequence.
Figure 12: the molecular weight of escherichia coli expression BS-Amy, BC-Amy, BL16-Amy, BL-Amy and the coded α-Dian Fenmei of BP-Amy sequence.
Embodiment
The inventor separates acquisition one class first and has the excellent hydrolysis glycogen or the α-Dian Fenmei of starch effect through deep research, and it has high and stability (particularly thermostability) the good characteristics of enzymic activity.Finish the present invention on this basis.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and albumen, but same polynucleotide or albumen as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " isolating α-Dian Fenmei " is meant that α-Dian Fenmei is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying α-Dian Fenmei of standard.Basically pure albumen can produce single master tape on non-reduced polyacrylamide gel.The purity of α-Dian Fenmei can be used amino acid sequence analysis.
α-Dian Fenmei of the present invention can be recombinant protein (polypeptide), native protein, synthetic proteins, preferred recombinant protein.α-Dian Fenmei of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, α-Dian Fenmei of the present invention can be glycosylated, maybe can be nonglycosylated.α-Dian Fenmei of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises fragment, derivative and the analogue of α-Dian Fenmei.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of natural α-Dian Fenmei of the present invention or active albumen basically.α-Dian Fenmei fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted albumen of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has the albumen of substituted radical, or (iii) maturation protein and another compound (such as the compound that prolongs the albumen transformation period, polyoxyethylene glycol for example) merges formed albumen, or (iv) additional aminoacid sequence is fused to this protein sequence and the albumen that forms (as leader sequence or secretion sequence or be used for this proteic sequence of purifying or proteinogen sequence, or with the fusion rotein of the segmental formation of antigen I gG).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.
In the present invention, term " α-Dian Fenmei " refers to have the albumen of the SEQ ID NO:11 sequence of alpha-amylase activity.This term also comprises having and the variant form α-Dian Fenmei identical function, SEQ ID NO:11 sequence.These variant forms comprise (but being not limited to): one or more (it is individual to be generally 1-30, preferably 1-20, more preferably 1-10,1-5 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises the active fragments and the reactive derivative of α-Dian Fenmei.
The variant form of this α-Dian Fenmei comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of the DNA hybridization of α-Dian Fenmei and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-α-Dian Fenmei to obtain.The present invention also provides other albumen, as comprises α-Dian Fenmei or its segmental fusion rotein.Except the albumen of total length almost, the present invention has also comprised the soluble fragments of α-Dian Fenmei.Usually, this fragment have the α-Dian Fenmei sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
Invention also provides the analogue of α-Dian Fenmei.The difference of these analogues and natural α-Dian Fenmei can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These albumen comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that α-Dian Fenmei of the present invention is not limited to the above-mentioned representational albumen that exemplifies.
(the not changing primary structure usually) form of modification comprises: interior or external proteic chemically derived form such as the acetylize or carboxylated of body.Modify and also to comprise glycosylation, as those in proteic synthetic and processing or further carry out glycosylation modified and albumen that produce in the procedure of processing.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by albumen is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the albumen that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
In the present invention, " α-Dian Fenmei conservative property variant protein (polypeptide) " refers to compare with the aminoacid sequence of SEQ ID NO:11, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed albumen at the most best.These conservative property variant protein are preferably carried out the amino acid replacement according to table 1 and are produced.
Table 1
Initial residue Representational replacement The preferred replacement
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
As optimal way of the present invention, described α-Dian Fenmei is the α-Dian Fenmei of cloning from subtilis, bacillus cereus, Bacillus licheniformis (2 strain) and bacillus pumilus, be referred to as BS-Amy, BC-Amy, BL16-Amy, BL-Amy, BP-Amy respectively, they have the aminoacid sequence shown in the SEQ ID NO:6-10 respectively.On amino acid sequence level, the homogeny between them is 99.5%.
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The proteic coding region sequence of encoding mature can be identical with the coding region sequence shown in SEQ ID NO:1-5 is arbitrary or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ IDNO:11, but with SEQ ID NO:1-5 the differentiated nucleotide sequence of coding region sequence shown in arbitrary.
The polynucleotide of the maturation protein of coding SEQ ID NO:11 comprise: the proteic encoding sequence of an encoding mature; The encoding sequence of maturation protein and various additional code sequence; Encoding sequence of maturation protein (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of proteins encoded " can be to comprise these proteic polynucleotide of coding, also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, it is encoded albumen or proteic fragment, analogue and the derivative of identical aminoacid sequence with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded protein in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and .1%SDS, 0 ℃; Or (2) hybridization the time is added with denaturing agent, 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the albumen of interfertile polynucleotide encoding has identical biological function and activity with the maturation protein shown in the SEQID NO:11.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or separation coding for alpha-diastatic polynucleotide.
As optimal way of the present invention, the polynucleotide of the described α-Dian Fenmei of encoding separate from subtilis, bacillus cereus, Bacillus licheniformis (2 strain) and bacillus pumilus, and they have the nucleotide sequence shown in the SEQ ID NO:1-5 respectively.
Albumen among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
α-Dian Fenmei Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
Use method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to obtain the cDNA of total length from the library, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.
As one of embodiment, the inventor extracts the genomic dna of subtilis, bacillus cereus, Bacillus licheniformis (2 strain) and bacillus pumilus respectively, is cloned into coding for alpha-diastatic full length sequence by the method for the good screening active ingredients of genomic library construction.The result obtains the nucleotide sequence shown in SEQ ID NO:1-5.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or α-Dian Fenmei encoding sequence, and produce proteic method of the present invention through recombinant technology.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), polymerized nucleoside acid sequence of the present invention can be used to express or produce the α-Dian Fenmei of reorganization.In general following steps are arranged:
(1). with coding for alpha of the present invention-diastatic polynucleotide (or varient), or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, the α-Dian Fenmei polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: escherichia coli plasmid.As long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains α-Dian Fenmei DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage PL promotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, to cultivate Tetrahydrofolate dehydrogenase, the neomycin resistance of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
In one embodiment, with behind NcoI and EcoRI double digestion, not being connected, obtain large intestine recombinant expressed year pTrcHis2-BSAmy, pTrcHis2-BCAmy, pTrcHis2-BL16Amy, pTrcHis2-BLAmy and pTrcHis2-BPAmy with the pTrcHis2 carrier of NcoI and EcoRI double digestion with the α-Dian Fenmei encoding sequence of the present invention of endogenous signal coding sequence.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc.The preferred various cells that are beneficial to gene product expression or fermentative production of described host cell, this type of cell has been well known and commonly used, for example various Bacillus coli cells and yeast cell.In one of embodiments of the present invention, select the reconstitution cell of e. coli bl21 construction expression α-Dian Fenmei for use.
Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results 2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl 2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses α-Dian Fenmei of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant protein in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
As one of embodiment, come the production α-Dian Fenmei by intestinal bacteria (for example e. coli bl21) fermentation that comprises α-Dian Fenmei encoding sequence of the present invention, and by ammonium sulfate precipitation, ion exchange chromatography and gel chromatography have obtained the target protein of pure enzyme form.
The purposes of α-Dian Fenmei of the present invention includes, but is not limited to: be used for hydrolysis glycogen or starch.α-Dian Fenmei of the present invention has the excellent hydrolysis glycogen or the effect of starch, and has good thermostability and pH stability, and application prospect is good.
The present invention also provides a kind of composition, and it contains on the α-Dian Fenmei of significant quantity and the bromatology or industrial acceptable carrier or vehicle.This class carrier comprises (but being not limited to): damping fluid, water etc.It can be made into solution or pulvis etc.Described " significant quantity " is meant the function that can bring into play α-Dian Fenmei or active and can received amount.In use, described significant quantity can be determined easily according to the enzymic activity of described α-Dian Fenmei in this area.
The present invention utilizes genetic engineering means to prepare can express alpha-diastatic recombinant bacterial strain, and has obtained the fine α-Dian Fenmei.The present invention identifies by zymologic property and has carried out the optimum temperature of enzyme, the suitableeest action pH value, pH stability, thermostability and than the analysis of physico-chemical properties such as vigor, prove that α-Dian Fenmei of the present invention has good pH stability, good thermostability and protease inhibitor hydrolysis ability.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring HarborLaboratory Press, 1989); Zhao Yongfang etc., Measurement for Biochemistry principle and application thereof (second edition); Zhu Jian etc., the condition described in the Biochemistry Experiment [M], or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
I. experiment material and reagent
1. bacterial strain and carrier
E. coli bl21, JM109 and expression vector pTrcHis2 all available from Invitrogen company (Carlsbad, CA, USA).
2. enzyme and other biochemical reagents
Restriction enzyme, DNAMaker, protein Maker are all available from Fermentas (MBI), and effect substrate Zulkovsky starch is available from Chemical Reagent Co., Ltd., Sinopharm Group (China, Shanghai), and other conventional reagent are given birth to the worker available from Shanghai.
3. substratum
The substratum that uses: the LB substratum, YPD, YPAD, BMDY, BNNY, MM, MD substratum are all with reference to Invitrogen pichia yeast operational manual.
4. method
4.1 α-Dian Fenmei
All involved enzyme work, enzyme activity, enzymic activity all are meant alpha-amylase activity in the various embodiments of the present invention, and available Zulkovsky starch detects and measures as substrate.Wherein, international DNS (3, the 5-dinitrosalicylic acid) method is adopted in the enzyme activity quantitative assay.
Specifically, the Zulkovsky starch solution (pH6.0,50mM Sodium phosphate dibasic-SODIUM PHOSPHATE, MONOBASIC) that accurately pipettes 250 μ l 1% (m/v) is put into 70 ℃ of water-bath preheating 3min in the 2ml centrifuge tube.(pH6.0 50mM) does 10 times of gradient dilutions with enzyme liquid sample, selects the gratifying extent of dilution of color developing effect with the PBS damping fluid.250 μ l are joined in the centrifuge tube according to the good enzyme liquid of selected extent of dilution (N) dilution, continuation is reacted 10min in 70 ℃ of water-baths, add 1mlDNS reagent termination reaction again, centrifuge tube is boiled 5min in boiling water, be cooled to room temperature with flowing water immediately, room temperature is placed the 10min colour developing, measures absorbance then in the 540nm place.
Blank is for showing the same dilution enzyme liquid of 250 μ l in the 2ml centrifuge tube, add 1ml DNS reagent, add 250 μ l substrates again, after vibration mixes, place 70 ℃ of water-baths to react 10min, centrifuge tube is boiled 5min in boiling water, be cooled to room temperature with flowing water immediately, room temperature is placed the 10min colour developing, measures absorbance then in the 540nm place.
