CN103290039A - Alpha-amylase derived from animal feces metagenome and gene of the alpha-amylase - Google Patents

Abstract

The invention relates to alpha-amylase derived from animal feces metagenome and a gene of the alpha-amylase. The alpha-amylase gene is 1458 bp in length and the sequence of the alpha-amylase gene is shown as SEQIDNO.1. The gene encodes 485 amino acids and the sequence of the amino acids is shown as SEQIDNO.2. The alpha-amylase provided by the invention has characters that the optimal operative pH is 5.6; the enzyme activity remains larger than 65% when the pH is ranging from 4.6 to 6.6; the enzyme activity remains larger than 70% after the alpha-amylase is treated at a pH ranging from 4.0 to 8.0 for 1 h; the optimal operative temperature is 50 DEG C; and the alpha-amylase is relatively stable at 37 DEG C, and the enzyme activity remains larger than 80% after the enzyme is kept at 37 DEG C for 50 min. As a novel enzyme preparation, the alpha-amylase provided by the invention can be widely used in forage processing industry, starch processing industry, and the like.

Description

A kind of from the grand genomic α-Dian Fenmei of animal excrement and gene thereof

Technical field

The invention belongs to microorganism and gene engineering technology field, be specifically related to a kind of from the grand genomic α-Dian Fenmei of animal excrement and gene thereof.

Background technology

α-Dian Fenmei (EC 3.2.1.1) is a kind of inscribe glucuroide, α-1 in the energy hydrolyzed starch, the 4-glucoside bond, generate short chain dextrin different in size and a spot of low molecule carbohydrate, thereby the viscosity of starch paste is descended rapidly, namely play the effect that reduces denseness and " liquefaction ", so be called Ye Huamei again.α-Dian Fenmei extensively is present in the various biologies such as people, animal, plant, fungi and bacterium, reported first was separated α-Dian Fenmei so far in 1956, the various countries investigator has separated and has identified and surpassed 120 kinds of α-Dian Fenmei, wherein mainly be the bacillus that derives from microorganism ( Bacillus), Aspergillus ( Aspergillus), streptomyces ( Streptomyces) and yeast.

Because the animal ingestion kind is plant and animal food widely, make the microorganism that exists in its gi tract possess corresponding special physiological, metabolic characteristic, can produce various enzymes to decompose the materials such as starch, pectin, Mierocrystalline cellulose, protein and fat in the food.Therefore, the animal gastrointestinal tract microorganism itself is huge, a undeveloped enzyme genetic resources storehouse, is containing a large amount of potential enzyme genetic resourceses.

Yet, traditional microorganism pure culture technigne makes that accounting for microbe species can not culturing micro-organisms can't separate acquisition more than 99%, therefore especially the anaerobion cultivation property in the enteron aisle is lower, and the traditional method of screening novel enzyme by the separation and Culture microorganism has limited popularity and the validity of screening greatly.Grand genomics has been avoided the problem of microorganism separation and Culture, has greatly expanded the space that utilizes of Microbial resources, for seeking and finding that new functional gene and biological catalyst---enzyme provides new research strategy.At present, the investigator has utilized grand genome-based technologies successfully to screen a plurality of amylases genes, but research field mainly concentrates on water body and soil (Delavat et al. Sci Rep.2012,2:354; Liu et al. Mar Biotechnol (NY) .2012,14 (3): 253-260; Sharma et al. Appl Microbiol Biotechnol. 2010,86 (6): 1821-1828), be research less (Tasse et al. Genome Res. 2010, the 20:1605-1612 that object carries out with the animal gastrointestinal tract microorganism; Ferrer et al. Biotechnol J. 2007,2:207-213).

Summary of the invention

The purpose of this invention is to provide a kind of new for the grand genomic α-Dian Fenmei of animal excrement.

A further object of the present invention provides the gene of the above-mentioned α-Dian Fenmei of coding.

Another object of the present invention provides the recombinant expression vector that comprises said gene.

Another object of the present invention provides the recombinant bacterial strain with described recombinant expression vector transformed host cell gained.

Of the present invention a kind of from the grand genomic alpha-amylase gene of animal excrement, its nucleotide sequence is shown in SEQ ID NO.1, and the size of this gene is 1458bp.

A kind of α-Dian Fenmei of the present invention, its aminoacid sequence shown in SEQ ID NO.2, totally 485 amino acid, its theoretical molecular is 55.439 kDa.

