CN105296444B

CN105296444B - Pilot-scale fermentation method for expressing recombinant acetylcholinesterase in pichia methanolica

Info

Publication number: CN105296444B
Application number: CN201510878108.5A
Authority: CN
Inventors: 翁海波; 李倩; 孙召伟; 田柳杨
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2015-12-04
Filing date: 2015-12-04
Publication date: 2020-02-18
Anticipated expiration: 2035-12-04
Also published as: CN105296444A

Abstract

The invention discloses a pilot fermentation process for expressing recombinant acetylcholinesterase in pichia methanolica, which comprises the following steps: firstly, culturing an activated recombinant pichia pastoris strain to prepare a fermentation working seed solution, then transferring the fermentation working seed solution into a basic salt culture medium, adjusting the pH of a fermentation system by ammonia water or phosphoric acid, inoculating, adding a proper amount of trace element solution, growing to a certain stage, adding a mixed solution of glycerol and trace elements, finally adding a methanol solution containing trace elements in a flowing manner to perform protein induction fermentation, continuously maintaining the temperature at 29 ℃ during the fermentation process, maintaining the DO at more than 20%, inducing the methanol for 120h, and finishing the whole fermentation process. The invention has simple fermentation process, convenient operation, high protein solubility expression and expression quantity, high enzyme activity and lower protein expression cost. The invention firstly performs large-scale fermentation tank expression on the acetylcholinesterase in a yeast expression system, and lays a foundation for the large-scale production and final application of AChE.

Description

Pilot-scale fermentation method for expressing recombinant acetylcholinesterase in pichia methanolica

Technical Field

The invention relates to the technical field of biology, in particular to a pilot-scale fermentation method for expressing recombinant acetylcholinesterase in pichia methanolica.

Background

The pesticide can be widely used in the prevention and treatment process of diseases, pests and weeds of agricultural and forestry crops, has a very important effect on agricultural production increase, but has partial pesticide residues on the crops in a certain period, and can affect human health if the crops with residual pesticide are eaten for a long time. The pesticide has the insecticidal effect by inhibiting cholinesterase in central nerve of insect to kill it, and has the inhibiting effect on cholinesterase in human body to block neurotransmitter transmission, so that after reaching a certain amount, muscle paralysis can be caused, and poisoning, carcinogenesis, teratogenesis, mutagenesis and other hazards can be caused. In recent years, with the continuous and deep research on pesticide residues, the detection method of pesticide residues is improved, and the detection method is developed towards simple, rapid, sensitive, multi-residue detection, low cost and easy popularization. The enzymes used for detecting the residual pesticide include carboxylesterase, butyrylcholinesterase and acetylcholinesterase, the former two enzymes are subjected to a plurality of interference factors during detection, the false positive rate is high, and the specificity is low. Acetylcholinesterase (AChE) is a hydrolase enzyme present in the central nervous system, and the classical action is to hydrolyze the neurotransmitter acetylcholine (ACh), terminating the conduction of nerve impulses. Since the common pesticide residues are mainly organophosphorus and carbamate drugs, the pesticide residues are killed by inhibiting cholinesterase in central nerves of insects. Therefore, AChE is widely used as the optimal reaction substrate of the pesticide in the rapid detection of pesticide residue, and especially, the AChE-based biosensor becomes a hot spot in the research of pesticide residue detection.

Because AChE is mainly extracted from insect or animal blood, the yield is low, the cost is high, the sensitivity change amplitude is large, and the application of AChE in pesticide residue detection is seriously hindered. With the development of genetic engineering technology, AChE gene can be cloned to be expressed efficiently in an exogenous expression system. It is found that AChE is not suitable for prokaryotic expression, and has no biological activity after expression, but can maintain its intact biological activity by using eukaryotic expression system (such as yeast, insect, etc.).

