CN105002150A

CN105002150A - Alpha-amylase gene and application thereof

Info

Publication number: CN105002150A
Application number: CN201510538994.7A
Authority: CN
Inventors: 马永凯; 王毅; 李建凯; 魏云林; 季秀玲; 苏杨; 宋鹏飞
Original assignee: China Tobacco Yunnan Industrial Co Ltd
Current assignee: China Tobacco Yunnan Industrial Co Ltd
Priority date: 2015-08-29
Filing date: 2015-08-29
Publication date: 2015-10-28

Abstract

The invention discloses an alpha-amylase, a gene coding the amylase, a recombinant carrier containing the coding gene thereof, genetically engineered bacteria and a recombinant amylase preparation produced from the alpha amylase. The amino acid sequence of the amylase having endo-alpha-1,4 glycosidic bond activity is as shown by SEQ ID NO.1, and the coding sequence of the gene of the alpha amylase is a nucleotide sequence shown by SEQ ID NO.2. Through constructing the recombinant carrier and expressing that a expression product has an endo-alpha-1,4 glycosidic bond function in colon bacillus, the amylase gene provided by the invention derives from microbial genomics on the surfaces of flue-cured tobacco, and the produced amylase can acts on the purification process of the flue-cured tobacco, so that the purification progress of the flue-cured tobacco is changed, and the quality of tobacco leaves is promoted.

Description

A kind of alpha-amylase gene and application thereof

Technical field

The invention belongs to biotechnology and technical field of enzyme engineering, be specifically related to a kind of α-amylase, the gene of this enzyme of encoding, the recombinant vectors containing its encoding gene, genetic engineering bacterium and application thereof.

Background technology

Starch can be divided into amylose starch and amylopectin two kinds according to the difference of structure.The two be all D-glucopyranoside by α-l, 4 glycosidic bonds be connected forms; With amylose starch unlike, amylopectin, every 20 ~ 25 glucose units, just has one with α-l, 6 glycosidic bonds be connected side chain.In the hydrolysis pathway of starch, need the acting in conjunction of the smart enzyme of α-amylase, P-amylase and limit Hu etc.

α-amylase is amylolytic initial enzyme, can the α-Isosorbide-5-Nitrae glycosidic link of random degenerate amylose starch and amylopectin non-end, is called endo-amylase; The product that it acts on amylose starch is maltose and trisaccharide maltose, acts in the product of amylopectin except glucose, maltose, also has a series of oligose with α-1,6 glycosidic link.Beta-amylase also acts on α-1, 4 glycosidic links, but its effect is from non reducing end, by two glucose unit degraded amylose starchs, be called as exo-amylase enzyme, the product that it acts on amylose starch is maltose, when acting on amylopectin, α-the l of peripheral carbochain can only be hydrolyzed, 4 glycosidic links, and the α-1 of tapping point can not be surmounted, 6 glycosidic links anhydrate the α-l separated inside tapping point, 4 glycosidic links, so it is incomplete to the hydrolysis of amylopectin, when effect is to α-l, limit dextrin is generated during 4 glycosidic link, need the further effect of the smart enzyme of limit Hu.The effective object of limit dextrinase is α-l, 6 glycosidic links, after can amylopectin being changed into linear form again by α-amylase, beta-amylase hydrolysis and also it require that at least respectively there is a α-l both sides of α-1,6 glycosidic link, 4 keys connect D-Glucose residue.Limit dextrinase is to α-l, and 6 glycosidic links play specificity effect, but it can not direct degraded starch grain, only has when just working after α-amylase effect.

Current amylase in food, papermaking, weave, brewage fermentation industry, pharmaceutical industries and tobacco industry etc. and all play an important role.

Summary of the invention

The object of the invention is in order to solve the deficiencies in the prior art, to provide a kind of α-amylase, the gene of this enzyme of encoding, the recombinant vectors containing its encoding gene, genetic engineering bacterium and application thereof.

The technical solution used in the present invention is as follows:

The present invention aims to provide a kind of from genus bacillus bacillussp. be separated the alpha-amylase gene obtained in 65#, the fragment of this gene nucleotide series or nucleotide sequence as shown in SEQ ID NO.2, or with the nucleotide sequence of SEQ ID NO.2 complementation, this gene order length is 1980bp(base).

