CN101531987A - Medium temperature Alpha-amylase high yielding strain and building method thereof - Google Patents
Medium temperature Alpha-amylase high yielding strain and building method thereof Download PDFInfo
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- CN101531987A CN101531987A CN200910067922A CN200910067922A CN101531987A CN 101531987 A CN101531987 A CN 101531987A CN 200910067922 A CN200910067922 A CN 200910067922A CN 200910067922 A CN200910067922 A CN 200910067922A CN 101531987 A CN101531987 A CN 101531987A
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Abstract
The invention relates to a medium temperature Alpha-amylase strain and a building method thereof. Bacillus subtilis AS 1.108 medium temperature Alpha-amylase gene obtained by PRC is cloned to bacillus subtilis expression plasmid pWB980 and is switched to WB600 to facilitate medium temperature Alpha-amylase to obtain high expression. The medium temperature Alpha-amylase gene has 1431 basic groups and codes 447 amino acids. The molecular mass of recombinase is 49.1KDa by SDS-PAGE detection, the optimal operation temperature and pH value are 60 DEG C and 6.0 respectively. The activity of the enzyme is about 2 times of the activity of the original strain, thus the problems of stink generated by the original strain and long fermentation period are solved. The method not only saves grains for industrial uses, reduces cost and provides better condition for deep processing of starch material, but also greatly reduces energy consumption and better suits industrialized production and application, thus having important economic benefits and obvious social benefits.
Description
Technical field
The invention belongs to bioengineering field, especially a kind of in warm α-Dian Fenmei superior strain and construction process thereof.
Background technology
α-Dian Fenmei (α-1,4-glucan-glucanhydrolase EC 3.2.1.1) is called liquefaction type amylase again, and it can cut α-1,4 glycosidic link arbitrarily from the inside of starch molecule, thereby produces the less Star Dri 5 of molecular weight ratio.Starch is under the effect of α-Dian Fenmei, and starch molecule is degraded rapidly, and viscosity degradation is promptly finished liquefaction.α-Dian Fenmei has sizable commercial value, is widely used in starch deep processing industries, alcohol industry, brewing industry, citric acid industry, monosodium glutamate and mashing industry, pharmaceutical industry and textile industry.α-Dian Fenmei can be produced by microbial fermentation, also can extract by plant, animal, at present on the industrial production mostly with microbe fermentation method scale operation α-Dian Fenmei.Bacillus subtilus, Bacillus licheniformis, stearothermophilus bud pole bacterium, aspergillus oryzae, aspergillus niger etc. all are that α-Dian Fenmei with practical value produces bacterium, and the middle temperature α-Dian Fenmei of being produced by subtilis AS 1.108 is widely used in starch deep processing industry.
At present, the main flow direction in the existing heavy industrialization starch hydrolysis process carries out moment high-temperature injection liquefaction for using high temperature resistant α-Dian Fenmei to starch, and the internal temperature of starch wine with dregs reaches 105-110 ℃ during liquefaction.Its application method is: 1, in the brewage process, treat that auxiliary material and water mix after, once add amylase, heat up rapidly, at 95-97 ℃, about insulation 30min.2, in Alcohol Production, add amylase, pH6.5-7.0 stirs evenly the back with being pumped into steam cooker or continuously cooking well heater, and Controllable Temperature is built in 100 ± 5 ℃, and the time is 100min, cooling back saccharification.3, when monosodium glutamate, Dian Fentang sector application, adjust pH to 6.0-6.5, add amylase.As adopt liquefaction at interval, in liquefied pot, can be warming up to 100 ± 5 ℃ rapidly, more than 95-100 ℃ of insulation 30min; As adopting steam ejection liquefaction, injection temperature can be about 105 ℃, and at 95 ℃ of insulation 60-120min.High temperature resistant α-Dian Fenmei is because its Heat stability is good is suitable for liquefying starch at high temperature, and industries such as food, pharmacy therefore are widely used.
But, in hydrolytic process, owing to need long-time insulation, in hydrolysis and fermenting process, be sizable to the consumption of industrial electro and vapour.Current, China's energy-saving and emission-reduction work enters the key stage, as a vital task in China's Eleventh Five-Year Plan, has been not only an action target of government, but also be the social responsibility that each enterprise ought to bear, allow the city resident can obtain a living environment preferably.Energy-saving and emission-reduction are a human the only way which must be passed that solves environmental problem especially.
By tackling key problem, for China unit's leavened prod water loss, energy consumption are significantly reduced, at the defective that zymin exists at aspects such as industrial application, intelligent evaluation technique, research biological enzyme directional transformation and molecular designing technology, research enzyme gene efficient expression technology, enzyme production downstream gordian technique and the utilisation technology of enzyme engineering in starch refine dsugar, biological products production etc. of carrying out exploitation, research enzyme genescreen and the enzyme function of new product specific enzymes preparation are extremely important.
