CN103710325A - Alpha-amylase AmyASS and application thereof in raw starch degradation - Google Patents
Alpha-amylase AmyASS and application thereof in raw starch degradation Download PDFInfo
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- 102000004139 alpha-Amylases Human genes 0.000 title claims abstract description 82
- 229940024171 alpha-amylase Drugs 0.000 title claims abstract description 82
- 229920002472 Starch Polymers 0.000 title claims abstract description 70
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- 230000015556 catabolic process Effects 0.000 title claims description 13
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- 238000000034 method Methods 0.000 claims abstract description 22
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 19
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- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
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- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 235000003969 glutathione Nutrition 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
- C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01001—Alpha-amylase (3.2.1.1)
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Abstract
The invention discloses an alpha-amylase, the gene sequence of which is shown as SEQ ID NO. 2; the amino acid sequence coded by the gene sequence is shown as SEQ ID NO. 1; the gene sequence SEQ ID NO. 2 can successfully express the alpha-amylase in the form of inclusion body in the escherichia coli. The alpha-amylase obtained by the inclusion body renaturation method has the protein purity of more than 85 percent and the renaturation rate of more than 50 percent; the alpha-amylase can be used for quickly degrading raw starch, and is particularly suitable for degrading rice raw starch.
Description
Technical field
The present invention relates to biological technical field, specifically recombinant expressed, the renaturing inclusion bodies of the inclusion body form of the α-amylase AmyASS in a kind of marine bacteria source obtain α-amylase and the application in the raw starch degradation of rice thereof with catalytic activity.
Background technology
α-amylase is a kind ofly on α-Isosorbide-5-Nitrae glycosidic link, to cut off sugar chain, the enzyme of degraded starch.α-amylase at each industrial circle as brewageed, fiber, pharmacy, washing composition and foodstuffs industry has a wide range of applications.Natural raw starch granules has complicated fine and close structure.Glucose molecule forms long-chain by glycosidic link, and long-chain is folded into crystallization and unformed two kinds of structures, and then these two kinds of structures interweave and form natural raw starch granules.Therefore, natural raw starch granules can not directly be degraded by common starch degrading enzyme.Traditional starch degradation all needs to comprise starch pasting and amylorrhexis two steps.Only have the structure of first utilizing high temperature gelatinization to destroy raw starch granules, impel farinose crystalline texture to open, namely raw starch becomes ripe starch, and starch long-chain could effectively be cut off by starch degrading enzyme.Starch pasting is a very process for power consumption, therefore, if adopt a kind of special farinose amylase of directly degrading to degrade to starch, save starch pasting link, energy efficient, reduces production costs greatly, traditional technology can also be united two into one, shorten the treatment time.It is active that approximately 10% α-amylase has the raw starch degradation of this uniqueness, and their overwhelming majority are from terrestrial life.These only have three α-amylase from marine bacteria to be in the news to have raw starch degradation active so far, are respectively from Bacillus sp.ALSHL3 and B.aquimaris MKSC6.2 and from the α-amylase (AmyP) of belonging to of the grand genomic library in ocean of the most newly-established glycoside hydrolase subfamily GH13_37.
Although had been found that the α-amylase much with raw starch degradation ability, these α-amylase are mainly all that raw wheat starch and Treatment are had to good degradation capability.Rice is the staple food crop of our country in Southeast Asia, and Starch rice is with a wide range of applications in industries such as food, chemical industry and medicine.But the Study on degradation of Starch rice and practical application are far from, wheat starch and W-Gum are deeply.Important reason is that to have the α-amylase of uncooked rice starch degradation ability more rare.In current bibliographical information, the raw starch of rice that only has altogether 10 α-amylase directly to degrade natural.But the degradation efficiency of these enzymes is low, and need above long-time reaction in 24 hours.
Summary of the invention
The object of this invention is to provide a kind of can advantage the farinose α-amylase of degraded rice, the weak point existing to make up prior art.
