CN105296444A - Pilot fermentation process for expressing recombinant acetylcholinesterase (AChE) in pichia methanolica - Google Patents

Pilot fermentation process for expressing recombinant acetylcholinesterase (AChE) in pichia methanolica Download PDF

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CN105296444A
CN105296444A CN201510878108.5A CN201510878108A CN105296444A CN 105296444 A CN105296444 A CN 105296444A CN 201510878108 A CN201510878108 A CN 201510878108A CN 105296444 A CN105296444 A CN 105296444A
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翁海波
李倩
孙召伟
田柳杨
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Zhengzhou University
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Abstract

The invention discloses a pilot fermentation process for expressing recombinant acetylcholinesterase (AChE) in pichia methanolica. The process is characterized by firstly culturing an activated recombinant pichia pastoris strain, thus preparing a fermentation working seed liquid, then transferring the fermentation working seed liquid to a basal salt culture medium, regulating the pH value of a fermentation system by utilizing ammonia water or phosphoric acid, then carrying out inoculation, adding proper amount of trace element solution, adding a mixed solution of glycerin and trace elements after growing to a certain stage, finally feeding a methanol solution containing trace elements to carry out protein induced fermentation, continuously maintaining the temperature at 29 DEG C and maintaining DO more than 20% in the fermentation process and carrying out induction with methanol for 120 hours, thus finishing the whole fermentation process. The pilot fermentation process has the advantages that the fermentation process is simple and is convenient to operate; the proteins are subjected to soluble expression and the expression quantities are high and the enzymatic activity is high but the protein expression cost is relatively low; large scale fermentation tank expression is carried out on AChE in a yeast expression system for the first time and lays the foundation for large scale production and final application of AChE.

Description

The pilot scale fermentation technique that restructuring acetylcholinesterase is expressed in pichia methanolica
Technical field
The present invention relates to biological technical field, especially relate to the pilot scale fermentation technique that a kind of acetylcholinesterase of recombinating is expressed in pichia methanolica.
Background technology
Agricultural chemicals is prevented and treated in process at farm-forestry crop disease pest and weed and is widely used, very important effect is had to agricultural produce, but farm crop have part pesticide residue over a period to come, if the farm crop of remains of pesticide of ingesting for a long time, can impact HUMAN HEALTH.Agricultural chemicals plays insecticidal action by suppressing the Pseudocholinesterase in insect nervus centralis to make it dead, also there is restraining effect to the Pseudocholinesterase in human body simultaneously, the transmission of energy block nerves mediator, reach a certain amount of after can cause muscular paralysis, cause the harm such as poisoning, carcinogenic, teratogenesis, mutagenesis.In recent years, along with deepening continuously of pesticide residue research, the detection method of pesticide residue is also gradually improved, and to simple, quick, sensitive, multi-residue determination, low cost, the easy future development promoted.The enzyme detected for remains of pesticide has Procaine esterase, butyrylcholine esterase and acetylcholinesterase, and be disturbed factor when first two enzyme detects many, false positive rate is high, and specificity is low.Acetylcholinesterase (Acetycholinesterase, AChE) is a kind of lytic enzyme be present in central nervous system, and the function of classics is hydrolysis neurotransmitter acetylcholine (ACh), stops the conduction of nerve impulse.Due to common remains of pesticide mainly Organophosphorus and carbamate pesticides class medicament, make it dead by suppressing the Pseudocholinesterase in insect nervus centralis.Therefore, AChE, as the optimal reaction substrate of this type of agricultural chemicals, is widely applied in the rapid detection of pesticide residue, has especially become the focus in the residual detect delay of agriculture based on the biosensor of AChE.
Because AChE mainly extracts from insect or animal blood, yield poorly, cost is high, sensitivity drift amplitude is large, seriously hinder the application of AChE in Detecting Pesticide.Along with the development of genetic engineering technique, by clone AChE gene, its high expression in external source expression system can be made.Research finds, AChE is unsuitable for prokaryotic expression, and after expressing, abiology is active, but utilizes eukaryotic expression system (as yeast, insect etc.) then can keep its intact biologic activity.
