CN103224920A - Method for separation purification of high temperature-resistant beta-amylase from bacillus subtilis - Google Patents
Method for separation purification of high temperature-resistant beta-amylase from bacillus subtilis Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering enzyme preparations and relates to a method for separation purification of high temperature-resistant beta-amylase from a wild-type bacillus subtilis 6-7 fermentation broth. Bacillus subtilis is preserved in the China center for type culture collection and has the preservation number of CTCC M2009200. The method is characterized in that wild-type B.subtilis6-7, and cassava powder and soybean meal as carbon and nitrogen sources are prepared into a fermentation broth; and the fermentation broth is subjected to refrigeration centrifugation, ammonium sulfate precipitation, dialysis desalting, HiTrap Qff ion column exchange, ultrafiltration centrifuge tube-based ultrafiltration desalting, monoQ5/50GL ion column exchange and Sephadex G-75 separation. The high temperature-resistant beta-amylase obtained by the method has a yield of 18.66%, purification fold of 4.83 and enzyme activity of 245395U/mg. An enzymatic property research shows that the purified high temperature-resistant beta-amylase has good heat resistance, is stable at the pH of 5-8 and is identified as the beta-amylase by mass spectrum identification. The high temperature-resistant beta-amylase has high purity, is colorless and satisfies food-grade beta-amylase standard requirements. The method provided by the invention has simple processes, is convenient for operation, has a high yield and realizes enzyme activity of 240000 U/mg.
Description
Technical field
The present invention relates to the high temperature resistant beta-amylase production technique of a kind of wild-type subtilis Bacillus subtilis6-7, relate to a kind of fermented liquid method of separation and purification high enzymatic activity beta-amylase efficiently particularly, belong to fermentation and enzyme engineering field.
Background technology
Beta-amylase (1,4-α-D-glucan maltohydrolase, EC3.2.1.2), it is the circumscribed-type saccharifying enzyme, when acting on starch or glycogen, downcut maltose unit successively from the non reducing end of α-1,4 glycosidic link, hydrolysate has maltose, trisaccharide maltose, β-limit dextrin and a spot of glucose.Walden translocation reaction (Walden inversion) takes place in maltose simultaneously, becomes β-type by α-type, so claim beta-amylase.In theory beta-amylase all amylose starchs can be changed into maltose and with about 60% amylopectin change into maltose remaining change dextrin into.
Beta-amylase extensively is present in the plants such as barley, wheat, oat, soybean, sweet potato, and in the microorganisms such as bacillus (Bacillus), heat-resisting Clostridium (Clostridium) and mould.Beta-amylase is mainly used in foodstuffs industry, as the production of high maltose syrup, superhigh maltose syrup, maltose alcohol.Starch is hydrolyzed to maltose, and maltose can be used as the precursor of sweeting agent, quality improver, sanitas, stablizer, maltose alcohol and the intravenous injection for the treatment of diabetes.Beta-amylase also can be used for beer industry, is the ideal replacement product of Fructus Hordei Germinatus.An amount of beta-amylase acts on different W-Gums, can increase resistant starch content, as the HylonV W-Gum through the beta-amylase hydrolysis after 4 hours Resistant starch RS content up to 70.7%.
Present domestic beta-amylase mainly directly extracts from plant, as soybean, barley, wheat, sweet potato, belongs to natural product, safety, but still have many deficiencies.Earlier plant material is cleaned, grinds or pulverizes, carry out screenings again and separate, water or damping fluid extract, complicated operation, cost height.This method yield of directly extracting pure beta-amylase from plant is low, the yielding poorly of enzyme, and enzyme activity is low, wastes time and energy, and contains some other soluble componentss, and quality is coarse.And the beta-amylase of plant origin is subjected to seasonal effect, and it is limited to originate, and can not realize industrial large scale continuous prod, is difficult to realize the commercial profit of ensured sustained development.And price such as barley, soybean continues surging, and its cost is more and more big, does not satisfy the demand to beta-amylase.The beta-amylase thermotolerance of plant origin is poorer than microbe-derived, so it is extremely important to develop microbe-derived beta-amylase separation purification method.The microorganism submerged fermentation is produced, and can realize the heavy industrialization automatic production.