Therefore, it is imperative to adopt in the low pressure ejector will heating-type warm continuous steaming-boiling technology equipment that starch is hydrolyzed.Bacillus subtilus AS 1.108 α-Dian Fenmei are the widest a kind of efficient liquefaction type amylase of the maximum purposes of China's output, it is by the high Bacillus subtilus of resistance toheat enlarged culturing step by step after mutagenesis, the Powdered zymin frowzy of a kind of terra brown that obtains through purification again, main effect is the liquefaction of the starchiness in the raw material can be dextrin, it is comparatively stable below 60 ℃, optimum temperature 55-60 ℃, between 70-90 ℃ along with raise its speed of response of temperature is accelerated, but inactivation is also accelerated, react 10min or 90 ℃ of 5min that work down down at 75 ℃, enzyme activity loses seldom.But when 75 ℃ of reaction 30min, enzyme activity forfeiture 1/3; When working 30min under 90 ℃, enzyme activity is total loss almost.Enzyme is comparatively stable at pH6.0-7.0, the suitableeest action pH 6.0-6.5, and the following inactivation of pH5.0 is more serious.
Temperature-amylase during recently people use gradually, because its optimum temperature is about 50-70 ℃, so it has activity when starch pasting, inactivation gradually then in the process of baking is finally baking loss of activity when finishing; And α-Dian Fenmei can generate the dextrin of the polymerization degree at 4-9 for hydrolyzed starch in the course of processing, and these dextrin also have resistance to deterioration.But, now in warm α-Dian Fenmei only can from some few microorganisms, extract.In starch industry, because α-Dian Fenmei has stronger hydrolysis ability to starch under optimum conditions, the condition of control reaction can be controlled the percent hydrolysis of starch, thereby starch is hydrolyzed into cavernous porous-starch.Gelatinization takes place in starch under hot conditions, therefore produce warm α-Dian Fenmei in the many employings of porous-starch; In addition, α-Dian Fenmei and other amylase such as collaborative use such as saccharifying enzyme, Pullulanase can be improved reaction efficiency and starch pore-forming effect.Preparation of Porous Starch adopts α-Dian Fenmei and other enzyme concerted reaction more now, and when being the textiles destarch of slurry with starch, the silk that middle temperature α-Dian Fenmei is particularly useful for non-refractory is thick, the desizing of chemical fibre cotton woolen knitwear.
China has made extensive work to the research of bacterial.Nineteen sixty-five, China began using Bacillus subtilis AS1.108 production α-Dian Fenmei, fermentation unit is about 200U/mL, later on through protracted and unremitting efforts, He Jichun etc. set out with AS 1.108 variants 209, through UV and NTG complex mutation, obtain mutagenic fungi 8a5, produce the strain of setting out of enzyme activity ratio and improve 17-35%, and having, product enzyme Zao advantage faster than former strain growth, pilot scale and industrial scale alpha-amylase activity reach 529U/mL and 505U/mL, remarkable in economical benefits respectively.
China's zymin industry in recent years is flourish, and variety yield also constantly increases, but with external zymin industry one section gap is arranged more still.Warm α-Dian Fenmei fermentation level reaches 900U/mL (measuring down for 60 ℃) in external, and warm α-Dian Fenmei fermentation level is 300-500U/mL in China, therefore need badly and improve China's α-Dian Fenmei fermentation level, particularly in order to save grain for industrial uses, just need the utilization genetic engineering means, the efficiently expressing of warm α-Dian Fenmei in the acquisition is to significantly improve economic benefit and social benefit.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, provide a kind of in warm α-Dian Fenmei bacterial strain and construction process thereof; Be to be cloned on the subtilis expression plasmid pWB980 through warm alpha-amylase gene among the subtilis AS 1.108 of PCR acquisition, and change WB600 over to, warm α-Dian Fenmei obtains to efficiently express in making.