Nucleotide sequence of the present invention is DNA form, comprises the DNA of cDNA, genomic dna or synthetic, can be strand or double-strand.Due to the singularity of nucleotide sequence, any have more than 80% homology with the nucleotide sequence shown in SEQ ID NO.2 and have the polynucleotide variant of identical function, all within protection scope of the present invention.Described polynucleotide variant refers to a kind of polynucleotide sequence having one or more Nucleotide and change, and can use replacement known in the art, disappearance or insert variation to obtain.

The aminoacid sequence of the alpha-amylase gene coding in the present invention is polypeptide or the protein with the amino acid residue sequence shown in SEQ ID NO.1.Due to the singularity of aminoacid sequence; as long as the polypeptide fragment containing aminoacid sequence shown in SEQ ID NO.1 or its variant; amino acid residue sequence as shown in its examples of conservative variations, bioactive fragment or derivative with SEQ ID NO.1 has more than 90% homology and has the protein of identical activity, all within protection scope of the present invention.These methods comprise the disappearance of amino-acid residue in aminoacid sequence, insertion, chemically modified or replacement, and described albumen can be recombinant protein, native protein or synthetic proteins.

Another object of the present invention is to provide a kind of recombinant expression vector containing alpha-amylase gene, be gene shown in SEQ ID NO.2 is directly connected from different expression vector constructed by recombinant vectors.

In the present invention, coding for alpha-diastatic polynucleotide sequence can be inserted in carrier, to form containing recombinant vectors of the present invention.Carrier refers to plasmid known in the art, virus or other carriers, advises preferred pET carrier families and other prokaryotic expression carriers.Can build containing coding for alpha-diastatic nucleotide sequence by method well known to those skilled in the art and suitable to transcribe/expression vector of translational control element.These methods comprise recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc.The nucleotide sequence of described encode starch enzyme can be effectively connected in the suitable promotor in expression vector, to instruct the synthesis of mRNA.The representative example of these promotors has: colibacillary lacor trppromotor; The PL promotor of lambda particles phage; Eukaryotic promoter comprise CMV early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, the LTRs of retrovirus and some other known can the promotor expressed in prokaryotic cell prokaryocyte or eukaryotic cell or its virus of controlling gene.Expression vector also comprises the ribosome bind site and transcription terminator etc. of translation initiation.Inserting enhancer sequence in the carrier will make its transcribing in higher eucaryotic cells be enhanced.Enhanser is the cis-acting factors that DNA expresses, and usual nearly 10-300bp, acts on promotor transcribing with enhancing gene, as adenovirus cancers.

In the present invention, coding for alpha-diastatic polynucleotide or the recombinant vectors containing these polynucleotide can transform by method well-known to those having ordinary skill in the art or import in host cell, this cell can be bacterial cell, fungal cell, vegetable cell or zooblast, or the offspring of these host cells, representative example has: intestinal bacteria; Fungal cell or yeast; Vegetable cell is as rape, tobacco, soybean; Insect cell is as fruit bat S2 or Sf9; Zooblast is as CHO, COS or Bowes melanoma cell etc.

The invention still further relates to the application of described encoding gene in preparation recombinant alpha-amylases, by the recombinant DNA technology of routine, utilize polynucleotide sequence of the present invention can the α-amylase of construction expression or Restruction.In general can be realized by following steps: build recombinant vectors, be transformed into constructing host cell reconstitution cell, in suitable substratum, cultivate host cell, analysis, protein purification from substratum or cell.

compared with prior art, its beneficial effect is in the present invention:

The present invention utilizes escherichia expression system to construct the engineering bacteria of α-amylase, compared with this bacterium producing multi enzyme preparation itself, can be quicker, a large amount of give expression to target protein.And required substratum and culture condition are simple, technique is simple, and the cycle is short, greatly can reduce production cost in industrial application.In addition when building recombinant vectors used carrier end with Histidine (His) label, can with nickel post specific binding, be convenient to purifying.Compared with bacterium producing multi enzyme preparation itself, this project bacterium institute α-amylase Producer enzyme amount is larger, purity is higher, enzymatic properties is higher, more stable, more effectively can be applied to tobacco leaf industry, improve quality of tobacco.

Accompanying drawing explanation

Fig. 1 is e. coli bl21 (DE3) the expression vector 65#-pET-30a that the present invention builds;

Fig. 2 is the bacterium colony PCR proof diagram of recombinant plasmid object fragment in the present invention, the Maker of 2000bp sized by M in figure; Fragment amplification product (parallel repetition) for the purpose of swimming lane 1,2 is equal.

