CN102154140A - High-efficiency preparation method of beta-amylase - Google Patents
High-efficiency preparation method of beta-amylase Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title abstract description 9
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- 241000235058 Komagataella pastoris Species 0.000 claims abstract description 34
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Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to a high-efficiency preparation method of beta-amylase, belonging to the technical fields of enzyme engineering and biological engineering. The preparation method comprises the following steps: respectively positioning a sweet potato-derived beta-amylase gene 0BBA or a signal peptide-deleted beta-amylase gene 28BBA in four different secretion signal peptides (pPIC9K-A, pPIC9K-B, pPIC9K-C or pPIC9K-D) to construct 8 different recombinant plasmids, converting into two Pichia pastoris (GS115 or KM71) incapable of producing beta-amylase through an electroporation method, and screening to obtain the Pichia pastoris CCTCC NO:M2011014 which is converted from KM71 and contains pPIC9K-A-0BBA; carrying out high-density fermentation on the Pichia pastoris CCTCC NO:M2011014, wherein the enzyme activity of the fermentation liquid can be 500U/mL; and carrying out centrifugation, ultrafiltration, microfiltration and freeze-drying to obtain the beta-amylase, wherein the protein concentration of the finished product is up to 3-6g/L, and the enzyme activity is up to 2000U/g. The invention can be used for the high-efficiency production of beta-amylase for starch processing, sugar making and food processing industries.
Description
Technical field
The present invention relates to the pichia pastoris phaff genetic engineering bacterium and produce the method for beta-amylase, belong to enzyme engineering and technical field of bioengineering.
Background technology
Beta-amylase (EC 3.2.1.2), promptly (1 → 4)-alpha-glucan Fructus Hordei Germinatus lytic enzyme is a kind of excision enzyme, it is from the starch non-reducing end, press the hydrolysis successively of maltose unit, the Walden translocation reaction takes place simultaneously, make product change β-type maltose into by α-type.This enzyme can not the hydrolyzed starch bifurcation α-1,6 glycosidic links, 2 ~ 3 the glucosyl residue places of the decomposition meeting of starch before 1,6 key stop, so decomposing the product of amylose starch mainly is maltose, the product that decomposes amylopectin mainly is maltose and macromolecular β-limit dextrin.Beta-amylase extensively is present in the high plants such as barley, wheat, corn, soybean, sweet potato, and some microorganisms also can produce beta-amylase.
Beta-amylase has important use and is worth in industry such as grain processing, food mfg, fermentation industry and medicine.This enzyme is widely used in beer, maltose, and high maltose syrup, crystal maltitol etc. are the sugar refining technology of product with maltose, are mainly used in the conversion coefficient and the output capacity of further raising maltose; Beta-amylase also can be used for the starch liquefacation of fermentation industry and weaving, dye-printing desizing etc.In addition, this enzyme as digestive ferment, is used for poor appetite, maldigestion, mycogastritis etc. clinically.
Zhang Jian etc. have studied the extraction process condition of beta-amylase in the analysis for soybean powder, influence to beta-amylase extracted amount in the analysis for soybean powder such as tensio-active agent, reductive agent has been discussed respectively, and further optimized extraction conditions by orthogonal test, ultimate capacity improves 29.8%.Wang Hui is superfine to extract beta-amylase from the germination broad bean, with 1.0 g/L Na
2SO
4Being reductive agent, serves as to extract damping fluid with pH 6.5 potassium phosphate buffers, and it is better to extract germination broad bean beta-amylase effect.Ceng Ying etc. provide the preparation technology of high-activity beta-amylase, and the defatted soy flour vat liquor obtains the product of excellent property through ultrafiltration and concentration, secondary sedimentation and precipitation freeze thawing treatment.The method of extracting beta-amylase from analysis for soybean powder, sweet potato has been studied by Jiangsu Province Agriculture Science Institute processing of farm products institute, Hubei Province's agricultural process engineering Technical Research Center, Agricultural University Of Shenyang's Foodstuffs Academy, HeFei University of Technology etc., but only is confined to the laboratory.Zhengzhou jintudi Energy Technology Co., Ltd. proposes to extract the production route of beta-amylase first from the sweet potato cell, raw material obtains beta-amylase through cleaning, fragmentation, screenings separation, suction strainer, adsorption bleaching, filtration, membrane sepn.The beta-amylase that extracts from plant has characteristics such as enzyme activity height, thermotolerance is better, action pH value scope is wide, but this beta-amylase production technique is restricted by condition influence such as raw material, the place of production, weather and population resource.
At present, the beta-amylase vigor that China's microbial fermentation is produced is low, poor heat resistance, and the cost height is studied less relatively.
The pichia pastoris phaff expression system of this patent utilization has unique advantages such as the easy purifying of post-treatment product of high expression level, high stable, expressing protein, make up the gene recombined Pichia pastoris that produces beta-amylase, by its high density fermentation is had the favorable industrial using value.
Summary of the invention
One of the object of the invention provides a kind of bacterial strain that produces beta-amylase.
Two of the object of the invention provides the construction process of the pichia pastoris phaff genetic engineering bacterium of efficient secretory expression Ipomoea batatas beta-amylase.