The technical scheme that the present invention takes is:
Warm α-Dian Fenmei superior strain in a kind of, warm alpha-amylase gene nucleotide sequence is as follows among its bacterium subtilis AS 1.108 that sets out:
SAETANKSNELTAPSIKSGTILHAWNWSFNTLKHNMKDIHDAGYTAIQTSPINQVKEGNQGDKSMSNWYWLYHPTSYQIGNRYLGTEQEFKEMCAAAEEYGIKVIVDAVINHTTSDYAAISNEVKSIPNWTHGNTQIKNWSDRWDVTQNSLLGLYDWNTQNTQVQSYLKRFLERALNDGADGFRFDAAKHIELPDDGSYGSQFWPNITNTSAEFQYGEILQDSASRDAAYANYMDVTASNYGHSIRSALKNRNLGVSNISHYASDVSADKLVTWVESHDTYANDDEESTWMSDDDIRLGWAVIASRSGSTPLFFSRPEGGGNGVRFPGKSQIGDRGSALFEDQAITAVNRFHNVMAGQHEELSNPNGNNQIFMNQRGSHGVVLANAGSSSVSINTATKLPDGRYDNKAGAGSFQVNDGKLTGTINARSVAVLYPDDIEIRCNTFFQ。
The construction process of warm α-Dian Fenmei superior strain the steps include: in a kind of
(1) acquisition and the construction of prokaryotic expression vector of warm alpha-amylase gene among the subtilis AS 1.108;
1. obtain: according to oneself the subtilis amylase gene of report among the genebank is foundation, the design primer, utilize PCR obtain in the mature peptide gene of warm α-Dian Fenmei;
Upstream primer P1 is: 5-CGCG
GATCCCTTACAGCACCGTCGATCAA-3, underscore partly are the BamHI restriction enzyme site;
Downstream primer P2:5-CCC
AAGCTTAC TCAATGGGGAAGAGAACCG-3, underscore partly are the HindIII restriction enzyme site;
Template is subtilis AS 1.108 genomic dnas, and the reaction conditions of its amplification is: 95 ℃ of pre-sex change 2min, and with 95 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 1min30s, and 30 circulations of increasing extend 20 with 72 ℃ again
Min, pcr amplification product downcut the purpose band through the 8g/L agarose gel electrophoresis, reclaim purifying, the mature peptide gene of warm α-Dian Fenmei in obtaining with the DNA test kit;
2. the structure of recombinant plasmid pET22b-amy and evaluation: after the mature peptide gene of warm α-Dian Fenmei and carrier pET22b use BamHI and HindIII double digestion in will obtaining, reclaim purifying enzyme with the DNA test kit respectively and cut product, the directed amy to pET22b that connects under the effect of T4DNA ligase enzyme, to connect the product electric shock is transformed in the DH5a competence bacterium, screening and culturing in LA (Amp) substratum, the picking positive colony, cultivate the back and extract plasmid, adopt BamHI and HindIII dibit to put single, double enzyme and cut evaluation, order-checking;
(2) clone and the expression of warm alpha-amylase gene among the subtilis AS1.108;
1.. order-checking obtains complete amylase mature peptide gene, and the redesign primer is changed restriction enzyme site;
Upstream primer P3 is: 5-CCC
AAGCTTAC CTTACAGCACCGTCGATCAA-3, underscore partly are the Hind1II restriction enzyme site;
Downstream primer P4 is: 5-CGCG
GATCCTCAATGGGGAAGAGAACCG-3, underscore partly are the BamHI restriction enzyme site;
Template is recombinant plasmid pET22b-amy; The reaction conditions of amplification is: 95 ℃ of pre-sex change 2min, with 95 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 1min30s, and 30 circulations of increasing are extended 20min with 72 ℃ again, pcr amplification product is through the 8g/L agarose gel electrophoresis, the purpose band is downcut, reclaim purifying, obtain the PCR purified product with the DNA test kit;
2.. the structure of recombinant plasmid pWB980-amy and evaluation: behind PCR purified product and carrier pWB980 usefulness BamHI and HindIII double digestion, reclaim purifying enzyme with the DNA test kit respectively and cut product, the directed amy to pWB980 that connects under the effect of T4DNA ligase enzyme, to connect the product chemical conversion goes in the WB600 competence bacterium, screening and culturing in LA (Kan) substratum, the picking positive colony is cultivated the back and is extracted plasmid, adopt BamHI and HindIII dibit to put single, double enzyme and cut evaluation, order-checking.
And the described product that will connect is transformed into the method that the electric shock in the DH5a competence bacterium transforms and is:
(1) in-80 ℃ of refrigerators, takes out competent cell and put thawing on ice, simultaneously electric revolving cup is also placed in precooling on ice;
(2) open the electric shock instrument, adjust to the Ec1 shelves, voltage is 1800V;
(3) above-mentioned connection product 10 μ L are mixed with competence bacterium 40 μ L, shake electric revolving cup on ice and be positioned at electric revolving cup bottom to guarantee cell and DNA suspension;
(4) wipe the water of condensation on the electric revolving cup and ice slag with paper handkerchief, put into electroporation, start the electricimpulse of pair cell;
(5) behind the end-of-pulsing, take out electric revolving cup as quickly as possible, add 900 μ LSOC nutrient solutions under the room temperature rapidly in electric revolving cup, change over to behind the mixing in the Ep pipe, in 37 ℃ of thermostat containers, cultivate 1h; Be coated on LA (Amp) flat board with the electricity of the cultivating 1h thing of changing the line of production, be inverted plate and in 37 ℃ of thermostat containers, cultivate 12-16h.
And, describedly will connect the method that product is transformed into the chemical conversion in the WB600 competence bacterium and be:
(1) activatory WB600 is forwarded in the 3ml LB substratum 37 ℃, 250r/min overnight incubation; Get the above-mentioned nutrient solution of 160 μ l and be forwarded to 8ml SPI substratum, 37 ℃, 250r/min is cultured to logarithmic growth latter stage;
(2) nutrient solution of getting 0.2ml step (1) acquisition is to 2ml SPII substratum, and 37 ℃, 100r/min cultivates 90min;
(3) add 20ul10mmol/L EGTA, 37 ℃, 100r/min cultivates 10min;
(4) be distributed into the every pipe of 0.5ml, each adds 5ul DNA, and 37 ℃, 250r/min cultivates 90min, gets bacterium liquid coating screening flat board.