Along with the raising of people's living standard, the developing rapidly of food service industry and beer industry production, the high temperature resistant beta-amylase of Development and Production high quality is the emphasis of current research work.Produce beta-amylase at above-mentioned plant origin and have problems such as cost height, product foreign matter content height, poor heat resistance, the invention provides a kind of wild-type subtilis Bacillus subtilis6-7 fermented liquid separation and purification refractory beta-diastatic method efficiently.Simultaneously, the present invention utilizes and originates widely that Tapioca Starch, bean cake powder have significantly reduced production cost, for suitability for industrialized production lays the foundation as medium component fermentative production beta-amylase.
Summary of the invention
Technology to be solved by this invention is the enforcement by a series of separation and purification means, provide a kind of from B.subtilis6-7 fermented liquid separation and purification refractory beta-diastatic method, this bacterial strain is (related application number: 201010167019.7) disclose, and submitted preservation proof and survival proof before this in applicant's patent application before this.This method can access the beta-amylase of electrophoresis level purity.And pure enzyme can be used for the research of zymologic property, can be used in further mass spectrum identification and analysis and other physico-chemical property research.
For solving the problems of the technologies described above, the present invention adopts following technical scheme: with Tapioca Starch, bean cake powder is culture medium raw material, and the preparation substratum utilizes the condition of enzyme production of optimizing to carry out shake flask fermentation and cultivates.The separation purifying technique feature is:
(1) fermented liquid separates through 4 ℃ of frozen centrifugations, collects supernatant liquor, is crude enzyme liquid;
(2) ammonium sulfate precipitation: in crude enzyme liquid, add ammonium sulfate to saturation ratio and be 30%, 4 ℃ and leave standstill 4h that frozen centrifugation is removed precipitation, collects supernatant liquor; Continuation interpolation ammonium sulfate to saturation ratio is 60%, 4 ℃ and leaves standstill 4h, frozen centrifugation, and the removal supernatant liquor, collecting precipitation is with the Na of pH8.0,20mmol/L
2HPO
4-NaH
2PO
4Damping fluid redissolves;
(3) dialysis desalination: at same pH8.0, the Na of 20mmol/L
2HPO
4-NaH
2PO
4Damping fluid is dialysed, and is the dialysis tubing of 10KDa with the average molecular interception;
(4) HiTrap Q Fast Flow: sample concentrates to the accumulation of HiTrap Q Fast Flow post anion-exchange chromatography on the sample of will dialysing, and A liquid is pH8.0,20mmol/LNa
2HPO
4-NaH
2PO
4Damping fluid, B liquid are pH8.0,20mmol/LNa
2HPO
4-NaH
2PO
4/ 1mol/L NaCl damping fluid carries out linear gradient elution with the NaCl of 0～1mol/L, is 20%～30% o'clock at B liquid, and the target protein beta-amylase elutes (as accompanying drawing 1);
(5) ultrafiltration desalination: use ultra-filtration centrifuge tube centrifugal ultrafiltration desalination under freezing low temperature low speed, 4 ℃, 3800r/min are centrifugal;
(6) MonoQ5/50GL: will keep liquid and further separate (as accompanying drawing 2) by high efficiency MonoQ5/50GL post ion-exchange;
(7) gel molecular screening from: the further gel molecular sieve of the elutant of collecting Sephadex G-75 post is separated, removes little foreign protein.
The enzyme liquid of purifying is carried out SDS-PAGE electrophoretic analysis purity (as accompanying drawing 3), zymologic property research and ground substance assistant laser parsing flight time mass spectrum identification and analysis.