Three of the object of the invention provides the method for utilizing pichia pastoris phaff genetic engineering bacterium fermentative preparation beta-amylase.
The structure and the fermentation method for producing thereof of the pichia pastoris phaff genetic engineering bacterium of a kind of efficient secretory expression beta-amylase of the present invention, it mainly is the beta-amylase gene that derives from Ipomoea batatas by reverse transcription PCR method clone, insert then and contain in the Pichia anomala expression plasmid of different secreting signal peptides, obtain different recombinant plasmids, recombinant plasmid is imported screening acquisition genetic engineering bacterium among the pichia pastoris phaff host.The genetic engineering bacterium that contains the unlike signal peptide, comprise pPIC9K self alpha factor homing sequence signal peptide A, acid phosphatase lipase gene signal peptide B, saccharase gene signal peptide C and Killer toxin gene signal peptide D, all can effectively mediate beta-amylase albumen from emiocytosis to fermented liquid.
For realizing technical purpose of the present invention, the present invention takes following technical scheme: a kind of bacterial strain that produces beta-amylase, classification called after pichia pastoris phaff RYP 20110110 has been deposited in Chinese typical culture collection center, deposit number CCTCC NO:M 2011014.
Described pichia pastoris phaff CCTCC NO:M 2011014, its construction step:
(1) utilize Trizol reagent to extract total RNA of Ipomoea batatas;
(2) be template with total RNA, under the effect of reverse transcription primer oligdT-18 and reversed transcriptive enzyme, a synthetic chain cDNA;
(3) design a pair of primer according to the Ipomoea batatas beta-amylase gene order of having announced, the primer is:
BBA-F: 5′-a
agatctccatggctccaatccccggt-3′，
BBA-R: 5′-a
agatctgctcctccttcaccttcag-3′；
With a chain cDNA is the template pcr amplification, obtains amplification and contains complete beta-amylase gene BBA, is connected into the Teasy carrier and obtains Teasy-BBA.With the Teasy-BBA plasmid is template, and pcr amplification contains the goal gene 0BBA of complete beta-amylase gene open reading frame sequence or removes the beta-amylase gene 28BBA of signal peptide coding region;
Described pcr amplification 0BBA, the primer is:
0BBA-F: 5′-atggctccaatccccggt-3′，
0BBA-R: 5′-tcaatcaaacgggtttgagccatc-3′；
Described pcr amplification 28BBA, the primer is:
28BBA-F: 5′-gtaatgctcccgttgggagttgt-3′，
28BBA-R: 5′-tcaatcaaacgggtttgagccatc-3′；
(4) goal gene 0BBA and pichia pastoris phaff secretion expression carrier are used respectively
BglII and
BamHI carries out double digestion and spends the night, and enzyme is used T after cutting product purification
4Dna ligase obtains the ligation thing at 3 ~ 5 ℃ of reaction 14 ~ 16 h;
Or with goal gene 28BBA and warp
SnaThe BI enzyme cut spend the night, the pichia pastoris phaff secretion expression carrier T behind the purifying
4Dna ligase connects, and 3 ~ 5 ℃ of reaction 14 ~ 16 h obtain the ligation thing;
Described pichia pastoris phaff secretion expression carrier is meant pPIC9K-A, and himself secreting signal peptide A pichia pastoris phaff secretion expression carrier pPIC9K-B, pPIC9K-C, pPIC9K-D after being replaced by acid phosphatase lipase signal peptide B, sucrase signal peptide C, Killer toxin signal peptide D;
Described signal peptide A, B, C, D give birth to worker's bio tech ltd by Shanghai and synthesize restriction enzyme
BglII,
BamHI and
SnaBI is available from Shanghai brilliant U.S. Bioisystech Co., Ltd;
(5) the ligation thing is transformed the host bacterium
E. coliThe JM109 competent cell, coating contains the LB flat board of 50 μ g/mL penbritins, selects positive colony, and cultivates the back and extract plasmid usefulness in a small amount
SalI carries out enzyme and cuts checking, obtains inserting by correct direction the recombinant expression plasmid of goal gene;
Recombinant expression plasmid comprises: pPIC9K-A-0BBA, pPIC9K-B-0BBA, pPIC9K-C-0BBA, pPIC9K-D-0BBA, pPIC9K-A-28BBA, pPIC9K-B-28BBA, pPIC9K-C-28BBA and pPIC9K-D-28BBA;
(6) step (5) gained recombinant expression plasmid is used
BglII carries out linearization for enzyme restriction, 36 ~ 38 ℃ of reaction 4 h, then enzyme is cut product purification, transform pichia pastoris phaff host bacterium GS115 or KM71 with electroporation, coating MD culture medium flat plate, picking list bacterium colony, identify positive colony by colony polymerase chain reaction (PCR) method, promptly obtain to have the pichia pastoris phaff genetic engineering bacterium of goal gene, test screening by the enzyme biopsy and obtain enzyme the highest transformant alive, the pichia pastoris phaff KM71 that promptly contains recombinant plasmid pPIC9K-A-0BBA, its deposit number are CCTCC NO:M 2011014;
Described electroporation transform with reference to Invitrogen company " pichia spp transforms handbook to carry out, and its condition is that voltage is 1500 V, and the time is 5 ms, and resistance is 200 Ω; The MD nutrient media components is counted with mass concentration: 2% glucose, and 1.34% YNB, 2% agar powder supplies 100% with distilled water;
The described pcr amplification of step (6), the primer is 0BBA-F and 0BBA-R when transforming pPIC9K-(A/B/C/D)-0BBA, the primer is 28BBA-F and 28BBA-R when transforming pPIC9K-(A/B/C/D)-28BBA.