The gene order that the invention provides the described α-amylase of coding, sequence is SEQ ID NO:2; The aminoacid sequence of this gene order coding is SEQ ID NO:1.This aminoacid sequence only than the aminoacid sequence of sequence number YP_001143410 in NCBI few 26 amino acid of N end, similarity is 98.8%; But the gene order similarity that gene order SEQ ID NO:2 is corresponding with the aminoacid sequence of sequence number YP_001143410 in NCBI is only 64.5%.This gene order SEQ ID NO:2 can successfully give expression to the α-amylase of inclusion body form in intestinal bacteria.
The invention provides the recombinant plasmid of expressing above-mentioned α-amylase inclusion body, is by gene order, to be SEQ ID NO:2 nucleotide fragments is inserted in expression vector pColdIII.
The invention provides and carry the recombinant bacterium of expressing above-mentioned α-amylase inclusion body.
The invention provides the abduction delivering condition that above-mentioned recombinant bacterium gives expression to α-amylase inclusion body.
The invention provides above-mentioned α-amylase renaturing inclusion bodies method.Make the α-amylase inclusion body of lifeless matter activity change the α-amylase with catalytic activity into, the Zulkovsky starch of take can detect enzyme during as substrate and live.The washings that comprises inclusion body, the composition of lysate and dialyzate, and the concentration of the temperature in whole renaturation process and the front α-amylase of renaturation.
Refolding method:
1) by washings washing 1-2 time for α-amylase inclusion body, deionized water wash 1 time, described washings consists of final concentration 50mM Tris-HCl buffer, the final concentration 5mM EDTA of pH8.0, and final concentration 1%(v/v) Triton X-100;
2) the α-amylase inclusion body after step 1) washing is dissolved with lysate, making the protein concentration after solubilization of inclusion bodies is 280-320 μ g/ml, described lysate consist of final concentration 6M GdmCl, the final concentration 100mM Tris-HCl buffer of pH6.0, final concentration 100mM NaCl, final concentration 100mM dithiothreitol and final concentration 1mM EDTA; Then under 4 ℃ of conditions, place 24 hours, therebetween every concussion in 3 hours once, to promote the dissolving of inclusion body; After dissolving, centrifugal 20 minutes of 12000r/min, removes the inclusion body precipitation that still cannot dissolve on a small quantity, gets supernatant liquor;
3) by step 2) supernatant liquor that obtains packs dialysis tubing into, carries out dialysis renaturation; Dialysis adopts 4 kinds of different dialyzates, every 12 hours, changes a kind of dialyzate, and whole dialysis procedure is carried out at 4 ℃; The dialyzate using for the first time consists of the final concentration 50mM Tris-HCl buffer of pH8.0, final concentration 10% (v/v) glycerine, final concentration 50mM NaCl, final concentration 0.5mM EDTA and final concentration 4M GdmCl; The dialyzate using for the second time consists of final concentration 50mM Tris-HCl buffer, final concentration 10% (v/v) glycerine, final concentration 50mM NaCl, final concentration 0.5mM EDTA and the final concentration 2M GdmCl of pH8.0; The dialyzate using for the third time consists of final concentration 50mM Tris-HCl buffer, final concentration 10% (v/v) glycerine, final concentration 50mM NaCl, final concentration 0.5mM EDTA, final concentration 1M GdmCl, final concentration 2%L-arginine, final concentration 1mM GSSG and the final concentration 5mM GSH of pH8.0; The dialyzate using for the 4th time consists of final concentration 50mM Tris-HCl buffer, final concentration 10% (v/v) glycerine, final concentration 50mM NaCl, final concentration 0.5mM EDTA, final concentration 2%L-arginine, final concentration 1mM GSSG and the final concentration 5mM GSH of pH8.0; After dialysis finishes, protein liquid in dialysis tubing is taken out, centrifugal 20 minutes of 12000r/min, 4 ℃, remove a small amount of inclusion body precipitation that still cannot renaturation, gets supernatant and be the α-amylase of having recovered catalytic activity.