Described subtilis is high temperature resistant beta-amylase separation purification method, described optimization are cultivated and are obtained high yield beta-amylase fermented liquid and comprise:
(1) the used bacterial classification of the present invention is subtilis B.subtilis6-7, and seed culture medium is the LB substratum: 1% Tryptones, 0.5% yeast extract, 1%NaCl.
(2), be forwarded to shaking of fresh 50mL/250mL again and cultivate 12h in bottle LB substratum as secondary seed with bacterial strain activation culture 12h in the LB of 10mL/50mL substratum of glycerine frozen pipe preservation.
(3) optimize substratum: 2% Tapioca Starch, 4% bean cake powder, 0.1% Secondary ammonium phosphate, 0.00139% ferrous sulfate, 0.6% Trisodium Citrate, 0.4% potassium primary phosphate, 0.0005% zinc sulfate, 0.0123% sal epsom, 0.0111% calcium chloride, pH7.0.It is the 250mL substratum that 500mL shakes bottled liquid measure, 121 ℃ of sterilization 20min.
(4) secondary seed is inoculated in the fermentation optimization substratum by inoculum size 4%.Rotating speed 160r/min, 37 ℃ of shake-flask culture 60h of culture temperature.
The pure beta-amylase that beta-amylase separation and purification that described subtilis is high temperature resistant obtains carries out zymologic property research and comprises:
(1) optimal reactive temperature and temperature stability:, determine optimal reactive temperature measuring the beta-amylase vigor at 30,35,40,45,50,55,60,65,70,75,80 ℃ respectively; Treat enzyme liquid different time under 50,55,60,65,70,75 ℃ of differing tempss is measured residual enzyme vigor (as accompanying drawing 4).
(2) optimal reaction pH and pH stability: enzyme liquid is measured the vigor of beta-amylase in pH3.0,4.0,5.0,6.0,7.0,8.0 citric acids-Sodium phosphate dibasic damping fluid, determine optimal reaction pH; At pH3.0,4.0,5.0,6.0,7.0,8.0 times treat enzyme liquid different times, measure residual enzyme vigor (as accompanying drawing 5).
(3) metal ion and EDTA influence that enzyme is lived: the Na that in the enzyme reaction system, adds 1mmol/L respectively
+, K
+, Ca
2+, Mg
2+, Fe
2+, Mn
2+, Ni
2+, Cu
2+Deng 8 metal ion species and EDTA.And setting does not add metal ion blank group, each enzyme of organizing of mensuration (as accompanying drawing 6) alive then.
By the present invention, the success separation and purification has been set up a kind of wild-type subtilis B.subtilis6-7 fermented liquid method of separation and purification beta-amylase efficiently to electrophoretically pure beta-amylase, and the rate of recovery is up to 18.66%, the purifying multiple is 4.83, and enzyme work reaches 245395U/mg.
Description of drawings
Accompanying drawing 1 is HiTrap Q Fast Flow (reinforcing yin essence ion exchange resin) ion exchange chromatography spectrogram, and peak A is a beta-amylase
Accompanying drawing 3 is SDS-PAGE electrophorograms, and path 1～10 is respectively: the 1.B10-54 fermented liquid; 2. ammonium sulfate precipitation; 3.DEAEpH8.0; 4.HiTrap Q FF pH8.0; 5.HiTrap Q FF pH7.5; 6.SP pH6.0; 7.marker; 8.sephadex G-75; 9.monoQ pH8.0; 10.marker
Accompanying drawing 4 is beta-amylase temperature stabilities
Accompanying drawing 5 is a beta-amylase pH stability
Accompanying drawing 6 is that metal ion and EDTA influence enzyme is alive
Embodiment
Subtilis B.subtilis6-7 is high temperature resistant beta-amylase separation purification method, the present invention will further describe in detail with embodiment, but not limit the present invention in any way.