Restriction enzyme described in Teasy carrier described in reverse transcription primer oligdT-18 described in Trizol reagent described in the above-mentioned construction process step (1), the step (2), the step (3), the step (4) is respectively
BglII and
BamHI all derives from reagent company of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Beta-amylase gene described in the above-mentioned steps (3) is the beta-amylase gene of plant origin or the beta-amylase gene that obtains through methods such as sudden changes, rearrangement, disappearance, with from the consensus dna sequence of the beta-amylase gene of Ipomoea batatas more than or equal to 70%, the protein sequence consistence is more than or equal to 70%.
The purifying that the described enzyme of above-mentioned steps (4) is cut product can adopt the PCR product to reclaim purification process commonly used in test kit or other molecular cloning.
Pichia spp host bacterium described in the above-mentioned construction process step (6) is meant GS115 or KM71, preferably
Pichia pastorisGS115, more preferably
Pichia pastorisKM71.Described bacterial strain is all bought from Invitrogen company.
Method with described bacterial strain CCTCC NO:M 2011014 efficient production beta-amylases, adopt reorganization pichia pastoris phaff RYP 20110110 CCTCC NO:M 2011014 as producing bacterial strain, produce beta-amylase through high density fermentation, the freeze-drying behind ultrafiltration micro-filtration of gained fermented liquid obtains beta-amylase enzyme powder, and step is:
(1) seed culture: will be in the CCTCC of 4 ℃ of preservations NO:M 2011014 bacterium be inserted seed culture medium, be to cultivate 24 ~ 36 h under the condition of 150 ~ 250 r/min at 28 ~ 30 ℃, shaking speed, nutrient solution is used as seed liquor and is inoculated in fermention medium;
The composition of described seed culture medium is counted with mass concentration: 1% yeast extract paste, and 2% peptone, 1% ~ 4% glycerine supplies 100% with distilled water;
(2) fermentation culture: the volume ratio according to 6% ~ 10% is inoculated into the seed liquor of step (1) in the 15 L fermentor tanks that 10 L fermention mediums are housed, at 28 ~ 30 ℃, pH 5.5 ~ 6.0, shaking speed is to cultivate under the condition of 150 ~ 250 r/min, timing sampling detects glycerol concentration, when glycerol concentration drops to when being lower than 0.5%, change the methanol induction cultivation stage over to;
The composition of described fermention medium is counted with mass concentration: 1% yeast extract paste, and 2% peptone, 1% methyl alcohol supplies 100% with distilled water;
(3) methanol induction is cultivated: the fermentation culture that obtains by step (2), and when glycerol concentration drops to when being lower than 0.5%, begin stream and add methyl alcohol, flow velocity is 1 mL/min; Control methyl alcohol stream and add by the control dissolved oxygen again, when system's dissolved oxygen＜20%, stop stream and add methyl alcohol, this step incubation time is 80 ~ 100 h, obtains fermented liquid;
(4) centrifugal: collect the fermented liquid of step (3), 4 ℃ of following centrifugal 10 min of 8000 g collect supernatant liquor; Described fermentation broth enzyme work can reach 500 U/mL;
(5) micro-filtration: the supernatant liquor of step (4) is carried out micro-filtration with 0.45 μ m ceramic membrane Microfilter, collect filtrate;
(6) ultrafiltration: the filtrate of step (5) is used ultrafiltration membrance filter, obtain beta-amylase and concentrate enzyme liquid;
(7) freeze-drying: the concentrated enzyme liquid of step (6) is carried out lyophilize, obtain Powdered beta-amylase goods;
Ceramic membrane material described in the above-mentioned steps (5) is a column aluminum oxide material, and the flow velocity of supernatant liquor can be by regulating the reverse flow valve control of ceramic membrane outer end, thus with the pressure-controlling of film end in 0.01 ~ 0.02 MPa, the flow velocity of this moment is 0.2 ~ 0.4 L/min;
Ultra-filtration membrane described in the above-mentioned steps (6) is the rolling ultra-filtration membrane, and its material is a polyethersulfone, and molecular weight cut-off is 10 kD, and the temperature of ultra-filtration membrane can be controlled by the speed of circulation of regulating water coolant;
Lyophilize condition described in the above-mentioned steps (7) is that condenser temperature maintains about-50 ℃, and freeze-drier is a U.S. Labcon company, and model is FreeZone 12 L.
Every min starch-splitting generated 1 mg maltose and is defined as 1 U when fermentation broth enzyme work described in the above-mentioned steps (3) can reach 500 U/mL(enzyme work and is defined as 50 ℃).
The protein concentration of the beta-amylase of above-mentioned fermentative production preparation has reached 3 ~ 6 g/L, and enzyme work can reach 2000 U/g.