And described SPI substratum is 50% glucose solution, 1% 100%CAYE for the SP salts solution adds 1% concentration, and wherein CAYE comprises 2% peptone and 10% yeast extract paste.
And described SPII substratum is the 50mmol/L CaCl in SPI substratum adding 1%
2Solution, 1% 250mmol/LMgCl
2Solution.
And, described AS1.108 amylase gene prokaryotic expression carrier pWB980, size is 1112bp, has the P43 promotor, sacB signal peptide sequence, kalamycin resistance sign.
Advantage of the present invention and positively effect are:
The present invention utilizes expression vector pWB980, has realized going warm alpha-amylase gene (amy) efficiently expressing in subtilis WB600 among the subtilis AS 1.108 of signal peptide first.This gene contains 1,431 base, 447 amino acid of encoding.Detect through SDS-PAGE, the molecular mass of recombinase (AMY) is 49.1KDa, and the suitableeest operative temperature and pH are respectively 60 ℃ and 6.0.The enzyme work of this enzyme is about 2 times of starting strain, and has solved a stink and the long difficult problem of fermentation period that starting strain produces during the fermentation.Not only saved grain for industrial uses, reduced cost, better condition is provided for the deep processing of starch material, and reduced the consumption of the energy greatly, more be applicable to industrialized production and application, not only have important economic benefit, also have obvious social.
Description of drawings
Fig. 1 is the structure synoptic diagram of alpha-amylase gene of the present invention and prokaryotic expression carrier pET22b;
Fig. 2 is the structure synoptic diagram of alpha-amylase gene of the present invention and prokaryotic expression carrier pWB980;
Fig. 3 measures graphic representation for recombinase optimum temperuture of the present invention;
Fig. 4 measures graphic representation for recombinase thermotolerance of the present invention;
Fig. 5 measures graphic representation for recombinase optimal pH of the present invention;
Fig. 6 measures graphic representation for recombinase pH stability of the present invention.
Embodiment
Below in conjunction with embodiment, the present invention is further described, and following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
Warm α-Dian Fenmei superior strain in a kind of, warm alpha-amylase gene nucleotide sequence is as follows among its bacterium subtilis AS1.108 that sets out:
SAETANKSNELTAPSIKSGTILHAWNWSFNTLKHNMKDIHDAGYTAIQTSPINQVKEGNQGDKSMSNWYWLYHPTSYQIGNRYLGTEQEFKEMCAAAEEYGIKVIVDAVINHTTSDYAAISNEVKSIPNWTHGNTQIKNWSDRWDVTQNSLLGLYDWNTQNTQVQSYLKRFLERALNDGADGFRFDAAKHIELPDDGSYGSQFWPNITNTSAEFQYGEILQDSASRDAAYANYMDVTASNYGHSIRSALKNRNLGVSNISHYASDVSADKLVTWVESHDTYANDDEESTWMSDDDIRLGWAVIASRSGSTPLFFSRPEGGGNGVRFPGKSQIGDRGSALFEDQAITAVNRFHNVMAGQHEELSNPNGNNQIFMNQRGSHGVVLANAGSSSVSINTATKLPDGRYDNKAGAGSFQVNDGKLTGTINARSVAVLYPDDIEIRCNTFFQ。
The construction process of warm α-Dian Fenmei superior strain the steps include: in a kind of
1. the acquisition and the order-checking of the gene of warm α-Dian Fenmei among the subtilis AS1.108
(1). according to the subtilis amylase gene of having reported among the genebank is foundation, the design primer, utilize PCR obtain in the mature peptide gene of warm α-Dian Fenmei.