Pichia pastoris (Pichia pastoris) is a unicellular eukaryote, grows fast, and is easy for molecular genetic manipulation; the promoter of Alcohol Oxidase (AOXI) gene of Pichia pastoris has strong inductivity and strong startability, and is suitable for high-level induction expression of exogenous genes; the pichia pastoris has strong aerobic growth preference, can be used for high-density cell culture, and is beneficial to large-scale industrial production; the pichia pastoris can secrete and express exogenous egg yolk at high level, and because the protein secreted into the culture medium by the pichia pastoris is little, the later purification is more facilitated; meanwhile, the exogenous gene is not present in a plasmid which autonomously replicates but is integrated on a yeast chromosome together with an expression vector, replicates and inherits along with the chromosome, and the loss phenomenon of the exogenous gene cannot occur. Due to the advantages of this expression system, it is increasingly used as an expression system for foreign genes to produce proteins of interest. However, large-scale fermentative expression of acetylcholinesterase in yeast expression systems has not been reported to date.

Disclosure of Invention

The invention aims to provide a pilot-scale fermentation method for expressing recombinant acetylcholinesterase in pichia methanolica, which is simple to operate, low in protein expression cost, high in expression level and high in enzyme activity.

In order to achieve the purpose, the invention can adopt the following technical scheme:

the pilot fermentation method for expressing the recombinant acetylcholinesterase in the pichia methanolica comprises the following steps:

firstly, culturing an activated and preserved recombinant pichia pastoris strain to prepare a fermentation working seed solution, then transferring the fermentation working seed solution into a basic salt culture medium according to a certain inoculation amount, adjusting the temperature of a fermentation tank to 29 ℃, the pressure of the fermentation tank to 0.06MPa and the rotation speed to 600rpm, adjusting the pH of a fermentation system to 5.2 by using ammonia water or phosphoric acid, and starting fermentation after correcting dissolved oxygen DO to 100 percent:

adding glycerol solution to perform enrichment fermentation, after the glycerol is exhausted, enabling the OD600 of the bacterial liquid to reach about 90.0, performing protein induction fermentation in three stages by adopting methanol solution, continuously maintaining the temperature at 29 ℃ and the DO at more than 20% (because the rotating speed before induction is 600rpm, the rotating speed after induction is automatically cascaded with the dissolved oxygen lower limit of 20%, and the dissolved oxygen in the tank is maintained at more than 20% in a coordinated manner), and finishing the whole fermentation process after the methanol induction is performed for 120 h.

When the glycerol solution is added for enrichment fermentation, the adding flow rate of the glycerol solution is 1.67 ml/min.

When the methanol solution is adopted for protein induction fermentation, the methanol solution is slowly added in three stages: in the first stage, the methanol is supplemented at the flow rate of 0.2ml/min for 4 hours, and the methanol is stopped for 2 hours; the replenishing flow rate of the methanol in the second stage is 0.4ml/min, and the replenishing time is 6 hours; the replenishing flow rate of the methanol in the third stage is 1.5ml/min, and the replenishing time is 108 hours; the total induction fermentation time is 120 h.

The glycerol and methanol solutions each contained 12ml of PTM1 (trace element salt solution) per liter.

The recombinant pichia pastoris strain is a Chinese red forest ant Acetylcholinesterase recombinant pichia pastoris strain X33/pPICZ α A-Acetylcholinesterase, the construction method comprises the steps of carrying out enzyme digestion and connection on pMD-19T/Acetylcholinesterase and an expression vector pPICZ α A to obtain an expression vector pPICZ α A-Acetylcholinesterase, transforming the expression vector into escherichia coli DH5 α, selecting single clone, identifying positive plasmid, transforming yeast X33, screening out multi-copy recombinant strain X33/pPICZ α A-Acetylcholinesterase, and storing for later use after identification.

The invention has the advantages of simple fermentation process, convenient operation, high protein solubility expression and expression quantity, high enzyme activity and lower protein expression cost. The invention firstly performs large-scale fermentation tank expression on the acetylcholinesterase in a yeast expression system, and lays a foundation for the large-scale production and final application of AChE.

Drawings

FIG. 1 shows the wet weight (mg/mL) of cells measured at different stages of culture in the fermentation process of the present invention.