It is host strain that the present invention adopts pichia spp, makes up the engineering strain of secreting, expressing Ipomoea batatas beta-amylase, by optimizing the fermentation level of the secreting signal peptide raising engineering strain in the carrier, can realize the efficient production of large zymin beta-amylase.
Beneficial effect of the present invention: (1) gained pichia pastoris gene engineering bacterial strain of the present invention beta-amylase secreting, expressing level height; (2) beta-amylase enzyme running water of the present invention is flat high, Heat stability is good; (3) the beta-amylase quality height of the inventive method production, cost is low, is suitable for suitability for industrialized production.
The biological material specimens preservation: a kind of bacterial strain that produces beta-amylase, classification called after pichia pastoris phaff RYP 20110110 has been deposited in Chinese typical culture collection center, deposit number CCTCC NO:M 2011014, preservation date on January 11st, 2011.
Description of drawings
Fig. 1 utilizes the beta-amylase secreting, expressing plasmid pPIC9K-A-0BBA physical map of alpha factor signal peptide sequence.
Fig. 2 utilizes the beta-amylase secreting, expressing plasmid pPIC9K-A-28BBA physical map of alpha factor signal peptide sequence.
Embodiment
The Ipomoea batatas RNA that embodiment 1 Trizol method is extracted
(1) Ipomoea batatas is cleaned up, be cut into fine grained chippings, in liquid nitrogen, clay into power, change about 50 ~ 100 mg samples over to 1.5 mL centrifuge tubes, add 1 mL Trizol reagent, put upside down mixing and fully suspend sample in reagent.
(2) the suspension room temperature adds 0.2 mL chloroform after placing 5 min, concuss centrifuge tube 15 seconds, and room temperature is placed 5 min, and 4 ℃ then, centrifugal 10 min of 12000 rpm.
(3) carefully draw the upper strata water in a new centrifuge tube, add 0.5 mL Virahol, room temperature is placed 10 min behind the mixing, and 4 ℃ then, centrifugal 10 min of 12000 rpm.
(4) abandoning supernatant adds the 75% ethanol washing and precipitating of 1 mL, and 4 ℃, centrifugal 5 min of 12000 rpm.
(5) abandoning supernatant, drying at room temperature precipitation 5 ~ 10 min.Adding 50 μ L does not have RNase water, and room temperature is placed 5 ~ 10 min fully dissolves RNA, detected through gel electrophoresis RNA quality.
The amplification of the synthetic and beta-amylase gene of embodiment 2 cDNA
(1) the synthetic cDNA of reverse transcription
Reaction system (20 μ L): 1 μ g RNA, reverse transcription primer 10 mM oligdT-18 1 μ L, 5 * Buffer, 4 μ L, 10M dNTPs 1 μ L, M-MLV ThermoScript II 1 μ L, the no RNase water polishing of residual volume.
Reaction conditions: add RNA and reverse transcription primer in the reaction tubes earlier, no RNase water polishing volume 10 μ L, 70 ℃, 5 min place 5 min on ice; Add residue reagent again, no RNase water polishing volume 20 μ L, 42 ℃, 60 min.
(2) pcr amplification contains complete beta-amylase gene BBA, and the primer is:
BBA-F: 5′-a
agatctccatggctccaatccccggt-3′
BBA-R: 5′-a
agatctgctcctccttcaccttcag-3′
Use the ExTaq archaeal dna polymerase amplification of Takara company
Reaction system:
| Volume (μ L) | |
| The sterilization distilled water | 33.5 |
| 10×Ex TaqBuffer | 5 |
| dNTP （2 mM） | 5 |
| Upstream primer (10 mM) | 2 |
| Downstream primer (10 mM) | 2 |
| Template cDNA | 2 |
| Polysaccharase ExTaq | 0.5 |
| Cumulative volume | 50 |
Reaction conditions: 94 ℃, 2 min; 94 ℃, 40 sec, 55 ℃, 40 sec, 72 ℃, 1 min, 30 circulations; 72 ℃, 5 min.
Pcr amplification obtains the beta-amylase gene cDNA fragment and be connected into the Teasy carrier after glue recovery purifying is cut in gel electrophoresis, after filtering out correct Teasy-BBA transformant, order-checking shows that obtaining segmental length is 1618 bp, comprise beta-amylase gene complete open reading frame sequence 0BBA, its length is 1500bp, shown in SEQ ID NO:7.
The structure of embodiment 3 secretion expression carrier pPIC9K-(A/B/C/D)-0BBA
Teasy-BBA plasmid with 10 pg is a template, pcr amplification purpose fragment 0BBA, and the primer is:
0BBA-F: 5′-atggctccaatccccggt-3′，
0BBA-R: 5′-tcaatcaaacgggtttgagccatc-3′；
Reaction system and reaction conditions are with embodiment 2.
Amplified production is removed open reading frame sequence 1500 bp in signal peptide coding region territory for the beta-amylase gene.
