CN114181958B

CN114181958B - Alpha-amylase gene amy1, protein coded by same and application thereof

Info

Publication number: CN114181958B
Application number: CN202110775430.0A
Authority: CN
Inventors: 沈其荣; 刘东阳; 刘志颖; 马磊; 孔志坚
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2021-07-08
Filing date: 2021-07-08
Publication date: 2023-06-09
Anticipated expiration: 2041-07-08
Also published as: CN114181958A

Abstract

The invention discloses an alpha-amylase gene amy1, a protein coded by the alpha-amylase gene amy1 and application of the alpha-amylase gene amy1. Firstly, total RNA is extracted by taking compost samples as raw materials, and then alpha-amylase genes are obtained by reverse transcriptionamy1, a cDNA; using cDNA as template, and PCR amplification methodamy1a complete sequence of the gene; then adopting double enzyme digestion and enzyme linking method to make the wholeamy1 gene is inserted into pPICZ alpha A expression vector to obtain recombinant expression plasmid pPICZ alpha A-amy1, a step of; then the recombinant expression plasmid pPICZalpha A-amy1, transforming into pichia pastoris competence, and screening high-efficiency engineering bacterium transformants; finally, after the engineering bacteria are subjected to induced culture by a liquid fermentation culture medium, the alpha-amylase AMY1 is secreted. Analysis of the enzymatic results showed that: at 40 ℃, the content of reducing sugar produced by the AMY1 hydrolysis of the soluble starch per minute is 0.55 mug/mL, and at the pH of 6-8, the highest content of reducing sugar produced by the AMY1 hydrolysis of the soluble starch per minute is 0.78 mug/mL. The invention provides theoretical basis and technical guarantee for the resource utilization of agricultural wastes and the development of ecological agriculture.

Description

Alpha-amylase gene amy1, protein coded by same and application thereof

Technical Field

The invention belongs to the field of enzymology engineering, and particularly relates to an alpha-amylase gene amy1, a protein coded by the alpha-amylase gene amy1 and application of the alpha-amylase gene amy1.

Background

Environmental and resource problems are one of the most important and serious crisis faced by humans in today's society. The solid waste produced in the agricultural production process mainly comprises various straws and livestock manure produced by large-scale cultivation. The kinds of various agricultural wastes are complex, and if the agricultural wastes are directly piled on the soil without treatment, the stability and productivity of the soil are adversely affected. Various household garbage produced in rural areas can lead to the entry of a large number of harmful substances and pathogens into the soil if not properly and effectively treated. The growth of crops can absorb various harmful substances through root systems, so that the spread of various diseases is easily caused, and even the human health is seriously endangered because of biological amplification. With the continuous development of modern technology, the past agricultural solid waste technology cannot adapt to the actual treatment of the solid waste at the present stage. In the long term, the enzymolysis method has the advantages of low energy consumption, mild reaction condition, environmental friendliness and the like, so that the enzymolysis method gradually becomes the focus of attention of researchers in various countries. Starch is one of the main components in agricultural solid waste.

The alpha-amylase is a product widely applied in enzyme preparations, has early application history and high yield, accounts for about 25% of the market of the enzyme preparations, and is mainly applied to the aspects of textile desizing, beer and alcohol production, starch processing, food processing, fermentation industry, medicine industry, petroleum exploitation industry and the like. Alpha-amylase takes glycogen or starch as a substrate, and cuts alpha-1, 4 glucosidic bonds from the inside of molecules, so that the substrate is hydrolyzed into dextrin and a small amount of glucose and maltose. Most of the alpha-amylase strains used in domestic industrial production at present are mutant strains obtained by mutagenesis of wild strains. The wild-type strain of alpha-amylase, which is usually isolated directly from nature, has a low enzyme production capacity and cannot meet the needs of industrial production, so how to obtain a strain with a higher enzyme production capacity has become a key to the current solution of the amylase preparation industry.

