CN109182304B

CN109182304B - Alpha-amylase gene and application thereof

Info

Publication number: CN109182304B
Application number: CN201811089417.4A
Authority: CN
Inventors: 吴丽君; 白晓莉; 王毅; 朱杰; 杨德中; 段如敏; 孙万万; 魏云林; 季秀玲
Original assignee: China Tobacco Yunnan Industrial Co Ltd
Current assignee: China Tobacco Yunnan Industrial Co Ltd
Priority date: 2018-09-18
Filing date: 2018-09-18
Publication date: 2021-05-25
Anticipated expiration: 2038-09-18
Also published as: CN109182304A

Abstract

The invention discloses an alpha-amylase gene, which is derived from bacillusBacillus sp.48-1, recombinant vector containing the same, genetic engineering bacteria and recombinant amylase preparation produced by the same. The amino acid sequence of the amylase with the activity of the endo alpha-l, 4-glycosidic bond is shown as SEQ ID NO.1, and the coding sequence of the gene is the nucleotide sequence shown as SEQ ID NO. 2. Through constructing a recombinant vector and expressing in escherichia coli, an expression product has the function of an inscribed alpha-l, 4-glycosidic bond, and the amylase described by the invention plays an important role in promoting tobacco mellowing and the like.

Description

Alpha-amylase gene and application thereof

Technical Field

The invention relates to an amylase, in particular to an alpha-amylase, a gene for coding the amylase, a recombinant vector containing the coding gene, a construction method of the recombinant vector, a genetically engineered bacterium and application of the genetically engineered bacterium, and belongs to the field of biotechnology and enzyme engineering.

Background

The starch is prepared by polymerizing glucose molecules, and can be divided into amylose and amylopectin, and has a general formula of (C6H10O5) n. The amylose is an unbranched helical structure, the amylopectin is formed by connecting 24-30 glucose residues end to end through alpha-1, 4-glycosidic bonds, and the alpha-1, 6-glycosidic bonds are at the branched chain. Starch is a nutrient stored in plants, is stored in seeds and tubers, and has high starch content in various plants. Besides eating, starch can also be used as raw materials for preparing dextrin, maltose, glucose and alcohol, and can also be used for preparing printing paste, textile sizing, paper sizing, medicine tablet pressing and the like, and meanwhile, the starch content in tobacco leaves also has great influence on the quality of tobacco leaves.

Amylases are a generic term for enzymes that hydrolyze starch and glycogen, and are classified as alpha-amylases and beta-amylases, depending on the type of isomerization of the enzymatic hydrolysate. Alpha-amylase is present in plants, animals, microorganisms, and is the starting enzyme for starch hydrolysis, and the amylase of microorganisms is generally secreted; alpha-amylase acts on both amylose and amylopectin to randomly cleave alpha-1, 4-chains within sugar chains indiscriminately, and the final product mainly contains glucose when amylose is decomposed, and in addition, a small amount of maltotriose and maltose; when amylopectin is decomposed, alpha-dextrin is generated in addition to maltose, glucose and maltotriose; beta-amylase is found mainly in higher plants, but is found in bacteria, cow's milk and mold, and acts on alpha-1, 4 glycosidic bonds as well as alpha-amylase, but its action starts from a non-reducing end, so that it does not completely hydrolyze amylopectin, and when it acts on alpha-l, 4 glycosidic bonds, limit dextrins are formed, requiring further action of limit piceidase. The limiting dextrinase plays a specific role in alpha-l, 6 glycosidic bonds, but the limiting dextrinase cannot directly degrade starch granules and only plays a role after the alpha-amylase acts.

At present, a few reports of the alpha-amylase for promoting the alcoholization of tobacco exist, and although reports also show that the amylase gene is cloned from bacillus, the obtained alpha-amylase has insufficient purity, poor enzyme activity and instability due to the reasons of strain selection, expression plasmid selection, plasmid construction method and the like.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides alpha-amylase, a gene for coding the alpha-amylase, a recombinant vector containing the coding gene, a construction method of the recombinant vector, a genetically engineered bacterium and application of the genetically engineered bacterium.