The typical curve of following drafting enzymolysis product (glucose) colour developing: with the glucose reference liquid PBS (pH6.0 of 500 μ mol/L, 50mM phosphoric acid salt) be diluted to the solution of 0,25,50,100,150,200,250 μ mol/L, react by the operation steps one of above-mentioned sample determination.(C, μ mol/L) is ordinate zou with glucose concn, is X-coordinate with light absorption value (A), and the drawing standard curve is listed equation of linear regression (C=KA+b).
The alpha-amylase activity unit definition: it is 1 unit of activity (U) that the amylase per minute starch-splitting of unit vol produces 1 μ mol glucose.
The alpha-amylase activity U of sample calculates according to following formula:
U = K × ( A - A 0 ) + b t × V × V 0 × N
In the formula:
U---sample alpha-amylase activity, U/mL;
The slope of K---typical curve;
The light absorption value of A---sample solution;
A 0---the light absorption value of blank;
The intercept of b---typical curve;
T---the reaction times, min;
V---the add-on of enzyme liquid in the reaction system, ml;
V 0---the cumulative volume of reaction system, ml;
N---sample extension rate;
Each mensuration is all got two parallel test results' arithmetical av, keeps integer.
The ratio vigor of α-Dian Fenmei calculates by following formula:
U c＝U/c
Wherein:
U c---sample α-Dian Fenmei specific activity, U/mg;
U---sample alpha-amylase activity, U/ml;
C---the protein content in the sample solution, mg/ml.
Purifying multiple X calculates according to following formula:
X＝U C/U C0
Wherein:
X---purifying multiple;
U C---the ratio vigor of α-Dian Fenmei sample behind the purifying, U/mg;
U C0---the ratio vigor of α-Dian Fenmei sample before the purifying, U/mg.
II. embodiment
The clone of embodiment 1, α-Dian Fenmei encoding sequence
The extraction of genomic dna: get 37 ℃ and cultivated cell concentration OD 2 days 600nmSubtilis to 0.5～0.8 (Bacillus subtilis, CICC 10090, available from Chinese industrial microorganism strains preservation administrative center (CICC)) bacterium liquid 50ml, the centrifugal 10min of 10000rpm, get the 50mg thalline and add 500 μ l sterile water wash, the centrifuging and taking precipitation.Precipitation is suspended in the lysozyme soln of 500 μ l 1mg/ml again, bathe 30min in 37 ℃ of temperature, add N,O-Diacetylmuramidase liquid 100 μ l again and continue insulation 30min in 40-50 ℃, to bacterium liquid transparent after, add 10%SDS to final concentration 2% (m/v), stir about 5min significantly descends to the bacterium fluid viscosity, and the centrifugal 10min of 15000rpm removes fragment.Supernatant is used equal-volume phenol, phenol successively: chloroform (1: 1), chloroform extracting.Get the Virahol normal temperature precipitation 10min that chloroform extracting gained upper solution adds 0.6-1 times of volume.The centrifugal 15min of 16000rpm.Precipitation is cleaned with 70% (v/v) ethanol, and low-speed centrifugal dries the back with the dissolving of 30 μ l sterilized waters with precipitation, and is standby.
Get the genomic dna 5 μ g of extraction, utilize the BamHI enzyme to cut, be connected,, make up gene library with gained recombinant plasmid transformed bacillus coli DH 5 alpha (available from precious biotechnology (Dalian) company limited) with the pUC19 carrier that same enzyme is cut.The positive colony of picking white (the LB substratum contains 100 μ g/ml Amp, 0.5% (m/v) X-gal and 1.5mMIPTG) is transferred to screening active ingredients flat board (agar and 1% (m/v) Zulkovsky starch that contains 3% (m/v) in the LB substratum) and is gone up incubated overnight.Thalline on the flush away flat board is used 1% iodine staining, again with the decolouring of 0.5M NaCl solution washing.The clone who contains the α-Dian Fenmei encoding sequence can produce the degraded circle.Picking produces the clone of degraded circle, extract this clone's plasmid, check order, obtain the encoding sequence BS-Amy of α-Dian Fenmei, this sequence comprises 1452bp (Fig. 1, SEQ ID NO:1), wherein the 1450-1452 position is the maturation protein (Fig. 1 and Fig. 6) that terminator codon TAG, 1-1449 position coding do not contain signal peptide, and this maturation protein contains 483 amino acid (SEQ ID NO:6).
Use bacillus cereus (Bacillus respectively CereusCGMCC 1.260, available from Chinese common micro-organisms bacterial strain preservation administrative center (CGMCC)), Bacillus licheniformis (Bacillus licheniformis, ATCC21610, available from American type culture collection (ATCC)), Bacillus licheniformis (Bacillus licheniformis, ATCC27811, available from American type culture collection (ATCC)) and bacillus pumilus (Bacillus pumilus, CGMCC1.0271, available from Chinese common micro-organisms bacterial strain preservation administrative center (CGMCC)) repeat above process, obtain the complete encoding sequence BC-Amy (SEQ ID NO:2) of α-Dian Fenmei, BL16-Amy (SEQ ID NO:3), maturation protein aminoacid sequence (SEQ ID NO:7 to SEQ ID NO:10) (Fig. 2 of BL-Amy (SEQ ID NO:4) and BP-Amy (SEQ ID NO:5) and coding thereof, 3,4 and Fig. 5).
Embodiment 2, structure intestinal bacteria recombinant expression vector reach in the expression in escherichia coli α-Dian Fenmei
Design primer according to embodiment 1 gained coding for alpha-diastatic nucleotide sequence:
Primer 1:5 '-CCATGGGCAAATCTTAATGGGACGC-3 ' (SEQ ID NO:12);
Primer 2: 5 '-GAATTCCTATCTTTGAACATAAATTGAAACCGAC-3 ' (SEQ ID NO:13);
Primer 3:5 '-GAATTCTTATCTTTGAACATAAATTGAAACCGAC-3 ' (SEQ ID NO:14).
Be template to select with separating the pUC19 recombinant plasmid that contains BS-Amy, BC-Amy, BL16-Amy, BL-Amy and BP-Amy that obtains among the embodiment 1 respectively, the encoding sequence of 5 kinds of α-Dian Fenmei of pcr amplification.The pcr amplification condition is: 94 ℃ of 5min; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, 32 circulations; 72 ℃ of 10min.Carry out double digestion with NcoI and EcoRI, be connected on the carrier pTrcHis2, obtain to contain recombinant plasmid pTrcHis2-BSAmy, pTrcHis2-BCAmy, pTrcHis2-BL16Amy, pTrcHis2-BLAmy and the pTrcHis2-BPAmy of 5 kinds of α-Dian Fenmei encoding sequences respectively.
Get recombinant plasmid pTrcHis2-BSAmy, pTrcHis2-BCAmy, pTrcHis2-BL16Amy, pTrcHis2-BLAmy and pTrcHis2-BPAmy that 10 μ l build, join respectively in the competent cell (e. coli bl21 and JM109) that 100 μ l prepare, shake up and place on ice ice bath 30min; Place 42 ℃ of water-bath thermal shock 90s; Centrifuge tube is moved to ice bath 2min in the mixture of ice and water fast; Every pipe adds 400 μ l SOC substratum (2% peptone, 0.5% yeast powder (m/v), 10mM NaCl, 2.5mM KCl, 10mM MgCl 2, 10mM MgSO 4, 20mM glucose, pH7.0～7.2), break up back recovery 1h (80rpm～200rpm) on 37 ℃ of shaking tables with light suction of pipettor; Centrifugal, 4000rpm * 5min removes 400 μ l supernatants, the remainder mixing; Be coated with flat board (the LB-agar flat board contains 80 μ g/ml Amp), 37 ℃ just putting 1h after, be inverted overnight incubation, on resistant panel, grow for containing positive colony of recombinant plasmid.
Get the recombinant escherichia coli strain JM109 at 5 kinds of recombinant plasmid positive colony places respectively, be inoculated in (250ml triangular flask in the 50ml LB nutrient solution, contain 50 μ g/ml Amp), 37 ℃ of 250rpm shaking culture 2-2.5h, get the centrifugal 10min of nutrient solution 10000rpm, collect thalline, extract plasmid, enzyme cuts back to close target DNA fragment (plasmid extracts and glue reclaims E.Z.N.A.Plasmid Mini Kit I and the E.Z.N.A.Gel ExtractionKit test kit of using OMEGA company respectively), check order, through comparison, measured sequence is consistent with sequence among the embodiment 1, and this illustration purpose gene correctly inserts at plasmid pTrcHis2.
Get the recombinant escherichia coli strain BL21 of 5 kinds of recombinant plasmid positive colonies respectively, be inoculated in (250ml triangular flask in the 50ml LB nutrient solution, contain 50 μ g/ml Amp), 37 ℃ of 250rpm shaking culture 1-1.5h, add IPTG and induce (final concentration is 2 μ mol/ml), 37 ℃ of 250rpm are shaking culture 3-3.5h again.Get the centrifugal 10min of nutrient solution 10000rpm, collect thalline, add isopyknic sterilized water thalline that suspends again again, the centrifugal 10min of 12000rpm gets precipitation with 1/5 volume pH6.0, the PBS suspension thalline of 50mM, carry out ultrasonic disruption, broken condition is: 60% power, the broken 10min of 5s at interval, stop 10min, broken again 10min.12000rpm is centrifugal, collect supernatant liquor, measure alpha-amylase activity, the result shows that the coded α-Dian Fenmei of the encoding gene of five kinds of α-Dian Fenmei all can be at expression in escherichia coli, and alpha-amylase activity is all arranged, record BS-Amy, BC-Amy, BL16-Amy, BL-Amy and the coded alpha-amylase activity of BP-Amy sequence and can reach 81U/ml, 115U/ml, 280U/ml, 85U/ml and 95.7U/ml respectively.
In addition, top collected cytoclasis supernatant liquor is carried out SDS-PAGE target protein matter electrophoretic analysis (seeing shown in Figure 11), and according to the purifying target protein matter from supernatant liquor of method described in the embodiment, respectively resulting pure target protein being carried out the N terminal amino acid sequence measures, resulting N terminal amino acid sequence is consistent with amino acid sequence corresponding among SEQ ID NO:6 to the SEQ ID NO:10, and this shows that target protein has obtained correct expression.