To solve these problems, some researchers have attempted to design Trichoderma strains using genetic engineering techniques to increase the proportion of the required enzyme content, and to manually interfere with control to achieve the desired effect. With the continuous application and development of genetic engineering technology, exogenous expression of various enzymes is emphasized. The recombinant protein expression system mainly comprises: coli, yeast, insect cells, mammalian cells, transgenic plant or animal expression systems and in vitro translation systems. However, yeast expression systems are becoming more and more important and utilized because of the characteristics of fast growth, easy culture, simple genetic manipulation, and capability of processing, modifying and folding expression proteins. Among them, the Pichia pastoris expression system is a relatively perfect methanol nutritional type yeast expression system which has been developed for several years and has the most widely application.

The patent realizes the high-efficiency expression of the alpha-amylase gene amy1 by constructing recombinant pichia pastoris, has important significance for improving the comprehensive utilization of agricultural solid wastes, and provides a theoretical basis for actual production.

The invention comprises the following steps:

in view of the shortcomings of the prior art, it is an object of the present invention to provide an alpha-amylase gene amy1; it is an object of the present invention to provide a protein encoded by the above gene;

it is an object of the present invention to provide a recombinant expression vector comprising the above-mentioned alpha-amylase gene amy1;

the 4 th object of the present invention is to provide a genetically engineered bacterium containing the above-mentioned alpha-amylase gene amy1;

the 5 th object of the invention is to provide the application of the alpha-amylase gene amy1, protein, recombinant expression vector and genetic engineering bacteria. The invention realizes the expression of the alpha-amylase gene in pichia pastoris, constructs excellent genetic engineering bacteria, further promotes the industrialization of the results, and creates good social and economic benefits.

The aim of the invention can be achieved by the following technical scheme:

an alpha-amylase gene amy1, the nucleotide sequence of which is shown in SEQ ID NO. 1.

The invention also protects the protein encoded by the alpha-amylase gene AMY1 described above, namely alpha-amylase AMY1.

Preferably, the amino acid sequence of the alpha-amylase AMY1 is shown as SEQ ID NO. 2.

The optimal hydrolysis temperature of the alpha-amylase AMY1 of the invention is 40 ℃ but has 60-80% of enzyme activity at 30-50 ℃ through test; the optimal pH for the alpha-amylase AMY1 is 7, but 90% of the enzyme activity can be maintained at pH 6-8.

The invention also protects a recombinant expression vector comprising the alpha-amylase gene amy1 as described above.

Preferably, the recombinant expression vector is a recombinant plasmid pPICZalpha A-amy1.

Preferably, the coding region sequence of the alpha-amylase gene amy1 is obtained by first amplifying, and then the recombinant plasmid pPICZ alpha A-amy1 is constructed by using a double enzyme digestion and enzyme ligation method.

Specifically, total RNA is extracted by taking a straw compost sample as a raw material, and cDNA of an alpha-amylase gene amy1 is obtained through reverse transcription; the cDNA is used as a template, and the complete sequence of the amy1 gene obtained by a PCR amplification method is utilized; the complete amy1 gene is inserted into pPICZ alpha A expression vector by adopting double enzyme digestion and enzyme ligation method to obtain recombinant expression plasmid pPICZ alpha A-amy1.

The invention also protects recombinant engineering bacteria containing the alpha-amylase gene amy1. The engineering bacteria can be escherichia coli, yeast, lactobacillus and the like; preferably yeast, more preferably pichia pastoris.

Specifically, the recombinant plasmid pPICZ alpha A-amy1 is transformed into Pichia pastoris to obtain engineering bacteria capable of expressing the alpha-amylase gene amy1.

Preferably, high yields of the protein alpha-amylase AMY1 are obtained under conditions of the induction medium.

Specifically, the recombinant expression vector is selected to transform host cells to obtain recombinant engineering bacteria, and then the recombinant engineering bacteria are cultured to induce amylase expression.

