CN102838655B - Solid-liquid separation method for Pichia pastoris fermentation broth - Google Patents
Solid-liquid separation method for Pichia pastoris fermentation broth Download PDFInfo
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Abstract
The invention discloses a solid-liquid separation method for Pichia pastoris fermentation broth. The method comprises the following steps: uniformly mixing chitosan solution and Pichia pastoris fermentation broth, adjusting pH and stirring, subjecting chitosan to flocculation in the Pichia pastoris fermentation broth, and finally removing strains and macromolecule impurity through centrifugation or filtering to obtain solution comprising recombinant protein. By adopting the method disclosed by the invention, the removal rate of Pichia pastoris strains and the recovery rate of recombinant protein are both significantly improved, the time for separation is short, the cost for separation is low, the obtained solution has high clarity, and the method is more suitable for industrial production.
Description
Technical field
The invention belongs to bioengineering field, be specifically related to the separation purification method of Pichia anomala expression recombinant protein fermentation liquor.
Background technology
Pichia yeast expression system is a kind of novel exogenous protein expression system grown up the phase at the beginning of the eighties in last century, have operation simple and easy, be easy to that cultivation, fast growth, expression amount are high, low cost and other advantages.This system has become the production being widely used in recombinant protein at present, as recombinant human serum albumin and fusion rotein thereof, and industrial enzymes etc.
Along with maturation and the development of pichia spp high density fermentation technology, improve fermentation density and become a kind of Critical policies improving expressing quantity, current pichia spp fermentation thalli content can reach 40 ~ 50% of total fermented liquid, even higher, this solid-liquid separation being the total fermented liquid of pichia spp is had higher requirement, particularly in commercial process.Pichia spp solid-liquid separating method conventional at present mainly contains filter press method, membrane separation process, expanding bed chromatography, centrifuging etc.Filter press method mainly adopts plate-and-frame filter press, the method that filtration resistance realizes roughing out is reduced by adding flocculating aids, there is the advantages such as the rate of recovery is high, easy and simple to handle, cost is low, but the method equipment used floor space is large, clarity is low, filter effect is unstable, not easily " locked in " operation, and need the flocculating aids adding complicated ingredient, be unfavorable for Control of Impurities; Membrane separation process is the new technology occurring in 20 beginnings of the century, emerge rapidly after the sixties, pichia spp roughing out is carried out by the method, clarity is higher, easy to operate, be easy to automatization, but need to operate after diluted sample 3 ~ 4 times, operating time is long, shearing force is bigger than normal, is not suitable for separation [Guo Zhenyou etc., the application of tubular fibre millipore filtration (PVDF) in fermentation industry is produced of labile protein, Chinese biological engineering magazine, 2004,24 (4), 81 ~ 85]; Expanding bed chromatography is as pichia spp initial gross separation method, the three steps operations such as solid-liquid separation, concentrated and preliminary purification can be replaced, there is raising the efficiency, shorten the advantages such as operating time, be applied in the industrialization of pichia spp is produced at present, but the method is high to equipment requirements, investment large, high [the Sumi A.et al Purification of recombinant human serum albumin efficient purification using STREAMLINE of cost, Bioseparation, 1999; 8 (1-5): 195 ~ 200]; Centrifuging is the most frequently used solid-liquid separating method of current pichia spp, and can meet the separation requirement of various scale with different centrifugation apparatus, but this method energy consumption is bigger than normal, the cycle is long, the rate of recovery is low, is separated thorough, often needs dilution or repeatedly centrifugal.Thus in the urgent need to a kind of novel solid-liquid separating method, for suitability for industrialized production.