Fig. 3 is the single, double restriction enzyme mapping of recombinant plasmid in the present invention, in figure: M is Marker; Swimming lane 1 is that recombinant plasmid adopts restriction enzyme hinthe single endonuclease digestion product of d III; Swimming lane 2 is recombinant plasmid double digestion results; Swimming lane 3 is the fragments obtained that increase in embodiment 1.

Fig. 4 is that SDS-PAGE of the present invention detects inducing effect schematic diagram, and in figure, M is Marker, and 1 is the expressing protein of not inducing; 2 is the target proteins of expressing after induction.Arrow instruction is target protein band.

Embodiment

Below in conjunction with embodiment, the present invention is described in further detail.

It will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by buying the conventional products obtained.

Embodiment 1: from subtilis ( bacillus subtilis) be separated the nucleotide sequence of the alpha-amylase gene obtained in 65#.

CTAB method is adopted to extract the genomic dna of subtilis, getting 2 μ l is that template carries out polymerase chain reaction (PCR), according to the conserved regions of the amylase homologous sequence delivered, in conjunction with used carrier design primer (primers F and primer R), in this PCR reaction process, the primer, component and amplification condition are as follows:

Primers F: 5 '-CGGGATCCATGTTTGAAAAACGATTCAAAAC-3 ' (SEQ ID NO.3)

Primer R:5 '-CCCAAGCTT ATGCGGAAGATAACCATTCAA-3 ' (SEQ ID NO.4)

PCR amplification system composition is as shown in table 1.

Table 1

10×Taq Buffer	5μl
		dNTP(2.5mmol/L)	4μl
Template (genomic dna)	2μl
		Primers F	2μl
Primer R	2μl
		Taq DNA polymerase	0.5μl
ddH ₂O	Complement to 50 μ l

Amplification condition: 94 DEG C of denaturation 4min, 94 DEG C of sex change 45S, 57 DEG C of annealing 45S, 72 DEG C extend 90S, 72 DEG C of polishing end 10min, and sex change, annealing, extension three step carry out 30 circulations.Pcr amplification terminates rear agarose gel electrophoresis and detects, result shows, obtain the fragment that size is about 2000bp, purify with multifunctional dna and reclaim test kit (centrifugal column type) (Beijing hundred Tyke Bioisystech Co., Ltd) recovery, recovery fragment is subcloned into pMD-18T(TaKaRa Products) in, connect product conversion to using CaCl ₂the bacillus coli DH 5 alpha of method process, cultivating containing on the LB solid plate of penbritin (100 μ g/ml), the white colony of picking grow on plates, positive colony is verified by bacterium colony PCR, as shown in Figure 2, in figure, swimming lane 1 and swimming lane 2 are the parallel repetition of identical object fragment to the result.LB liquid nutrient medium (adding 100 μ g/ml penbritins) is linked into by being verified as positive clone, 37 DEG C of incubated overnight, plasmid is extracted with high pure plasmid Mini Kit (centrifugal column type) (Beijing hundred Tyke Bioisystech Co., Ltd), through order-checking (the prosperous bio tech ltd of Beijing AudioCodes), sequencing result display gained clip size is 1980bp, i.e. nucleotide sequence shown in SEQ ID NO.2, i.e. coding for alpha-diastatic gene.

Embodiment 2: the structure of recombinant expression vector

Increase in embodiment 1 fragment (size is 1980bp) obtained, because 5 ' end of its primers F contains respectively bam5 ' the end of HI restriction enzyme site (the 3rd base is to the 8th base) and primer R contains respectively hind III restriction enzyme site (the 4th base is to the 9th base), so use these two enzymes to carry out double digestion to fragment, the identical enzyme of pET30a is carried out double digestion, electrophoresis reclaims enzyme and cuts large fragment simultaneously, and connecting 6h with T4 ligase enzyme at 16 DEG C, the carrier schematic diagram successfully constructed is as shown in Figure 1.Connecting product chemical transformation is transformed in e. coli bl21 (DE3), cultivating containing on the LB solid plate of kantlex (100 μ g/ml), the white colony of picking grow on plates, cut (as Fig. 3) by bacterium colony PCR and the single, double enzyme of extraction plasmid and screen positive colony and qualification of checking order, successfully build the intestinal bacteria recombinant bacterial strain containing alpha-amylase gene.