Goal gene 0BBA and pichia pastoris phaff secretion expression carrier pPIC9K-A are used respectively
BglII and
BamHI carries out double digestion, and 37 ℃ of reactions are spent the night; Behind plasmid enzyme restriction product and PCR product usefulness purification kit purifying, T
4Dna ligase connects 14 h for 4 ℃.To connect product transformed into escherichia coli JM109, coating contains the LB flat board of 50 μ g/mL kantlex, selects positive colony, carries out shake-flask culture 12 h, and harvested cell extracts plasmid and uses
SalI carries out enzyme and cuts and the electrophoresis checking, obtains the correct pPIC9K-A-0BBA recombinant plasmid that inserts of target gene fragment.With
BglII linearization for enzyme restriction pPIC9K-A-0BBA with PCR product purification test kit purifying, transforms to be ready for use on pichia spp behind the plasmid linearization.
PPIC9K-B-0BBA, pPIC9K-C-0BBA, the building process of pPIC9K-D-0BBA is identical with pPIC9K-A-0BBA.
Described signal peptide A, B, C, D give birth to worker's bio tech ltd by Shanghai and synthesize.
The structure of embodiment 4 secretion expression carrier pPIC9K-(A/B/C/D)-28BBA
Teasy-BBA with 10 pg is a template, pcr amplification purpose fragment 28BBA, and the primer is:
28BBA-F: 5′-gtaatgctcccgttgggagttgt-3′，
28BBA-R: 5′- tcaatcaaacgggtttgagccatc-3′；
Reaction system and reaction conditions are with embodiment 2.
Amplified production is for the goal gene 28BBA of the open reading frame sequence of beta-amylase gene removal signal peptide coding region, shown in SEQ ID NO:8.
With
SnaBI digested plasmid pPIC9K-A, 37 ℃ of reaction overnight; Behind plasmid enzyme restriction product and PCR product usefulness purification kit purifying, T
4Dna ligase connects 14 h for 4 ℃.To connect product transformed into escherichia coli JM109, coating contains the LB flat board of 50 μ g/mL kantlex, selects positive colony, carries out shake-flask culture 12 h, and harvested cell extracts plasmid and uses
SalI carries out enzyme and cuts and the electrophoresis checking, obtains the correct pPIC9K-A-28BBA recombinant plasmid that inserts of target gene fragment.With
BglII linearization for enzyme restriction pPIC9K-A-28BBA with PCR product purification test kit purifying, transforms to be ready for use on pichia spp behind the plasmid linearization.
PPIC9K-B-28BBA, pPIC9K-C-28BBA, the building process of pPIC9K-D-28BBA is identical with pPIC9K-A-0BBA.
The pichia yeast genetic engineering bacteria of embodiment 5 secreting, expressing beta-amylases makes up
Inoculate pichia pastoris phaff list bacterium colony in 5 mL YPD substratum, 30 ℃ of incubated overnight; Get overnight culture 0.5 mL and be inoculated into the fresh YPD substratum of 50 mL, 30 ℃ of shaking tables are cultivated and are made OD
600Reach 1.3 ~ 1.5, collecting cell, 4 ℃, centrifugal 5 min of 5000 r/min; Abandon supernatant liquor, resuspended, the washing of ice-cold 1 mol/L Sorbitol Powder is 2 times behind resuspended, centrifugal 2 times of the sterilized water with precooling, at last cell is resuspended in the 1 ice-cold mol/L Sorbitol Powder of 100 μ L, every pipe 80 μ L remain in the ice bath with the competent cell packing; With 80 μ L competent cells with by behind the plasmid 10 μ g mixings after the linearizing of embodiment 3 acquisitions, move in the 2 mm specifications electric shock cup of people's precooling, 1.5 kv, burst lengths 5 ms, shock by electricity 2 times, the soft suspension cell of 1 mol/L sorbyl alcohol that adds 1 mL precooling then rapidly, 30 ℃ leave standstill cultivation 1 h.Concentrate nutrient solution, it evenly is coated with in the MD substratum, 30 ℃ of lucifuges are cultivated picking positive colony behind 2 ~ 3 d.
Embodiment 5 pichia yeast genetic engineering bacteria fermentative preparation beta-amylases
To insert in the seed culture medium (1% yeast extract paste, 2% peptone, 1% ~ 4% glycerine) at the pichia yeast genetic engineering bacteria of 4 ℃ of preservations, be to cultivate 24 ~ 36 h under the condition of 150 ~ 250 r/min at 28 ~ 30 ℃, shaking speed; Volume ratio according to 6% ~ 10% is inoculated into above-mentioned nutrient solution 10 L fermention medium (1% yeast extract pastes is housed, 2% peptone, 1% methyl alcohol) in the 15 L fermentor tanks, be controlled at 5.5 ~ 6.0 at 28 ~ 30 ℃, pH, shaking speed is to cultivate 24 ~ 36 h under the condition of 150 ~ 250 r/min; Detect glycerol concentration in the fermented liquid, when being lower than concentration 0.5% when glycerine exhausts, changing methanol induction over to and cultivate, begin stream and add methyl alcohol, flow velocity is 1 mL/min; Control methyl alcohol stream and add by the control dissolved oxygen again, when dissolved oxygen＜20%, stop stream and add methyl alcohol, this step incubation time is 80 ~ 100 h, obtains fermented liquid.The enzyme running water of beta-amylase is flat in the fermented liquid can reach 500 U/mL.