The suitableeest action pH of α-Dian Fenmei of the present invention is 5.6, can keep 65% above enzyme to live between pH 4.6-6.6; Handle 1h in pH 4.0-8.0 scope after, enzyme is lived residue more than 70%; Optimum temperature is 50 ℃, and is comparatively stable under 37 ℃ of conditions, and tolerance 50min still keeps the enzyme more than 80% to live.

The present invention at first extracts the preparation microbe genome DNA from the ight soil of Japan honeybee monkey, made up grand genomic library with this DNA, fosmid carrier pCC1FOS and intestinal bacteria then, by the functional screening method, screening obtains can form transparent circle and the higher strains A my4-7-2 of amylase activity on starch-containing substrate flat board from the library.Extract the fosmid plasmid from strains A my4-7-2, transformed into escherichia coli has made up the subclone library with being connected also with carrier PUC118 behind this fosmid plasmid fragmentation then, obtains to form the strains A myC-1-5 of transparent circle from the library by functional screening.From strains A myC-1-5, extract plasmid, with this plasmid encoding gene of α-Dian Fenmei that has been the template method separating clone that passes through PCR PlA.With this alpha-amylase gene PlA is connected with plasmid pEASY-E1 and obtains recombinant expression vector, then transformed into escherichia coli BL21(DE3) obtain recombinant bacterial strain.Through the BLAST of NCBI website comparison, derive from human gi-tract among this alpha-amylase gene amino acid sequence coded and the GenBank Bifidobacterium pseudocatenulatumImaginary albumen have the highest consistence (70%).

The method for preparing α-Dian Fenmei of the present invention is carried out according to the following steps:

1) cultivates above-mentioned recombinant bacterial strain, induce recombinant alpha-amylases to express;

2) reclaim the also expressed α-Dian Fenmei of purifying.

α-Dian Fenmei of the present invention has following character: the suitableeest action pH is 5.6, can keep 65% above enzyme to live between pH 4.6-6.6; Handle 1h in pH 4.0-8.0 scope after, enzyme is lived residue more than 70%; Optimum temperature is 50 ℃, and is comparatively stable under 37 ℃ of conditions, and tolerance 50min still keeps the enzyme more than 80% to live.Above character shows α-Dian Fenmei of the present invention as a kind of novel enzyme preparation, can be widely used in feed, starch processing industry etc.

Description of drawings

Fig. 1: the SDS-PAGE in the recombinant alpha-amylases of expression in escherichia coli analyzes, wherein, and M: low molecular weight protein Marker; The recombinant alpha-amylases of purifying under 1～4:NTA-300, NTA-200, NTA-100 and the NTA-80 elution requirement; 5: the intestinal bacteria culture supernatant that contains the recombinant alpha-amylases gene.

Fig. 2: the optimal pH of recombinant alpha-amylases.

Fig. 3: the pH stability of recombinant alpha-amylases.

Fig. 4: the optimum temperuture of recombinant alpha-amylases.

Fig. 5: the thermostability of recombinant alpha-amylases.

Embodiment

Test materials and reagent

1, bacterial strain and carrier: bacterial strain Escherichia coliEPI300 and carrier pCC1FOS purchase the company in EPICENTRE, subcloning vector PUC118 and host E. coliDH10B purchases the company in TaKaRa, E.coliBL21(DE3) purchase the company in Novagen, coli expression carrier pEASY-E1 purchases in the Beijing Quanshijin Biotechnology Co., Ltd.

2, genetically engineered operation enzyme, test kit and other biochemical reagents: restriction enzyme, archaeal dna polymerase, ligase enzyme and dNTP, Blunting Kination Ligation(BKL) Kit is available from TaKaRa company; Other all is domestic reagent (all can buy from common biochemical reagents company and obtain).

3, substratum:

TB substratum: A component: Tryptones 12g, yeast powder 24g, glycerine 4mL, agar 2% is dissolved in the 900mL distilled water; B component (PBS damping fluid): KH ₂PO ₄9.5g and KH ₂PO ₄3H ₂O 82.16g is dissolved in the 100mL distilled water; A, B component respectively at 120 ℃ of sterilization 30min after, to be cooledly both are mixed during to 65 ℃ of left and right sides.Solid medium adds 2.0%(w/v on this basis) agar.

The LB substratum: Peptone 10g, Yeast extract 5g, NaCl 10g, adding distil water are to 1000mL, and pH is (being about 7) naturally.Solid medium adds 2.0%(w/v on this basis) agar.