More specifically, a recombinant expression plasmid pPICZ alpha A-amy1 is transformed into pichia pastoris competence by adopting an electric shock transformation method, and efficient engineering bacteria transformants are screened; after the engineering bacteria are subjected to induced culture by a liquid fermentation culture medium, a large amount of alpha-amylase AMY1 can be secreted, and then the engineering bacteria are recovered and purified.

The invention also protects the application of the alpha-amylase gene amy1, the protein and the recombinant expression vector in degrading starch or other glycogen.

Advantageous effects

Compared with the prior art, the alpha-amylase gene amy1 and the encoded protein and application thereof provided by the invention have the following beneficial effects:

(1) The invention realizes the expression of the alpha-amylase gene amy1 in pichia pastoris, constructs excellent genetic engineering bacteria, further promotes the industrialization of achievements and creates good social and economic benefits;

(2) The optimal hydrolysis temperature of the alpha-amylase AMY1 is 40 ℃, and the content of reducing sugar produced by hydrolyzing soluble starch by AMY1 per minute is 0.55 mug/mL; has an enzyme activity of 60-80% at 30-50deg.C; the optimal pH of the alpha-amylase AMY1 is 7, but the pH of the alpha-amylase AMY1 can maintain 90 percent of enzyme activity at the pH of 6 to 8, and the maximum content of reducing sugar which can be produced by hydrolyzing soluble starch per minute of the AMY1 is 0.78 mug/mL;

(3) The invention aims to comprehensively improve the decomposition efficiency of starch, establishes an efficient recombinant protein expression system through a proper genetic engineering technology, obtains the genetic engineering strain of the enzyme, improves the degradation of alpha-starch by improving the activity of alpha-amylase, and has important significance for improving the recycling utilization of agricultural solid wastes.

Drawings

FIG. 1 is a graph showing the effect of different temperatures on the alpha-amylase AMY1;

FIG. 2 is a graph showing the effect of different pH on the alpha-amylase AMY1;

FIG. 3 is the thermostability of the alpha-amylase AMY1;

FIG. 4 is the effect of different metal ions on the alpha-amylase AMY1; wherein, 1: no metal ion treatment is added; 2: co (Co) ²⁺ 3：Fe ³⁺ ；4：Ca ²⁺ ；5：K ⁺ ；6：Zn ²⁺ ；7：Cu ²⁺ ；8：Mg ²⁺ ；9：Mn ²⁺ ；

FIG. 5 is an SDS-PAGE electrophoresis of purified alpha-amylase AMY1; wherein 1 is AMY1 crude enzyme solution, and 2 is purified AMY1.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to be limiting, and are generally by the means well known in the art.

EXAMPLE 1 cloning and expression of amy1 Gene

1. Construction of expression plasmid

Primer design: according to the known sequence information of the body signal peptide gene of the alpha-amylase gene, the primer sequence capable of heterologously expressing the gene in Pichia pastoris is designed by combining with the known different polyclonal localization points and gene characteristics on the expression vector of the alpha-amylase gene, and the sequence is shown in table 1.

Table 1: alpha-amylase gene amy1 primer sequence for heterologous expression in pichia pastoris

Total RNA was extracted from the compost samples using Trizol reagent from Qiagen, and the first strand cDNA was synthesized using RT-PCR kit (supplied by Takara). The cDNA synthesized by the reverse transcription is used as a template, the primers in the table 1 are used for PCR amplification to obtain complete amy1 gene fragments, and amy1 genes without signal peptide sequences are obtained after sequencing, and the procedure used for PCR is shown in the table 2.