With fermented liquid centrifugal 10 min of 8000 g under 4 ℃, remove yeast cell, collect supernatant liquor; Successively through being installed on placed in-line two ceramic membrane filter cores in the stainless steel cylinder, the ceramic membrane material is a column aluminum oxide material to supernatant liquor under the conveying of impeller pump, and the aperture is 0.45 μ m.Liquid and film surface friction heating when preventing that flow velocity is too high and the albumen inactivation that causes, in 0.01 ~ 0.02 MPa, the flow velocity of this moment is 0.2 ~ 0.4 L/min to the reverse flow valve by regulating storage tank with the pressure-controlling of film end.The supernatant liquor that micro-filtration is obtained goes into to have the rolling ultra-filtration membrane of water cooling plant through infusion, removes small molecular weight impurity, holds back macromolecular albumen, and beta-amylase is concentrated purification.The ultra-filtration membrane material of selecting for use is a polyethersulfone, and molecular weight cut-off is 10 kD.To concentrate enzyme liquid pre-freeze in-70 ℃ Ultralow Temperature Freezer at last and be placed on freeze drier, the control condenser temperature maintains about-50 ℃, and moisture is distillation rapidly in a vacuum, can guarantee the activity of beta-amylase simultaneously again.Finally obtain pulverous beta-amylase, protein concentration has reached 3 ~ 6 g/L, and enzyme work can reach 2000 U/g.
＜110〉the sharp positive bio tech ltd in Jiangsu
＜120〉a kind of high efficiency preparation method of beta-amylase
<160> 8
<210> 1
<211> 27
<212> DNA
＜213〉primer BBA-F
<400> 1
aagatctcca tggctccaat ccccggt
<210> 2
<211> 26
<212> DNA
＜213〉primer BBA-R
<400> 2
aagatctgct cctccttcac cttcag
<210> 3
<211> 18
<212> DNA
＜213〉primer 0BBA-F
<400> 3
atggctccaa tccccggt
<210> 4
<211> 24
<212> DNA
＜213〉primer 0BBA-R
<400> 4
tcaatcaaac gggtttgagc catc
<210> 5
<211> 23
<212> DNA
＜213〉primer 2 8BBA-F
<400> 5
gtaatgctcc cgttgggagt tgt
<210> 6
<211> 24
<212> DNA
＜213〉primer 2 8BBA-R
<400> 6
tcaatcaaac gggtttgagc catc
<210> 7
<211> 1500
<212> DNA
＜213〉beta-amylase gene 0BBA
<400> 7
agctgaagca ggtgaaagca ggggggtgcg atggggtgat ggtggatgtg tggtggggga 60
tcatcgaggc caaagggcca aagcagtacg attggtctgc ttacagggag ttattccagt 120
tggttaagaa atgtgggctc aaaatccagg cgatcatgtc ttttcaccaa tgcggcggca 180
atgtcggcga cgccgtcttc atccccatcc ctcaatggat tctccaaatc ggcgacaaaa 240
accccgatat cttctacacc aaccgggccg gtaaccgaaa ccaggagtac ctctctctcg 300
gcgtcgacaa tcaacgtctc ttccagggcc gcaccgctct cgagatgtac agggatttca 360
tggaaagttt cagggataat atggcagact ttttgaaggc tggagatatt gtagacattg 420
aagtagggtg tggggctgcc ggagagcttc ggtatccctc gtatcccgag actcaaggat 480
gggtttttcc cggcatcgga gaatttcagt gctatgacaa gtacatggtg gcagactgga 560
aggaggctgt gaagcaagct gggaatgcag attgggagat gccgggaaaa ggcaccggga 620
cttacaacga cacgccggac aagacggaat tcttccgccc aaacgggact tacaagacgg 680
atatgggcaa gtttttcttg acctggtact ccaacaagct catcatccat ggcgatcaag 720
tcctcgaaga agccaacaaa gttttcgttg ggctccgcgt caacatagct gccaaagttt 780
ctggaattca ctggtggtac aaccatgtga gccatgcggc ggagctcacc gccggattct 840
acaatgtggc gggaagagac ggttatcggc ctattgccag gatgctggca aggcaccatg 900
ccactctgaa tttcacttgc cttgagatga gagactccga acagcctgcc gaggccaaga 960
gtgctcctca agaactcgtt caacaggtgt tgagcagcgg atggaaagag tatatcgatg 1020
tggcaggtga gaatgcactg ccaagatatg atgccacggc atacaaccaa atacttctga 1080
acgtcaggcc aaacggcgtc aaccttaacg gccctcccaa gctcaagatg tccggcttga 1140
catatctccg tttgtcggac gatctgttac agacagacaa cttcgaactc ttcaagaaat 1200
tcgtcaagaa gatgcacgct gatctggatc catccccgaa cgctatctct ccggcggtgt 1260
tggagagatc aaactcggcg atcaccattg atgaactgat ggaagccacc aaaggtagca 1320
ggccattccc atggtatgac gtcacagaca tgccggtgga tggctcaaac ccgtttgatt 1380
gattaggatg acatcaccct cctccctgga ctactgcatg tggaacaata ataagttaat 1440
gcataaatat tataaataaa tgaataatcc agctttcagt gctgaaggtg aaggaggagc 1500
<210> 8
<211> 1416
<212> DNA
＜213〉beta-amylase gene 28BBA
<400> 8
agtacgattg gtctgcttac agggagttat tccagttggt taagaaatgt gggctcaaaa 60
tccaggcgat catgtctttt caccaatgcg gcggcaatgt cggcgacgcc gtcttcatcc 120
ccatccctca atggattctc caaatcggcg acaaaaaccc cgatatcttc tacaccaacc 180
gggccggtaa ccgaaaccag gagtacctct ctctcggcgt cgacaatcaa cgtctcttcc 240
agggccgcac cgctctcgag atgtacaggg atttcatgga aagtttcagg gataatatgg 300