Microorganism subtilis B.subtilis6-7 fermentative preparation fermented liquid crude enzyme liquid.With bacterial strain B.subtilis6-7 activation culture 12h in the LB of 10mL/50mL substratum of glycerine frozen pipe preservation, be forwarded to shaking of fresh 50mL/250mL again and cultivate 12h in bottle LB substratum as secondary seed.Optimize substratum: 2% Tapioca Starch, 4% bean cake powder, 0.1% Secondary ammonium phosphate, 0.00139% ferrous sulfate, 0.6% Trisodium Citrate, 0.4% potassium primary phosphate, 0.0005% zinc sulfate, 0.0123% sal epsom, 0.0111% calcium chloride, pH7.0.It is the 250mL substratum that 500mL shakes bottled liquid measure, 121 ℃ of sterilization 20min.Secondary seed is inoculated in the fermentation optimization substratum by inoculum size 4%.Rotating speed 160r/min, 37 ℃ of shake-flask culture of culture temperature.Behind the fermentation culture 60h, collect fermented liquid, carry out 4 ℃, the centrifugal 20min of 8000r/min, collect supernatant liquor as crude enzyme liquid.
Microorganism subtilis B.subtilis6-7 refractory beta-diastatic separation and purification.At first draw the ammonium sulfate precipitation curve.Get 7 bottles, adorn equivalent 100ml crude enzyme liquid respectively, press the ammonium sulfate saturation table and add different ammonium sulfate solids gram numbers, reach 10%, 20%, 30%, 40%, 50%, 60%, 70% saturation ratio respectively, standing over night, centrifugal collecting precipitation, citrate buffer solution with pH6.0 redissolves, the mensuration enzyme is lived, and is 100% with prima facies to enzyme work, draws the curve of saltouing.Add ammonium sulfate in the crude enzyme liquid, according to saltouing curve, the centrifugal precipitation of abandoning of 30% saturation ratio is removed foreigh protein removing, and 60% saturation ratio centrifugal collecting precipitation is precipitated out the target protein beta-amylase.Na with pH8.0,20mmol/L
2HPO
4-NaH
2PO
4Damping fluid redissolves and precipitates, and the molecular retention amount of packing into is in the dialysis tubing of 5K, is immersed in the same damping fluid, continuous exchange buffering liquid, dialysis desalination.With sample on the dialyzed sample in HiTrap Q Fast Flow reinforcing yin essence ion exchange column, A liquid: pH8.0,20mmol/LNa
2HPO
4-NaH
2PO
4Damping fluid, B liquid: contain the A liquid balance pillar of the NaCl of 1mol/L in the A liquid with 10 column volumes, with 1mL injection annulus sample introduction, flow velocity is 1mL/min, then wash post with the A liquid of 5 column volumes, remove unconjugated foreign protein, according to A liquid, B liquid proportioning, carry out the linear gradient elution of 0～100%B liquid then.Every pipe effluent liquid of collecting is detected enzyme live, determine the wash-out feature of target protein.Then the effluent liquid to target protein is placed in the pipe, carries out the centrifugal desalination of ultra-filtration centrifuge tube.To keep the separation that sample on the liquid carries out further Gao Zhuxiao in the MonoQ5/50GL, operation steps and HiTrap Q Fast Flow are similar.The enzyme liquid of collecting is carried out the gel molecular sieve element, is 6.0 20mmol/LNa with pH
2HPO
4-NaH
2PO
4Buffer solution elution is convenient under the buffered environment that enzyme liquid is stored in pH6.0.Above-mentioned purification step is calculated enzyme work, protein content, the rate of recovery, the purifying multiple in each step.The beta-amylase rate of recovery that obtains is 18.66%, and the purifying multiple is 4.83, and enzyme work reaches 245395U/mg.