The technical scheme adopted by the invention is as follows:

the invention aims to provide an amylase gene separated from Bacillus sp.48-1 (the strain preservation number is CGMCC No.5311), the nucleotide sequence or the fragment of the nucleotide sequence is shown as SEQ ID No.2, or the nucleotide sequence which is complementary with the SEQ ID No.2, and the length of the gene sequence is 1977 bp.

The nucleotide sequences of the present invention are in the form of DNA, including cDNA, genomic DNA, or synthetic DNA, and may be single-stranded or double-stranded. Due to the specificity of the nucleotide sequence, any polynucleotide variant with more than 80% homology and the same function with the nucleotide sequence shown in SEQ ID NO.2 is within the protection scope of the invention. The polynucleotide variant refers to a polynucleotide sequence having one or more nucleotide changes, which may be obtained using substitution, deletion or insertion variations well known in the art.

The amino acid sequence encoded by the amylase gene of the present invention is a polypeptide or protein having the amino acid residue sequence shown in SEQ ID NO. 1. Due to the particularity of the amino acid sequence, any polypeptide fragment containing the amino acid sequence shown in SEQ ID NO.1 or its variant, such as conservative variant, bioactive fragment or derivative thereof, has more than 90% homology with the amino acid residue sequence shown in SEQ ID NO.1 and has the same activity, and is within the protection scope of the invention. These methods include deletion, insertion, chemical modification or substitution of amino acid residues in the amino acid sequence, which may be a recombinant protein, a natural protein or a synthetic protein.

Another objective of the invention is to provide a recombinant expression vector containing amylase gene, which is constructed by directly connecting the gene shown in SEQ ID NO.2 with different expression vectors.

In the present invention, the polynucleotide sequence encoding the amylase may be inserted into a vector to constitute a vector containing the recombinant vector of the present invention. The vector refers to a plasmid, virus or other vector well known in the art, and pET28a is selected for use in the present invention. Methods well known to those skilled in the art can be used to construct expression vectors containing amylase-encoding nucleotide sequences and appropriate transcription/translation regulatory elements. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The amylase-encoding nucleotide sequence may be operably linked to an appropriate promoter in an expression vector to direct the synthesis of mRNA. Representative examples of such promoters are: lac or trp promoter of E.coli; the PL promoter of lambda phage; eukaryotic promoters include CMV early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retrovirus, and other known promoters capable of controlling the expression of genes in prokaryotic or eukaryotic cells or viruses. The expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. The insertion of enhancer sequences into vectors will enhance transcription in higher eukaryotic cells. Enhancers are cis-acting elements of DNA expression, usually about 10-300bp, that act on a promoter to enhance gene transcription, such as adenovirus enhancers.

According to the invention, Nde I and BamH I are used as enzyme cutting sites at two ends of a gene shown in SEQ ID NO.2, and when the alpha-amylase is expressed by induction, histidine (His) tags are connected to two ends of the alpha-amylase, so that the purification of target protein is facilitated.

In the present invention, the polynucleotide encoding the amylase or the recombinant vector containing the polynucleotide may be transformed or introduced into a host cell, which may be a bacterial cell, a fungal cell, a plant cell or an animal cell, or a progeny of such host cells, by methods well known to those skilled in the art, and representative examples are: e.coli; fungal cells or yeast; plant cells such as rape, tobacco, soybean; insect cells such as Drosophila S2 or Sf 9; animal cells such as CHO, COS or Bowes melanoma cells.

The invention also relates to the application of the coding gene in recombinant gene engineering bacteria expression recombinant alpha-amylase, and the recombinant amylase can be constructed and expressed or produced by utilizing the polynucleotide sequence of the invention through the conventional recombinant DNA technology. This can generally be achieved by: constructing recombinant vector, transforming into host cell to construct recombinant cell, culturing host cell in proper culture medium, and analyzing and purifying protein from the culture medium or cell.

Compared with the prior art, the invention has the beneficial effects that:

(1) the amylase gene described by the invention is cloned from bacillus separated from tobacco leaves, and can directly act on the tobacco leaf alcoholization process without worrying about the influence of biological factors on the tobacco leaf quality.