Flocculation separation technology is by adding flocculation agent, make the particle aggregation in solution, the neutralizing effect of compression with electric charge of electrostatic double layer, bridging effect, throw out net is utilized to catch the mechanism such as effect and small colloid, particle and the suspended substance in solution is removed and the technology be separated [Hao Shaoli etc., the application of precipitated separation technology in protein process, grain and foodstuffs industry, 2007,14 (1), 20 ~ 22].Flocculation agent is mainly divided into inorganic flocculating agent and polymeric flocculant, conventional iron, aluminium inorganic flocculating agent and be that the organic polymer class flocculation agent of representative exists the problems such as effect is bad or toxic with polyacrylamide, therefore should not adopt.Chitosan is the deacetylation product of chitin, is extensively present in the cell walls of insect, shrimp, crab shell animal shell and fungi, algae.The chemical name of chitosan is Isosorbide-5-Nitrae-2-amido-2-deoxidation-B-D-glucose, because of the different relative molecular weights of material and preparation method from hundreds thousand of to millions of not etc.Containing a large amount of free amine groups in chitosan molecule, under proper condition, can show cationic polyelectrolyte, therefore have strong throwing out, be a kind of rising natural macromolecule flocculating agent.Because it has the characteristics such as natural, nontoxic, tasteless, chitosan is widely used in drinking water purification, heavy metal recovery, molasses and fruit clarification of juice etc.In leavened prod flocculation, accidental employing chitosan carries out the report of fermentable small molecules product solid-liquid separation as flocculation agent, but because flocculate with chitosan has certain adsorption for protein, institute is not used widely in this approach in the roughing out of pichia spp fermented liquid.
Zhang Mingzheng etc. disclose a kind of method adopting chitosan to flocculate to bacillus thuringiensis fermented liquid, add 0.0025% chitosan, pH5.0 ~ 6.0, during temperature 30 ~ 35 DEG C, 550rpm stirs 5min, most of water and foreign pigment in fermented liquid can be removed, and make the insecticidal protein crystal in fermented liquid and gemma obtain well concentrated [Zhang Mingzheng etc., chitosan is to the throwing out of BT fermented liquid, Chinese biological is prevented and treated, 2009,25 (2), 133 ~ 137].
Liu Bingtao etc. disclose a kind of method adopting treatment with chitosan to contain proteinic wastewater, and add the chitosan of 0.01 ~ 0.02%, when pH is 6.0, flocculate, effectively can remove the protein in proteinic wastewater, clearance can reach more than 95%.[Liu Bingtao etc., chitosan is to the throwing out of BT fermented liquid, and Chinese biological is prevented and treated, and 2009,25 (2), 133 ~ 137].
Wang Qiujing etc. disclose a kind of method adopting chitosan to remove the thalline in soybean protein fermented liquid, and result display chitosan flocculating effect when pH value is 4 is best, and the soybean polypeptide concentration simultaneously in fermented liquid does not significantly reduce.[Wang Qiujing etc., chitosan is studied the flocculation of soybean protein fermented liquid, and soybean is circulated a notice of, and 2007,1 (86), 21-24].
The present invention adopts chitosan to be flocculation agent, by controlling solution temperature and pH value, flocculation agent addition, hydrophilic organic solvent addition, define a kind of solid-liquid separating method being applicable to the pichia spp fermented liquid of suitability for industrialized production of uniqueness, improve flocculation efficiency, greatly reduce separating difficulty, decrease operating time and cost, method of the present invention is while guarantee target protein high-recovery simultaneously, and the removal for macromolecule impurity albumen also has good effect.
Summary of the invention
The invention provides a kind of solid-liquid separating method of brand-new pichia spp fermented liquid, effectively can solve thalline in pichia spp high density fermentation liquid and remove difficulty, solid-liquid separation high in cost of production problem.
The present invention solves the problem by the following technical programs:
A solid-liquid separating method for pichia spp fermented liquid, comprises the following steps:
1) a kind of solution being dissolved with chitosan is provided;
2) by step 1) solution mix with the pichia spp fermented liquid containing recombinant protein tunning;
3) with alkaline matter regulating step 2) pH value to 6 ~ 12 of gained mixed solution, stir and make flocculate with chitosan;
4) by step 3) gained solution centrifugal or filtration, obtain the solution containing recombinant protein.