The above-mentioned α-amylase of the present invention various raw starch that can be used for degrading, as uncooked rice starch, raw wheat starch, Treatment, raw potato starch, raw pea starch and raw green starch, when degraded uncooked rice starch, its activity is higher than the more than a times of the raw starch activity of other kinds.
During the raw starch of α-amylase degraded of the present invention: the pH value of the raw starch reaction system of degraded is 4.5-9.5, and optimal pH is 6.0; Farinose concentration is 12-25%, and optimal concentration is 12%; Temperature of reaction is 10-70 ℃, and optimum temperuture is 40 ℃; Reaction times is 6 hours.The CaCl that contains final concentration 5mM in reaction system
2time, when substrate is the raw starch of rice, it is 230% that its enzyme work significantly improves.
Accompanying drawing explanation
Fig. 1 has represented the pColdIII-AmyASS plasmid map of α-amylase of the present invention
Fig. 2 has represented the reaction pH curve of α-amylase of the present invention
Fig. 3 has represented the temperature of reaction curve of α-amylase of the present invention
Temperature-stable linearity curve when Fig. 4 has represented 40 ℃ of α-amylase of the present invention
Fig. 5 has represented that α-amylase of the present invention is to various farinose degrading activity comparison diagrams
Fig. 6 has represented the degrading activity comparison diagram of α-amylase of the present invention to the uncooked rice starch of different concns
Fig. 7 has represented the degrade time curve of 12% uncooked rice starch of α-amylase of the present invention
Fig. 8 has represented that α-amylase of the present invention is at the CaCl of different concns
2time the degrade active comparison diagram of 12% uncooked rice starch
Embodiment
The chemical reagent using in following examples is existing known reagent, and chemical reagent abbreviation table is as follows: EDTA(disodium EDTA); Triton X-100(Triton X-100) GdmCl(Guanidinium hydrochloride); NaCl(sodium-chlor); Dithiothreitol(DTT, dithiothreitol (DTT)) L – arginine(L-arginine); GSSG(Sleep-promoting factor B); GSH(reduced glutathion).
Embodiment 1
The gene of α-amylase AmyASS is synthetic
According to the pathogenic bacteria aeromonas salmonicida of the marine fishes of announcing in NCBI, kill the full genomic information of salmon subspecies (Aeromonas salmonicida ssp.salmonicida), find one " supposition glycoside hydrolase family protein " (aminoacid sequence YP_001143410).The amino acid of this AmyASS albumen and the α-amylase AmyP(Liu et al.2012 of experimental identification) similarity is 50%, is presumed to a α-amylase.26 amino acid that are predicted as signal peptide of AmyASS albumen n end are removed, and redesigned gene order according to colibacillary codon preference.This gene order is only 64.5% with gene order (gene order NC_009348.1) similarity that " supposition glycoside hydrolase family protein " announced in NCBI.At these gene two ends, add NdeI and XhoI restriction site.The text sequence of this gene is delivered to biotech firm (Sheng Gong bio tech ltd, Shanghai) synthetic.It is upper that synthetic AmyASS gene order is cloned in carrier pUC57, and insertion point is T-A clone, and recipient bacterium is e.colistraindh5α.Wherein the nucleotides sequence of α-amylase AmyASS of the present invention is classified SEQ ID NO:2 as, and aminoacid sequence is SEQ ID NO:1.