cagactttttg aaggctgga gatattgtag acattgaagt agggtgtggg gctgccggag 360
agcttcggta tccctcgtat cccgagactc aaggatgggt ttttcccggc atcggagaat 420
ttcagtgcta tgacaagtac atggtggcag actggaagga ggctgtgaag caagctggga 480
atgcagattg ggagatgccg ggaaaaggca ccgggactta caacgacacg ccggacaaga 540
cggaattctt ccgcccaaac gggacttaca agacggatat gggcaagttt ttcttgacct 600
ggtactccaa caagctcatc atccatggcg atcaagtcct cgaagaagcc aacaaagttt 660
tcgttgggct ccgcgtcaac atagctgcca aagtttctgg aattcactgg tggtacaacc 720
atgtgagcca tgcggcggag ctcaccgccg gattctacaa tgtggcggga agagacggtt 780
atcggcctat tgccaggatg ctggcaaggc accatgccac tctgaatttc acttgccttg 840
agatgagaga ctccgaacag cctgccgagg ccaagagtgc tcctcaagaa ctcgttcaac 900
aggtgttgag cagcggatgg aaagagtata tcgatgtggc aggtgagaat gcactgccaa 960
gatatgatgc cacggcatac aaccaaatac ttctgaacgt caggccaaac ggcgtcaacc 1020
ttaacggccc tcccaagctc aagatgtccg gcttgacata tctccgtttg tcggacgatc 1080
tgttacagac agacaacttc gaactcttca agaaattcgt caagaagatg cacgctgatc 1140
tggatccatc cccgaacgct atctctccgg cggtgttgga gagatcaaac tcggcgatca 1200
ccattgatga actgatggaa gccaccaaag gtagcaggcc attcccatgg tatgacgtca 1260
cagacatgcc ggtggatggc tcaaacccgt ttgattgatt aggatgacat caccctcctc 1320
cctggactac tgcatgtgga acaataataa gttaatgcat aaatattata aataaatgaa 1380
taatccagct ttcagtgctg aaggtgaagg aggagc 1416
Claims (3)
1. bacterial strain that produces beta-amylase, classification called after pichia pastoris phaff RYP 20110110 has been deposited in Chinese typical culture collection center, deposit number CCTCC NO:M 2011014.
2. the construction process of the described pichia pastoris phaff CCTCC of claim 1 NO:M 2011014 is characterized in that construction step is:
(1) utilize Trizol reagent to extract total RNA of Ipomoea batatas;
(2) be template with total RNA, under the effect of oligdT-18 and reversed transcriptive enzyme, a synthetic chain cDNA;
(3) design a pair of primer according to the Ipomoea batatas beta-amylase gene order of having announced, the primer is:
BBA-F: 5′- a
agatctccatggctccaatccccggt-3′，
BBA-R: 5′-a
agatctgctcctccttcaccttcag -3′；
With a chain cDNA is the template pcr amplification, obtain amplification and contain complete beta-amylase gene BBA, be connected into the Teasy carrier and obtain Teasy-BBA, with the Teasy-BBA plasmid is template, and pcr amplification contains the goal gene 0BBA of complete beta-amylase gene open reading frame sequence or removes the beta-amylase gene 28BBA of signal peptide coding region;
Described pcr amplification 0BBA, the primer is:
0BBA-F: 5′-atggctccaatccccggt-3′，
0BBA-R: 5′-tcaatcaaacgggtttgagccatc -3′；
Described pcr amplification 28BBA, the primer is:
28BBA-F: 5′-gtaatgctcccgttgggagttgt-3′，
28BBA-R: 5′-tcaatcaaacgggtttgagccatc-3′；
(4) goal gene 0BBA and pichia pastoris phaff secretion expression carrier are used respectively
BglII and
BamHI carries out double digestion and spends the night, and enzyme is used T after cutting product purification
4Dna ligase obtains the ligation thing at 3 ~ 5 ℃ of reaction 14 ~ 16 h;
Or with goal gene 28BBA and warp
SnaThe BI enzyme cut spend the night, the pichia pastoris phaff secretion expression carrier T behind the purifying
4Dna ligase connects, and 3 ~ 5 ℃ of reaction 14 ~ 16 h obtain the ligation thing;
Described pichia pastoris phaff secretion expression carrier is meant pPIC9K-A, and himself secreting signal peptide A pichia pastoris phaff secretion expression carrier pPIC9K-B, pPIC9K-C or pPIC9K-D after being replaced by acid phosphatase lipase signal peptide B, sucrase signal peptide C, Killer toxin signal peptide D;
(5) the ligation thing is transformed the host bacterium
E. coliThe JM109 competent cell, coating contains the LB flat board of 50 μ g/mL penbritins, selects positive colony, and cultivates the back and extract plasmid usefulness in a small amount
SalI carries out enzyme and cuts checking, obtains inserting by correct direction the recombinant expression plasmid of goal gene;
Recombinant expression plasmid comprises: pPIC9K-A-0BBA, pPIC9K-B-0BBA, pPIC9K-C-0BBA, pPIC9K-D-0BBA, pPIC9K-A-28BBA, pPIC9K-B-28BBA, pPIC9K-C-28BBA and pPIC9K-D-28BBA;
(6) step (5) gained recombinant expression plasmid is used