Embodiment 3: the preparation of α-amylase

The recombinant bacterial strain obtained in embodiment 2 is received 100ml LB liquid nutrient medium [containing 1 ‰ (v/v) 50mg/ml kantlex] by 1% inoculum size, and 37 DEG C, 150rpm cultivates OD ₆₀₀=0.6, add the 1mM IPTG(isopropyl-beta D-thio galactopyranoside of 1 ‰ (v/v)), forward 16 DEG C to, after 80rpm induces 10h, collected by centrifugation thalline, suspends the imidazole buffer (pH8.2) of thalline with 5ml 50mmol/l, ultrasonication.Centrifuging and taking supernatant, then the imidazole buffer (pH8.2) of 5ml 50mmol/l is used to suspend, get 50 μ l respectively and carry out sds polyacrylamide gel electrophoresis (SDS-PAGE), band occurs, in precipitation after centrifugation, illustrating that the recombinant protein obtained is inclusion body (as Fig. 4).

Embodiment 4: the mensuration that the sex change renaturation of inclusion body and enzyme are lived

By the recombinant bacterial strain that obtains in the embodiment 2 method abduction delivering by embodiment 3, collect thalline, ultrasonication, removes supernatant after centrifugal, by urea soln (pH8.0) the resuspended precipitation of 5ml 8mol/l, spends the night make inclusion body sex change in 37 DEG C.Solution after sex change is encased in dialysis tubing, put into dialyzate [Tutofusin tris (Tis Base) 12.14g/l, adjust pH to 8 with hydrochloric acid, then often liter adds 200 μ l beta-mercaptoethanols] middle dialysis 24h renaturation, wherein change liquid two to three times.

The definition of enzyme activity unit: 1 ml liquid enzymes (or 1 gram of solid enzyme powder), under 40 DEG C of pH ﹦ 4.6 conditions, per minute hydrolysis Zulkovsky starch, produces the maltose of 1.0 μ g, is 1 Ge Meihuo unit, with U/g(U/ml) represent.

The mensuration that enzyme is lived: adopt DNS colorimetry.

Embodiment 5: the purifying of α-amylase

Nickel post on protein liquid after sex change in embodiment 4 again renaturation is carried out purifying, the single nickel salt in nickel post can with have His(Histidine) protein binding of label, also can be combined with imidazoles.First protein liquid is made protein hanging column with certain speed by nickel post, then carry out wash-out with the imidazole buffer of different concns, elutriant SDS-PAGE electrophoresis detection.

More than show and describe ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.

The aminoacid sequence of α-amylase of the present invention, nucleotide sequence and primer sequence are as follows.