(2) The pET28a is selected as an expression plasmid, the plasmid background is clear, lactose operon can be used for induction expression, the expression quantity and the expression time of amylase are convenient to control, NdeI and BamHI are selected as enzyme cutting sites at two ends of a gene shown in SEQ ID NO.2 when the plasmid is constructed, and histidine (His) tags are connected at two ends of the alpha-amylase when the alpha-amylase is induced and expressed, so that the specific combination of the alpha-amylase and a nickel column is easier and the purification is convenient compared with the prior art that the histidine (His) tag is added at the C-terminal of the amylase only.

(3) The invention adopts an escherichia coli expression system, can be used for quickly expressing amylase in large quantity, the recombinant amylase produced by the recombinant technology can obtain the alpha-amylase with the purity of more than 95 percent only by two simple purification steps, and the specific enzyme activity of the fermentation liquor unit can exceed 10³The enzyme activity of IU is nearly 100 times higher than that of the original strain fermentation product, the enzyme activity is stable, 80% of activity is kept at pH5.0-pH8.0 and the reaction temperature is 30-45 ℃, and the enzyme activity is higher than that of the alpha-amylase expressed by the existing recombinant engineering bacteria.

(4) The invention has the advantages of simple culture medium and culture conditions, simple process, short period and greatly reduced production cost. The amylase gene is derived from the microbial genome on the surface of the flue-cured tobacco, can act on the alcoholization process of the flue-cured tobacco, further changes the purification process of the flue-cured tobacco, is favorable for improving the quality of the tobacco and has better safety.

Drawings

FIG. 1 shows the E.coli BL21(DE3) expression vector Amy48-pET-28a constructed in the present invention;

FIG. 2 is a colony PCR verification diagram of the target fragment of the recombinant plasmid of the present invention, in which M is marker of 2000bp in size;

lanes

1, 2, 3, 4, 5 are amplification products of the target fragment.

FIG. 3 shows single and double restriction maps of the recombinant plasmid of the present invention, wherein: m is maker, lane 1 is recombinant plasmid, lane 2 is double digested plasmid, lane 3 is Nde I single digested, lane 4 is BamH I single digested.

FIG. 4 is a schematic diagram of the purification effect of SDS-PAGE according to the present invention, in which M is Marker, and lane 1 is a cell disruption solution; lane 2 is 20% to 60% ammonium sulfate precipitation; lane 3 is ammonium sulfate precipitation after dialysis; lane 4 is the dialysate passed through a nickel column; lane 5 is purified amylase.

FIG. 5 is the effect of pH on recombinant alpha-amylase activity and stability;

FIG. 6 is a graph of the effect of temperature on recombinant α -amylase activity and stability.

Detailed Description

The present invention will be described in further detail with reference to examples.

It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available by purchase.

Example 1: the nucleotide sequence of amylase gene separated from Bacillus subtilis 48-1.

Extracting genome DNA of bacillus subtilis by a CTAB method, taking 2 mu l as a template to perform Polymerase Chain Reaction (PCR), and designing primers (a primer F and a primer R) by combining the used carriers according to a published conserved region of an amylase homologous sequence, wherein the primers, components and amplification conditions used in the PCR reaction process are as follows:

F:5’-GGAATTCCATATGGTGAGAAGCAAAAAA-3’(SEQ ID NO.3)

R:5’-CGGGATCCTTATTGTGCAGCTGCTTGTA-3’(SEQ ID NO.4)

the PCR amplification system composition is shown in Table 1.

TABLE 1 reaction parameters of PCR

10×Taq Buffer	5μl
		dNTP(2.5mmol/L)	4μl
Stencil (genome DNA)	2μl
		Primer F	2μl
Primer R	2μl
		Taq DNA polymerase	0.5μl
ddH₂O	Make up to 50. mu.l

Amplification conditions: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 45S, annealing at 57 ℃ for 45S, extension at 72 ℃ for 90S, and tail end completion at 72 ℃ for 10min, wherein 30 cycles of denaturation, annealing and extension are carried out. PCR amplification junctionAfter the completion of the ligation, the fragment of about 2000bp was obtained by agarose gel electrophoresis, and the fragment was recovered by a multifunctional DNA purification recovery kit (centrifugal column type) (Bekkacter Biotech Co., Ltd., Beijing), and the recovered fragment was subcloned into pMD-18T (product of TaKaRa), and the ligation product was transformed into a plasmid using CaCl₂The treated Escherichia coli DH5 alpha is cultured on LB solid plate containing ampicillin (100 mug/ml), white colony growing on the plate is picked, positive clone is verified through colony PCR, the clone verified to be positive is inoculated into LB liquid medium (added with 100 mug/ml ampicillin), overnight culture is carried out at 37 ℃, plasmid is extracted by a high-purity plasmid small-quantity extraction kit (centrifugal column type) (Beijing Baitaike biotechnology limited), and the size of the obtained fragment is 1977bp as shown by a sequencing result (Beijing Okoku Sheng Biotechnology limited).