Wherein, can adopt and well known to a person skilled in the art method preparation steps 1) described in the solution being dissolved with chitosan, such as, the hydrochloric acid soln of the chitosan formed after chitosan being dissolved in hydrochloric acid or acetic acid or the acetum of chitosan, or by the solution of direct for water-soluble chitosan water-soluble formation.
Wherein, step 1) comprise further:
A) a kind of hydrophilic organic solvent is joined step 1) described in be dissolved with in the solution of chitosan.
The volume ratio of hydrophilic organic solvent and chitosan solution is 1: 9 ~ 1: 99, and hydrophilic organic solvent is selected from ethanol, n-propyl alcohol, Virahol, or their mixture, preferred alcohol.
Step 2) content of chitosan is 0.04% ~ 1% (w/v, g/ml) in gained mixed solution.
Step 3) described in alkaline matter be sodium hydroxide, sodium carbonate, sodium bicarbonate, or their mixture.PH value preferably 8 ~ 10.This step is at 2 DEG C ~ 25 DEG C, carries out under being preferably the temperature of 5 DEG C ~ 15 DEG C.Make flocculate with chitosan by stirring, the chitosan crosslinked thalline of flocculation forms larger particles, finally by filtration or centrifugal segregation particle, obtains the solution containing recombinant protein.
Described recombinant protein is recombinant human serum albumin and fusion rotein thereof, preferred recombinant human serum albumin/il-1 fusion rotein.
Advantage of the present invention is, by the throwing out of chitosan, makes thalline form larger particles, more easily realizes solid-liquid separation, meanwhile, adds a small amount of hydrophilic organic solvent and can increase thalline dispersity, improves protein recovery.Compared with existing isolation technique, method disengaging time of the present invention is short, separation costs is low, and operation controlled range is wide, and last gained clarity of solution is high, is more suitable for suitability for industrialized production.In addition, the present invention has obvious removal effect for the macromolecule impurity of some albumen, can reduce downstream purification operation pressure.
Accompanying drawing explanation
Fig. 1 is the HPLC analytical results of embodiment 1 gained supernatant liquor.
Fig. 2 is the HPLC analytical results of embodiment 4 gained supernatant liquor.
Fig. 3 is the HPLC analytical results of embodiment 7 gained sample.
Fig. 4 is under different chitosan content, thallus removing rate and recombinant protein rate of recovery comparison diagram, and wherein, X-coordinate represents chitosan content, and ordinate zou represents per-cent, "-◆-" represent the recombinant protein rate of recovery, "-■-" represents thallus removing rate.
Embodiment
Cell concentration p measuring method in embodiment: get the centrifugal 15min of 1ml fermented liquid 12000rpm, carry out HPLC analysis after supernatant liquid filtering, determines that protein content is C
1, separately get 500ul fermented liquid, accurately add after 500ul purified water mixes, the centrifugal 15min of 12000rpm, carries out HPLC analysis after supernatant liquid filtering, determines that protein content is C
2, cell concentration p=(C
1-2C
2)/(C
1-C
2) × 100%.
In embodiment, Tot Prot method of calculation are: C
1× V × (1-p), wherein V is fermentating liquid volume.
In embodiment, protein recovery method of calculation are: Tot Prot × 100% in Tot Prot/fermented liquid in supernatant liquor after solid-liquid separation.
Thallus removing rate measures and adopts spectrophotometry, and under 600nm, measure the OD value of supernatant liquor after fermented liquid and solid-liquid separation respectively, thallus removing rate is, the difference of the OD value of fermented liquid and supernatant liquor and the ratio of fermented liquid OD value.
Adopt Agilent 1200 high performance liquid chromatography to carry out macromolecule impurity assay in embodiment, method is with reference to " Chinese Pharmacopoeia " version in 2010 three annex VI Q human serum albumin polymer measuring methods.
Embodiment 1 (comparative example)
Adopt own bacterial classification and fermentation technique to carry out recombinant human serum albumin production in 100L fermentor tank, the OD600 measuring fermented liquid is 421, target protein content C in centrifugal rear detection supernatant liquor
1for 9.5g/L, after dilution, detect C
2for 3.5g/L, calculating cell concentration p is 41.7%.