Illustrate: make the experimental methods of molecular biology specify in following examples, all carry out with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Sa nurse Brooker one book, perhaps carry out according to test kit and product description.

Embodiment one: the acquisition of alpha-amylase gene

1, the grand genomic library construction of Japan honeybee monkey fecal microorganism

(extracting method can be with reference to patent " a kind of genomic method of high molecular of extracting from animal excrement " to extract microbe genome DNA from Japan's honeybee monkey ight soil, publication number 102586234A), fragmentation is by the dna fragmentation that pulsed field gel electrophoresis separates, agarose gel electrophoresis reclaims the about 40kb of size, and the dna fragmentation of recovery is connected with fosmid carrier pCC1FOS and transfecting host bacterium E.coliEPI300 coats on the LB flat board that contains 12.5 μ g/mL paraxin, and 37 ℃ of overnight incubation obtain transformant, this clone library are stored in 96 orifice plates ,-80 ℃ of preservations.

2, produce the functional screening of amylase positive colony

The clone bacterium liquid of preserving in 96 orifice plates is suitably diluted, and it is dull and stereotyped to coat TB-paraxin (the 12.5 μ g/mL) screening that contains 0.1% Zulkovsky starch, uses 0.5% iodine staining behind 37 ℃ of cultivation 48h, selects and can form transparent circle and the higher bacterial strain of amylase activity.

3, subclone library construction and screening

Screening obtains to form transparent circle and the higher strains A my4-7-2 of amylase activity from above-mentioned grand genomic library, therefrom extracts fosmid plasmid and fragmentation.Plasmid DNA employing Blunting Kination Ligation(BKL behind the fragmentation) Kit carries out flush endization and 5 ' terminal phosphate processing, agarose gel electrophoresis detects also to be cut glue and reclaims dna fragmentation in 2.0～4.0kb scope, then the DNA that reclaims is connected with carrier PUC118 and transforms E. coliDH10B has made up the subclone library.Library bacterium liquid is suitably coated LB-penbritin (100 μ g/mL) the screening flat board that contains 0.1% Zulkovsky starch after the dilution, uses 0.5% iodine staining behind 37 ℃ of cultivation 48h, selects and can form transparent circle and the higher bacterial strain of amylase activity.

4, the clone of alpha-amylase gene

Screening obtains to form the strains A myC-1-5 of transparent circle from above-mentioned subclone library, therefrom extracts plasmid and checks order.According to sequencing result, the insertion fragment total length 2541bp of this plasmid utilizes ORF finder on-line analysis to obtain a length and is the complete open reading frame (ORF) of 1539bp, through the no matching sequence of BLAST comparison.Utilize SignalP 4.1 on-line prediction signal peptides, the 1st～27 amino acid of its N end has typical signal peptide feature.According to The sequencing results design amplification alpha-amylase gene ( PlA) primer of mature peptide:

plA F：5`-GCGACCAATCGCGACAGCTACG-3`

plA R：5`-CTAGAGTCTCACATAGGCCACC-3`

Be template with the plasmid that extracts among the strains A myC-1-5, with the encoding gene of above-mentioned primer by the pcr amplification α-Dian Fenmei PlA.

Embodiment two: the preparation of recombinant alpha-amylases

With this alpha-amylase gene PlA is connected with plasmid pEASY-E1 and obtains recombinant expression vector, then transformed into escherichia coli BL21(DE3) obtain recombinant bacterial strain.Get and contain recombinant expression vector E. coliBL21(DE3) bacterial strain, the inoculum size with 0.1% are inoculated in LB(and contain 100 μ g/mL Amp) in the nutrient solution, 37 ℃ of quick oscillation 16h.Then the bacterium liquid of this activation being inoculated into fresh LB(with 1% inoculum size and containing 100 μ g/mL Amp) in the nutrient solution, quick oscillation is cultivated about 2 – 3h(OD ₆₀₀Reach 0.6 – 1.0) after, the alpha-lactose that adds final concentration 0.5% is induced, and continues the about 20h of shaking culture or 26 ℃ of about 8h of shaking culture in 20 ℃.The centrifugal 5min of 12000rpm collects thalline.Behind an amount of pH7.0 Tris-HCl damping fluid suspension thalline, ultrasonic disruption thalline under the low temperature water-bath., behind the centrifugal 10min of 13,000rpm, draw supernatant and use Nickel-NTA Agarose purifying target protein with first enzyme liquid concentrated in the upper eye lid.SDS-PAGE result (Fig. 1) shows that recombinant alpha-amylases has obtained expression in intestinal bacteria, is single band behind purifying under the NTA-300 elution requirement.