Table 2: PCR program

The DNA gel block of amy1 gene is dyed by EB for 10min, then washed by water, absorbed and dried, the gel block containing amy1 gene part is cut under the irradiation of ultraviolet lamp, transferred to a 1.5mL centrifuge tube, the weight of the gel block is weighed, and the formula is shown as follows: 1mL = 1g as one gel volume, the gel volume was calculated;

adding binding Buffer with the same volume as the gel, and water-bathing at 65 ℃ until the gel is completely dissolved; after the dissolution is completed, the water is dissolved,transferring the mixed solution into a centrifuge tube with a DNA column for multiple times, centrifuging for 1min at 12000rpm, and discarding the filtrate; adding 300 μl binding Buffer into the DNA column, centrifuging at 12000rpm for 1min, and discarding the filtrate; adding 700 μl of wash Buffer into the DNA column, centrifuging at 12000rpm for 1min, discarding the filtrate, and repeating the steps; centrifuging the empty column for 1min at the rotating speed of 12000 rpm; the DNA column was transferred to a fresh 1.5mL centrifuge tube and 35. Mu.L of sterilized ddH was added ₂ O was applied to the DNA column matrix, allowed to stand at room temperature for 2min, and centrifuged at 12000rpm for 1min.

2. Transformation and screening

The pichia pastoris heterologously expressed alpha-amylase gene amy1 and a vector plasmid are respectively subjected to restriction enzyme XbaI and EcoRI step-by-step double enzyme digestion operation, and a total enzyme digestion system is designed to be 20 mu L. The total system included 2. Mu.L of 10 Xbuffer, 1. Mu.L of XbaI, 1. Mu.L of EcoRI, 6.75. Mu.L of the target gene for the cut gel recovery and 9.25. Mu.L of ddH ₂ O. Double-enzyme digestion is carried out on plasmid pPICZ alpha A and the recovered gel-cutting cDNA by using corresponding restriction endonuclease; the enzyme digestion products after double enzyme digestion are cleaned and recovered by using a PCR recovery kit; performing a second enzyme digestion operation on the recovered product, wherein the enzyme digestion temperature is controlled to be kept at 37 ℃ according to a total enzyme digestion reaction system designed before; and (3) cleaning and recycling the enzyme digestion product subjected to double enzyme digestion again by using a PCR recycling kit. The cloning vector and the double cleavage product alpha-amylase gene amy1 were subjected to an enzymatic ligation operation, wherein the enzymatic ligation reaction system is described in Table 3, the molar ratio of gene fragments to vector in the system is 10:1 proportion design.

Table 3: enzyme linked reaction system

100 mu L of Pichia pastoris competent cells and 5 mu g of linearized DNA are uniformly mixed and transferred to 2mm pre-cooling at-20 ℃ in advance for killingIn the bacterial electric rotating cup, the bacterial electric rotating cup is electrified for 4-10ms under the condition of 1500V,200 omega and 20 mu F. Immediately after the electrotransformation is finished, 1mL of 1M sorbitol solution precooled in a refrigerator at 4 ℃ is added on an ultra-clean workbench, the solution is blown uniformly by a pipetting gun, no bubbles are generated, and after uniform mixing, the mixed solution is transferred into a 2mL centrifuge tube. Resuscitates for 1h at 30℃and 100rpm in a shaker. 100. Mu.L of the suspension was pipetted and coated onto a solution containing 100. Mu.g.mL ^-1 On a YPDS plate of Zeocin, the culture is carried out in a light-proof incubator at 30 ℃ until single bacterial colony appears. A suitable single colony is selected from the culture medium from which the single colony is grown for preparing a PCR reaction template. In addition, PCR verification also requires the incorporation of the alpha-amylase gene amy1 primer.

3. Enzyme production and electrophoresis analysis

The obtained single colony of yeast transformant was inoculated into a 2.5L conical flask containing 1L BMGY, and cultured with shaking at 30℃and 180rpm to OD ₆₀₀ After about 1.0, methanol was added to the 2.5L Erlenmeyer flask at 24h intervals to induce protein expression in 1% by volume (10 mL) of the total volume of the medium, and the culture was continued for 5-7 days. Centrifuging at 4deg.C and 10000rpm for 5min with Beckmann centrifuge, removing precipitate, collecting supernatant, obtaining crude enzyme solution, measuring enzyme activity of crude enzyme solution, and collecting part of crude enzyme solution for storage at-80deg.C. Crude enzyme solution and SDS-PAGE protein loading buffer were added at 5:1, shaking uniformly, and carrying out boiling water bath for 5min to further analyze the yield and solubility of the protein by using a protein electrophoresis technology.