FIG. 2 shows the wet weight (mg/mL) of cells in the fermentation culture process of the present invention and the prior art.

FIG. 3 is a SDS-PAGE electrophoresis of the recombinant Pichia pastoris X33 high density fermentation supernatant of Ant Waltheria AChE of the present invention.

FIG. 4 shows the AChE activity (U/mL) measured in the fermentation supernatants at different stages of culture in the present invention.

Detailed Description

The fermentation process of the present invention is described in more detail below by way of specific examples.

First, preparing genetically engineered bacteria

The bacterial strain adopts Chinese red forest ant Acetylcholinesterase recombined Pichia pastoris X33/pPICZ α A-Acetylcholinesterase bacterial strain, and the construction method comprises the following steps:

the pMD-19T/Acetylcholinesterase and the expression vector pPICZ α A are subjected to double enzyme digestion by EcoRI and NotI respectively, a target product is recovered and is connected by T4 DNAllase to obtain the expression vector pPICZ α A-Acetylcholinesterase, the expression vector is transformed into Escherichia coli DH5 α, and a single clone is selected to identify a positive plasmid.

The method comprises the steps of performing single enzyme digestion linearization on pPICZ α A-Acetylcholinesterase by SacI, uniformly mixing the linearized pPICZ α A-Acetylcholinesterase with Pichia pastoris X33 competent cells, performing electrotransformation, uniformly coating a transformant on a YPD (100 mu g/mL) culture medium plate with resistance to Zeocin, placing the YPD culture medium plate at 29 ℃ for constant temperature culture for 2-4 days, and selecting a colony growing on the culture medium plate as a His + transformant for inducible expression, so that a multi-copy recombinant strain X33/pPICZ α A-Acetylcholinesterase is screened out and stored for later use after identification.

Second, seed liquid preparation

Taking out and activating the recombinant yeast engineering bacteria preserved in the first step, streaking on a YPD solid culture medium with resistance of Zeocin (100 mu g/mL), and after the bacteria grow out, selecting a monoclonal recombinant bacteria from the plate to culture in a YPD liquid culture medium of first-level seeds for about 24 hours until the bacteria concentration OD600 reaches 5.0-6.0, so as to obtain a first-level seed culture solution; then, the mixture was inoculated into BMGY medium (total 600 mL) at an inoculum size of 10% and cultured for about 20 hours until the OD600 of the cell concentration reached 40.0, to obtain a fermentation seed solution.

YPD liquid medium used therein: weighing 1% yeast powder, 2% peptone and 2% glucose, adding water to required volume, mixing well, and sterilizing at 115 deg.C for 20 min. Cooling, and storing at 4 deg.C.

BMGY liquid medium used: sterilizing appropriate amount of water solution containing 1% yeast powder and 2% peptone, adding 1.34% YNB, 0.1M phosphate buffer (pH 6.0), 410-5% biotin and 10% glycerol, filtering with 0.22 μ M filter membrane, mixing, and storing at 4 deg.C.

Third step, inoculation

1) Preparation before inoculation

Preparing a BSM culture medium: 26.7mLH3PO4 (85%), 0.93g CaSO4,18.2g K2SO4,14.9g MgSO4 & 7H2O,4.13g KOH and 40.0g glycerol were weighed, added with water to 1L and mixed uniformly, and then sterilized at 121 ℃ and stored for later use.

Preparing a trace element salt solution PTM 1: 6.0g of CuSO4 & 5H2O, 0.08g of NaI,3.0g of MnSO4 & H2O,0.2g of NaMoO4 & 2H2O,0.02g of H3BO4,0.5g of CoCl2,20.0g of ZnCl2,65.0g of FeSO4 & 7H2O,0.2g of biotin and 5.0mL of sulfuric acid, adding water to 1L, filtering and sterilizing the mixture with a 0.22 mu m filter membrane, and storing the mixture at room temperature.