Upstream primer P1 is: 5-CGC
GGATCCCTTACAGCACCGTCGATCAA-3 (underscore partly is the BamHI restriction enzyme site)
Downstream primer P2:5-CCC
AAGCTTAC TCAATGGGGAAGAGAACCG-3 (underscore partly is the HindIII restriction enzyme site).Template is a subtilis AS1.108 genomic dna; The reaction conditions of amplification is: 95 ℃ of pre-sex change 2min, with 95 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 1min30s, and 30 circulations of increasing are extended 20min with 72 ℃ again, pcr amplification product is through the 8g/L agarose gel electrophoresis, the purpose band is downcut, reclaim purifying with the DNA test kit, with warm α-Dian Fenmei mature peptide gene in obtaining;
(2). the structure of recombinant plasmid pET22b-amy and evaluation (Fig. 2): behind the PCR product and carrier pET22b usefulness BamHI and HindIII double digestion with purifying in the step (1), reclaim purifying enzyme with the DNA test kit respectively and cut product, the directed amy to pET22b that connects under the effect of T4DNA ligase enzyme, to connect the product electric shock is transformed in the DH5a competence bacterium, screening and culturing in LA (Amp) substratum, the picking positive colony, cultivate the back and extract plasmid, adopt BamHI and HindIII dibit to put single, double enzyme and cut evaluation, order-checking;
AS 1.108 amylase gene construction of prokaryotic expression vector methods, the method steps that this electric shock transforms is:
(1). in-80 ℃ of refrigerators, take out competent cell and put thawing on ice, simultaneously electric revolving cup is also placed in precooling on ice;
(2). open the electric shock instrument, adjust to the Ec1 shelves, voltage is 1800V;
(3). above-mentioned connection product 10 μ L are mixed with competence bacterium 40 μ L, shake electric revolving cup on ice and be positioned at electric revolving cup bottom to guarantee cell and DNA suspension;
(4). wipe the water of condensation on the electric revolving cup and ice slag with paper handkerchief, put into electroporation, start the electricimpulse of pair cell;
(5). behind the end-of-pulsing, take out electric revolving cup as quickly as possible, add 900 μ LSOC nutrient solutions under the room temperature rapidly in electric revolving cup, change over to behind the mixing in the Ep pipe, in 37 ℃ of thermostat containers, cultivate 1h; Be coated on LA (Amp) flat board with the electricity of the cultivating 1h thing of changing the line of production, be inverted plate and in 37 ℃ of thermostat containers, cultivate 12-16h.
2, the clone and the expression of the gene of warm α-Dian Fenmei among the subtilis AS1.108
(1). order-checking obtains complete amylase mature peptide gene, and the redesign primer is changed restriction enzyme site
Upstream primer P3 is: 5-CCC
AAGCTTAC CTTACAGCACCGTCGATCAA-3 (underscore partly is the HindIII restriction enzyme site)
Downstream primer P4 is: 5-CGC
GGATCCTCAATGGGGAAGAGAACCG-3 (underscore partly is the BamHI restriction enzyme site).Template is recombinant plasmid pET22b-amy; The reaction conditions of amplification is: 95 ℃ of pre-sex change 2min, and with 95 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 1min30s, and 30 circulations of increasing are extended 20min with 72 ℃ again, pcr amplification product downcuts the purpose band through the 8g/L agarose gel electrophoresis, reclaims purifying with the DNA test kit;
(2). the structure of recombinant plasmid pWB980-amy and evaluation (Fig. 3): behind the PCR product and carrier pWB980 usefulness BamHI and HindIII double digestion with purifying, reclaim purifying enzyme with the DNA test kit respectively and cut product, the directed amy to pWB980 that connects under the effect of T4DNA ligase enzyme, to connect product is transformed in the WB600 competence bacterium, screening and culturing in LA (Kan) substratum, the transformant that obtains and control strain corresponding points are connected on two LB (20 μ g/ml kantlex) flat boards that contain 1% Zulkovsky starch, cultivate 24h for 37 ℃.Get one flat plate I2/KI solution (0.088g I
220.176g KI1-1) dyeing, the big bacterium colony of picking transparent circle on another piece flat board adopts BamHI and HindIII dibit to put single, double enzyme and cuts evaluation, order-checking;
AS 1.108 amylase gene construction of prokaryotic expression vector, the method steps of chemical conversion is:
(1). activatory WB600 is forwarded in the 3ml LB substratum, 37 ℃, the 250r/min overnight incubation.Get 160 μ l nutrient solutions and be forwarded to 8ml SPI substratum [SP salts solution (0.2% (NH
4)
2SO
4, 1.4% K
2HPO
4, 0.6% KH
2PO
4, 0.02% MgsO
47H
2O, 0.1% Trisodium Citrate) adding 1% concentration is 50% glucose solution, 1% 100%CAYE (2% peptone, 10% yeast extract paste)] in, 37 ℃, 250r/min is cultured to logarithmic growth latter stage (about 5h).
(3). (the SPI substratum adds 1% 50mmol/L CaCl to 2ml SPII to get the 0.2ml nutrient solution
2Solution, 1% 250mmol/LMgCl
2Solution) in the substratum, 37 ℃, 100r/min cultivates 90min.
(4). add 20 μ l 10mmol/L EGTA, 37 ℃, 100r/min cultivates 10min.
(5). be distributed into the every pipe of 0.5ml, each adds 5 μ l DNA, and 37 ℃, 250r/min cultivates 90min, gets bacterium liquid coating screening flat board.