MET Phe Glu Lys Arg Phe Lys Thr Ser Leu Leu Pro Leu Phe Ala

1 5 10 15

Gly Phe Leu Leu Leu Phe His Leu Val Leu Ser Gly Pro Ala Ala

16 20 25 30

Ala Asn Ala Glu Thr Ala Asn Lys Ser Asn Glu MET Ala Ala Ser

31 35 40 45

Ser Val Lys Asn Gly Thr Ile Leu His Ala Trp Asn Trp Ser Phe

46 50 55 60

Asn Thr Leu Thr Gln Asn MET Lys Glu Ile Arg Asp Ala Gly Tyr

61 65 70 75

Ala Ala Ile Gln Thr Ser Pro Ile Asn Gln Val Lys Glu Gly Asn

76 80 85 90

Gln Gly Asp Lys Ser MET Arg Asn Trp Tyr Trp Leu Tyr Gln Pro

91 95 100 105

Thr Ser Tyr Gln Ile Gly Asn Arg Tyr Leu Gly Thr Glu Gln Glu

106 110 115 120

Phe Lys Asp MET Cys Ala Ala Ala Glu Lys Tyr Gly Val Lys Val

121 125 130 135

Ile Val Asp Ala Val Val Asn His Thr Thr Ser Asp Tyr Gly Ala

136 140 145 150

Ile Ser Asp Glu Ile Lys Arg Ile Pro Asn Trp Thr His Gly Asn

151 155 160 165

Thr Gln Ile Lys Asn Trp Ser Asp Arg Trp Asp Ile Thr Gln Asn

166 170 175 180

Ala Leu Leu Gly Leu Tyr Asp Trp Asn Thr Gln Asn Thr Glu Val

181 185 190 195

Gln Ala Tyr Leu Lys Gly Phe Leu Glu Arg Ala Leu Asn Asp Gly

196 200 105 210

Ala Asp Gly Phe Arg Tyr Asp Ala Ala Lys His Ile Glu Leu Pro

211 215 220 225

Asp Asp Gly Asn Tyr Gly Ser Gln Phe Trp Pro Asn Ile Thr Asn

226 230 235 240

Thr Ser Ala Glu Phe Gln Tyr Gly Glu Ile Leu Gln Asp Ser Ala

241 245 250 255

Ser Arg Asp Thr Ala Tyr Ala Asn Tyr MET Asn Val Thr Ala Ser

256 260 265 270

Asn Tyr Gly His Ser Ile Arg Ser Ala Leu Lys Asn Arg Asn Leu

271 275 280 285

Ser Val Ser Asn Ile Ser His Tyr Ala Ser Asp Val Ser Ala Asp

286 290 295 300

Lys Leu Val Thr Trp Val Glu Ser His Asp Thr Tyr Ala Asn Asp

301 305 310 315

Asp Glu Glu Ser Thr Trp MET Ser Asp Asp Asp Ile Arg Leu Gly

316 320 325 330

Trp Ala Val Ile Gly Ser Arg Ser Gly Ser Thr Pro Leu Phe Phe

331 335 340 345

Ser Arg Pro Glu Gly Gly Gly Asn Gly Val Arg Phe Pro Gly Lys

346 350 355 360

Ser Gln Ile Gly Asp Arg Gly Ser Ala Leu Phe Lys Asp Gln Ala

361 365 370 375

Ile Thr Ala Val Asn Gln Phe His Asn Glu MET Ala Gly Gln Pro

376 380 385 390

Glu Glu Leu Ser Asn Pro Asn Gly Asn Asn Gln Ile Phe MET Asn

391 395 400 405

Gln Arg Gly Ser Lys Gly Val Val Leu Ala Asn Ala Gly Ser Ser

406 410 415 420

Ser Val Thr Val Asn Thr Ser Thr Lys Leu Pro Asp Gly Arg Tyr

421 425 430 435

Asp Asn Arg Ala Gly Ala Gly Ser Phe Gln Val Ala Asn Gly Lys

436 440 445 450

Leu Thr Gly Thr Ile Asn Ala Arg Ser Ala Ala Val Leu Tyr Pro

451 455 460 465

Asp Asp Ile Gly Asn Ala Pro His Val Phe Leu Glu Asn Tyr Gln

466 470 475 480

Thr Gly Ala Val His Ser Phe Asn Asp Gln Leu Thr Val Thr Leu

481 485 490 495

Arg Ala Asn Ala Lys Thr Thr Lys Ala Val Tyr Gln Ile Asn Asn

496 500 505 510

Glu Gln Gln Thr Ala Phe Lys Asp Gly Asp Arg Leu Thr Ile Gly

511 515 520 525

Lys Glu Asp Pro Ile Gly Thr Thr Tyr Asn Ile Lys Leu Thr Gly

526 530 535 540

Thr Asn Gly Glu Gly Thr Ala Arg Thr Gln Glu Tyr Thr Phe Val

541 545 550 555

Lys Lys Asp Pro Ser Gln Thr Asn Ile Ile Gly Tyr Gln Asn Pro

556 560 565 570

Asp His Trp Ser Gln Val Asn Ala Tyr Ile Tyr Lys His Asp Gly

571 575 580 585

Gly Arg Ala Ile Glu Leu Thr Gly Ser Trp Pro Gly Lys Ala MET

586 590 595 600

Thr Lys Asn Ala Asp Gly MET Tyr Thr Leu Thr Leu Pro Glu Asn

601 605 610 615

Ala Asp Thr Ala Asn Ala Lys Val Ile Phe Asn Asn Gly Ser Ala

616 620 625 630