α-amylase AmyASS inclusion body recombinant expressed
The pUC57 carrier that comprises α-amylase AmyASS gene and cold shock shock expression vector pCold III have been carried out respectively, after the double digestion in NdeI and XhoI site, AmyASS gene being connected to pCold
on body, obtain recombinant expression plasmid pColdIII-AmyASS, as shown in Figure 1.Plasmid pColdIII-AmyASS is proceeded to e. coli bl21 cell, in the LB liquid nutrient medium that contains 50ug/ml penbritin, 200r/min, 37 ℃ of cultivations.When the OD600 of intestinal bacteria nutrient solution reaches 0.5 left and right, be cooled to 16 ℃ by nutrient solution is hurried, and add 1mM IPTG, then 16 ℃, 120rpm incubated overnight.In this culturing process, the α-amylase AmyASS of inclusion body form will be produced.8000r/min collects the Bacillus coli cells of 16 ℃ of incubated overnight for centrifugal 5 minutes, and intestinal bacteria are resuspended in to 50mM Tris-HCl buffer(pH8.0), ultrasonication, until muddy Bacillus coli cells suspension becomes limpid liquid.Centrifugal 5 minutes of 3000r/min, gets supernatant and abandons precipitation.Supernatant liquid centrifugal 20 minutes at 12000r/min, the precipitation of acquisition is α-amylase AmyASS inclusion body.
Attempted the method that various optimum combinations are expressed, the gene order that all cannot realize this α-amylase AmyASS is the soluble proteins with catalytic activity at expression in escherichia coli.This gene order can only be expressed as the inclusion body of catalytically inactive.In addition, the gene order (gene order NC_009348.1) that " supposition glycoside hydrolase family protein " announced in NCBI is cloned in expression vector pET20b and pColdIII, all cannot go out any albumen at expression in escherichia coli.
The renaturation of α-amylase AmyASS inclusion body
Renaturing inclusion bodies refers to the albumen of the inclusion body form of lifeless matter activity is being changed into bioactive albumen.By α-amylase AmyASS washings (50mM Tris-HCl buffer for inclusion body, pH8.0,5mM EDTA, 1%Triton X-100) washing is 2 times, deionized water wash 1 time, dissolves inclusion body with lysate, requires the protein concentration after solubilization of inclusion bodies to be about 300 μ g/ml, according to this protein concentration requirement, add a certain amount of lysate.The composition of lysate is 6M GdmCl, 100mM Tris-HCl buffer, pH6.0, pH6.0,100mM NaCl, 100mM dithiothreitol, 1mM EDTA.Then under 4 ℃ of conditions, place 24 hours, therebetween every concussion in 3 hours once, to promote the dissolving of inclusion body.After dissolving, centrifugal 20 minutes of 12000r/min, removes the inclusion body precipitation that still cannot dissolve on a small quantity, gets supernatant, packs dialysis tubing into, carries out dialysis renaturation.Adopted altogether 4 kinds of different dialyzates, every 12 hours, changed a kind of dialyzate, whole dialysis procedure is carried out at 4 ℃.The dialyzate composition using is for the first time 50mM Tris-HCl buffer(pH8.0), 10% (v/v) glycerine, 50mM NaCl, 0.5mM EDTA and 4M GdmCl; The dialyzate composition using is for the second time 50mM Tris-HCl buffer(pH8.0), 10% (v/v) glycerine, 50mM NaCl, 0.5mM EDTA and 2M GdmCl; The dialyzate composition using is for the third time 50mM Tris-HCl buffer(pH8.0), 10% (v/v) glycerine, 50mM NaCl, 0.5mM EDTA, 1M GdmCl, 2%L-arginine, 1mM GSSG and 5mM GSH; The dialyzate composition using for the 4th time is 50mM Tris-HCl buffer(pH8.0), 10% (v/v) glycerine, 50mM NaCl, 0.5mM EDTA, 2%L-arginine, 1mM GSSG and 5mM GSH.After dialysis finishes, protein liquid in dialysis tubing is taken out, centrifugal 20 minutes of 12000r/min, 4 ℃, remove a small amount of inclusion body precipitation that still cannot renaturation, gets supernatant and be the α-amylase AmyASS that has recovered catalytic activity.
The α-amylase AmyASS that adopts aforesaid method to obtain, purity of protein reaches more than at least 85%, and renaturation yield reaches more than at least 50%.