400ml recombinant human serum albumin fermented liquid (calculating Tot Prot is 2.2g) is got after fermentation ends, be placed in Centrifuge Cup, at 4 DEG C in Beckman whizzer the centrifugal 5min of 10000rpm, obtain supernatant liquor 210ml, detecting protein content in supernatant liquor is 8.1g/L (Tot Prot is 1.70g), macromolecule impurity content is 7.5% (in accompanying drawing 1 RT=8.853 and 9.451 two impurity peaks), centrifugal secondary fermentation liquid OD600 is 22.3, calculating thallus removing rate is 94.7%, and protein recovery is 77.3%.
Embodiment 2
0.176g chitosan is dissolved in the acetum of 39.6ml 1%, then 0.4ml dehydrated alcohol is added, stir, then join in the fermented liquid of 400ml recombinant human serum albumin described in embodiment 1, red-tape operati temperature 15 DEG C, stirs 30min, adds sodium bicarbonate, regulate pH to 9.5, stir and make flocculate with chitosan.
Above-mentioned mixed solution is placed in Centrifuge Cup, at 4 DEG C in Beckman whizzer the centrifugal 5min of 10000rpm, obtain supernatant liquor 250ml, detecting protein content in supernatant liquor is 7.06g/L (Tot Prot is 1.765g), macromolecule impurity content is 3.0%, centrifugal secondary fermentation liquid OD600 is 2.8, and calculating thallus removing rate is 99.3%, and protein recovery is 80.2%.
Embodiment 3
0.44g chitosan is dissolved in the acetum of 40ml 1%, then joins in the fermented liquid of 400ml recombinant human serum albumin described in embodiment 1, red-tape operati temperature 15 DEG C, stir 30min, add sodium carbonate, regulate pH to 8.5, stir and make flocculate with chitosan.
Above-mentioned mixed solution is placed in Centrifuge Cup, at 4 DEG C in Beckman whizzer the centrifugal 5min of 10000rpm, obtain supernatant liquor 250ml, detecting protein content in supernatant liquor is 7.2g/L (Tot Prot is 1.8g), macromolecule impurity content is 3.3%, centrifugal secondary fermentation liquid OD600 is 1.1, and calculating thallus removing rate is 99.7%, and protein recovery is 81.8%.
Embodiment 4
0.44g chitosan is joined in the acetum of 38ml 1%, then 2ml dehydrated alcohol is added, stirring and evenly mixing, then join in the fermented liquid of 400ml recombinant human serum albumin described in embodiment 1, red-tape operati temperature 15 DEG C, stirs 30min, adds sodium carbonate, regulate pH to 8.5, stir and make flocculate with chitosan.
Above-mentioned mixed solution is placed in Centrifuge Cup, at 4 DEG C in Beckman whizzer the centrifugal 5min of 10000rpm, obtain supernatant liquor 250ml, detecting protein content in supernatant liquor is 7.5g/L (Tot Prot is 1.875g), macromolecule impurity content is 3.1% (accompanying drawing 2RT=8.866 and 9.447 two impurity peaks), centrifugal secondary fermentation liquid OD600 is 1.2, and calculating thallus removing rate is 99.7%, and protein recovery is 85.2%.
Embodiment 5
2.2g chitosan is dissolved in the acetum of 36ml 1%, then 4ml Virahol is added, stirring and evenly mixing, then join in the fermented liquid of 400ml recombinant human serum albumin described in embodiment 1, red-tape operati temperature 5 DEG C, stirs 30min, adds sodium carbonate, regulate pH to 8.0, stir and make flocculate with chitosan.
Above-mentioned mixed solution is placed in Centrifuge Cup, at 4 DEG C in Beckman whizzer the centrifugal 5min of 10000rpm, obtain supernatant liquor 245ml, detecting protein content in supernatant liquor is 7.8g/L (Tot Prot is 1.91g), macromolecule impurity content is 2.8%, centrifugal secondary fermentation liquid OD600 is 1.0, and calculating thallus removing rate is 99.8%, and protein recovery is 86.9%.