The fermenter was assembled and cleaned, and the pH electrode probe of the fermenter was calibrated with a pH4.0, pH6.8 standard solution, and the flow rate was calibrated with a peristaltic pump. Setting fermentation tank parameters: the temperature is 29 ℃, the rotating speed is 600rpm, the pH value is 5.2, the dissolved oxygen is 20 percent, and the tank pressure is 0.06 MPa. The prepared fermenter medium BSM (15L) was poured into a fermenter (20L) and autoclaved at 121 ℃ for 30 min. The dissolved oxygen DO is corrected to be 0% at the beginning of high pressure, stirring is well set when the temperature is reduced to 29 ℃ and the sterilization is about to be finished, and the dissolved oxygen DO is corrected to be 100% after ventilation.

2) Inoculation of

After cooling to 29 ℃ after sterilization, the inoculation opening is opened under the protection of a flame ring, 5mL of antifoaming agent and 65.25mL of PTM1 solution with the concentration of 4.35mL/L are added, and the pH of the fermentation tank medium is adjusted to 5.2 by using 28% ammonia water. Inoculating the fermentation working seed liquid into a full-automatic control fermentation tank (the liquid loading amount is 20L) under the protection of a flame ring by using the inoculation amount of 4 percent, automatically feeding ammonia water or phosphoric acid to control the pH value to be 5.2, adjusting the rotating speed, the air flow and the tank pressure of the fermentation equipment to be 0.06MPa, and correcting the dissolved oxygen DO to be 100 percent (note: DO is relative dissolved oxygen level, namely the saturated dissolved oxygen level at the moment of inoculation is set to be 100 percent, and the dissolved oxygen level displayed in the fermentation process is a numerical value relative to the saturated dissolved oxygen).

The fourth step, supplement material growth (enrichment fermentation)

Inoculating for about 21-24 h, wherein the DO value tends to rise after the glycerol in the fermentation tank is exhausted, the OD600 is about 70.00, and the wet weight is about 120 mg/mL. 600mL of feed (50% glycerol in water with 1.2% PTM 1) was added at a flow rate of 1.67mL/min and stopped after 6h, at which time the OD600 was approximately 100.00. And after culturing for 4-6 h, the DO value of the dissolved oxygen is greatly increased, the glycerol is in a depletion state at the moment, the OD600 value of a culture system is about 120.00, the wet weight of the thallus is 242mg/mL, and the thallus amount reaches the preset requirement at the moment.

As can be seen from FIG. 1, in the fermentation process, after a short adaptation period, the thalli enters the logarithmic growth phase, the thalli grow very rapidly in 12-30 h, the wet weight of the thalli is increased from 37mg/mL to 242mg/mL, and the wet weight of the thalli is only increased from 118mg/mL to 147mg/mL in 18h to 24h, because the glycerol in the culture medium of the fermentation tank is almost exhausted at the moment, and after 50% of glycerol is added to 30h, the wet weight of the thalli can reach 242mg/mL, and at the moment, the bacterial amount reaches the preset requirement by supplementing glycerol.

Step five, methanol induction expression of acetylcholinesterase recombinant protein stage

After the glycerol is stopped being supplemented, the thalli are maintained in a starvation state for 2-4 hours, when the glycerol is completely consumed and the wet weight of the thalli reaches a preset value, methanol solution containing 1.2 percent of PTM1 is supplemented in three stages to start inducing the expression of recombinant AChE: in the first stage, the replenishing flow rate of the methanol is 0.2ml/min, the DO value is maintained to be more than 20%, the replenishing is carried out for 4 hours, and the methanol is stopped for 2 hours; the replenishing flow rate of the methanol for replenishing in the second stage is 0.4ml/min and the methanol for replenishing is 6 hours; the feeding speed of methanol in the third stage is 1.5ml/min, and the feeding time is 108h, so that the whole fermentation process is finished after 120h of induction, the temperature in the fermentation process is 29 ℃, and the DO value is maintained to be more than 20%.