BglII carries out linearization for enzyme restriction, 36 ~ 38 ℃ of reaction 4 h, then enzyme is cut product purification, transform pichia pastoris phaff host bacterium GS115 or KM71 with electroporation, coating MD culture medium flat plate, picking list bacterium colony, identify positive colony by colony polymerase chain reaction (PCR) method, promptly obtain to have the pichia pastoris phaff genetic engineering bacterium of goal gene, test screening by the enzyme biopsy and obtain enzyme the highest transformant alive, the pichia pastoris phaff KM71 that promptly contains recombinant plasmid pPIC9K-A-0BBA, its deposit number are CCTCC NO:M 2011014;
Described electroporation transforms, and condition is that voltage is 1500 V, and the time is 5 ms, and resistance is 200 Ω; The MD nutrient media components is counted with mass concentration: 2% glucose, and 1.34% YNB, 2% agar powder supplies 100% with distilled water;
The described pcr amplification of step (6), the primer is 0BBA-F and 0BBA-R when transforming pPIC9K-(A/B/C/D)-0BBA, the primer is 28BBA-F and 28BBA-R when transforming pPIC9K-(A/B/C/D)-28BBA.
3. method with the described bacterial strain CCTCC of claim 1 NO:M 2011014 efficient production beta-amylases, it is characterized in that adopting pichia pastoris phaff CCTCC NO:M 2011014 as producing bacterial strain, produce beta-amylase through high density fermentation, the freeze-drying behind ultrafiltration micro-filtration of gained fermented liquid obtains beta-amylase enzyme powder, and step is:
(1) seed culture: will be in the CCTCC of 4 ℃ of preservations NO:M 2011014 bacterium be inserted seed culture medium, be to cultivate 24 ~ 36 h under the condition of 150 ~ 250 r/min at 28 ~ 30 ℃, shaking speed, nutrient solution is used as seed liquor and is inoculated in fermention medium;
The composition of described seed culture medium is counted with mass concentration: 1% yeast extract paste, and 2% peptone, 1% ~ 4% glycerine supplies 100% with distilled water;
(2) fermentation culture: the volume ratio according to 6% ~ 10% is inoculated into the seed liquor of step (1) in the 15 L fermentor tanks that 10 L fermention mediums are housed, be to cultivate under the condition of 150 ~ 250 r/min at 28 ~ 30 ℃, pH 5.5 ~ 6.0, shaking speed, timing sampling detects glycerol concentration, when glycerol concentration drops to when being lower than 0.5%, change the methanol induction cultivation stage over to;
The composition of described fermention medium is counted with mass concentration: 1% yeast extract paste, and 2% peptone, 1% methyl alcohol supplies 100% with distilled water;
(3) methanol induction is cultivated: the fermentation culture that obtains by step (2), and when glycerol concentration drops to when being lower than 0.5%, begin stream and add methyl alcohol, flow velocity is 1 mL/min; Control methyl alcohol stream and add by the control dissolved oxygen again, when system's dissolved oxygen＜20%, stop stream and add methyl alcohol, this step incubation time is 80 ~ 100 h, obtains fermented liquid;
(4) centrifugal: collect the fermented liquid of step (3), 4 ℃ of following centrifugal 10 min of 8000 g collect supernatant liquor; Described fermentation broth enzyme work can reach 500 U/mL;
(5) micro-filtration: the supernatant liquor of step (4) is carried out micro-filtration with 0.45 μ m ceramic membrane Microfilter, collect filtrate;
(6) ultrafiltration: the filtrate of step (5) is used ultrafiltration membrance filter, obtain beta-amylase and concentrate enzyme liquid;
(7) freeze-drying: the concentrated enzyme liquid of step (6) is carried out lyophilize, obtain Powdered beta-amylase goods.
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| CN107201351A (en) * | 2017-03-23 | 2017-09-26 | 中国农业科学院饲料研究所 | A kind of height is than amylase mutant preparation method and applications living |
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| CN107058263B (en) * | 2017-01-26 | 2020-11-06 | 福建福大百特生物科技有限公司 | Efficient preparation method of novel beta-amylase |
| CN107201351A (en) * | 2017-03-23 | 2017-09-26 | 中国农业科学院饲料研究所 | A kind of height is than amylase mutant preparation method and applications living |
| CN107201351B (en) * | 2017-03-23 | 2020-01-21 | 中国农业科学院饲料研究所 | Preparation method and application of amylase mutant |
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