Embodiment 3, recombinant alpha-amylases fermentative preparation
Get the recombinant escherichia coli strain BL21 of 5 kinds of prepared among the embodiment 2 α-Dian Fenmei recombinant plasmid positive colonies respectively, respectively be inoculated in (250ml triangular flask in 2 bottles of 50ml LB nutrient solutions, contain 50 μ g/ml Amp), 37 ℃ of 250rpm shaking culture are to OD600nm=0.3～0.5 (about 2-3hr), respectively 2 bottles of seeds are inoculated in separately 3L fermentation minimum medium (10g/L peptone then, the 5g/L yeast powder, 1g/L NaCl, 6g/L Na 2HPO 412H 2O, 3g/L KH 2PO 4, 6g/L (NH 4) 2SO 4, 1g/L MgSO 47H 2O, 0.01g/L CaCl 2, 15g/L glucose, 0.05g/L Amp, 0.1g/L FeSO 4) in, in the 5L fermentor tank, ferment.
At initial period---in the thalli growth stage, the ammoniacal liquor with 25% (v/v) in the fermenting process is regulated pH, makes it maintain 7.0-7.2, and the velocity flow with 2.8ml/hr adds trace element solution (3.5mM copper sulfate, 0.06mM sodium iodide, 1.8mM manganous sulfate, 0.08mM Sodium orthomolybdate, 0.04mM boric acid, 0.5mM cobalt chloride, 0.02mM zinc chloride, 0.03mM ferrous sulfate, 0.17mM vitamin H) carry out continuous flow feeding, until OD600=15 (8-10hr).
Enter induction period, add the IPTG that a certain amount of concentration is 1mol/L, making its final concentration is 1mmol/L, begins to induce, and adds TY (500g/L glucose, 12.5g/L MgSO with the velocity flow of 15-20ml/hr 47H 2O), the velocity flow with 10-15ml/hr adds DY (100g/L yeast powder, 10g/L (NH 4) 2SO 4), the final concentration that makes reducing sugar and amino nitrogen in the substratum is between 0.4-0.6% and 0.05-0.15% (m/v).In the fermenting process, from inducing, every the 3hr sampling once, obtain enzyme liquid, and measure alpha-amylase activity, when enzyme activity does not have obvious increases, stop to ferment (18-21hr) according to the method for being narrated among the embodiment 2.After the fermentation ends (18hr), record BS-Amy, BC-Amy, BL16-Amy, BL-Amy and the coded alpha-amylase activity of BP-Amy sequence and can reach 202U/ml, 230U/ml, 430U/ml, 175U/ml and 220U/ml respectively.
The molecular weight identification of escherichia coli expression BS-Amy, BC-Amy, BL16-Amy, BL-Amy and the coded α-Dian Fenmei of BP-Amy sequence is seen Figure 12.
The purifying of embodiment 4, recombinant alpha-amylases
Respectively that embodiment 3 is the prepared centrifugal 10min of 5 kinds of fermentation cultures (reclaiming gained behind the 18hr) 10000rpm, collect thalline separately, with 4 times of pH6.0 to the thalline volume, the PBS damping fluid of the 50mM thalline that suspends again, after thorough mixing is even, the centrifugal 10min of 10000rpm, the collecting precipitation thing is used the pH6.0 of 4 times of volumes once more, the PBS damping fluid of 50mM fully suspends it, the centrifugal 10min of 10000rpm, the collecting precipitation thing continues the pH6.0 with 4 times of volumes, the PBS damping fluid of 50mM fully suspends it, carry out cell wall breaking with high pressure homogenizer then, the broken wall condition is: pressure 800-1000bar, cool off with 0-4 ℃ of frozen water in the process.With the centrifugal 10min of suspension 10000rpm after the cytoclasis, 4 ℃ of preservations of collection supernatant liquor are stand-by.
Get supernatant liquor as crude enzyme liquid, crude enzyme liquid is placed ice bath, slowly add ammonium sulfate to 70% (w/v) while stirring, the centrifugal 15min of 13000rpm, get precipitation, use pH6.0, the PBS damping fluid of 50mM dissolves again, placing molecular weight cut-off is the dialysis tubing of 6000Da, with pH6.0, the PBS damping fluid of 20mM is an extracellular fluid dialysis, and the volume ratio of extracellular fluid dialysis and interior liquid is greater than 50,4 ℃ of dialysis 12-16h, extracellular fluid dialysis is changed once every 4h in the centre, after having dialysed, gets dialyzed solution and concentrates with the rotary evaporation in vacuo instrument, after carrying out lyophilize again, place-20 ℃ cryogenic refrigerator to preserve stand-by.
Get above the 50mg resulting lyophilized powder in centrifuge tube, add 2ml pH8.0, the PBS damping fluid of 50mM, it is fully dissolved after, last TOSOH Toyopearl EDAE-650C anion column.Earlier use pH8.0,50mM PBS damping fluid balance pillar, stream adds sample then, and with 5 column volumes of 0-1.0mol/L NaCl gradient elution of same buffer configuration, flow velocity is 1ml/min again, collects every pipe 3ml with Fraction Collector.Then to measured in solution alpha-amylase activity in the collection tube and protein electrophoresis analysis.
Collect the active peak after normal pressure ion-exchange separates, concentrate, after the desalination, freeze-drying, use pH7.0 again, the dissolving of 50mM PBS damping fluid, (Φ 1.6 * 100cm) for last Sephadex G-75 gel column.Earlier use pH7.0,50mM PBS damping fluid balance pillar is gone up sample then, uses pH7.0,1.5 column volumes of 50mM PBS buffer solution elution, and flow velocity is 0.2ml/min, collects every pipe 3ml with Fraction Collector.Then to measured in solution alpha-amylase activity in the collection tube and protein electrophoresis analysis.
Collect the active peak after the normal pressure gel-filtration separates, concentrate, after the desalination, freeze-drying, use pH7.0 again, the dissolving of 50mM PBS damping fluid, last Mono Q HPLC post, earlier use pH8.0,50mM PBS damping fluid balance pillar is gone up sample then, gradient is: 0mol/L NaCl 20min, 0-0.8mol/L NaCl 90min, 0.8-1.0mol/L NaCl20min, 1.0mol/L NaCl 20min.Flow velocity 1.0ml/min presses the peak and collects, then to the sample determination alpha-amylase activity collected and carry out the protein electrophorese analysis.
Active peak after collection of ions exchange separates concentrates, after the desalination, freeze-drying, uses pH7.0 again, the dissolving of 20mM PBS damping fluid, last Superdex 75HPLC post.Earlier with pH7.0,20mM PBS damping fluid balance pillar, go up sample then, with pH7.0,1.5 column volumes of 20mM PBS buffer solution elution, flow velocity is 0.25ml/min, press the peak and collect, then to the sample determination alpha-amylase activity collected and carry out the protein electrophorese analysis.SDS-PAGE result (Figure 13) shows that the α-Dian Fenmei albumen behind the α-Dian Fenmei purifying in 5 kinds of sources only has single band, and molecular weight all is about 56kDa.
After purifying was finished, the α-Dian Fenmei specific activity of BS-Amy coding was brought up to the 20300U/mg of pure enzyme from the 326U/mg of crude enzyme liquid, and the purifying multiple is 62.3; The α-Dian Fenmei specific activity of BC-Amy coding is brought up to the 17500U/mg of pure enzyme from the 345U/mg of crude enzyme liquid, and the purifying multiple is 50.7; The α-Dian Fenmei specific activity of BL16-Amy coding is brought up to the 31200U/mg of pure enzyme from the 640U/mg of crude enzyme liquid, and the purifying multiple is 48.8; The α-Dian Fenmei specific activity of BL-Amy coding is brought up to the 33000U/mg of pure enzyme from the 283U/mg of crude enzyme liquid, and the purifying multiple is 116.6; The α-Dian Fenmei specific activity of BP-Amy coding is brought up to the 24000U/mg of pure enzyme from the 416U/mg of crude enzyme liquid, and the purifying multiple is 57.7.
Embodiment 5, recombinant alpha-amylases zymologic property are analyzed
Embodiment 4 prepared α-Dian Fenmei enzyme liquid (cytoclasis supernatant liquor) are carried out enzymatic reaction to measure its optimal pH under different pH.Used damping fluid is the Britton-Robinson damping fluid (citric acid, potassium primary phosphate, boric acid, sodium hydroxide, veronal) of pH4.0-10.0.α-Dian Fenmei in the damping fluid of different pH, 70 ℃ of suitable property results that measure down pH.The result shows that the optimal pH of the α-Dian Fenmei of BS-Amy, BC-Amy, BL16-Amy, BL-Amy and BP-Amy coding all between 6.0-7.0, is seen Fig. 7.
α-Dian Fenmei enzyme liquid is left standstill 60min in the Britton-Robinson of different pH values damping fluid, measure the pH stability of residual enzyme activity again under room temperature with the research α-Dian Fenmei.The results are shown in Figure 9, show in the pH5.0-10.0 scope, the residual activity of the α-Dian Fenmei of BS-Amy coding is more than 80%; In the pH5.0-10.0 scope, the residual activity of the α-Dian Fenmei of BC-Amy coding is more than 80%; In the pH6.0-10.0 scope, the residual activity of the α-Dian Fenmei of BL16-Amy coding is more than 90%; In the pH5.0-10.0 scope, the residual activity of the α-Dian Fenmei of BL-Amy coding is more than 85%; In the pH5.0-10.0 scope, the residual activity of the α-Dian Fenmei of BP-Amy coding is more than 80%; The α-Dian Fenmei of this explanation BS-Amy, BC-Amy, BL16-Amy, BL-Amy and BP-Amy coding all has good pH stability.
(pH6.0,50mM) (40 ℃-90 ℃) carry out the PBS that is determined at of optimal reactive temperature under buffer system and the differing temps.Carry out enzymatic reaction and vitality test according to aforementioned DNS method.The results are shown in Figure 8, the optimal reactive temperature that shows the α-Dian Fenmei that BC-Amy, BL16-Amy and BP-Amy encode is about 70 ℃, the optimal reactive temperature of the α-Dian Fenmei of BS-Amy coding is about 75 ℃, and the optimal reactive temperature of the α-Dian Fenmei of BL-Amy coding is about 85 ℃.