The determination of activity of α-amylase
The activity of α-amylase adopts 3,5-dinitrosalicylic acid (DNS) method to measure.The method is to detect the reducing sugar amount discharging after Starch Hydrolysis, and typical curve be take reducing sugar glucose as standard test.A standard reaction system comprises 1% Zulkovsky starch or the raw starch (raw starch concentration is according to different situations setting) of 40 μ l enzyme liquid and 560 μ l.Starch is all configured in 100mM citric acid-sodium citrate damping fluid (pH6.0).Enzyme reacts 10 minutes at 40 ℃ with starch, adds immediately 300 μ l DNS solution, is placed in the 0.3M Na of precooling
2cO
3in solution 5 minutes.When substrate is Zulkovsky starch, directly in boiling water, boil 15 minutes, then at A540nm, measure light absorption value.When substrate is made a living starch, need centrifugal 5 minutes of first 4 ℃ of 12000r/min, get supernatant and in boiling water, boil 15 minutes, then at A540nm, measure light absorption value.A standard enzyme unit alive (U) is defined as per minute and discharges the required enzyme amount of 1 μ mol reducing sugar.Take 1% Zulkovsky starch during as substrate, and the specific activity of enzyme is 45U/mg; Take 12% uncooked rice starch during as substrate, and the specific activity of enzyme is 30U/mg.
α-amylase AmyASS is to various farinose degrading activity comparisons
Uncooked rice starch, raw wheat starch, Treatment, raw potato starch, raw pea starch and raw green starch are configured in 100mM citric acid-sodium citrate damping fluid (pH6.0), and concentration is 4%.The method of describing according to embodiment 4 is measured enzyme and is lived, and result as shown in Figure 6.α-amylase AmyASS can degrade uncooked rice starch, raw wheat starch and raw green starch, the Treatment of can not degrading, raw potato starch and raw pea starch.α-amylase AmyASS is the highest to the activity of uncooked rice starch, is at least 5 times and 2.5 times of raw green starch of raw wheat starch.
The degraded of α-amylase AmyASS to uncooked rice starch
(1) action pH scope and the suitableeest action pH
By Zulkovsky starch, respectively with the damping fluid configuration of different pH, concentration is 1%.What pH4.0-6.0 adopted is 100mM Ning Meng Suan – sodium citrate buffer solution; What pH7.0-8.0 adopted is 100mM Lin acid hydrogen Er Na – phosphate sodium dihydrogen buffer solution; What pH9.0-10.0 adopted is 100mM Gan An Suan – sodium hydrate buffer solution.40 ℃ of methods of describing according to embodiment 4, measure enzyme and live, result as shown in Figure 2.α-amylase AmyASS works at pH4.5-9.5, and the suitableeest action pH is 6.0.
(2) operative temperature scope and the best use of temperature
The method of describing according to embodiment 4, the 10-70 ℃ of activity of measuring respectively α-amylase AmyASS, result as shown in Figure 3.α-amylase AmyASS all works within the scope of 10-70 ℃, and optimum temperature is 40 ℃.
(3) temperature stability
α-amylase AmyASS is added in 100mM citric acid-sodium citrate damping fluid (pH6.0), at 40 ℃, hatch 24 hours, during this period, take out at regular intervals 40 μ l enzyme liquid, the method for describing according to embodiment 4 is measured enzyme and is lived, and result as shown in Figure 4.α-amylase AmyASS 40 ℃ highly stable, after within 24 hours, hatching, it shows and is not less than 70% residual enzyme and lives.