The expression of warm α-Dian Fenmei and zymin are made in the narration below:
At activation medium (beef powder 5.0g, NaCl 2.0g, yeast powder 2.0g, peptone 10g, thick starch 10g, glucose 50g (singly disappearing), agar powder 17g, H
2O 1000mL) goes up cultivation 36h, the single colony inoculation of picking subtilis pWB-amy/WB600 is to 50mL seed culture medium/250mL triangular flask (seed culture based formulas: beef powder 1.50g, yeast powder 1.50g, peptone 5.00g, potassium primary phosphate (anhydrous) 1.32g, dipotassium hydrogen phosphate 3.68g, glucose 1.00g, NaCl 3.00g, H
2O 1000mL), 37 ℃, 200r/min cultivates 16h.Be forwarded to 50mL fermention medium/250mL baffle flask (soybean cake powder 26g, cottonseed meal 21g, dipotassium hydrogen phosphate 28.8g, potassium primary phosphate (anhydrous) 6.7g, trisodium citrate 2.0g, corn steep liquor 20g, (NH by 2% inoculum size then
4)
2SO
45.0g, CaCl
20.45g, lactose 10g (singly disappearing), H
2O 1000mL), 42 ℃, 300r/min cultivates 96h.
Fermented liquid is centrifugal, removes thalline, decolorizing with activated carbon, and membrane filtration is removed small-molecule substance, and spraying drying or lyophilize obtain the powdery zymin.
Product performance are measured:
1. the mensuration of alpha-amylase activity
Enzyme activity unit definition: under 60 ℃, pH6.0 condition, 1min liquefaction 1mg Zulkovsky starch becomes the needed enzyme amount of dextrin, is 1 enzyme activity unit, represents with U/mL (U/g).(QB/T1803-93) is as follows for measuring method: 1. enzyme liquid preparation: be mixed with enzyme solution with damping fluid, its final enzyme concn is controlled within the 65U/mL-70U/mL scope.2. measure: (1) absorption Zulkovsky starch solution (20g/L) 20mL and phosphoric acid buffer (pH=6.0) 5mL are in test tube.Preheating 3min-5min in 60 ℃ of waters bath with thermostatic control.(2) add the good enzyme liquid 1.00mL to be measured of dilution, timing immediately shakes up, accurate response 5min.(3) drawing the 1.00mL reaction solution immediately moves in the test tube that 0.1mol/L HCl 0.5mL and the rare iodine liquid of 5mL are housed in advance and shakes up.(4) make blank with the mixed solution of 0.1mol/L HCl 0.5mL and the rare iodine liquid of 5mL, under the 660nm wavelength, with 10mm cuvette rapid test absorbancy (A).Table look-up according to absorbancy, try to achieve the concentration (C) of tested enzyme liquid.3. calculate: in X=C * N formula: the enzyme activity U/mL (U/g) of X-sample; The enzyme liquid concentration U/mL of C-test; N-dilution of sample multiple; Gained is the result represent to integer.
The fermented liquid of will recombinating, the centrifugal 10min of 12000r/min removes cell, measures enzyme in the supernatant liquor live (being designated as perienzyme lives).
. the research of α-Dian Fenmei character
With the centrifugal removal thalline of fermented liquid, decolorizing with activated carbon, membrane filtration are removed the concentrated enzyme liquid that obtains after the small molecules, carry out the research of zymologic property.
(1). temperature is to the influence of enzyme activity
1. the mensuration of this enzyme optimal reactive temperature: at differing temps (30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, 100 ℃), measure the enzyme of recombinase liquid under the condition of pH6.0 and live, the highest vigor is decided to be 100% (Fig. 4).
2. the thermal stability determination of this enzyme: recombinase liquid is placed respectively under the condition of differing temps (50 ℃, 60 ℃, 70 ℃, 80 ℃) pH6.0 and be incubated 2h, take out every 20min, measure its residual enzyme activity separately, with uninsulated enzyme liquid vigor under the relevant temperature is 100%, draws temperature-stable linearity curve (Fig. 5).
The optimum temperuture of recombinant alpha-amylases is 60 ℃, and enzyme work is more than 80% relatively between 50 ℃ to 70 ℃.50 to 70 ℃ of insulation 120min, enzyme is lived and is not lost substantially, and remnant enzyme activity remains on more than 80%, with the character basically identical of protoenzyme.
(2) .pH is to the influence of enzyme activity
1. the mensuration of this enzyme optimal reaction pH: the enzyme at the following mensuration recombinase liquid of 60 ℃ of different pH values (3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0) is lived, and the highest vigor is decided to be 100% (Fig. 6).
2. the pH of this enzyme stability is measured: recombinase liquid is incubated 60min at 60 ℃ under different pH (3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0) condition, measure its residual enzyme activity separately, with uninsulated enzyme activity under the corresponding pH is 100%, draws pH beta stability line (Fig. 7).
The optimal pH of original α-Dian Fenmei is 6.0, and between pH5.5-6.5, relatively enzyme work is more than 90%, the following complete deactivation of pH3.5.The recombinant alpha-amylases optimal pH is 6.0, and relative enzyme work reaches 80% when pH5.5.Original α-Dian Fenmei is more stable in pH 5.5-6.5 scope, and remnant enzyme activity is more than 80%, and recombinant alpha-amylases is more stable in pH 5.5-6.5 scope, and remnant enzyme activity is more than 80%.The result shows that the enzymatic property of original α-Dian Fenmei gained recombinant alpha-amylases after cloning does not change.