The activation analysis of recombinant alpha-amylases:

Activity determination method adopts 3,5-dinitrosalicylic acid (DNS) method: Zulkovsky starch is dissolved in the 0.1M damping fluid, and it is 0.2%(w/v that preparation obtains final concentration) substrate solution; Get 900 μ L substrate solution preheating 5min under temperature of reaction, add 100 μ L enzyme liquid, accurately react 5min, add 1.5mL DNS termination reaction then, boiling water boils 5min, is cooled to and measures the OD value after the room temperature under the 540nm wavelength.1 enzyme unit alive (U) is defined as the required enzyme amount of per minute bottom exploded deposits yields 1 μ mol reducing sugar under given condition.

The property testing of recombinant alpha-amylases:

1, the measuring method of the optimal pH of recombinant alpha-amylases and pH stability is as follows:

The optimal pH of enzyme is measured: the recombinant alpha-amylases of embodiment two purifying is carried out enzymatic reaction under the damping fluid of 50 ℃ and pH3.0 – 8.0.The pH of enzyme stability is measured: the enzyme liquid of purifying is placed the 0.1M damping fluid of pH2.0 – 11.0, and room temperature is placed 1h, carries out enzymatic reaction then under pH5.6 and 50 ℃, with untreated enzyme liquid in contrast.Damping fluid is: 0.1M citric acid-Sodium phosphate dibasic damping fluid (pH2.0 – 8.0), 0.1M Tris-HCl(pH8.0 – 9.0) and 0.1M glycine-NaOH damping fluid (pH9.0 – 11.0).Be substrate with the Zulkovsky starch, reaction 5min measures the zymologic property of the recombinant alpha-amylases of purifying.The result shows: the optimal pH of recombinant alpha-amylases is 5.6, can keep 65% above enzyme (Fig. 2) alive between pH 4.6-6.6; Handle 1h in pH 4.0-8.0 scope after, enzyme is lived residue more than 70% (Fig. 3).

2, the optimum temperuture of recombinant alpha-amylases and thermal stability determination method are as follows:

The optimum temperuture of enzyme is measured: in the damping fluid of pH5.6, carry out enzymatic reaction under 80 ℃ of 0 –.The thermal stability determination of enzyme: after placing the temperature (37 ℃, 50 ℃ or 60 ℃) of setting to handle 0 – 50min the enzyme liquid of same enzyme amount, under pH5.6 and 50 ℃, carry out enzymatic reaction, with untreated enzyme liquid in contrast.Be substrate with the Zulkovsky starch, reaction 5min measures the zymologic property of the recombinant alpha-amylases of purifying.The result shows: the optimum temperuture of recombinant alpha-amylases is 50 ℃, can keep 65% above enzyme to live in 35-60 ℃ of scope, and 60 ℃ of enzyme work later on are with temperature rising decline (Fig. 4) rapidly.Tolerate 50min under 37 ℃ of conditions, enzyme work remains on more than 80%; Tolerate 10min under 60 ℃ of conditions, enzyme is lived loss rapidly to about 15%(Fig. 5).

＜110〉Yunnan Normal University

＜120〉a kind of from the grand genomic α-Dian Fenmei of animal excrement and gene thereof

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＜213〉the grand genome of animal excrement

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gcgaccaatc gcgacagcta cgccgacacg ctgggtgacg ccaccttcga ggcggcgcgc 60

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tcgttcaaca cgatcaccgc caacatgaag gacatcgccg aggccggcta cacctcgatc 180

cagaccgagc cgatgagcca tatcaagacc aacacggcca acggcaagaa gttcaccgag 240

aactggtact acgtctacca gcccacctcc acctcgatcg gcaacttcgt ggtcggcacc 300

caggatgacc tcaagcgcat gtgcgccgag gcccataaat acggcatccg catcatcgtg 360

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aaccagaccg cggcgaacaa gatgaaggag ttcctcgtgc aggccgtcaa cgacggtgtg 600

gacggcttcc gcttcgacgc cgccaagcat gtggagcttc cggacgagct gggcgagcat 660

tcggtctact gggacacgat cctggagaac ggcgcccagt accagtacgg cgaggtcctg 720

cagggagact ccggcctcga ctacagggcc tatgccaacc tcatgacgaa ccactcctcg 780

cgcggcggcg gcaacaccgc ctccgactac ggccgcaccg tgcgctcggc cgtcaagagc 840

cgcaacctct ccgcgggctc cctaagcaac atgagcaagg gcggcgtggc cgacgaccaa 900

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Ala Thr Asn Arg Asp Ser Tyr Ala Asp Thr Leu Gly Asp Ala Thr Phe