Example 2: construction of recombinant expression vectors

The fragment amplified in example 1 contains Nde I cleavage site (8 th to 13 th bases) at 5 'end of primer F and BamH I cleavage site (3 rd to 8 th bases) at 5' end of primer R, so that the fragment is double-cleaved with the same enzyme, pET28a is double-cleaved with the same enzyme, the large fragment is recovered by electrophoresis, and ligated with T4 ligase at 16 ℃ for 6h, and the schematic diagram of the successfully constructed vector is shown in FIG. 1. The connecting product is transformed into Escherichia coli BL21(DE3) by a chemical transformation method, cultured on an LB solid plate containing kanamycin (100 mu g/ml), white colonies growing on the plate are picked, positive clones are screened by colony PCR (figure 2) and single and double enzyme digestion (figure 3) of extracted plasmid, sequencing and identification are carried out, and the Escherichia coli recombinant engineering bacteria containing the alpha-amylase gene are successfully constructed.

Example 3: preparation of Amylase

The recombinant engineered bacteria obtained in example 2 were inoculated at 1% inoculum size to 100ml LB liquid medium (containing 1 ‰ 50mg/ml kanamycin), cultured at 37 ℃ at 150rpm to OD600 ═ 0.6, 1 ‰ 1mM IPTG (isopropyl-. beta. -D-thiogalactopyranoside) was added, transferred to 16 ℃, induced at 80rpm for 10 hours, centrifuged to collect cells, suspended in 5ml of 50mmol/l imidazole buffer (pH8.2), and disrupted by sonication. The supernatant and the pellet were collected by centrifugation, and the pellet was suspended in 5ml of 50mmol/l imidazole buffer (pH8.2), and 50. mu.l of the suspension was subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE), and bands appeared in the pellet, indicating that the resulting recombinant protein was inclusion bodies, as shown in lane 3 of FIG. 4. Example 4: denaturation and renaturation of inclusion body and determination of enzyme activity

The engineered bacterium of example 2 was expressed by induction as described in example 3, and the cells were collected and resuspended in 40mL Lysis attenuation Buffer (LE Buffer:50mM NaH2PO4,300mM NaCl, pH 8.0); the cells were disrupted using a sonicator (SONICS Autotune) under ice bath conditions: the power is 70%, the operation is performed for 5s and the operation is stopped for 5s, the crushing is performed until the liquid becomes clear, the use time is about 30min, 1200g is centrifuged for 10min after the crushing is completed, then a 0.45 mu m filter membrane is used for ultrafiltration to remove cell debris, the supernatant is reserved, and the volume is measured; adding an isovolumetric 20% thiamine solution while stirring to precipitate the protein, and stirring the solution on a magnetic stirrer for 2-3 hours; centrifuging at 11000g for 15min (4 deg.C), and retaining supernatant; adding 40% thiamine solution into the supernatant, centrifuging again to obtain supernatant, adding 60% thiamine solution into the supernatant, centrifuging again to obtain supernatant, adding 80% thiamine solution into the supernatant, centrifuging again to obtain precipitate, respectively measuring enzyme activity of the precipitate and the supernatant after adding thiamine solution each time, and measuring the enzyme activity by using a DNS colorimetric method, wherein the enzyme activity can reach 3.5 × 10 at most⁴U/mL, and the enzyme activity is kept stable between pH5.0 and pH8.0, and the temperature is 30-45 ℃, and the enzyme activity can keep more than 80 percent of the activity, as shown in figure 5-6.

Definition of enzyme activity unit: the amount of enzyme that released 1mg of maltose per minute from a 2% soluble starch solution at 60 ℃ and pH5.6 was defined as 1 enzyme activity unit (U) in terms of U/g or (U/ml).