Embodiment 6
4.4g chitosan is dissolved in the hydrochloric acid soln of 38ml 0.5%, then 2ml dehydrated alcohol is added, stirring and evenly mixing, then join in the fermented liquid of 400ml recombinant human serum albumin described in embodiment 1, red-tape operati temperature 10 DEG C, stirs 30min, adds sodium carbonate, regulate pH to 10, stir and make flocculate with chitosan.
Above-mentioned mixed solution is placed in Centrifuge Cup, at 4 DEG C in Beckman whizzer the centrifugal 5min of 10000rpm, obtain supernatant liquor 240ml, detecting protein content in supernatant liquor is 8.0g/L (Tot Prot is 1.92g), macromolecule impurity content is 2.7%, centrifugal secondary fermentation liquid OD600 is 1.0, and calculating thallus removing rate is 99.8%, and protein recovery is 87.3%.
Embodiment 7
The supernatant liquor containing recombinant human serum albumin embodiment 5 obtained carries out purification process according to method disclosed in CN201010154987.4, obtain recombinant human serum albumin concentrated solution, its purity of HPLC methods analyst is adopted to be 99.6% (accompanying drawing 3, RT=14.808), host protein < 0.01ppm, analyzes chitosan with Phenol-sulphate acid method residual not higher than 0.2%.
Result shows, the present invention effectively can remove thalline, and through downstream purification operation, can obtain high-quality recombinant protein, and the chitosan molecule added also is easy to remove.
Embodiment 8
The recombination yeast of construction expression recombinant human serum albumin/il-1 fusion rotein produces bacterial strain, ferment in 100L fermentor tank, obtain the fermented liquid containing recombinant human serum albumin/il-1 fusion rotein, the OD600 measuring fermented liquid is 330, macromolecule impurity content is 6%, target protein content C in supernatant liquor
1for 0.2g/L, after dilution, detect protein content C
2for 0.082g/L, calculating cell concentration is 30.5%.
2.2g water-soluble chitosan is dissolved in 38ml purified water, then 2ml dehydrated alcohol is added, stirring and evenly mixing, then join above-mentioned 400ml and contain (target protein total amount 55.6mg) in the fermented liquid of recombinant human serum albumin/il-1 fusion rotein, red-tape operati temperature 15 DEG C, stirs 30min, adds sodium carbonate, regulate pH to 8.5, stir and make flocculate with chitosan.
Above-mentioned mixed solution is placed in Centrifuge Cup, at 4 DEG C in Beckman whizzer the centrifugal 5min of 10000rpm, obtain supernatant liquor 275ml, detecting protein content in supernatant liquor is 0.17g/L (Tot Prot is 46.75mg), macromolecule impurity content is 2.0%, centrifugal secondary fermentation liquid OD600 is 1.2, and calculating thallus removing rate is 99.6%, and protein recovery is 84.1%.
Embodiment 9
Adopt own bacterial classification and fermentation technique to carry out recombinant human serum albumin production in 5000L fermentor tank, obtain fermented liquid 3800L after fermentation ends, the OD600 measuring fermented liquid is 450, and macromolecule impurity content is 8.4%, target protein content C in supernatant liquor
1for 9.8g/L, after dilution, detect C
2for 3.6g/L, calculating cell concentration in fermented liquid is 41.9%, and in fermented liquid, target protein total amount is 21.64kg.
41.8kg chitosan is dissolved in the acetum of 361L 1%, then 19L dehydrated alcohol is added, stirring and evenly mixing, 3800L chitosan solution is proceeded in above-mentioned recombinant human serum albumin fermented liquid, control fermentation jar temperature 15 DEG C, stir 30min, add sodium bicarbonate, regulate pH to 9.5, stir and make flocculate with chitosan.