THERMAL STABILITY is carried out enzyme assay again for to handle 10-60min under differing temps.The results are shown in Figure 10, show in 30 ℃-60 ℃ scope to be incubated 60min, the residual enzyme activity of 5 kinds of α-Dian Fenmei all can maintain more than 85%; Be incubated 30min down at 70 ℃, the residual enzyme activity of 5 kinds of α-Dian Fenmei all can maintain more than 80%; Be incubated 10min down at 80 ℃, the residual enzyme activity of 5 kinds of α-Dian Fenmei all can maintain more than 60%.Illustrate that 5 kinds of source α-Dian Fenmei all have good thermostability.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Fujian Fudabaite Sci-Tech Devpt Co., Ltd.
＜120〉α-Dian Fenmei, its encoding gene and expression thereof
<130>093845
<160>14
<170>PatentIn?version?3.3
<210>1
<211>1452
<212>DNA
＜213〉subtilis (Bacillus subtilis)
<400>1
gcaaatctta?atgggacgct?gatgcagtat?tttgaatggt?acatgcccaa?tgacggccaa 60
cattggaagc?gcttgcaaaa?cgactcggca?tatttggctg?aacacggtat?tactgccgtc 120
tggattcccc?cggcatataa?gggaacgagc?caagcggatg?tgggctacgg?tgcttacgac 180
ctttatgatt?taggggagtt?tcatcaaaaa?gggacggttc?ggacaaagta?cggcacaaaa 240
ggagagctgc?aatctgcgat?caaaagtctt?cattcccgcg?acattaacgt?ttacggggat 300
gtggtcatca?accacaaagg?cggcgctgat?gcgaccgaag?atgtaaccgc?ggttgaagtc 360
gatcccgctg?accgcaaccg?cgtaatttca?ggagaacacc?gaattaaagc?ctggacacat 420
tttcattttc?cggggcgcgg?cagcacatac?agcgatttta?aatggcattg?gtaccatttt 480
gacggaaccg?attgggacga?gtcccgaaag?ctgaaccgca?tctataagtt?tcaaggagag 540
gcttgggatt?gggaagtttc?caatgaaaac?ggcaactatg?attatttgat?gtatgccgac 600
atcgattatg?accatcctga?tgtcgcagca?gaaattaaga?gatggggcac?ttggtatgcc 660
aatgaactgc?aattggacgg?tttccgtctt?gatgctgtca?aacacattaa?attttctttt 720
ttgcgggatt?gggttaatca?tgtcagggaa?aaaacgggga?aggaaatgtt?tacggtagct 780
gaatattggc?agaatgactt?gggcgcgctg?gaaaactatt?tgaacaaaac?aaattttaat 840
cattcagtgt?ttgacgtgcc?gcttcattat?cagttccatg?ctgcatcgac?acagggaggc 900
ggctatgata?tgaggaaatt?gctgaacggt?acggtcgttt?ccaagcatcc?gttgaaagcg 960
gttacatttg?tcgataacca?tgatacacag?ccggggcaat?cgcttgagtc?gactgtccaa 1020
acatggttta?agccgcttgc?ttacgctttt?attctcacaa?gggaatctgg?ataccctcag 1080
gttttctacg?gggatatgta?cgggacgaaa?ggagaccccc?agcgcgaaat?tcctgccttg 1140
aaacacaaaa?ttgaaccgat?cttaaaagcg?agaaaacagt?atgcgtacgg?agcacagcat 1200
gattatttcg?accaccatga?cattgtcggc?tggacaaggg?aaggcgacag?ctcggttgca 1260
aattcaggtt?tggcggcatt?aataacagac?ggacccggtg?gggcaaagcg?aatgtatgtc 1320
ggccggcaaa?acgccggtga?gacatggcat?gacattaccg?gaaaccgttc?ggagccggtt 1380
gtcatcaatt?cggaaggctg?gggagagttt?cacgtaaacg?gcgggtcggt?ttcaatttat 1440
gttcaaagat?ag 1452
<210>2
<211>1452
<212>DNA
＜213〉bacillus cereus (Bacillus cereus)
<400>2
gcaaatctta?atgggacgct?gatgcagtat?tttgaatggt?acatgcccaa?tgacggccaa 60
cattggaagc?gcttgcaaaa?cgactcggca?tatttggctg?aacacggtat?tactgccgtc 120
tggattcccc?cggcatataa?gggaacgagc?caagcggatg?tgggctacgg?tgcttacgac 180
ctttatgatt?taggggagtt?tcatcaaaaa?gggacggttc?ggacaaagta?cggcacaaaa 240
ggagagctgc?aatctgcgat?caaaagtctt?cattcccgcg?acattaacgt?ttacggggat 300
gtggtcatca?accacaaagg?cggcgctgat?gcgaccgaag?atgtaaccgc?ggttgaagtc 360
gatcccgctg?accgcaaccg?cgtaatttca?ggagaacacc?aaattaaagc?ctggacacat 420
tttcattttc?cggggcgcgg?cagcacatac?agcgatttta?aatggcattg?gtaccatttt 480
gacggaaccg?attgggacga?gtcccgaaag?ctgaaccgca?tctataagtt?tcaaggaaag 540
gcttgggatt?gggaagtttc?taatgaaaac?ggcaactatg?attatttgat?gtatgccgac 600
atcgattatg?accatcctga?tgtcgcagca?gaaattaaga?gatggggcac?ttggtatgcc 660
aatgaactgc?aattggatgg?tttccgtctt?gatgctgtca?aacacattaa?attttctttt 720
ttgcgggatt?gggttaatca?tgtcagggaa?aaaacgggga?aggaaatgtt?tacggtagct 780
gaatattggc?agaatgactt?gggcgcgctg?gaaacctatt?tgaacaaaac?aaattttaat 840
cattcagtgt?ttgacgtgcc?gcttcattat?cagttccatg?ctgcatcgac?acagggaggc 900
ggctatgata?tgaggaaatt?gctgaacggt?acggtcgttt?ccaagcaccc?gttgaaagcg 960
gttacatttg?tcgataacca?tgatacacag?ccggggcaat?cgcttgagtc?gactgtccaa 1020
acatggttta?agccgcttgc?ttacgctttt?attctcacaa?gggaatctgg?ataccctcag 1080
gttttctacg?gggatatgta?cgggacgaaa?ggagactccc?agcgcgaaat?tcctgccttg 1140
aaacacaaaa?ttgaaccgat?cttaaaagcg?agaaaacagt?atgcgtacgg?agcacagcat 1200
gattatttcg?accaccatga?cattgtcggc?tggacaaggg?aaggcgacag?ctcggttgca 1260
aattcaggtt?tggcggcatt?aataacagac?ggacccggtg?gggcaaagcg?aatgtatgtc 1320
ggccggcaaa?acgccggtga?gacatggcat?gacattaccg?gaaaccgttc?ggagccggtt 1380
gtcatcaatt?cggaaggctg?gggagagttt?cacgtaaacg?gcgggtcggt?ttcaatttat 1440
gttcaaagat?ag 1452
<210>3
<211>1452
<212>DNA
＜213〉Bacillus licheniformis (Bacillus licheniformis)
<400>3
gcaaatctta?atgggacgct?gatgcagtat?tttgaatggt?acatgcccaa?tgacggccaa 60
cattggaagc?gcttgcaaaa?cgactcggca?tatttggctg?aacacggtat?tactgccgtc 120
tggattcccc?cggcatataa?gggaacgagc?caagcggatg?tgggctacgg?tgcttacgac 180
ctttatgatt?taggggagtt?tcatcaaaaa?gggacggttc?ggacaaagta?cggcacaaaa 240
ggagagctgc?aatctgcgat?caaaagtctt?cattcccgcg?acattaacgt?ttacggggat 300
gtggtcatca?accacaaagg?cggcgctgat?gcgaccgaag?atgtaaccgc?ggttgaagtc 360
gatcccgcta?accgcaaccg?cgtaatttca?ggagaacacc?gaattaaagc?ctggacacat 420
tttcattttc?cggggcgcgg?cagcacatac?agcgatttta?aatggcattg?gtaccatttt 480
gacggaaccg?attgggacga?gtcccgaaag?ctgaaccgca?tctataagtt?tcaaggaaag 540
gcttgggatt?gggaagtttc?caatgaaaac?ggcaactatg?attatttgat?gtatgccgac 600
atcgattatg?accatcctga?tgtcgcagca?gaaattaaga?gatggggcac?ttggtatgcc 660
aatgaactgc?aattggacgg?tttccgtctt?gatgctgtca?aacacattaa?attttctttt 720
ttgcgggatt?gggttaatca?tgtcagggaa?aaaacgggga?aggaaatgtt?tacggtagct 780
gaatattggc?agaatgactt?gggcgcgctg?gaaaactatt?tgaacaaaac?aaattttaat 840
cattcagtgt?ttgacgtgcc?gcttcattat?cagttccatg?ctgcatcgac?acagggaggc 900
ggctatgata?tgaggaaatt?gctgaacggt?acggtcgttt?ccaagcatcc?gttgaaagcg 960
gttacatttg?tcgataacca?tgatacacag?ccggggcaat?cgcttgagtc?gactgtccaa 1020
acatggttta?agccgcttgc?ttacgctttt?attctcacaa?gggaatctgg?ataccctcag 1080