Pichia spp (PichiaPastoris) is unicellular eukaryote, grows soon, is easy to molecular genetics operation; The promotor of alcohol oxidase (AOXI) gene of pichia spp has strong inducibility and strong startability, is suitable for the high-level abduction delivering of foreign gene; Pichia spp has strong aerobic growth preferences, can carry out high cell densities cultivation, be beneficial to large-scale industrial production; Pichia spp can high-level secretory expression external source egg from, and due to pichia spp self, to be secreted into albumen in substratum little, is more conducive to later-period purification; Foreign gene is not be present in the plasmid of self-replicating simultaneously, but has been incorporated on yeast chromosomal together with expression vector, copies and heredity, the Loss of foreign gene can not occur with karyomit(e).Due to the plurality of advantages of this expression system, people more and more profit use it as the expression system of foreign gene to produce target protein.But even to this day, in yeast expression system, large scale fermentation expression is carried out to acetylcholinesterase and but have no report.
Summary of the invention
The object of the present invention is to provide the pilot scale fermentation technique that a kind of acetylcholinesterase of recombinating is expressed in pichia methanolica, this technological operation is simple, and protein expression cost is low and expression amount is high, enzymic activity is high.
For achieving the above object, the present invention can take following technical proposals:
The pilot scale fermentation technique that restructuring acetylcholinesterase of the present invention is expressed in pichia methanolica is:
First the recombinant pichia yeast strain that activation is preserved is cultivated, obtained fermentation work seed liquor, then this fermentation work seed liquor is transferred in basal salt media by certain inoculum size, adjustment fermentation jar temperature 29 DEG C, tank pressure 0.06MPa, rotating speed 600rpm, utilizes ammoniacal liquor or phosphoric acid adjustment fermentation system pH to be 5.2, corrects after dissolved oxygen DO reaches 100% and start to ferment:
First add glycerine solution and carry out the fermentation of increasing bacterium, treat glycerol depletion, bacterium liquid OD600 reaches about 90.0, adopt methanol solution to carry out protein induced fermentation in three stages again, in fermenting process, constant temperature maintains 29 DEG C, and it (is 600rpm owing to inducing front rotating speed that DO maintains more than 20%, rotating speed and dissolved oxygen lower limit 20% tandem automatically after induction, in collaborative maintenance tank body, dissolved oxygen remains on more than 20%), after methanol induction 120h, whole fermenting process terminates.
When adding glycerine solution and carrying out the fermentation of increasing bacterium, the flow velocity that adds of glycerine solution is 1.67ml/min.
When adopting methanol solution to carry out protein induced fermentation, methanol solution divides three phases slowly to add: the flow velocity of adding of first stage methyl alcohol is 0.2ml/min, adds 4h, stops adding methyl alcohol 2h; The flow velocity of adding of subordinate phase methyl alcohol is 0.4ml/min, adds 6h; The flow velocity of adding of phase III methyl alcohol is 1.5ml/min, adds 108h; Total induction fermentation duration is 120h.
In described glycerine and methanol solution, often liter of PTM1(trace element salts solution all containing 12ml).
Recombinant pichia yeast strain of the present invention is Chinese red woods ant Acetylcholinesterase recombinant yeast pichia pastoris X33/pPICZ α A-Acetylcholinesterase bacterial strain, its construction process is: pMD-19T/Acetylcholinesterase and expression vector pPICZ α A is carried out enzyme and cuts, connect, obtain expression vector pPICZ α A-Acetylcholinesterase, by its transformation of E. coli DH5 α, choose mono-clonal, identify positive plasmid, transformed yeast X33, filter out multiple copied recombinant bacterial strain X33/pPICZ α A-Acetylcholinesterase, save backup after qualification.