(4) the farinose concentration of rice
The raw starch of rice is configured in 100mM citric acid-sodium citrate damping fluid (pH6.0), and concentration is respectively 4,8,12,16 and 20%, and the method for describing according to embodiment 4 is measured enzyme and lived, and result as shown in Figure 6.When the raw starch concentration of lower rice, the activity of α-amylase AmyASS increases along with the increase of the raw starch concentration of rice, and best degraded concentration is 12%.After the raw starch concentration of rice surpasses 12%, declining appears in the activity of α-amylase AmyASS, shows the retarding effect of high concentration of substrate.The raw starch concentration of the highest rice that α-amylase AmyASS can degrade is 25%.
(5) reaction times
The raw starch of rice is configured in 8ml 100mM citric acid-sodium citrate damping fluid (pH6.0), concentration is 12%, add 0.2% toluene to prevent microbial contamination, add 600 μ l enzyme liquid, 40 ℃ of reactions 8 hours, during this period, take out at regular intervals 600 μ l reaction solutions, add immediately 300 μ l DNS solution, the method for describing according to embodiment 4 is measured enzyme and is lived, and result as shown in Figure 7.In the starting stage of reaction, the reducing sugar amount that the raw starch of degraded rice produces is along with the time sharply increases; After 30 minutes, gathering way of reducing sugar amount obviously slows down; After 6 hours, reducing sugar amount remains unchanged.This shows, α-amylase AmyASS completed the hydrolysis to uncooked rice starch in 6 hours.
(6) add CaCl
2
The raw starch of rice is configured in 100mM citric acid-sodium citrate damping fluid (pH6.0), and concentration is 12%, adds respectively 0,3,5,8,10mM CaCl in reaction system
2, the method for describing according to embodiment 4 is measured enzyme and is lived, and result is as shown in Figure 8.CaCl
2existence can significantly improve the farinose activity of α-amylase AmyASS degraded rice, CaCl
2the best to add concentration be 5mM, the activity of α-amylase AmyASS at least rises to 2.3 times when adding.
(7) effect
The raw starch solution of rice with 100mM citric acid-sodium citrate damping fluid (pH6.0) configuration 12%, adds 0.2% toluene to prevent microbial contamination, adds enzyme liquid, 40 ℃ of reactions 6 hours.Centrifugal 5 minutes of 8000r/min, gets supernatant and is the farinose hydrolysate of rice.The analysis of hydrolysate adopts thin layer chromatography to carry out.Exhibition coating systems is the Virahol of volume ratio 3:1:1: ethyl acetate: H
2o.After chromatography finishes, evenly spray aniline-pentanoic phosphoric acid, 85 ℃ of baking sheets 10 minutes.Result shows that the farinose product of α-amylase AmyASS hydrolysed rice is glucose, maltose and trisaccharide maltose.
Claims (10)
1. a α-amylase, is characterized in that, described diastatic gene order is as shown in SEQ ID NO:2.
2. the amino acid of alpha-amylase gene sequence described in coding claim 1, is characterized in that, described aminoacid sequence is as shown in SEQ ID NO:1.
3. for express alpha-diastatic recombinant expression plasmid, this plasmid at least comprises gene claimed in claim 1.
4. recombinant plasmid according to claim 3, the carrier that it is characterized in that this recombinant plasmid is pCold III.
5. produce a recombinant bacterium for α-amylase inclusion body, in this recombinant bacterium, imported gene claimed in claim 2.
6. recombinant bacterium according to claim 5, is characterized in that this recombinant bacterium is e. coli strain bl21.