Example 5: purification of amylases

The denatured and renatured protein solution in example 4 was purified on a nickel column, and nickel sulfate in the nickel column was bound to a protein having a His (histidine) tag or imidazole. The protein solution was first passed through a nickel column at a constant rate to allow the protein to be suspended, then the eluate was eluted with imidazole buffers of different concentrations, and the eluate was detected by SDS-PAGE, and as shown in lane 5 of FIG. 4, only a single protein band with a molecular weight of 72kD was present.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

<110> tobacco industry Limited liability company in Yunnan

<120> alpha-amylase gene and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

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<213> Bacillus sp.48-1

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Met Phe Ala Lys Arg Phe Lys Thr Ser Leu Leu Pro Leu Phe Ala Gly

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Phe Leu Leu Leu Phe His Leu Val Leu Ala Gly Pro Ala Ala Ala Asn

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Ala Glu Thr Ala Asn Lys Ser Asn Glu Leu Thr Ala Pro Ser Ile Lys

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

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His Asn Met Lys Asp Ile His Asp Ala Gly Tyr Thr Ala Ile Gln Thr

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Ser Pro Ile Asn Gln Val Lys Glu Gly Asn Gln Gly Asp Lys Ser Met

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Ser Asn Trp Tyr Trp Leu Tyr Gln Pro Thr Ser Tyr Gln Ile Gly Asn

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Arg Tyr Leu Gly Thr Glu Gln Glu Phe Lys Glu Met Cys Ala Ala Ala

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Glu Glu Tyr Gly Ile Lys Val Ile Val Asp Ala Val Ile Asn His Thr

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Thr Ser Asp Tyr Ala Ala Ile Ser Asn Glu Ile Lys Ser Ile Pro Asn

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

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Val Thr Gln Asn Ser Leu Leu Gly Leu Tyr Asp Trp Asn Thr Gln Asn

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Thr Gln Val Gln Ser Tyr Leu Lys Arg Phe Leu Glu Arg Ala Leu Asn

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

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

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

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

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

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Asn Ile Ser His Tyr Ala Ser Asp Val Pro Ala Asp Lys Leu Val Thr

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

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Trp Met Ser Asp Asp Asp Ile Arg Leu Gly Trp Ala Val Ile Ala Ser

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Arg Ser Gly Ser Thr Pro Leu Phe Phe Ser Arg Pro Glu Gly Gly Gly

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Asn Gly Val Arg Phe Pro Gly Lys Ser Gln Ile Gly Asp Arg Gly Ser

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Ala Leu Phe Glu Asp Gln Ala Ile Thr Ala Val Asn Arg Phe His Asn

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Val Met Ala Gly Gln Pro Glu Glu Leu Ser Asn Pro Asn Gly Asn Asn

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Gln Ile Phe Met Asn Gln Arg Gly Ser His Gly Val Val Leu Ala Asn

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

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

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

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Pro Asp Asp Ile Ala Lys Ala Pro His Ala Phe Leu Glu Asn Tyr Lys

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Thr Gly Val Thr His Ser Phe Asn Asp Gln Leu Thr Ile Thr Leu Arg

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Ala Asp Ala Asn Thr Thr Lys Ala Val Tyr Gln Ile Asn Asn Gly Pro

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

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Pro Phe Gly Lys Thr Tyr Thr Ile Met Leu Lys Gly Thr Asn Ser Asp

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Gly Val Thr Arg Thr Glu Glu Tyr Ser Phe Val Lys Arg Asp Pro Ala

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Asn Ala Tyr Ile Tyr Lys His Asp Gly Gly Gly Ala Ile Glu Leu Thr

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Gly Ser Trp Pro Gly Lys Pro Met Thr Lys Asn Ala Asp Gly Ile Tyr

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Phe Asn Asn Gly Ser Ala Gln Val Pro Gly Gln Asn Gln Pro Gly Phe