The invention has the advantages that zymotechnique is simple, easy to operate, solubility expression of protein and expression amount is high, enzymic activity is high, but protein expression cost is lower.The present invention has carried out large-scale fermentor tank to acetylcholinesterase first and has expressed in yeast expression system, for the scale operation of AChE is laid a good foundation with final application.
Accompanying drawing explanation
Fig. 1 is the thalline weight in wet base (mg/mL) recording different cultivation stage in fermenting process of the present invention.
Fig. 2 is the thalline weight in wet base (mg/mL) under the present invention and prior art fermentating culturing process.
Fig. 3 is the SDS-PAGE electrophoretogram of the recombinant yeast pichia pastoris X33 high density fermentation supernatant liquor of the red woods ant AChE of the present invention China.
Fig. 4 is the AChE activity (U/mL) in the different cultivation stage fermentation supernatant recorded in the present invention.
Embodiment
Below by specific examples, more detailed explanation is done to zymotechnique of the present invention.
The first step, prepares genetic engineering bacterium
Bacterial strain adopts Chinese red woods ant Acetylcholinesterase recombinant yeast pichia pastoris X33/pPICZ α A-Acetylcholinesterase bacterial strain, and its construction process is:
PMD-19T/Acetylcholinesterase and expression vector pPICZ α A is carried out double digestion with EcoRI, NotI respectively, reclaim object product T4DNAligase to connect, obtain expression vector pPICZ α A-Acetylcholinesterase, by its transformation of E. coli DH5 α, choose mono-clonal, identify positive plasmid.
By pPICZ α A-Acetylcholinesterase with after the linearizing of SacI single endonuclease digestion, mix with pichia spp X33 competent cell and carry out electricity and transform, conversion product even spread is to Zeocin(100 μ g/mL) the YPD substratum plate of resistance, put 29 DEG C of constant temperature culture 2 ~ 4 days, the bacterium colony that this culture medium flat plate grows is His+ transformant.In picking flat board, the good bacterium colony of growing way carries out abduction delivering, thus filters out multiple copied recombinant bacterial strain X33/pPICZ α A-Acetylcholinesterase, saves backup after qualification.
Second step, prepared by seed liquor
Take out the recombination yeast engineering bacteria of the first step preservation and activate, at Zeocin(100 μ g/mL) the YPD solid medium of resistance is rule, after growing bacterium colony, from this plate, picking mono-clonal recombinant bacterium cultivates 24 hours in first order seed YPD liquid nutrient medium, reach 5.0 ~ 6.0 to cell concentration OD600, obtain first order seed nutrient solution; Then, 10% inoculum size is seeded in BMGY substratum (altogether 600mL), cultivates 20 hours, reaches 40.0, namely obtain fermentation seed liquid to cell concentration OD600.
YPD liquid nutrient medium wherein used: take the yeast powder of 1%, the peptone of 2% and the glucose of 2%, add water to volume required, mixes rear 115 DEG C of sterilizing 20min.Cool rearmounted 4 DEG C to save backup.
BMGY liquid nutrient medium used: will containing the yeast powder of 1%, the suitable quantity of water solution sterilization of the peptone of 2%, then add 0.22 μm of membrane filtration degerming 1.34% YNB, 0.1M phosphate buffered saline buffer (pH6.0), 410 -5% vitamin H and 10% glycerine, mix latter 4 DEG C and save backup.
3rd step, inoculation
1) prepare before inoculation
Preparation BSM substratum: weigh 26.7mLH 3pO 4(85%), 0.93gCaSO 4, 18.2gK 2sO 4, 14.9gMgSO 47H 2o, 4.13gKOH, 40.0g glycerine, adds water to after 1L mixes, saves backup after 121 ° of C high-temperature sterilizations.