4. Extraction and purification of amylase AMY1

By ammonium sulfate precipitation, the (NH) ₄ ) ₂ SO ₄ Powder, using a mill (NH) ₄ ) ₂ SO ₄ Grinding to finer powder, and dissolving. Wherein 52g (NH) of crude enzyme was added per 100mL of crude enzyme solution ₄ ) ₂ SO ₄ And (3) powder. In the ice bath state, the (NH) ₄ ) ₂ SO ₄ Slowly adding the powder into the crude enzyme solution, and stirring until all (NH) ₄ ) ₂ SO ₄ The powder was completely dissolved. Standing in a refrigerator at 4deg.C for 8-12 hr. The mixture was centrifuged at 10000rpm at 4℃for 10min to obtain a protein precipitate.The supernatant was decanted and protein pellet resuspended using Tris-HCl buffer pH 8. The alpha-amylase gene of the invention carries a histidine tag, and the nickel ions in the Ni column can be combined with proteins containing the histidine tag, but have stronger affinity with imidazole. The crude enzyme solution can be firstly passed through Ni column to make it be adsorbed on the Ni column, then the imidazole with different concentration can be used for eluting it so as to attain the goal of purification.

Example 2 Effect of different conditions on the enzymatic Activity Properties of alpha-Amylase AMY1

1. Influence of different metal ions on the enzyme activity of alpha-amylase AMY1

Into a 2mL centrifuge tube were added 100. Mu.L of purified diluted enzyme solution containing 1. Mu.M enzyme, 0.5% 200. Mu.L of soluble starch, 600. Mu.L of phosphate buffer (pH 7.0) and 100. Mu.L of a 100mM solution of different metal ions: co (Co) ²⁺ ，Fe ³⁺ ，Ca ²⁺ ，K ⁺ ，Zn ²⁺ ，Cu ²⁺ ，Mg ²⁺ ，Mn ²⁺ Mixing, treating in water bath at 40deg.C for 1 hr, and setting three repeated experiments for each gradient treatment. Enzyme activity was measured using DNS and OD values were measured using an enzyme-labeled instrument. The data were measured and a graphical analysis was made. The results are shown in FIG. 4, mn ²⁺ Has obvious promotion effect on the enzyme activity of alpha-amylase AMY1; fe (Fe) ³⁺ 、Zn ²⁺ 、Cu ²⁺ Has obvious inhibiting effect on the enzyme activity of alpha-amylase AMY1, fe ³⁺ The inhibition effect is most remarkable.

2. Response of alpha-amylase AMY1 to different temperatures

100. Mu.L of purified diluted enzyme solution containing 1. Mu.M enzyme, 0.5% 200. Mu.L of soluble starch solution and 700. Mu.L of phosphate buffer (pH 7.0) were added to a 2mL centrifuge tube, and the mixture was homogenized, treated in water baths at different temperatures for 1 hour, and the temperature treatment was set as follows: 30 ℃,40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃. Enzyme activity was determined using the DNS method and OD values were determined with an enzyme-labeled instrument, and three replicates were performed for each experimental design. The data were measured and a graphical analysis was made. As a result, as shown in FIG. 1, the optimal hydrolysis temperature of the alpha-amylase AMY1 is 40 ℃, but has an enzyme activity of 60-80% at 30-50 ℃; the enzyme activity decreased sharply after 50℃and was substantially lost at 70 ℃.

3. Response of alpha-amylase AMY1 to different pH

Into a 2mL centrifuge tube were added 100. Mu.L of purified diluted enzyme solution containing 1. Mu.M enzyme, 0.5% 200. Mu.L of soluble starch and 700. Mu.L of buffers of different pH: acetic acid-sodium acetate buffer at pH 4.0,5.0,6.0 and Tris-HCl buffer at pH 7.0,8.0,9.0, 10.0 were mixed well and treated in a water bath at 40℃for 1h with three replicates per gradient treatment. Enzyme activity was determined using the DNS method and OD values were determined with an enzyme-labeled instrument, and three replicates were performed for each experimental design. The data were measured and a graphical analysis was made. As a result, as shown in FIG. 2, the optimal pH of the alpha-amylase AMY1 was 7, but 90% of the enzyme activity was maintained at pH 6-8; both peracid and overbase conditions lead to a dramatic decrease in the enzyme activity or even inactivation.