gtcttctacg?gggatatgta?cgggacgaaa?ggagactccc?agcgcgaaat?tcctgccttg 1140
aaacacaaaa?ttgaaccgat?cttaaaagcg?agaaaacagt?atgcgtacgg?agcacagcat 1200
gattatttcg?accaccatga?cattgtcggc?tggacaaggg?aaggcgacag?ctcggttgca 1260
aattcaggtt?tggcggcatt?aataacagac?ggacccggtg?gggcaaagcg?aatgtatgtc 1320
ggccggcaaa?acgccggtga?gacatggcat?gacattaccg?gaaaccgttc?ggagccggtt 1380
gtcatcaatt?cggaaggctg?gggagagttt?cacgtaaacg?gcgggtcggt?ttcaatttat 1440
gttcaaagat?aa 1452
<210>4
<211>1452
<212>DNA
＜213〉Bacillus licheniformis (Bacillus licheniformis)
<400>4
gcaaatctta?atgggacgct?gatgcagtat?tttgaatggc?acatgcccaa?tgacggccaa 60
cattggaagc?gcttgcaaaa?cgactcggca?tatttggctg?aacacggtat?tactgccgtc 120
tggattcccc?cggcatataa?gggaacgagc?caagcggatg?tgggctacgg?tgcttacgac 180
ctttatgatt?taggggagtt?tcatcaaaaa?gggacggttc?ggacaaagta?cggcacaaaa 240
ggagagctgc?aatctgcgat?caaaagtctc?cattcccgcg?acattaacgt?ttacggggat 300
gtggtcatca?accacaaagg?cggcgctgat?gcgaccgaag?atgtaaccgc?ggttgaagtc 360
gatcccgctg?accgcaaccg?cgtaatttca?ggagaacgcc?gaattaaagc?ctggacacat 420
tttcattttc?cggggcgcgg?cagcacatac?agcgatttta?aatggcattg?gtaccatttt 480
gacggaaccg?attgggacga?gtcccgaaag?ctgaaccgca?tctataagtt?tcaaggaaag 540
gcttgggatt?gggaagtttc?caatgaaaac?ggcaactatg?attatttgat?gtatgccgac 600
atcgattatg?accatcctga?tgtcgcagca?gaaattaaga?gatggggcac?ttggtatgcc 660
aatgaactgc?aattggacgg?tttccgtctt?gatgctgtca?aacacattaa?attttctttt 720
ttgcgggatt?gggttaatca?tgtcagggaa?aaaacgggga?aggaaatgtt?tacggtagct 780
gaatattggc?cgaatgactt?gggcgcgctg?gaaaactatt?tgaacaaaac?aaattttaat 840
cattcagtgt?ttgacgtgcc?gcttcattat?cagttccatg?ctgcatcgac?acagggaggc 900
ggctatgata?tgaggaaatt?gctgaacagt?acggtcgttt?ccaagcatcc?gttgaaagcg 960
gttacatttg?tcgataacca?tgatacacag?ccggggcaat?cgcttgagtc?gactgtccaa 1020
acatggttta?agccgcttgc?ttacgctttt?attctcacaa?gggaatctgg?ataccctcag 1080
gttttctacg?gggatatgta?cgggacgaaa?ggagactccc?agcgcgaaat?tcctgccttg 1140
aaacacaaaa?ttgaaccgat?cttaaaagcg?agaaaacagt?atgcgcacgg?agcacagcat 1200
gattatttcg?accaccatga?cattgtcggc?tggacaaggg?aaggcgacag?ctcggttgca 1260
aattcaggtt?tggcggcatt?aataacagac?ggacccggtg?gggcaaagcg?aatgtatgtc 1320
ggccggcaaa?acgccggtga?gacatggcat?gacattaccg?gaaaccgttc?ggagccggtt 1380
gtcatcaatt?cggaaggctg?gggagagttt?cacgtaaacg?gcgggtcggt?ttcaatttat 1440
gttcaaagat?ag 1452
<210>5
<211>1452
<212>DNA
＜213〉bacillus pumilus (Bacillus pumilus)
<400>5
gcaaatctta?atgggacgct?gatgcagtat?tttgaatggt?acatgcccaa?tgacggccaa 60
cattggaagc?gcttgcaaaa?cgactcggca?tatttggctg?aacacggtat?tactgccgtc 120
tggattcccc?cggcatataa?gggaacgagc?caagcggatg?tgggctacgg?tgcttacgac 180
ctttatgatt?taggggagtt?tcatcaaaaa?gggacggttc?ggacaaagta?cggcacaaaa 240
ggagagctgc?aatctgcgat?caaaagtctt?cattcccgcg?acattaacgt?ttacggggat 300
gtggtcatca?accacaaagg?cggcgctgat?gcgaccgaag?atgtaaccgc?ggttgaagtc 360
gatcccgctg?accgcaaccg?cgtaatttca?ggagaacacc?gaattaaagc?ctggacacat 420
tttcattttc?cggggcgcgg?cagcacatac?agcgatttta?aatggcattg?gtaccatttt 480
gacggaaccg?gttgggacga?gtcccgaaag?ctgaaccgca?tctataagtt?tcaaggaaag 540
gcttgggatt?gggaagtttc?caatgaaaac?ggcaactatg?attatttgat?gtatgccgac 600
atcgattatg?accatcctga?tgtcgcagca?aaaattaaga?gatggggcac?ttggtatgcc 660
aatgaactgc?aattggacgg?tttccgtctt?gatgctgtca?aacacattaa?attttctttt 720
ttgcgggatt?gggttaatca?tgtcagggaa?aaaacgggga?aggaaatgtt?tacggtagct 780
gaatattggc?agaatgactt?gggcgcgctg?gaaaactatt?tgaacaaaac?aaattttaat 840
cattcagtgt?ttgacgtgcc?gcttcattat?cagttccatg?ctgcatcgac?acagggaggc 900
ggctatgata?tgaggaaatt?gctgaacggt?acggtcgttt?ccaagcatcc?gttgaaagcg 960
gttacatttg?tcgataacca?tgatacacag?ccggggcaat?cgcttgagtc?gactgtccaa 1020
acatggttta?agccgcttgc?ttacgctttt?attctcacaa?gggaatctgg?ataccctcag 1080
gttttctacg?gggatatgta?cgggacgaaa?ggagactccc?agcgcgaaat?tcctgccttg 1140
aaacacaaaa?ttgaaccgat?cttaaaagcg?agaaaacagt?atgcgtacgg?agcacagcat 1200
gattatttcg?accaccatga?cattgtcggc?tggacaaggg?aaggcgacag?ctcggttgca 1260
aattcaggtt?tggcggcatt?aataacagac?ggacccggtg?gggcaaagcg?aatgtatgtc 1320
ggccggcaaa?acgccggtga?gacatggcat?gacattaccg?gaaaccgttc?ggagccggtt 1380
gtcatcaatt?cggaaggctg?gggagagttt?cacgtaaacg?gcgggtcggt?ttcaatttat 1440
gttcaaagat?ag 1452
<210>6
<211>483
<212>PRT
＜213〉subtilis (Bacillus subtilis)
<400>6
Ala?Asn?Leu?Asn?Gly?Thr?Leu?Met?Gln?Tyr?Phe?Glu?Trp?Tyr?Met?Pro
1 5 10 15
Asn?Asp?Gly?Gln?His?Trp?Lys?Arg?Leu?Gln?Asn?Asp?Ser?Ala?Tyr?Leu
20 25 30
Ala?Glu?His?Gly?Ile?Thr?Ala?Val?Trp?Ile?Pro?Pro?Ala?Tyr?Lys?Gly
35 40 45
Thr?Ser?Gln?Ala?Asp?Val?Gly?Tyr?Gly?Ala?Tyr?Asp?Leu?Tyr?Asp?Leu
50 55 60
Gly?Glu?Phe?His?Gln?Lys?Gly?Thr?Val?Arg?Thr?Lys?Tyr?Gly?Thr?Lys
65 70 75 80
Gly?Glu?Leu?Gln?Ser?Ala?Ile?Lys?Ser?Leu?His?Ser?Arg?Asp?Ile?Asn
85 90 95
Val?Tyr?Gly?Asp?Val?Val?Ile?Asn?His?Lys?Gly?Gly?Ala?Asp?Ala?Thr
100 105 110
Glu?Asp?Val?Thr?Ala?Val?Glu?Val?Asp?Pro?Ala?Asp?Arg?Asn?Arg?Val
115 120 125
Ile?Ser?Gly?Glu?His?Arg?Ile?Lys?Ala?Trp?Thr?His?Phe?His?Phe?Pro
130 135 140
Gly?Arg?Gly?Ser?Thr?Tyr?Ser?Asp?Phe?Lys?Trp?His?Trp?Tyr?His?Phe
145 150 155 160
Asp?Gly?Thr?Asp?Trp?Asp?Glu?Ser?Arg?Lys?Leu?Asn?Arg?Ile?Tyr?Lys
165 170 175
Phe?Gln?Gly?Glu?Ala?Trp?Asp?Trp?Glu?Val?Ser?Asn?Glu?Asn?Gly?Asn
180 185 190
Tyr?Asp?Tyr?Leu?Met?Tyr?Ala?Asp?Ile?Asp?Tyr?Asp?His?Pro?Asp?Val
195 200 205
Ala?Ala?Glu?Ile?Lys?Arg?Trp?Gly?Thr?Trp?Tyr?Ala?Asn?Glu?Leu?Gln
210 215 220
Leu?Asp?Gly?Phe?Arg?Leu?Asp?Ala?Val?Lys?His?Ile?Lys?Phe?Ser?Phe
225 230 235 240
Leu?Arg?Asp?Trp?Val?Asn?His?Val?Arg?Glu?Lys?Thr?Gly?Lys?Glu?Met
245 250 255
Phe?Thr?Val?Ala?Glu?Tyr?Trp?Gln?Asn?Asp?Leu?Gly?Ala?Leu?Glu?Asn
260 265 270
Tyr?Leu?Asn?Lys?Thr?Asn?Phe?Asn?His?Ser?Val?Phe?Asp?Val?Pro?Leu
275 280 285
His?Tyr?Gln?Phe?His?Ala?Ala?Ser?Thr?Gln?Gly?Gly?Gly?Tyr?Asp?Met
290 295 300
Arg?Lys?Leu?Leu?Asn?Gly?Thr?Val?Val?Ser?Lys?His?Pro?Leu?Lys?Ala
305 310 315 320
Val?Thr?Phe?Val?Asp?Asn?His?Asp?Thr?Gln?Pro?Gly?Gln?Ser?Leu?Glu
325 330 335