As can be seen from FIG. 2, compared with the fermentation method adopted in the prior art (induction condition: pH5.0, first feeding the solution containing PTM112ml/L methanol at the speed of 0.115ml/min for 4 hours, then feeding the solution continuously at the speed of 0.23ml/min for 4 hours, and finally feeding the solution at the speed of 0.345ml/min, and finishing the fermentation after the induction expression is carried out for 72 hours), the protein activity induced by the process of the invention is higher and the yield is more than that induced by the process of the prior art. FIG. 2 shows the variation of wet weight of thallus with time under different culture conditions, the first series is the fermentation process of the present invention, and the second series is the fermentation process of the prior art. Therefore, the expression quantity of the Acetylcholinesterase recombinant protein in the Pichia pastoris can be greatly improved under different pH values, culture and induction conditions in the fermentation process.

The identification of the Acetylcholinesterase recombinant protein induced by the invention comprises the following steps:

(1) pouring 12% separation gel and 5% concentration gel, taking 20 μ l fermentation supernatant in different culture stages to perform SDS-PAGE electrophoresis, and observing the expression of Acetylcholinesterase recombinant protein after dyeing and decoloring, wherein the expression of Acetylcholinesterase recombinant protein (64 KD) is gradually increased along with the extension of induction time as shown in figure 3.

In fig. 3: protein molecular weight standard Marker (from top to bottom size: 99, 66, 44, 29, 20 and 14 kD); 1, fermentation supernatant of the X33 recombinant strain when the strain is not induced; 2, inducing fermentation supernatant for 12 hours by using methanol; 3, inducing the fermentation supernatant for 24 hours by using methanol; 4, inducing fermentation supernatant for 48 hours by using methanol; 5, inducing fermentation supernatant for 72 hours by using methanol; 6, inducing fermentation supernatant for 96 hours by using methanol;

(2) and (4) detecting the enzyme activity of the recombinant AChE.

The principle is as follows: the Ellman method is characterized in that acetylthiocholine is decomposed into thiocholine and acetate under the action of AChE, the thiocholine reacts with 5, 5-dithiobis-2-nitrobenzoic acid (DTNB) to generate a yellow compound 5-sulfur-2-nitrobenzoic acid, the maximum absorption peak exists at the position of 410-420 nm, and the concentration and the absorption value form a linear relation in a certain range. The method is subjected to colorimetric quantification, and the amount of the thiocholine generated by hydrolysis reflects the activity value of cholinesterase.

The method comprises the following steps:

(1) ph8.0 buffer solution: 11.9g of anhydrous dipotassium hydrogen phosphate and 3.2g of potassium dihydrogen phosphate were dissolved in 1L of distilled water, respectively.

(2) Thioacetyl iodocholine solution (substrate): weighing 25mg of thioacetyl iodocholine, dissolving in 3.0mL of double distilled water, shaking up, and storing in a refrigerator at 4 ℃.

(3) DTNB solution (color developer): DTNB160mg and 15.6mg of sodium bicarbonate were weighed, dissolved in 20mL of buffer, and stored in a refrigerator at 4 ℃.

(4) 2.5mL of buffer solution, 0.1mL of enzyme solution and 0.1mL of color developing agent were added to the test tube, and the test tube was shaken up and left at 37 ℃ for 15 min. 0.1mL of substrate was added, shaken well, immediately placed in a cuvette and the change in absorbance Δ A0 for 3min of reaction was recorded.

(5) The calculation formula of the enzyme activity unit is as follows:

wherein, Delta A is the difference value of absorbance before and after reaction;

v is the total volume of the reaction system, and the system is 2.810-3L;

v is the sample volume, the system is 0.1X 10-3L;

ε is the molar absorptivity of the yellow product, 1.36X104L/(mol cm);

t is the reaction time of 3 min;

106 is the conversion coefficient from mole to micromole;

l is the cuvette width (cm).

Because the calculation process of the enzyme activity is complex, sometimes the enzyme activity can be represented simply by the difference (delta A) between the absorbance before and after the reaction, and the larger the delta A value is, the larger the enzyme activity is.