4. Alpha-amylase AMY1 thermal stability analysis

The enzyme solutions were subjected to water bath at 40℃at 50℃at 60℃at 70℃at 80℃at 90℃for 1 hour, respectively, 0.5% 200. Mu.l of soluble starch and 700. Mu.l of phosphate buffer (pH 7.0) were added to a 2mL centrifuge tube, and 100. Mu.L of the purified diluted enzyme solutions containing 1. Mu.M of enzyme at each temperature were each preheated, mixed well, treated in water bath at 40℃for 1 hour, and three repeated experiments were set for each gradient treatment. Enzyme activity was measured using DNS and OD values were measured using an enzyme-labeled instrument. The data were measured and a graphical analysis was made. As shown in FIG. 3, the alpha-amylase AMY has poor heat stability, and the heat stability is drastically reduced at 40-50 ℃.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.

Sequence listing

<110> Nanjing agricultural university

<120> an alpha-amylase gene amy1, protein encoded by the same and use thereof

<160> 2

<170> SIPOSequenceListing 1.0

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tgtaatccag gcgcacccac ccagaaggaa gcgccgctta tcagtcagac ccctgtgccc 60

gaatggctta acgatgccgt aatttatgaa gtaaacctgc gccaatacac ccccgaaggc 120

agctttgaag ccttcgccaa ccacctgccc cgtttgcagg aactgggtgt agacatcctc 180

tggttcatgc ccatccaccc cattggacag gaaaaccgaa agggagaact gggttcctat 240

tactccatcc gtgattacaa agccgtcaac caggaattcg gcagctttga ggacttcaaa 300

gcgctggtgg ataaggccca tgaactgggc atgaaggtgg tgctcgactg ggtggccaac 360

catacggcct gggaccatgc ctgggtaagt gagcaccccg actggtacga aaaagacagc 420

actggaaaca tgtttgctcc gatggactgg agcgatgtgg tgcagctcga ctacgacaac 480

ccggaaatgc gtgcagccat gcaggatgct ttggcctttt gggttagaga ggcaaacata 540

gacggctacc gttgcgatgt ggcaggaatg gttccggttg acttctggga agaagccact 600

tcgaacctac agcaaataaa gccggtattt atgttggccg aagacgagga cgaaacagcc 660

ctgctcaaca aggcttttaa cgccaattac ggatgggcct tacaccacct gctcaacgga 720

cttgcccagg gcacagccaa agcctcggat ctgcaggccc acattgagga acagcagcaa 780

aaattgccac ggggcgcatt ctccatgcag tttaccacca accacgacga aaacagctgg 840

aacggcacgg tgttcgagcg tatgggcgct gcctacccca ccctggctgc actgaccttt 900

gtagtagaag gcatgcccct tatctacagt gggcaggaag ccggcctcaa caaacaactc 960

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ttcagtctgg agggccaata cgaagacacg cttaccggtg aaaagataaa tctctccaac 1260

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Cys Asn Pro Gly Ala Pro Thr Gln Lys Glu Ala Pro Leu Ile Ser Gln