7. a refolding method for α-amylase inclusion body, is characterized in that the α-amylase inclusion body obtaining in claim 5 to carry out as follows:
1) by washings washing 1-2 time for α-amylase inclusion body, deionized water wash 1 time, described washings consists of final concentration 50mM Tris-HCl buffer, the final concentration 5mM EDTA of pH8.0, and final concentration 1%(v/v) Triton X-100;
2) the α-amylase inclusion body after step 1) washing is dissolved with lysate, making the protein concentration after solubilization of inclusion bodies is 280-320 μ g/ml, described lysate consist of final concentration 6M GdmCl, the final concentration 100mM Tris-HCl buffer of pH6.0, final concentration 100mM NaCl, final concentration 100mM dithiothreitol and final concentration 1mM EDTA; Then under 4 ℃ of conditions, place 24 hours, therebetween every concussion in 3 hours once, to promote the dissolving of inclusion body; After dissolving, centrifugal 20 minutes of 12000r/min, removes the inclusion body precipitation that still cannot dissolve on a small quantity, gets supernatant liquor;
3) by step 2) supernatant liquor that obtains packs dialysis tubing into, carries out dialysis renaturation; Dialysis adopts 4 kinds of different dialyzates, every 12 hours, changes a kind of dialyzate, and whole dialysis procedure is carried out at 4 ℃; The dialyzate using for the first time consists of the final concentration 50mM Tris-HCl buffer of pH8.0, final concentration 10% (v/v) glycerine, final concentration 50mM NaCl, final concentration 0.5mM EDTA and final concentration 4M GdmCl; The dialyzate using for the second time consists of final concentration 50mM Tris-HCl buffer, final concentration 10% (v/v) glycerine, final concentration 50mM NaCl, final concentration 0.5mM EDTA and the final concentration 2M GdmCl of pH8.0; The dialyzate using for the third time consists of final concentration 50mM Tris-HCl buffer, final concentration 10% (v/v) glycerine, final concentration 50mM NaCl, final concentration 0.5mM EDTA, final concentration 1M GdmCl, final concentration 2%L-arginine, final concentration 1mM GSSG and the final concentration 5mM GSH of pH8.0; The dialyzate using for the 4th time consists of final concentration 50mM Tris-HCl buffer, final concentration 10% (v/v) glycerine, final concentration 50mM NaCl, final concentration 0.5mM EDTA, final concentration 2%L-arginine, final concentration 1mM GSSG and the final concentration 5mM GSH of pH8.0; After dialysis finishes, protein liquid in dialysis tubing is taken out, centrifugal 20 minutes of 12000r/min, 4 ℃, remove a small amount of inclusion body precipitation that still cannot renaturation, gets supernatant and be the α-amylase of having recovered catalytic activity.
8. α-amylase according to claim 1 or the application of the α-amylase with catalytic activity according to claim 7 in raw starch degradation.
9. the application of the α-amylase with catalytic activity that obtained according to Claim 8 in raw starch degradation, is characterized in that, described raw starch comprises uncooked rice starch, raw wheat starch and raw green starch.
10. the α-amylase with catalytic activity obtaining according to the claim 7 farinose method of degrading, is characterized in that: the pH value of the raw starch reaction system of degraded is 4.5-9.5; Farinose concentration is 12-25%; Temperature of reaction is 10-70 ℃; Reaction times is no more than 6 hours; The CaCl that contains final concentration 5mM in reaction system
2.
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| CN106566819A (en) * | 2016-06-03 | 2017-04-19 | 哈尔滨工业大学（威海） | Gene cloning, expression and application of low-temperature halophilic alpha-amylase |
| CN109679937A (en) * | 2019-01-07 | 2019-04-26 | 安徽大学 | A kind of produced amylolysis enzyme with high specific enzyme activity power, its encoding gene and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106566819A (en) * | 2016-06-03 | 2017-04-19 | 哈尔滨工业大学（威海） | Gene cloning, expression and application of low-temperature halophilic alpha-amylase |
| CN106566819B (en) * | 2016-06-03 | 2020-04-14 | 哈尔滨工业大学（威海） | Gene cloning, expression, separation and purification method of low-temperature halophilic α -amylase |
| CN109679937A (en) * | 2019-01-07 | 2019-04-26 | 安徽大学 | A kind of produced amylolysis enzyme with high specific enzyme activity power, its encoding gene and its application |
| CN109679937B (en) * | 2019-01-07 | 2022-06-07 | 安徽大学 | Raw starch hydrolase with high specific enzyme activity, encoding gene and application thereof |
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