Configure micro-salts solution PTM1:6.0gCuSO 45H 2o, 0.08gNaI, 3.0gMnSO 4h 2o, 0.2gNaMoO 42H 2o, 0.02gH 3bO 4, 0.5gCoCl 2, 20.0gZnCl 2, 65.0gFeSO 47H 2o, 0.2g vitamin H, 5.0mL sulfuric acid, adds water to 1L, degerming with 0.22 μm of membrane filtration, room temperature preservation.
Fermentor tank is carried out assembling and cleaning up, and corrects the pH electrode probe of fermentor tank with PH4.0, pH6.8 reference liquid, peristaltic pump traffic alignment.Fermentor tank parameter is set: temperature 29 ° of C, rotating speed 600rpm, pH5.2, dissolved oxygen 20%, tank pressure 0.06MPa.Fermentation tank culture medium BSM(15L by having prepared) substratum pours in fermentor tank (20L), 121 ° of C autoclaving 30min.Correcting dissolved oxygen DO when high pressure starts is 0%, until temperature be down to 29 DEG C of sterilizings close to an end time, set stirring, after ventilation, correcting dissolved oxygen DO is 100%.
2) inoculate
Sterilizing terminates to be cooled after 29 ° of C, opens inoculation mouth and add the PTM1 solution 65.25mL that the defoamer of 5mL and concentration are 4.35mL/L under the protection of flame ring, and with 28% ammoniacal liquor adjustment fermentation tank culture medium pH to 5.2.By fermentation work seed liquor with 4% inoculum size under the protection of flame ring, be inoculated in (liquid amount is for 20L) in full automatic control fermentor tank; auto-feeding ammoniacal liquor or phosphoric acid control ph are 5.2; regulate the rotating speed of fermentation equipment, air flow quantity, tank pressure 0.06MPa; correct dissolved oxygen DO and reach 100%(note: DO is relative dissolved oxygen level; saturated dissolved oxygen level when being about to just inoculation is set as 100%, and the dissolved oxygen level of fermenting process display is the numerical value relative to saturated dissolved oxygen).
4th step, feed supplement growth (increasing bacterium fermentation)
Meet bacterium about 21 ~ 24h, after the glycerol depletion in fermentor tank, DO value is on the rise, and now OD600 is about 70.00, and weight in wet base is about 120mg/mL.Stop feed supplement after adding feed supplement (50% aqueous glycerin solution containing 1.2%PTM1) 600mL, 6h with the flow velocity of 1.67mL/min, now OD600 is about 100.00.After cultivating 4 ~ 6h again, dissolved oxygen DO value significantly rises, and now glycerine is in spent condition, and culture system OD600 value should be about 120.00, and thalline weight in wet base is 242mg/mL, and now biomass reaches pre-provisioning request.
As can be seen from Figure 1, in this fermenting process, thalline enters logarithmic phase after the shorter adaptive phase, thalline is very fast in 12 ~ 30h growth, weight in wet base rises to 242mg/mL from 37mg/mL, and weight in wet base only rises to 147mg/mL from 118mg/mL during 18h to 24h, and this is because the glycerine in now fermentation tank culture medium is almost depleted, to 30h after adding 50% glycerine, thalline weight in wet base can reach 242mg/mL, and now by adding glycerine, biomass has reached pre-provisioning request.
5th step, methanol induction expresses the acetylcholinesterase recombinant protein stage
After stopping adding glycerine, thalline maintains starvation 2 ~ 4 hours, treat that glycerine thoroughly exhausts, when thalline weight in wet base reaches preset value, start the expression that the methanol solution added in three stages containing 1.2%PTM1 starts to induce restructuring AChE: the flow velocity of adding of first stage methyl alcohol is 0.2ml/min, DO value will maintain more than 20%, adds 4h, stops adding methyl alcohol 2h; The flow velocity of adding of the feed supplement methyl alcohol of subordinate phase is 0.4ml/min, adds 6h; The methanol feeding flow acceleration of phase III is 1.5ml/min, adds 108h, and so far after induction 120h, whole fermenting process terminates, and the temperature in fermenting process is 29 DEG C, and DO value will maintain more than 20%.