Microorganism subtilis B.subtilis6-7 refractory beta-diastatic zymologic property analysis.Optimal reactive temperature and temperature stability:, determine optimal reactive temperature measuring the beta-amylase vigor at 30,35,40,45,50,55,60,65,70,75,80 ℃ respectively; Treat enzyme liquid different time under 50,55,60,65,70,75 ℃ of differing tempss is measured the residual enzyme vigor.Optimal reaction pH and pH stability: enzyme liquid is measured the vigor of beta-amylase in pH3.0,4.0,5.0,6.0,7.0,8.0 citric acids-Sodium phosphate dibasic damping fluid, determine optimal reaction pH; At pH3.0,4.0,5.0,6.0,7.0,8.0 times treat enzyme liquid different times, measure the residual enzyme vigor.The influence that metal ion and EDTA live to enzyme: the Na that in the enzyme reaction system, adds 1mmol/L respectively
+, K
+, Ca
2+, Mg
2+, Fe
2+, Mn
2+, Ni
2+, Cu
2+Deng 8 metal ion species and EDTA.And be provided with and do not add the blank group of metal ion, measure enzyme of each group then and live.
The pure enzyme that obtains is run glue through SDS-PAGE, cut glue, use 100mmol/LNH
4HCO
3/ 30%ACN the cleaning of decolouring is with 100%ACN dehydration, 100mmol/LNH
4HCO
3, 100mmol/LDTT, 56 ℃ of hatching 30min crude protein also, go supernatant to dewater with 100%ACN, 20min is handled in 100mmol/LNH4HCO3,200mmol/LIAA dark place, go to reset and add the 100%ACN dehydration, freeze-drying adds 2.5ng/ μ L trypsin solution and handles 1h for 4 ℃, adds 25mmol/LNH again
4HCO
3Damping fluid, 37 ℃ of reactions are spent the night, sucking-off enzymolysis solution, some target plate.Last machine carries out mass spectroscopy.By mass-spectrogram, albumen of the same race when a following tiny band is with beta-amylase about the purpose band 65K of purifying as can be known is the degraded small protein of beta-amylase.
Claims (8)
1. the method for the high temperature resistant beta-amylase separation and purification of subtilis, described high temperature beta-amylase is from the subtilis 6-7 of high yield beta-amylase, be preserved in Chinese typical culture collection center, deposit number is: CTCC M2009200.
2. the method for claim 1 is characterized in that, wild type strain 6-7 utilizes Tapioca Starch, the outer beta-amylase of bean cake powder fermentative production born of the same parents, cultivates at the 100mL/500mL shake flask fermentation.
3. the method for claim 1 is characterized in that, the separation and purification concrete steps comprise:
(1) utilizes the high yield beta-amylase training method fermentation of optimizing, the fermented liquid 8000r/min of acquisition, 4 ℃ of frozen centrifugation 20min;
(2) centrifuged supernatant is added ammonium sulfate to 30% saturation ratio, leaves standstill 4 hours, and 8000r/min, 4 ℃ of frozen centrifugation 20min go precipitation, remove foreigh protein removing;
(3) continue to add ammonium sulfate to 60% saturation ratio, centrifugal collecting precipitation is precipitated out beta-amylase, with the Na of pH8.0,20mmol/L
2HPO
4-NaH
2PO
4Damping fluid redissolves;
(4) at same pH8.0, the Na of 20mmol/L
2HPO
4-NaH
2PO
4Damping fluid is dialysed, and is the dialysis tubing of 10KDa with the average molecular interception;
(5) will dialyse that sample concentrates to the accumulation of HiTrap Q Fast Flow post anion-exchange chromatography on the sample, A liquid is pH8.0,20mmol/LNa
2HPO
4-NaH
2PO
4Damping fluid, B liquid are pH8.0,20mmol/LNa
2HPO
4-NaH
2PO
4/ 1mol/LNaCl damping fluid carries out linear gradient elution with the NaCl of 0～1mol/L, is 20%～30% o'clock at B liquid, and the target protein beta-amylase elutes;
(6) wash-out being collected liquid is the freezing low temperature low-speed centrifugal of the ultra-filtration centrifuge tube desalination of 5K with the molecular retention amount, and the reduction electricity is directed at NaCl content and is lower than below the 50mmol/L, and sample is to the MonoQ5/50GL post on the processing reservation liquid, and A liquid is pH8.0,20mmol/LNa
2HPO
4-NaH
2PO
4Damping fluid, B liquid are pH8.0,20mmol/LNa
2HPO
4-NaH
2PO
4/ 1mol/LNaCl damping fluid, further high resolving power, high-bonding-ratio polishing purification;
(7), wash-out is collected on the liquid sample further remove the small molecules foreign protein to Sephadex G-75 according to selecting suitable gel column about target protein molecular size 65KDa;
(8) the enzyme liquid that is separated to is verified purity through the SDS-PAGE electrophoresis, the beta-amylase rate of recovery that obtains is 18.66%, and the purifying multiple is 4.83, and enzyme work reaches 245395U/mg.