Gln Val Pro Gly Gln Asn Gln Pro Gly Phe Asp Tyr Val Gln Asn

631 635 640 645

Gly Leu Tyr Asn Asn Ser Gly Leu Asn Gly Tyr Leu Pro His

646 650 655 659

SEQ ID NO.2

atgtttgaaa aacgattcaa aacctcttta ctgccgttat tcgccggatt tttattgctg 60

tttcatttgg ttttgtcagg cccggcggct gcaaacgctg aaactgcaaa caaatcgaat 120

gagatggccg cgtcatcggt caaaaacggg accatccttc atgcatggaa ttggtcattc 180

aatacgttaa cacaaaatat gaaagagatt cgtgatgcgg gttatgcagc cattcagacg 240

tctccgatta accaagtaaa ggaagggaac caaggagata aaagcatgag gaactggtac 300

tggctgtatc agccgacatc gtaccaaatc ggcaaccgtt acttaggcac tgaacaagaa 360

tttaaggaca tgtgtgcagc cgcggaaaaa tatggcgtaa aagtcattgt cgatgcggtt 420

gtcaatcata ccaccagcga ttatggcgcg atttctgatg agattaagcg tattccaaac 480

tggacccacg gaaacacaca aattaaaaat tggtcggacc gatgggacat cactcaaaat 540

gcattgcttg ggctgtatga ttggaatact cagaatactg aggtgcaagc ctacctgaaa 600

ggtttcttgg aaagagcatt gaatgacgga gcagacgggt tccgctatga tgccgccaag 660

catatagagc ttccggatga tgggaattac ggcagccaat tttggccgaa tatcacaaat 720

acatcggcgg agttccaata cggagaaatc ctgcaagaca gcgcgtccag agatactgct 780

tatgcgaatt atatgaatgt gacggcttct aactacgggc attccatcag atccgcttta 840

aagaatcgta atctgagtgt gtcgaatatc tcccattatg catctgacgt gtctgcggac 900

aagttagtca catgggtgga atcacatgat acgtatgcca atgatgatga agagtccaca 960

tggatgagtg atgacgatat ccgtttaggc tgggcagtga ttggttcccg ctcaggaagc 1020

acgcctcttt tcttttccag acctgagggc ggaggaaatg gtgtcaggtt ccctggaaaa 1080

agtcaaatag gcgatcgcgg gagtgcctta tttaaagatc aggcgatcac tgcggtcaat 1140

caatttcaca atgaaatggc cgggcagcct gaggaactct caaatccgaa tggcaacaat 1200

caaatattta tgaatcagcg cggctcaaaa ggcgttgtgc tggcaaatgc aggatcgtct 1260

tctgtcaccg tcaatacttc aacgaaatta cctgacggca ggtatgataa tagggccggc 1320

gccggttcat ttcaagtagc gaacggcaaa ctgacaggta cgatcaatgc cagatccgcg 1380

gctgttcttt atcctgacga cattggaaat gcgcctcatg tcttccttga gaattaccaa 1440

acgggggcag tccattcttt caatgatcag ctgacggtta ccctgcgtgc aaatgcgaaa 1500

acaacaaaag ccgtttacca aatcaataat gagcagcaga cagcatttaa ggatggagac 1560

cgattaacga tcgggaaaga agatccaatc ggcacgacat acaacatcaa attaaccgga 1620

acgaacggcg agggtacagc gagaacccaa gaatacacgt ttgtcaaaaa agacccgtcc 1680

caaaccaaca tcattggcta tcaaaatccg gatcattgga gccaggtaaa tgcttatatc 1740

tataaacatg atggaggcag ggccatagaa ttaaccggat cgtggccggg gaaagccatg 1800

actaagaatg cagatggaat gtacacgctg acgctgcctg agaatgcgga tacggccaac 1860

gccaaagtga tttttaacaa tggcagcgcc caagtgcccg gccagaacca gcccggcttt 1920

gattatgtgc agaatggttt gtataacaac tccggtttga atggttatct tccgcattaa 1980

SEQ ID NO.3

CGGGATCCAT GTTTGAAAAA CGATTCAAAA C 31

SEQ ID NO.4

CCCAAGCTTA TGCGGAAGAT AACCA TTCAA 30

Claims

1. a α-amylase, is characterized in that, described α-amylase is for being made up of aminoacid sequence shown in SEQ ID NO.1.

2. the gene of α-amylase described in claim 1 of encoding.

3. gene according to claim 2, is characterized in that, the encoding sequence of described gene is the nucleotide sequence shown in SEQ ID NO.2.

4. the recombinant vectors containing the gene shown in Claims 2 or 3.

5. one kind transforms the recombination engineering bacteria obtained with recombinant vectors described in claim 4.

6. the application of the gene described in Claims 2 or 3 in preparation recombinant alpha-amylases.