1 5 10 15

Thr Pro Val Pro Glu Trp Leu Asn Asp Ala Val Ile Tyr Glu Val Asn

20 25 30

Leu Arg Gln Tyr Thr Pro Glu Gly Ser Phe Glu Ala Phe Ala Asn His

35 40 45

Leu Pro Arg Leu Gln Glu Leu Gly Val Asp Ile Leu Trp Phe Met Pro

50 55 60

Ile His Pro Ile Gly Gln Glu Asn Arg Lys Gly Glu Leu Gly Ser Tyr

65 70 75 80

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

85 90 95

Glu Asp Phe Lys Ala Leu Val Asp Lys Ala His Glu Leu Gly Met Lys

100 105 110

Val Val Leu Asp Trp Val Ala Asn His Thr Ala Trp Asp His Ala Trp

115 120 125

Val Ser Glu His Pro Asp Trp Tyr Glu Lys Asp Ser Thr Gly Asn Met

130 135 140

Phe Ala Pro Met Asp Trp Ser Asp Val Val Gln Leu Asp Tyr Asp Asn

145 150 155 160

Pro Glu Met Arg Ala Ala Met Gln Asp Ala Leu Ala Phe Trp Val Arg

165 170 175

Glu Ala Asn Ile Asp Gly Tyr Arg Cys Asp Val Ala Gly Met Val Pro

180 185 190

Val Asp Phe Trp Glu Glu Ala Thr Ser Asn Leu Gln Gln Ile Lys Pro

195 200 205

Val Phe Met Leu Ala Glu Asp Glu Asp Glu Thr Ala Leu Leu Asn Lys

210 215 220

Ala Phe Asn Ala Asn Tyr Gly Trp Ala Leu His His Leu Leu Asn Gly

225 230 235 240

Leu Ala Gln Gly Thr Ala Lys Ala Ser Asp Leu Gln Ala His Ile Glu

245 250 255

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

260 265 270

Thr Asn His Asp Glu Asn Ser Trp Asn Gly Thr Val Phe Glu Arg Met

275 280 285

Gly Ala Ala Tyr Pro Thr Leu Ala Ala Leu Thr Phe Val Val Glu Gly

290 295 300

Met Pro Leu Ile Tyr Ser Gly Gln Glu Ala Gly Leu Asn Lys Gln Leu

305 310 315 320

Glu Phe Phe Glu Lys Asp Thr Ile Asp Trp Ser Asp Leu Ser Leu Thr

325 330 335

Pro Phe Tyr Gln Gln Leu Ile Arg Leu Lys Lys Glu Asn Pro Ala Leu

340 345 350

Trp Asn Gly Asn Ala Gly Gly Ser Met Glu Phe Leu Asn Asn Thr Lys

355 360 365

Pro Glu Gln Ala Leu Val Phe Lys Arg Thr Arg Asp Asn Asn Ser Ile

370 375 380

Tyr Ala Ile Phe Asn Leu Ser Asp Gln Pro Leu Val Leu Pro Ile Asp

385 390 395 400

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

405 410 415

Asn Leu Ser Asn Glu Ser Leu Glu Leu Ala Pro Trp Ala Tyr His Leu

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Ile Ser Val Arg Asn

435

Claims

1. The key gene alpha-amylase gene amy1 for decomposing starch is characterized in that the nucleotide sequence is shown in SEQ ID NO. 1.

2. The protein encoded by the alpha-amylase gene amy1 of claim 1.

3. A recombinant expression vector comprising the alpha-amylase gene amy1 of claim 1.

4. The recombinant expression vector of claim 3, wherein the recombinant expression vector is ppiczαa-amy1.

5. The recombinant expression vector according to claim 3, wherein the coding region sequence of the alpha-amylase gene amy1 is obtained by amplification, and the recombinant plasmid pPICZ alpha A-amy1 is constructed by a double enzyme digestion and enzyme ligation method.

6. A recombinant engineering bacterium comprising the alpha-amylase gene amy1 of claim 1.

7. The engineering bacterium according to claim 6, wherein the recombinant plasmid pPICZ alpha A-amy1 of claim 3 is transformed into Pichia pastoris to obtain the engineering bacterium capable of expressing the alpha-amylase gene amy1 of claim 1.

8. The engineered bacterium of claim 6, which can obtain high yield of AMY1 protein under conditions of induction medium.

9. Use of the alpha-amylase gene amy1 of claim 1, the protein of claim 2, and the recombinant expression vector of claim 3 for degrading starch or other glycogen.