1 5 10 15

Glu Ala Ala Arg Gln Arg Tyr Gly Leu Thr Glu Asp Met Ser His Gly

20 25 30

Thr Ile Leu His Ala Trp Met Trp Ser Phe Asn Thr Ile Thr Ala Asn

35 40 45

Met Lys Asp Ile Ala Glu Ala Gly Tyr Thr Ser Ile Gln Thr Glu Pro

50 55 60

Met Ser His Ile Lys Thr Asn Thr Ala Asn Gly Lys Lys Phe Thr Glu

65 70 75 80

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

85 90 95

Val Val Gly Thr Gln Asp Asp Leu Lys Arg Met Cys Ala Glu Ala His

100 105 110

Lys Tyr Gly Ile Arg Ile Ile Val Asp Val Val Ala Asn His Phe Thr

115 120 125

Ser Asp Trp Asn Ala Ile Asp Ser Ser Trp Lys Asp Ala Ser Leu Phe

130 135 140

His Lys Arg Ser Asn Cys Ser Gly Ala Asn Gly Asp Asn Ile Asn Tyr

145 150 155 160

Gly Asn Arg Trp Gln Val Thr Gln Cys His Leu Leu Gly Leu Trp Asp

165 170 175

Ile Asn Thr Gln Asn Gln Thr Ala Ala Asn Lys Met Lys Glu Phe Leu

180 185 190

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

195 200 205

Lys His Val Glu Leu Pro Asp Glu Leu Gly Glu His Ser Val Tyr Trp

210 215 220

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

225 230 235 240

Gln Gly Asp Ser Gly Leu Asp Tyr Arg Ala Tyr Ala Asn Leu Met Thr

245 250 255

Asn His Ser Ser Arg Gly Gly Gly Asn Thr Ala Ser Asp Tyr Gly Arg

260 265 270

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

275 280 285

Ser Asn Met Ser Lys Gly Gly Val Ala Asp Asp Gln Leu Val Thr Trp

290 295 300

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

305 310 315 320

Leu Thr Asn Asp Gln Leu Arg Phe Gly Trp Ala Leu Val Ala Ser Arg

325 330 335

Ser Ala Gly Ala Pro Leu Tyr Phe Asn Arg Pro Val Gly Ser Gly Gly

340 345 350

Thr Asn Pro Gln Phe Ala Glu Val Ser Gln Leu Gly Asp Ala Gly Asp

355 360 365

Gly Met Trp Lys Asp Lys Ala Val Val Gln Val Asn Lys Phe His Asn

370 375 380

Lys Met Asp Gly Asn Ala Glu Tyr Leu Arg Asn Cys Pro Asn Asn Gly

385 390 395 400

Ser Cys Leu Met Val Glu Arg Tyr Ala Lys Asp Gly Gln Ser Ala Asn

405 410 415

Asp Gly Val Val Ile Thr Asn Met Gly Gly Asp Thr Ser Leu Ala Gly

420 425 430

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

435 440 445

Gly Ala Ile Thr Val Ser Gly Gly Lys Ile Thr Ala Gly Thr Ala Lys

450 455 460

Gly Asn Ala Val Ser Ala Tyr Tyr Ser Gln Glu Lys Ala Glu Gln Val

465 470 475 480

Ala Tyr Val Arg Leu

485

Claims (4)

1. one kind from the grand genomic alpha-amylase gene of animal excrement, it is characterized in that its nucleotide sequence is shown in SEQ ID NO.1.

2. one kind comprises the described recombinant expression vector from the grand genomic alpha-amylase gene of animal excrement of claim 1.

3. recombinant bacterial strain with the recombinant expression vector transformed host cell gained of the described alpha-amylase gene of claim 2.

4. one kind from the grand genomic α-Dian Fenmei of animal excrement, it is characterized in that its aminoacid sequence is shown in SEQ ID NO. 2.

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