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aatacgttaa aacacaatat gaaggatatt catgatgcag gatatacagc gattcagacg 240

tctccgatta accaagtaaa ggaagggaat caaggagata aaagcatgtc aaactggtac 300

tggctctatc agccgacatc gtaccaaatt ggcaaccgtt acttaggtac tgaacaagaa 360

tttaaagaaa tgtgtgcagc cgctgaagaa tatggcataa aggtcattgt tgacgctgtc 420

atcaatcata ccaccagtga ctatgccgcg atttccaatg agattaagag tattccaaac 480

tggacacatg gaaacacaca aattaaaaac tggtcggatc gatgggatgt cacgcagaat 540

tcattgctcg ggctctatga ctggaataca caaaatacac aagtacagtc ctatctgaaa 600

cggttcttag aaagagcatt gaatgacggg gcagacggtt ttcgctttga tgccgccaaa 660

catatagagc ttccggatga tgggagttac ggcagtcaat tttggccgaa tatcacaaat 720

acatctgcag agttccaata cggagaaatc ctgcaggata gtgcctccag agatgcttca 780

tatgcgaatt atatgaatgt gacagcgtct aactatgggc attccataag gtccgcttta 840

aagaatcgca atctgggcgt gtcgaatatc tcccactatg catctgatgt gcctgcggac 900

aagctagtga catgggtaga gtcgcatgat acgtatgcca atgatgatga agagtcgaca 960

tggatgagcg atgatgatat ccgtttaggc tgggcggtga tagcttctcg ttcaggcagt 1020

acgcctcttt tcttttccag acctgaggga ggcggaaatg gtgtgagatt cccggggaaa 1080

agccaaatag gcgatcgcgg gagtgcttta tttgaagatc aggctatcac tgcggtcaat 1140

agatttcaca atgtgatggc tggacagcct gaggaactct cgaacccaaa tggaaacaac 1200

cagatattta tgaatcagcg cggctcacat ggcgttgtgc tggcaaatgc aggttcatcc 1260

tctgtttcta tcaatacgcc aacaaaattg cctgatggca ggtatgacaa taaagctggg 1320

gcaggttcat ttcaagtgaa tgatggtaaa ctgacaggca cgatcaatgc cagatctgta 1380

gctgtgcttt atcctgatga tattgcaaaa gcgcctcatg ctttccttga gaattacaaa 1440

acaggtgtaa cacattcttt caatgatcaa ctgacgatta ccttgcgtgc agatgcgaat 1500

acaacaaaag ccgtttatca aatcaataat ggaccagaga cagcgtttaa ggatggagat 1560

caattcacaa tcggaaaagg agatccattt ggcaaaacat acaccatcat gttaaaagga 1620

acgaacagtg atggtgtaac gaggaccgag gaatacagct ttgttaaaag agatccagct 1680

tcggccaaaa ccatcggcta tcaaaatccg aatcattgga gccaggtaaa tgcttatatc 1740

tataaacatg atgggggcgg ggcaattgaa ttgaccggat cttggcctgg aaaaccaatg 1800

actaaaaatg cagacggaat ttacacgctg acgctgcctg cggacacgga tacaaccaac 1860

gcaaaagtga tttttaataa tggcagcgcc caagtgcccg gtcagaatca gcctggcttt 1920

gattacgtgc taaatggttt atataatgac tcgggcttaa gcggttctct tcctcat 1977

<210> 3

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<213> Artificial sequence ()

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ggaattccat atggtgagaa gcaaaaaa 28

<210> 4

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<212> DNA

<213> Artificial sequence ()

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cgggatcctt attgtgcagc tgcttgta 28

Claims

1. An alpha-amylase, wherein said amylase gene is derived from a BacillusBacillus sp.48-1, and the amylase consists of an amino acid sequence shown in SEQ ID NO. 1.

2. A gene encoding the α -amylase of claim 1.

3. The gene of claim 2, wherein the coding sequence of the gene is the nucleotide sequence shown in SEQ ID No. 2.

4. A recombinant vector comprising the gene according to claim 2 or 3.

5. A recombinant genetically engineered bacterium transformed with the recombinant vector of claim 4.

6. The use of the recombinant genetically engineered bacterium of claim 5 in the preparation of recombinant alpha-amylase.

7. The method for constructing a recombinant vector according to claim 4, wherein: pET28a was selected as the expression plasmid.

8. The method for constructing a recombinant vector according to claim 7, wherein: when the alpha-amylase is induced and expressed, histidine tags are connected with two ends of the alpha-amylase.