As can be seen from Figure 2, fermentation process (the inductive condition: pH5.0 adopted with prior art, first add containing PTM112ml/L methanol solution 4 hours with the speed stream of 0.115ml/min, then continue stream with the speed of 0.23ml/min again and add 4 hours, finally add with the speed stream of 0.345ml/min again, fermentation is terminated after abduction delivering 72h) contrast, the protein-active that present invention process induces than prior art processes is higher, and output is more.Fig. 2 shows different culture condition hypothallus weight in wet base to scheme over time, and series one is zymotechnique of the present invention, and series two is prior art zymotechnique.Thus represent in zymotechnique, different pH value, cultivation and inductive condition significantly can improve the expression amount of Acetylcholinesterase recombinant protein in pichia spp.
Qualification to the Acetylcholinesterase recombinant protein that the present invention induces:
(1) separation gel of 12% and the concentrated glue of 5% is recorded, the fermented supernatant fluid getting the different cultivation stage of 20 μ l carries out SDS-PAGE electrophoresis, the rear expression observing Acetylcholinesterase recombinant protein of dyed and decolouring, see Fig. 3, along with the prolongation of induction time, the expression amount of Acetylcholinesterase recombinant protein (64KD) is raising gradually.
In Fig. 3: M. Protein Marker Marker(from top to bottom size is: 99,66,44,29,20 and 14KD); Fermentation supernatant when 1, X33 recombinant bacterium is not induced; 2, the fermentation supernatant of methanol induction 12h; 3, the fermentation supernatant of methanol induction 24h; 4, the fermentation supernatant of methanol induction 48h; 5, the fermentation supernatant of methanol induction 72h; 6, the fermentation supernatant of methanol induction 96h;
(2) Enzyme assay of restructuring AChE.
Principle: Ellman method utilizes acetylthiocholine to be decomposed into thiocholine and acetate under AChE effect, thiocholine and 5, two-2-nitrobenzoic acid (DTNB) reaction of 5-two sulphur generates yellow compound 5-sulphur-2-nitrobenzoic acid, have maximum absorption band at 410 ~ 420nm place, within the specific limits its concentration and absorption value linear.Colorimetric assay is carried out to it, the activity value of the thiocholine amount reflection Pseudocholinesterase that hydrolysis produces.
Step:
(1) pH8.0 buffered soln: get 11.9g anhydrous di-potassium hydrogen phosphate respectively and 3.2g potassium primary phosphate 1L distilled water dissolves.
(2) thioacetyl iodo choline solution (substrate): take thioacetyl iodo choline 25mg, be dissolved in 3.0mL distilled water, shakes up the preservation of 4 DEG C, rearmounted refrigerator.
(3) DTNB solution (developer): take DTNB160mg and sodium bicarbonate 15.6mg, use 20mL buffer solution, puts the preservation of 4 DEG C, refrigerator.
(4) in test tube, add 2.5mL buffered soln, then add 0.1mL enzyme liquid, 0.1mL developer, after shaking up, place 15min in 37 DEG C.Add 0.1mL substrate to shake up, put into cuvette immediately, the absorbancy changing value Δ A of record reaction 3min 0.
(5) calculation formula of enzyme activity unit is:
In formula: Δ A is the difference of absorbancy before and after reaction;
V is reaction system cumulative volume, and body is 2.810 -3l;
V is sample volume, and body is 0.1 × 10 -3l;
ε is the molar absorptivity of yellow product, is 1.36x10 4l/ (molcm);
T is reaction times 3min;
10 6for mole being converted into micromolar transformation ratio;
L is cuvette width (cm).
Because the computation process of enzyme activity is more complicated, the sometimes size of enzyme activity, simply can characterize the size of enzyme activity by the difference (Δ A) of the absorbancy before and after reaction, Δ A value is larger, represents that enzyme activity is larger.