Ser?Thr?Val?Gln?Thr?Trp?Phe?Lys?Pro?Leu?Ala?Tyr?Ala?Phe?Ile?Leu
340 345 350
Thr?Arg?Glu?Ser?Gly?Tyr?Pro?Gln?Val?Phe?Tyr?Gly?Asp?Met?Tyr?Gly
355 360 365
Thr?Lys?Gly?Asp?Pro?Gln?Arg?Glu?Ile?Pro?Ala?Leu?Lys?His?Lys?Ile
370 375 380
Glu?Pro?Ile?Leu?Lys?Ala?Arg?Lys?Gln?Tyr?Ala?Tyr?Gly?Ala?Gln?His
385 390 395 400
Asp?Tyr?Phe?Asp?His?His?Asp?Ile?Val?Gly?Trp?Thr?Arg?Glu?Gly?Asp
405 410 415
Ser?Ser?Val?Ala?Asn?Ser?Gly?Leu?Ala?Ala?Leu?Ile?Thr?Asp?Gly?Pro
420 425 430
Gly?Gly?Ala?Lys?Arg?Met?Tyr?Val?Gly?Arg?Gln?Asn?Ala?Gly?Glu?Thr
435 440 445
Trp?His?Asp?Ile?Thr?Gly?Asn?Arg?Ser?Glu?Pro?Val?Val?Ile?Asn?Ser
450 455 460
Glu?Gly?Trp?Gly?Glu?Phe?His?Val?Asn?Gly?Gly?Ser?Val?Ser?Ile?Tyr
465 470 475 480
Val?Gln?Arg
<210>7
<211>483
<212>PRT
＜213〉bacillus cereus (Bacillus cereus)
<400>7
Ala?Asn?Leu?Asn?Gly?Thr?Leu?Met?Gln?Tyr?Phe?Glu?Trp?Tyr?Met?Pro
1 5 10 15
Asn?Asp?Gly?Gln?His?Trp?Lys?Arg?Leu?Gln?Asn?Asp?Ser?Ala?Tyr?Leu
20 25 30
Ala?Glu?His?Gly?Ile?Thr?Ala?Val?Trp?Ile?Pro?Pro?Ala?Tyr?Lys?Gly
35 40 45
Thr?Ser?Gln?Ala?Asp?Val?Gly?Tyr?Gly?Ala?Tyr?Asp?Leu?Tyr?Asp?Leu
50 55 60
Gly?Glu?Phe?His?Gln?Lys?Gly?Thr?Val?Arg?Thr?Lys?Tyr?Gly?Thr?Lys
65 70 75 80
Gly?Glu?Leu?Gln?Ser?Ala?Ile?Lys?Ser?Leu?His?Ser?Arg?Asp?Ile?Asn
85 90 95
Val?Tyr?Gly?Asp?Val?Val?Ile?Asn?His?Lys?Gly?Gly?Ala?Asp?Ala?Thr
100 105 110
Glu?Asp?Val?Thr?Ala?Val?Glu?Val?Asp?Pro?Ala?Asp?Arg?Asn?Arg?Val
115 120 125
Ile?Ser?Gly?Glu?His?Gln?Ile?Lys?Ala?Trp?Thr?His?Phe?His?Phe?Pro
130 135 140
Gly?Arg?Gly?Ser?Thr?Tyr?Ser?Asp?Phe?Lys?Trp?His?Trp?Tyr?His?Phe
145 150 155 160
Asp?Gly?Thr?Asp?Trp?Asp?Glu?Ser?Arg?Lys?Leu?Asn?Arg?Ile?Tyr?Lys
165 170 175
Phe?Gln?Gly?Lys?Ala?Trp?Asp?Trp?Glu?Val?Ser?Asn?Glu?Asn?Gly?Asn
180 185 190
Tyr?Asp?Tyr?Leu?Met?Tyr?Ala?Asp?Ile?Asp?Tyr?Asp?His?Pro?Asp?Val
195 200 205
Ala?Ala?Glu?Ile?Lys?Arg?Trp?Gly?Thr?Trp?Tyr?Ala?Asn?Glu?Leu?Gln
210 215 220
Leu?Asp?Gly?Phe?Arg?Leu?Asp?Ala?Val?Lys?His?Ile?Lys?Phe?Ser?Phe
225 230 235 240
Leu?Arg?Asp?Trp?Val?Asn?His?Val?Arg?Glu?Lys?Thr?Gly?Lys?Glu?Met
245 250 255
Phe?Thr?Val?Ala?Glu?Tyr?Trp?Gln?Asn?Asp?Leu?Gly?Ala?Leu?Glu?Thr
260 265 270
Tyr?Leu?Asn?Lys?Thr?Asn?Phe?Asn?His?Ser?Val?Phe?Asp?Val?Pro?Leu
275 280 285
His?Tyr?Gln?Phe?His?Ala?Ala?Ser?Thr?Gln?Gly?Gly?Gly?Tyr?Asp?Met
290 295 300
Arg?Lys?Leu?Leu?Asn?Gly?Thr?Val?Val?Ser?Lys?His?Pro?Leu?Lys?Ala
305 310 315 320
Val?Thr?Phe?Val?Asp?Asn?His?Asp?Thr?Gln?Pro?Gly?Gln?Ser?Leu?Glu
325 330 335
Ser?Thr?Val?Gln?Thr?Trp?Phe?Lys?Pro?Leu?Ala?Tyr?Ala?Phe?Ile?Leu
340 345 350
Thr?Arg?Glu?Ser?Gly?Tyr?Pro?Gln?Val?Phe?Tyr?Gly?Asp?Met?Tyr?Gly
355 360 365
Thr?Lys?Gly?Asp?Ser?Gln?Arg?Glu?Ile?Pro?Ala?Leu?Lys?His?Lys?Ile
370 375 380
Glu?Pro?Ile?Leu?Lys?Ala?Arg?Lys?Gln?Tyr?Ala?Tyr?Gly?Ala?Gln?His
385 390 395 400
Asp?Tyr?Phe?Asp?His?His?Asp?Ile?Val?Gly?Trp?Thr?Arg?Glu?Gly?Asp
405 410 415
Ser?Ser?Val?Ala?Asn?Ser?Gly?Leu?Ala?Ala?Leu?Ile?Thr?Asp?Gly?Pro
420 425 430
Gly?Gly?Ala?Lys?Arg?Met?Tyr?Val?Gly?Arg?Gln?Asn?Ala?Gly?Glu?Thr
435 440 445
Trp?His?Asp?Ile?Thr?Gly?Asn?Arg?Ser?Glu?Pro?Val?Val?Ile?Asn?Ser
450 455 460
Glu?Gly?Trp?Gly?Glu?Phe?His?Val?Asn?Gly?Gly?Ser?Val?Ser?Ile?Tyr
465 470 475 480
Val?Gln?Arg
<210>8
<211>483
<212>PRT
＜213〉Bacillus licheniformis (Bacillus licheniformis)
<400>8
Ala?Asn?Leu?Asn?Gly?Thr?Leu?Met?Gln?Tyr?Phe?Glu?Trp?Tyr?Met?Pro
1 5 10 15
Asn?Asp?Gly?Gln?His?Trp?Lys?Arg?Leu?Gln?Asn?Asp?Ser?Ala?Tyr?Leu
20 25 30
Ala?Glu?His?Gly?Ile?Thr?Ala?Val?Trp?Ile?Pro?Pro?Ala?Tyr?Lys?Gly
35 40 45
Thr?Ser?Gln?Ala?Asp?Val?Gly?Tyr?Gly?Ala?Tyr?Asp?Leu?Tyr?Asp?Leu
50 55 60
Gly?Glu?Phe?His?Gln?Lys?Gly?Thr?Val?Arg?Thr?Lys?Tyr?Gly?Thr?Lys
65 70 75 80
Gly?Glu?Leu?Gln?Ser?Ala?Ile?Lys?Ser?Leu?His?Ser?Arg?Asp?Ile?Asn
85 90 95
Val?Tyr?Gly?Asp?Val?Val?Ile?Asn?His?Lys?Gly?Gly?Ala?Asp?Ala?Thr
100 105 110
Glu?Asp?Val?Thr?Ala?Val?Glu?Val?Asp?Pro?Ala?Asn?Arg?Asn?Arg?Val
115 120 125
Ile?Ser?Gly?Glu?His?Arg?Ile?Lys?Ala?Trp?Thr?His?Phe?His?Phe?Pro
130 135 140
Gly?Arg?Gly?Ser?Thr?Tyr?Ser?Asp?Phe?Lys?Trp?His?Trp?Tyr?His?Phe
145 150 155 160
Asp?Gly?Thr?Asp?Trp?Asp?Glu?Ser?Arg?Lys?Leu?Asn?Arg?Ile?Tyr?Lys
165 170 175
Phe?Gln?Gly?Lys?Ala?Trp?Asp?Trp?Glu?Val?Ser?Asn?Glu?Asn?Gly?Asn
180 185 190
Tyr?Asp?Tyr?Leu?Met?Tyr?Ala?Asp?Ile?Asp?Tyr?Asp?His?Pro?Asp?Val
195 200 205
Ala?Ala?Glu?Ile?Lys?Arg?Trp?Gly?Thr?Trp?Tyr?Ala?Asn?Glu?Leu?Gln
210 215 220
Leu?Asp?Gly?Phe?Arg?Leu?Asp?Ala?Val?Lys?His?Ile?Lys?Phe?Ser?Phe
225 230 235 240
Leu?Arg?Asp?Trp?Val?Asn?His?Val?Arg?Glu?Lys?Thr?Gly?Lys?Glu?Met
245 250 255
Phe?Thr?Val?Ala?Glu?Tyr?Trp?Gln?Asn?Asp?Leu?Gly?Ala?Leu?Glu?Asn
260 265 270
Tyr?Leu?Asn?Lys?Thr?Asn?Phe?Asn?His?Ser?Val?Phe?Asp?Val?Pro?Leu
275 280 285
His?Tyr?Gln?Phe?His?Ala?Ala?Ser?Thr?Gln?Gly?Gly?Gly?Tyr?Asp?Met
290 295 300
Arg?Lys?Leu?Leu?Asn?Gly?Thr?Val?Val?Ser?Lys?His?Pro?Leu?Lys?Ala
305 310 315 320
Val?Thr?Phe?Val?Asp?Asn?His?Asp?Thr?Gln?Pro?Gly?Gln?Ser?Leu?Glu
325 330 335
Ser?Thr?Val?Gln?Thr?Trp?Phe?Lys?Pro?Leu?Ala?Tyr?Ala?Phe?Ile?Leu
340 345 350
Thr?Arg?Glu?Ser?Gly?Tyr?Pro?Gln?Val?Phe?Tyr?Gly?Asp?Met?Tyr?Gly
355 360 365
Thr?Lys?Gly?Asp?Ser?Gln?Arg?Glu?Ile?Pro?Ala?Leu?Lys?His?Lys?Ile
370 375 380
Glu?Pro?Ile?Leu?Lys?Ala?Arg?Lys?Gln?Tyr?Ala?Tyr?Gly?Ala?Gln?His
385 390 395 400
Asp?Tyr?Phe?Asp?His?His?Asp?Ile?Val?Gly?Trp?Thr?Arg?Glu?Gly?Asp
405 410 415
Ser?Ser?Val?Ala?Asn?Ser?Gly?Leu?Ala?Ala?Leu?Ile?Thr?Asp?Gly?Pro
420 425 430
Gly?Gly?Ala?Lys?Arg?Met?Tyr?Val?Gly?Arg?Gln?Asn?Ala?Gly?Glu?Thr
435 440 445
Trp?His?Asp?Ile?Thr?Gly?Asn?Arg?Ser?Glu?Pro?Val?Val?Ile?Asn?Set
450 455 460
Glu?Gly?Trp?Gly?Glu?Phe?His?Val?Asn?Gly?Gly?Ser?Val?Ser?Ile?Tyr
465 470 475 480
Val?Gln?Arg
<210>9
<211>483
<212>PRT
＜213〉Bacillus licheniformis (Bacillus licheniformis)
<400>9
Ala?Asn?Leu?Asn?Gly?Thr?Leu?Met?Gln?Tyr?Phe?Glu?Trp?Tyr?Met?Pro
1 5 10 15
Asn?Asp?Gly?Gln?His?Trp?Lys?Arg?Leu?Gln?Asn?Asp?Ser?Ala?Tyr?Leu
20 25 30