SEQUENCE?LISTING
＜110〉University Of Science and Technology Of Tianjin
＜120〉warm α-Dian Fenmei superior strain and construction process thereof in a kind of
<130>20090224
<160>5
<170>PatentIn?version?3.3
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＜213〉warm alpha-amylase gene nucleotide sequence among the subtilis AS 1.108
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＜213〉upstream primer P1
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<210>5
<211>28
<212>DNA
＜213〉downstream primer P4
<400>5
Claims (7)
1, warm α-Dian Fenmei superior strain in a kind of, it is characterized in that: warm alpha-amylase gene nucleotide sequence is as follows among its bacterium subtilis AS1.108 that sets out:
SAETANKSNELTAPSIKSGTILHAWNWSFNTLKHNMKDIHDAGYTAIQTSPINQVKEGN
QGDKSMSNWYWLYHPTSYQIGNRYLGTEQEFKEMCAAAEEYGIKVIVDAVINHTTSD
YAAISNEVKSIPNWTHGNTQIKNWSDRWDVTQNSLLGLYDWNTQNTQVQSYLKRFLE
RALNDGADGFRFDAAKHIELPDDGSYGSQFWPNITNTSAEFQYGEILQDSASRDAAYA
NYMDVTASNYGHSIRSALKNRNLGVSNISHYASDVSADKLVTWVESHDTYANDDEES
TWMSDDDIRLGWAVIASRSGSTPLFFSRPEGGGNGVRFPGKSQIGDRGSALFEDQAITA
VNRFHNVMAGQHEELSNPNGNNQIFMNQRGSHGVVLANAGSSSVSINTATKLPDGRY
DNKAGAGSFQVNDGKLTGTINARSVAVLYPDDIEIRCNTFFQ。
2, a kind of as claimed in claim 1 in the construction process of warm α-Dian Fenmei superior strain, it is characterized in that: the steps include:
(1) acquisition and the construction of prokaryotic expression vector of warm alpha-amylase gene among the subtilis AS 1.108;
1. obtain: according to the subtilis amylase base of having reported among the genebank is foundation, the design primer, utilize PCR obtain in the mature peptide gene of warm α-Dian Fenmei;
Upstream primer P1 is: 5-CGC
GGATCCCTTACAGCACCGTCGATCAA-3, underscore partly are the BamHI restriction enzyme site;
Downstream primer P2:5-CCC
AAGCTTAC TCAATGGGGAAGAGAACCG-3, underscore partly are the HindIII restriction enzyme site;
Template is subtilis AS 1.108 genomic dnas, the reaction conditions of its amplification is: 95 ℃ of pre-sex change 2min, with 95 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 1min 30s, 30 circulations of increasing, extend 20min with 72 ℃ again, pcr amplification product downcuts the purpose band through the 8g/L agarose gel electrophoresis, reclaim purifying, the mature peptide gene of warm α-Dian Fenmei in obtaining with the DNA test kit;
2. the structure of recombinant plasmid pET22b-amy and evaluation: after the mature peptide gene of warm α-Dian Fenmei and carrier pET22b use BamHI and HindIII double digestion in will obtaining, reclaim purifying enzyme with the DNA test kit respectively and cut product, the directed amy to pET22b that connects under the effect of T4 dna ligase, to connect the product electric shock is transformed in the DH5a competence bacterium, screening and culturing in LA (Amp) substratum, the picking positive colony, cultivate the back and extract plasmid, adopt BamHI and HindIII dibit to put single, double enzyme and cut evaluation, order-checking;
(2) clone and the expression of warm alpha-amylase gene among the subtilis AS 1.108;
1.. order-checking obtains complete amylase mature peptide gene, and the redesign primer is changed restriction enzyme site;
Upstream primer P3 is: 5-CCC
AAGCTTAC CTTACAGCACCGTCGATCAA-3, underscore partly are the HindIII restriction enzyme site;
Downstream primer P4 is: 5-CGC
GGATCCTCAATGGGGAAGAGAACCG-3, underscore partly are the BamHI restriction enzyme site;
Template is recombinant plasmid pET22b-amy; The reaction conditions of amplification is: 95 ℃ of pre-sex change 2min, with 95 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 1min 30s, and 30 circulations of increasing are extended 20min with 72 ℃ again, pcr amplification product is through the 8g/L agarose gel electrophoresis, the purpose band is downcut, reclaim purifying, obtain the PCR purified product with the DNA test kit;
2.. the structure of recombinant plasmid pWB980-amy and evaluation: behind PCR purified product and carrier pWB980 usefulness BamHI and HindIII double digestion, reclaim purifying enzyme with the DNA test kit respectively and cut product, the directed amy to pWB980 that connects under the effect of T4DNA ligase enzyme, to connect the product chemical conversion goes in the WB600 competence bacterium, screening and culturing in LA (Kan) substratum, the picking positive colony is cultivated the back and is extracted plasmid, adopt BamHI and HindIII dibit to put single, double enzyme and cut evaluation, order-checking.