Above-mentioned mixed solution is added 1% diatomite, mixes, open transferpump, fermented liquid is pumped in plate-and-frame filter press and removes thalline, pass into pressurized air after filtration and the debris in sheet frame is dried up, keep filter pressure to be no more than 0.5MPa.Amount to after filtering and obtain supernatant liquor 2100L, detecting protein content in supernatant liquor is 8.6g/L (Tot Prot is 18.06kg), and macromolecule impurity content is 2.5%, and centrifugal secondary fermentation liquid OD600 is 3.6, calculating thallus removing rate is 99.2%, and protein recovery is 83.5%.
In embodiment 1-6 and embodiment 8,9, parameters detection architecture sees attached list 1.
Subordinate list 1
From the detected result of subordinate list 1, by adding solidifying wadding agent chitosan in pichia spp fermented liquid, effectively can improve the rate of recovery of thallus removing rate and target protein, reducing the content of macromolecule impurity.With the comparative result of embodiment 4, embodiment 3 shows that adding a small amount of hydrophilic organic solvent can improve protein recovery.
Claims (8)
1. a solid-liquid separating method for pichia spp fermented liquid, comprises the following steps:
1) a kind of solution being dissolved with chitosan is provided;
2) a kind of hydrophilic organic solvent is joined step 1) described in be dissolved with in the solution of chitosan;
3) by step 2) solution mix with the pichia spp fermented liquid containing recombinant protein tunning;
4) with alkaline matter regulating step 3) pH value to 8 ~ 10 of gained mixed solution, stir and make flocculate with chitosan;
5) by step 4) gained solution centrifugal or filtration, obtain the solution containing recombinant protein;
Wherein, the volume ratio of described hydrophilic organic solvent and chitosan solution is 1: 9 ~ 1: 99, step 3) content of chitosan is 0.04% ~ 1% (w/v) in gained mixed solution, described recombinant protein is recombinant human serum albumin and fusion rotein thereof.
2. the solid-liquid separating method of a kind of pichia spp fermented liquid according to claim 1, it is characterized in that, step 1) described in the solution being dissolved with chitosan be selected from: the hydrochloric acid soln of chitosan, the acetum of chitosan, or by the solution of direct for water-soluble chitosan water-soluble formation.
3. the solid-liquid separating method of a kind of pichia spp fermented liquid according to claim 1, it is characterized in that, described hydrophilic organic solvent is selected from ethanol, n-propyl alcohol, Virahol, or their mixture.
4. the solid-liquid separating method of a kind of pichia spp fermented liquid according to claim 1, it is characterized in that, described hydrophilic organic solvent is selected from ethanol.
5. the solid-liquid separating method of a kind of pichia spp fermented liquid according to claim 1, it is characterized in that, described alkaline matter is selected from sodium hydroxide, sodium carbonate, sodium bicarbonate, or their mixture.
6. the solid-liquid separating method of a kind of pichia spp fermented liquid according to claim 1, is characterized in that, step 4) be carry out at the temperature of 2 DEG C ~ 25 DEG C.
7. the solid-liquid separating method of a kind of pichia spp fermented liquid according to claim 1, is characterized in that, step 4) be carry out at the temperature of 5 DEG C ~ 15 DEG C.
8. the solid-liquid separating method of a kind of pichia spp fermented liquid according to claim 1, is characterized in that, described recombinant human serum albumin and fusion rotein thereof are recombinant human serum albumin/il-1 fusion rotein.
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| CN112301006B (en) * | 2020-11-17 | 2023-08-08 | 武汉新华扬生物股份有限公司 | Method for accelerating filtration speed of pichia pastoris fermentation broth |
| CN112375749A (en) * | 2020-11-17 | 2021-02-19 | 河北省微生物研究所 | Solid-liquid separation method for pichia pastoris fermentation liquor for producing glucose oxidase |
| CN115554978A (en) * | 2022-09-26 | 2023-01-03 | 武汉新华扬生物股份有限公司 | Compound for post-treatment of fermentation liquor and post-treatment method of fermentation liquor |
| CN117024567A (en) * | 2023-10-09 | 2023-11-10 | 健通(济南)生物科技有限公司 | Process for improving centrifugal yield of recombinant human serum albumin fermentation liquor |
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