Ala?Glu?His?Gly?Ile?Thr?Ala?Val?Trp?Ile?Pro?Pro?Ala?Tyr?Lys?Gly
35 40 45
Thr?Ser?Gln?Ala?Asp?Val?Gly?Tyr?Gly?Ala?Tyr?Asp?Leu?Tyr?Asp?Leu
50 55 60
Gly?Glu?Phe?His?Gln?Lys?Gly?Thr?Val?Arg?Thr?Lys?Tyr?Gly?Thr?Lys
65 70 75 80
Gly?Glu?Leu?Gln?Ser?Ala?Ile?Lys?Ser?Leu?His?Ser?Arg?Asp?Ile?Asn
85 90 95
Val?Tyr?Gly?Asp?Val?Val?Ile?Asn?His?Lys?Gly?Gly?Ala?Asp?Ala?Thr
100 105 110
Glu?Asp?Val?Thr?Ala?Val?Glu?Val?Asp?Pro?Ala?Asp?Arg?Asn?Arg?Val
115 120 125
Ile?Ser?Gly?Glu?His?Arg?Ile?Lys?Ala?Trp?Thr?His?Phe?His?Phe?Pro
130 135 140
Gly?Arg?Gly?Ser?Thr?Tyr?Ser?Asp?Phe?Lys?Trp?His?Trp?Tyr?His?Phe
145 150 155 160
Asp?Gly?Thr?Gly?Trp?Asp?Glu?Ser?Arg?Lys?Leu?Asn?Arg?Ile?Tyr?Lys
165 170 175
Phe?Gln?Gly?Lys?Ala?Trp?Asp?Trp?Glu?Val?Ser?Asn?Glu?Asn?Gly?Asn
180 185 190
Tyr?Asp?Tyr?Leu?Met?Tyr?Ala?Asp?Ile?Asp?Tyr?Asp?His?Pro?Asp?Val
195 200 205
Ala?Ala?Lys?Ile?Lys?Arg?Trp?Gly?Thr?Trp?Tyr?Ala?Asn?Glu?Leu?Gln
210 215 220
Leu?Asp?Gly?Phe?Arg?Leu?Asp?Ala?Val?Lys?His?Ile?Lys?Phe?Ser?Phe
225 230 235 240
Leu?Arg?Asp?Trp?Val?Asn?His?Val?Arg?Glu?Lys?Thr?Gly?Lys?Glu?Met
245 250 255
Phe?Thr?Val?Ala?Glu?Tyr?Trp?Gln?Asn?Asp?Leu?Gly?Ala?Leu?Glu?Asn
260 265 270
Tyr?Leu?Asn?Lys?Thr?Asn?Phe?Asn?His?Ser?Val?Phe?Asp?Val?Pro?Leu
275 280 285
His?Tyr?Gln?Phe?His?Ala?Ala?Ser?Thr?Gln?Gly?Gly?Gly?Tyr?Asp?Met
290 295 300
Arg?Lys?Leu?Leu?Asn?Gly?Thr?Val?Val?Ser?Lys?His?Pro?Leu?Lys?Ala
305 310 315 320
Val?Thr?Phe?Val?Asp?Asn?His?Asp?Thr?Gln?Pro?Gly?Gln?Ser?Leu?Glu
325 330 335
Ser?Thr?Val?Gln?Thr?Trp?Phe?Lys?Pro?Leu?Ala?Tyr?Ala?Phe?Ile?Leu
340 345 350
Thr?Arg?Glu?Ser?Gly?Tyr?Pro?Gln?Val?Phe?Tyr?Gly?Asp?Met?Tyr?Gly
355 360 365
Thr?Lys?Gly?Asp?Ser?Gln?Arg?Glu?Ile?Pro?Ala?Leu?Lys?His?Lys?Ile
370 375 380
Glu?Pro?Ile?Leu?Lys?Ala?Arg?Lys?Gln?Tyr?Ala?Tyr?Gly?Ala?Gln?His
385 390 395 400
Asp?Tyr?Phe?Asp?His?His?Asp?Ile?Val?Gly?Trp?Thr?Arg?Glu?Gly?Asp
405 410 415
Ser?Ser?Val?Ala?Asn?Ser?Gly?Leu?Ala?Ala?Leu?Ile?Thr?Asp?Gly?Pro
420 425 430
Gly?Gly?Ala?Lys?Arg?Met?Tyr?Val?Gly?Arg?Gln?Asn?Ala?Gly?Glu?Thr
435 440 445
Trp?His?Asp?Ile?Thr?Gly?Asn?Arg?Ser?Glu?Pro?Val?Val?Ile?Asn?Ser
450 455 460
Glu?Gly?Trp?Gly?Glu?Phe?His?Val?Asn?Gly?Gly?Ser?Val?Ser?Ile?Tyr
465 470 475 480
Val?Gln?Arg
<210>10
<211>483
<212>PRT
＜213〉bacillus pumilus (Bacillus pumilus)
<400>10
Ala?Asn?Leu?Asn?Gly?Thr?Leu?Met?Gln?Tyr?Phe?Glu?Trp?His?Met?Pro
1 5 10 15
Asn?Asp?Gly?Gln?His?Trp?Lys?Arg?Leu?Gln?Asn?Asp?Ser?Ala?Tyr?Leu
20 25 30
Ala?Glu?His?Gly?Ile?Thr?Ala?Val?Trp?Ile?Pro?Pro?Ala?Tyr?Lys?Gly
35 40 45
Thr?Ser?Gln?Ala?Asp?Val?Gly?Tyr?Gly?Ala?Tyr?Asp?Leu?Tyr?Asp?Leu
50 55 60
Gly?Glu?Phe?His?Gln?Lys?Gly?Thr?Val?Arg?Thr?Lys?Tyr?Gly?Thr?Lys
65 70 75 80
Gly?Glu?Leu?Gln?Ser?Ala?Ile?Lys?Ser?Leu?His?Ser?Arg?Asp?Ile?Asn
85 90 95
Val?Tyr?Gly?Asp?Val?Val?Ile?Asn?His?Lys?Gly?Gly?Ala?Asp?Ala?Thr
100 105 110
Glu?Asp?Val?Thr?Ala?Val?Glu?Val?Asp?Pro?Ala?Asp?Arg?Asn?Arg?Val
115 120 125
Ile?Ser?Gly?Glu?Arg?Arg?Ile?Lys?Ala?Trp?Thr?His?Phe?His?Phe?Pro
130 135 140
Gly?Arg?Gly?Ser?Thr?Tyr?Ser?Asp?Phe?Lys?Trp?His?Trp?Tyr?His?Phe
145 150 155 160
Asp?Gly?Thr?Asp?Trp?Asp?Glu?Ser?Arg?Lys?Leu?Asn?Arg?Ile?Tyr?Lys
165 170 175
Phe?Gln?Gly?Lys?Ala?Trp?Asp?Trp?Glu?Val?Ser?Ash?Glu?Asn?Gly?Asn
180 185 190
Tyr?Asp?Tyr?Leu?Met?Tyr?Ala?Asp?Ile?Asp?Tyr?Asp?His?Pro?Asp?Val
195 200 205
Ala?Ala?Glu?Ile?Lys?Arg?Trp?Gly?Thr?Trp?Tyr?Ala?Asn?Glu?Leu?Gln
210 215 220
Leu?Asp?Gly?Phe?Arg?Leu?Asp?Ala?Val?Lys?His?Ile?Lys?Phe?Ser?Phe
225 230 235 240
Leu?Arg?Asp?Trp?Val?Asn?His?Val?Arg?Glu?Lys?Thr?Gly?Lys?Glu?Met
245 250 255
Phe?Thr?Val?Ala?Glu?Tyr?Trp?Pro?Asn?Asp?Leu?Gly?Ala?Leu?Glu?Asn
260 265 270
Tyr?Leu?Asn?Lys?Thr?Asn?Phe?Asn?His?Ser?Val?Phe?Asp?Val?Pro?Leu
275 280 285
His?Tyr?Gln?Phe?His?Ala?Ala?Ser?Thr?Gln?Gly?Gly?Gly?Tyr?Asp?Met
290 295 300
Arg?Lys?Leu?Leu?Asn?Ser?Thr?Val?Val?Ser?Lys?His?Pro?Leu?Lys?Ala
305 310 315 320
Val?Thr?Phe?Val?Asp?Asn?His?Asp?Thr?Gln?Pro?Gly?Gln?Ser?Leu?Glu
325 330 335
Ser?Thr?Val?Gln?Thr?Trp?Phe?Lys?Pro?Leu?Ala?Tyr?Ala?Phe?Ile?Leu
340 345 350
Thr?Arg?Glu?Ser?Gly?Tyr?Pro?Gln?Val?Phe?Tyr?Gly?Asp?Met?Tyr?Gly
355 360 365
Thr?Lys?Gly?Asp?Ser?Gln?Arg?Glu?Ile?Pro?Ala?Leu?Lys?His?Lys?Ile
370 375 380
Glu?Pro?Ile?Leu?Lys?Ala?Arg?Lys?Gln?Tyr?Ala?His?Gly?Ala?Gln?His
385 390 395 400
Asp?Tyr?Phe?Asp?His?His?Asp?Ile?Val?Gly?Trp?Thr?Arg?Glu?Gly?Asp
405 410 415
Ser?Ser?Val?Ala?Asn?Ser?Gly?Leu?Ala?Ala?Leu?Ile?Thr?Asp?Gly?Pro
420 425 430
Gly?Gly?Ala?Lys?Arg?Met?Tyr?Val?Gly?Arg?Gln?Asn?Ala?Gly?Glu?Thr
435 440 445
Trp?His?Asp?Ile?Thr?Gly?Asn?Arg?Ser?Glu?Pro?Val?Val?Ile?Asn?Ser
450 455 460
Glu?Gly?Trp?Gly?Glu?Phe?His?Val?Asn?Gly?Gly?Ser?Val?Ser?Ile?Tyr
465 470 475 480
Val?Gln?Arg
<210>11
<211>483
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
<222>(14)..(14)
＜223〉Xaa=Tyr or His
<220>
<221>MISC_FEATURE
<222>(124)..(124)
＜223〉Xaa=Asp or Asn
<220>
<221>MISC_FEATURE
<222>(133)..(133)
＜223〉Xaa=His or Arg
<220>
<221>MISC_FEATURE
<222>(134)..(134)
＜223〉Xaa=Arg or Gln
<220>
<221>MISC_FEATURE
<222>(164)..(164)
＜223〉Xaa=Asp or Gly
<220>
<221>MISC_FEATURE
<222>(180)..(180)
＜223〉Xaa=Glu or Lys
<220>
<221>MISC_FEATURE
<222>(211)..(211)
＜223〉Xaa=Glu or Lys
<220>
<221>MISC_FEATURE
<222>(264)..(264)
＜223〉Xaa=Gln or Pro
<220>
<221>MISC_FEATURE
<222>(272)..(272)
＜223〉Xaa=Asn or Thr
<220>
<221>MISC_FEATURE
<222>(310)..(310)
＜223〉Xaa=Gly or Ser
<220>
<221>MISC_FEATURE
<222>(373)..(373)
＜223〉Xaa=Pro or Ser
<220>
<221>MISC_FEATURE
<222>(396)..(396)
＜223〉Xaa=Tyr or His
<400>11
Ala?Asn?Leu?Asn?Gly?Thr?Leu?Met?Gln?Tyr?Phe?Glu?Trp?Xaa?Met?Pro
1 5 10 15
Asn?Asp?Gly?Gln?His?Trp?Lys?Arg?Leu?Gln?Asn?Asp?Ser?Ala?Tyr?Leu
20 25 30
Ala?Glu?His?Gly?Ile?Thr?Ala?Val?Trp?Ile?Pro?Pro?Ala?Tyr?Lys?Gly
35 40 45
Thr?Ser?Gln?Ala?Asp?Val?Gly?Tyr?Gly?Ala?Tyr?Asp?Leu?Tyr?Asp?Leu
50 55 60
Gly?Glu?Phe?His?Gln?Lys?Gly?Thr?Val?Arg?Thr?Lys?Tyr?Gly?Thr?Lys
65 70 75 80