Taking fermentation supernatants at different culture stages, performing enzyme activity detection according to the enzyme activity detection method, calculating enzyme activity of the invention at different growth stages according to the formula, and finally obtaining 7686.27U/mL recombinant Acetylcholinesterase, which is shown in figure 4.

FIG. 4 shows that the activity of recombinant Acetylcholinesterase gradually increases with time, slowly increases before 84h, the activity of enzyme greatly increases from 84h to 108h, and then does not increase gradually, and finally 7686.27U/mL is obtained by the end of 120h fermentation.

Isolation of Acetylcholinesterase recombinant protein: after induced expression for 120 hours, the fermentation is finished, fermentation broth is centrifuged at 4 ℃ and 5000r/min to obtain fermentation supernatant, and after ultrafiltration concentration, the fermentation supernatant is frozen in vacuum, dried and stored at-20 ℃ for later use, and different methods can be adopted for purification according to requirements.

Claims

1. A pilot fermentation method for expressing recombinant acetylcholinesterase in pichia methanolica is characterized in that:

culturing the activated and preserved recombinant pichia pastoris strain to prepare a fermentation working seed solution, then transferring the fermentation working seed solution into a basic salt culture medium according to the inoculation amount of 4%, adjusting the temperature of a fermentation tank with the liquid loading amount of 20L to be 29 ℃, the pressure of the tank to be 0.06MPa and the rotation speed to be 600rpm, adjusting the pH of a fermentation system to be 5.2 by using ammonia water or phosphoric acid, and starting fermentation after correcting dissolved oxygen DO to reach 100%:

firstly, adding a glycerol solution for enrichment fermentation, wherein the adding flow rate of the glycerol solution is 1.67ml/min, when the glycerol is exhausted, the OD600 of a bacterial liquid reaches 90.0, then performing protein induction fermentation by using a methanol solution, wherein the temperature is continuously maintained at 29 ℃, the DO is maintained at more than 20% in the fermentation process, the methanol induction lasts 120 hours, and the whole fermentation process is finished;

when the methanol solution is adopted for protein induction fermentation, the methanol solution is slowly added in three stages: in the first stage, the methanol is supplemented at the flow rate of 0.1ml/min for 10h, and the methanol is stopped for 2 h; in the second stage, the replenishing flow rate of the methanol is 0.2ml/min, the replenishing is carried out for 22 hours, and the methanol is stopped for 2 hours; the replenishing flow rate of the methanol in the third stage is 0.5ml/min, and the replenishing time is 84 hours; the total induction fermentation time is 120 h;

the glycerol solution is a 50% glycerol aqueous solution containing 1.2% PTM1, and the methanol solution is a methanol solution containing 1.2% PTM 1;

the recombinant pichia pastoris strain is a Chinese red forest ant Acetylcholinesterase recombinant pichia pastoris strain X33/pPICZ α A-Acetylcholinesterase, the construction method comprises the steps of carrying out enzyme digestion and connection on pMD-19T/Acetylcholinesterase and an expression vector pPICZ α A to obtain an expression vector pPICZ α A-Acetylcholinesterase, transforming the expression vector into escherichia coli DH5 α, selecting single clones, identifying positive plasmids, transforming yeast X33, screening out a multi-copy recombinant strain X33/pPICZ α A-Acetylcholinesterase, and storing for later use after identification.

2. The pilot fermentation method of recombinant acetylcholinesterase expressed in pichia methanolica according to claim 1, wherein: each liter of the glycerol and methanol solution contains 12ml of trace element salt solution; the microelement salt solution comprises the following components: 6.0g CuSO₄·5H₂O，0.08g NaI, 3.0g MnSO₄·H₂O, 0.2g NaMoO₄·2H₂O,0.02g H₃BO₄, 0.5g CoCl₂, 20.0g ZnCl₂,65.0g FeSO₄· 7H₂O,0.2g biotin, 5.0mL sulfuric acid, water to 1L, filter sterilized with 0.22 μm filter, and stored at room temperature.