4. the electrophoresis level beta-amylase zymologic property of purifying is analyzed, its optimum temperuture is that 65 ℃, optimal pH are 6.0.
5. as measuring under the optimum condition as described in the claim 4, beta-amylase residual enzyme work behind 65 ℃ of processing 1h, 2h, 3h is respectively 72.34%, 33.2%, 9.36%, and residual enzyme work is respectively 100%, 99.91%, 98.86% behind 50 ℃ of processing 1h, 2h, 3h; Place enzyme almost not loss alive in several days at 37 ℃, 4 ℃.
6. as described in the claim 4, beta-amylase pH4,5,6,, 7,8 handle 15h after residual enzyme work be more than 90%, residual enzyme work is 84.95% after pH3 handles 15h.
7. as described in the claim 3, purified gained beta-amylase is adding Mg
2+, Ni
2+Back enzyme is lived to 125.34%, 123.10% of control group, and Mg is described
2+, Ni
2+Enzyme had activation; Adding Mn
2+, Cu
2+, behind the EDTA, enzyme is lived to 43.27%, 87.93%, 69% of control group, and Mn is described
2+, Cu
2+, EDTA has certain restraining effect to enzyme.
8. as described in the claim 3, the little band of a degraded is arranged under the beta-amylase purpose band of purifying, this band of ground substance assistant laser desorption ionization mass spectrum (MALDI-TOF) Analysis and Identification is the beta-amylase protein degradation.
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| CN104630084A (en) * | 2013-11-14 | 2015-05-20 | 邵素英 | Bacillus subtilis producing high-temperature-resistance alpha-amylase |
| CN104630180A (en) * | 2013-11-14 | 2015-05-20 | 邵素英 | Application of bacillus subtilis |
| CN116286746A (en) * | 2023-05-08 | 2023-06-23 | 南京农丰生物科技有限公司 | Method for purifying amylase by liquid chromatography |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630084A (en) * | 2013-11-14 | 2015-05-20 | 邵素英 | Bacillus subtilis producing high-temperature-resistance alpha-amylase |
| CN104630180A (en) * | 2013-11-14 | 2015-05-20 | 邵素英 | Application of bacillus subtilis |
| CN103951736A (en) * | 2014-05-09 | 2014-07-30 | 山东省农业科学院农产品研究所 | Antimicrobial protein product of bacillus amyloliquefaciens NCPSJ7 and preparation method of antimicrobial protein product |
| CN103951736B (en) * | 2014-05-09 | 2016-03-16 | 山东省农业科学院农产品研究所 | A kind of bacillus amyloliquefaciens NCPSJ7 antibacterial protein product and preparation method thereof |
| CN116286746A (en) * | 2023-05-08 | 2023-06-23 | 南京农丰生物科技有限公司 | Method for purifying amylase by liquid chromatography |
| CN116286746B (en) * | 2023-05-08 | 2023-08-15 | 南京农丰生物科技有限公司 | Method for purifying amylase by liquid chromatography |
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Application publication date: 20130731 |