Gly?Glu?Leu?Gln?Ser?Ala?Ile?Lys?Ser?Leu?His?Ser?Arg?Asp?Ile?Asn
85 90 95
Val?Tyr?Gly?Asp?Val?Val?Ile?Asn?His?Lys?Gly?Gly?Ala?Asp?Ala?Thr
100 105 110
Glu?Asp?Val?Thr?Ala?Val?Glu?Val?Asp?Pro?Ala?Xaa?Arg?Asn?Arg?Val
115 120 125
Ile?Ser?Gly?Glu?Xaa?Xaa?Ile?Lys?Ala?Trp?Thr?His?Phe?His?Phe?Pro
130 135 140
Gly?Arg?Gly?Ser?Thr?Tyr?Ser?Asp?Phe?Lys?Trp?His?Trp?Tyr?His?Phe
145 150 155 160
Asp?Gly?Thr?Xaa?Trp?Asp?Glu?Ser?Arg?Lys?Leu?Asn?Arg?Ile?Tyr?Lys
165 170 175
Phe?Gln?Gly?Xaa?Ala?Trp?Asp?Trp?Glu?Val?Ser?Asn?Glu?Asn?Gly?Asn
180 185 190
Tyr?Asp?Tyr?Leu?Met?Tyr?Ala?Asp?Ile?Asp?Tyr?Asp?His?Pro?Asp?Val
195 200 205
Ala?Ala?Xaa?Ile?Lys?Arg?Trp?Gly?Thr?Trp?Tyr?Ala?Asn?Glu?Leu?Gln
210 215 220
Leu?Asp?Gly?Phe?Arg?Leu?Asp?Ala?Val?Lys?His?Ile?Lys?Phe?Ser?Phe
225 230 235 240
Leu?Arg?Asp?Trp?Val?Asn?His?Val?Arg?Glu?Lys?Thr?Gly?Lys?Glu?Met
245 250 255
Phe?Thr?Val?Ala?Glu?Tyr?Trp?Xaa?Asn?Asp?Leu?Gly?Ala?Leu?Glu?Xaa
260 265 270
Tyr?Leu?Asn?Lys?Thr?Asn?Phe?Asn?His?Ser?Val?Phe?Asp?Val?Pro?Leu
275 280 285
His?Tyr?Gln?Phe?His?Ala?Ala?Ser?Thr?Gln?Gly?Gly?Gly?Tyr?Asp?Met
290 295 300
Arg?Lys?Leu?Leu?Asn?Xaa?Thr?Val?Val?Ser?Lys?His?Pro?Leu?Lys?Ala
305 310 315 320
Val?Thr?Phe?Val?Asp?Asn?His?Asp?Thr?Gln?Pro?Gly?Gln?Ser?Leu?Glu
325 330 335
Ser?Thr?Val?Gln?Thr?Trp?Phe?Lys?Pro?Leu?Ala?Tyr?Ala?Phe?Ile?Leu
340 345 350
Thr?Arg?Glu?Ser?Gly?Tyr?Pro?Gln?Val?Phe?Tyr?Gly?Asp?Met?Tyr?Gly
355 360 365
Thr?Lys?Gly?Asp?Xaa?Gln?Arg?Glu?Ile?Pro?Ala?Leu?Lys?His?Lys?Ile
370 375 380
Glu?Pro?Ile?Leu?Lys?Ala?Arg?Lys?Gln?Tyr?Ala?Xaa?Gly?Ala?Gln?His
385 390 395 400
Asp?Tyr?Phe?Asp?His?His?Asp?Ile?Val?Gly?Trp?Thr?Arg?Glu?Gly?Asp
405 410 415
Ser?Ser?Val?Ala?Asn?Ser?Gly?Leu?Ala?Ala?Leu?Ile?Thr?Asp?Gly?Pro
420 425 430
Gly?Gly?Ala?Lys?Arg?Met?Tyr?Val?Gly?Arg?Gln?Asn?Ala?Gly?Glu?Thr
435 440 445
Trp?His?Asp?Ile?Thr?Gly?Asn?Arg?Ser?Glu?Pro?Val?Val?Ile?Asn?Ser
450 455 460
Glu?Gly?Trp?Gly?Glu?Phe?His?Val?Asn?Gly?Gly?Ser?Val?Ser?Ile?Tyr
465 470 475 480
Val?Gln?Arg
<210>12
<211>25
<212>DNA
＜213〉primer
<400>12
ccatgggcaa?atcttaatgg?gacgc 25
<210>13
<211>34
<212>DNA
＜213〉primer
<400>13
gaattcctat?ctttgaacat?aaattgaaac?cgac 34
<210>14
<211>34
<212>DNA
＜213〉primer
<400>14
gaattcttat?ctttgaacat?aaattgaaac?cgac 34

Claims (10)

1. an isolating α-Dian Fenmei is characterized in that, the aminoacid sequence of described α-Dian Fenmei shown in SEQ IDNO:11, wherein,
The 14th amino acids is selected from Tyr or His; The 124th amino acids is selected from Asp or Asn;
The 133rd amino acids is selected from His or Arg; The 134th amino acids is selected from Arg or Gln;
The 164th amino acids is selected from Asp or Gly; The 180th amino acids is selected from Glu or Lys;
The 211st amino acids is selected from Glu or Lys; The 264th amino acids is selected from Gln or Pro;
The 272nd amino acids is selected from Asn or Thr; The 310th amino acids is selected from Gly or Ser;
The 373rd amino acids is selected from Pro or Ser; Or the 396th amino acids be selected from Tyr or His.
2. α-Dian Fenmei as claimed in claim 1 is characterized in that, the aminoacid sequence of described α-Dian Fenmei such as SEQ ID NO:6, and SEQ ID NO:7, SEQ ID NO:8 is shown in SEQ ID NO:9 or the SEQ ID NO:10.
3. isolating polynucleotide is characterized in that, described polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is selected from down group:
(a) polynucleotide of α-Dian Fenmei as claimed in claim 1 or 2 of encoding; Or
(b) with polynucleotide (a) complementary polynucleotide.
4. polynucleotide as claimed in claim 3 is characterized in that, the nucleotide sequence of described polynucleotide such as SEQ ID NO:1, and SEQ ID NO:2, SEQ ID NO:3 is shown in SEQ ID NO:4 or the SEQ ID NO:5.
5. a carrier is characterized in that, described carrier comprises the described polynucleotide of claim 2.
6. a cell is characterized in that, it comprises the described carrier of claim 5, or is integrated with claim 3 or 4 described polynucleotide in its genome.
7. a method of producing the described α-Dian Fenmei of claim 1 is characterized in that, comprising: cultivate the described cell of claim 6, isolate expression product from culture.
8. a composition is characterized in that, contains claim 1 or 2 described α-Dian Fenmei in the described composition; And acceptable carrier on the bromatology.
9. the purposes of the described α-Dian Fenmei of claim 1 is characterized in that, is used for hydrolysis glycogen or starch.
10. the method for hydrolysis glycogen or starch is characterized in that described method comprises: handle glycogen or starch with the described α-Dian Fenmei of claim 1.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103224920A (en) * 2013-02-04 2013-07-31 江南大学 Method for separation purification of high temperature-resistant beta-amylase from bacillus subtilis
CN106978409A (en) * 2017-04-26 2017-07-25 福建福大百特生物科技有限公司 A kind of high efficiency preparation method of α glucuroides
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1313608C (en) * 2004-01-18 2007-05-02 中国科学院微生物研究所 Basic alpha-diastase, and its coding gene and production method
CN101451115B (en) * 2008-12-30 2010-11-17 汪兵 Genetic engineering bacterial strain for expressing intermediate temperature alpha-amylase

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103224920A (en) * 2013-02-04 2013-07-31 江南大学 Method for separation purification of high temperature-resistant beta-amylase from bacillus subtilis
CN106978409A (en) * 2017-04-26 2017-07-25 福建福大百特生物科技有限公司 A kind of high efficiency preparation method of α glucuroides
CN106978409B (en) * 2017-04-26 2020-09-11 福建福大百特生物科技有限公司 Efficient preparation method of alpha-glucosidase
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections

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