Get the fermented supernatant fluid of different cultivation stage, carry out Enzyme assay according to above-mentioned detection enzyme activity method and go out the enzyme activity size of Different growth phases in the present invention according to above-mentioned formulae discovery, and finally obtain the restructuring Acetylcholinesterase of 7686.27U/mL, see Fig. 4.
Demonstrate in Fig. 4 that restructuring Acetylcholinesterase is active to be raised gradually along with the prolongation of time, increasesd slowly before 84h, from 84h to 108h, enzymic activity increases substantially, and afterwards without rising tendency, to 120h fermentation ends, finally obtains 7686.27U/mL.
The separation of Acetylcholinesterase recombinant protein: abduction delivering terminates fermentation after 120 hours, fermentation liquor 4 DEG C, obtain fermentation supernatant after 5000r/min is centrifugal, after ultrafiltration and concentration through vacuum freezing drain be stored in-20 DEG C for subsequent use, diverse ways can be adopted as required to carry out purifying.

Claims (5)

1. the pilot scale fermentation technique that acetylcholinesterase of recombinating is expressed in pichia methanolica, is characterized in that:
First the recombinant pichia yeast strain that activation is preserved is cultivated, obtained fermentation work seed liquor, then this fermentation work seed liquor is transferred in basal salt media by certain inoculum size, adjustment fermentation jar temperature 29 DEG C, tank pressure 0.06MPa, rotating speed 600rpm, utilizes ammoniacal liquor or phosphoric acid adjustment fermentation system pH to be 5.2, corrects after dissolved oxygen DO reaches 100% and start to ferment:
First add glycerine solution and carry out the fermentation of increasing bacterium, treat glycerol depletion, bacterium liquid OD600 reaches about 90.0, carry out protein induced fermentation with methanol solution again, in fermenting process, constant temperature maintains 29 DEG C, and DO maintains more than 20%, methanol induction 120h, whole fermenting process terminates.
2. the pilot scale fermentation technique expressed in pichia methanolica of restructuring acetylcholinesterase according to claim 1, is characterized in that: add glycerine solution when carrying out the fermentation of increasing bacterium, the flow velocity that adds of glycerine solution is 1.67ml/min.
3. the pilot scale fermentation technique expressed in pichia methanolica of restructuring acetylcholinesterase according to claim 1, it is characterized in that: when adopting methanol solution to carry out protein induced fermentation, methanol solution divides three phases slowly to add: the flow velocity of adding of first stage methyl alcohol is 0.1ml/min, add 10h, stop adding methyl alcohol 2h; The flow velocity of adding of subordinate phase methyl alcohol is 0.2ml/min, adds 22h, stops adding methyl alcohol and is about 2h; The flow velocity of adding of phase III methyl alcohol is 0.5ml/min, adds 84h; Total induction fermentation duration is 120h.
4. the pilot scale fermentation technique expressed in pichia methanolica of restructuring acetylcholinesterase according to claim 1, is characterized in that: in described glycerine and methanol solution, often liter of micro-salts solution all containing 12ml.
5. the pilot scale fermentation technique expressed in pichia methanolica of restructuring acetylcholinesterase according to claim 1, it is characterized in that: described recombinant pichia yeast strain is Chinese red woods ant Acetylcholinesterase recombinant yeast pichia pastoris X33/pPICZ α A-Acetylcholinesterase bacterial strain, its construction process is: pMD-19T/Acetylcholinesterase and expression vector pPICZ α A is carried out enzyme and cuts, connect, obtain expression vector pPICZ α A-Acetylcholinesterase, by its transformation of E. coli DH5 α, choose mono-clonal, identify positive plasmid, transformed yeast X33, filter out multiple copied recombinant bacterial strain X33/pPICZ α A-Acetylcholinesterase, save backup after qualification.
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