3, the construction process of warm α-Dian Fenmei superior strain in according to claim 2 is characterized in that: the described product that will connect is transformed into the method that the electric shock in the DH5a competence bacterium transforms and is:
(1) in-80 ℃ of refrigerators, takes out competent cell and put thawing on ice, simultaneously electric revolving cup is also placed in precooling on ice;
(2) open the electric shock instrument, adjust to the Ec1 shelves, voltage is 1800V;
(3) above-mentioned connection product 10 μ L are mixed with competence bacterium 40 μ L, shake electric revolving cup on ice and be positioned at electric revolving cup bottom to guarantee cell and DNA suspension;
(4) wipe the water of condensation on the electric revolving cup and ice slag with paper handkerchief, put into electroporation, start the electricimpulse of pair cell;
(5) behind the end-of-pulsing, take out electric revolving cup as quickly as possible, add 900 μ LSOC nutrient solutions under the room temperature rapidly in electric revolving cup, change over to behind the mixing in the Ep pipe, in 37 ℃ of thermostat containers, cultivate 1h; Be coated on LA (Amp) flat board with the electricity of the cultivating 1h thing of changing the line of production, be inverted plate and in 37 ℃ of thermostat containers, cultivate 12-16h.
4, the construction process of warm α-Dian Fenmei superior strain in according to claim 2 is characterized in that: describedly will connect the method that product is transformed into the chemical conversion in the WB600 competence bacterium and be:
(1) activatory WB600 is forwarded in the 3ml LB substratum 37 ℃, 250r/min overnight incubation; Get the above-mentioned nutrient solution of 160 μ l and be forwarded to 8ml SPI substratum, 37 ℃, 250r/min is cultured to logarithmic growth latter stage;
(2) nutrient solution of getting 0.2ml step (1) acquisition is to 2ml SPII substratum, and 37 ℃, 100r/min cultivates 90min;
(3) add 20 μ l10mmol/L EGTA, 37 ℃, 100r/min cultivates 10min;
(4) be distributed into the every pipe of 0.5ml, each adds 5 μ l DNA, and 37 ℃, 250r/min cultivates 90min, gets bacterium liquid coating screening flat board.
5, the construction process of warm α-Dian Fenmei superior strain in according to claim 4, it is characterized in that: described SPI substratum is 50% glucose solution, 1% 100%CAYE for the SP salts solution adds 1% concentration, and wherein CAYE comprises 2% peptone and 10% yeast extract paste.
6, the construction process of warm α-Dian Fenmei superior strain in according to claim 4 is characterized in that: described SPII substratum is for adding 1% 50mmol/L CaCl at the SPI substratum
2Solution, 1% 250mmol/L MgCl
2Solution.
7, the construction process of warm α-Dian Fenmei superior strain in according to claim 2, it is characterized in that: described AS 1.108 amylase gene prokaryotic expression carrier pWB980, size is 1112bp, has the P43 promotor, the sacB signal peptide sequence, the kalamycin resistance sign.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102134560A (en) * | 2011-01-13 | 2011-07-27 | 中国药科大学 | Strain for efficiently expressing intermediate temperature alpha-amylase |
| CN105524873A (en) * | 2016-02-02 | 2016-04-27 | 河南工业大学 | Bacterial strain capable of producing alpha-amylase and acid alpha-amylase generated by bacterial strain |
| CN105567602A (en) * | 2016-02-02 | 2016-05-11 | 河南工业大学 | Fermentation and enzyme production method of Leclercia strain capable of producing acidic alpha-amylase |
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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 |
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2009
- 2009-02-24 CN CN200910067922A patent/CN101531987A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134560A (en) * | 2011-01-13 | 2011-07-27 | 中国药科大学 | Strain for efficiently expressing intermediate temperature alpha-amylase |
| CN102134560B (en) * | 2011-01-13 | 2012-12-19 | 中国药科大学 | Strain for efficiently expressing intermediate temperature alpha-amylase |
| CN105524873A (en) * | 2016-02-02 | 2016-04-27 | 河南工业大学 | Bacterial strain capable of producing alpha-amylase and acid alpha-amylase generated by bacterial strain |
| CN105567602A (en) * | 2016-02-02 | 2016-05-11 | 河南工业大学 | Fermentation and enzyme production method of Leclercia strain capable of producing acidic alpha-amylase |
| CN105567602B (en) * | 2016-02-02 | 2018-11-02 | 河南工业大学 | A kind of enzymatic production method of the Le kirschner bacterial strain of production acid alpha-amylase |
| 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 |
| CN105950528A (en) * | 2016-06-13 | 2016-09-21 | 江南大学 | Genetically engineered bacterium for producing linear malt oligosaccharide generating enzyme and application of genetically engineered bacterium |
| CN105950528B (en) * | 2016-06-13 | 2019-10-25 | 江南大学 | A kind of genetic engineering bacterium and its application for producing linear maltooligosacchaeides and generating enzyme |
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