CN110343677B - Method for improving pichia pastoris catalase expression quantity - Google Patents

Abstract

The invention relates to a method for improving the expression level of pichia pastoris catalase, which comprises the following steps: s1, providing a pichia pastoris seed solution, a basic salt culture medium and a fermentation tank; s2, adding the basic salt culture medium into the fermentation tank, and then performing pretreatment and parameter setting on the fermentation tank; s3, inoculating the seed liquid into a basic salt culture medium in the fermentation tank for fed-batch fermentation, and continuously adding glycerol for fermentation culture after the glycerol in the basic salt culture medium is consumed in the fed-batch fermentation process; s4, after the glycerol added in the step S3 is consumed, adding methanol and a culture medium to continue the fed-batch fermentation. The method can obviously improve the expression quantity and the enzyme activity of catalase by optimizing the fermentation process of pichia pastoris.

Description

Method for improving pichia pastoris catalase expression quantity

Technical Field

The invention relates to a method for improving the expression level of pichia pastoris catalase, and belongs to the field of microorganisms.

Background

The biochemical processes of aerobic fermentation of compost are all enzymatic reactions carried out in the presence of enzymes. In the compost rotting stage, oxido-reducing enzymes such as catalase, dehydrogenase and polyphenol oxidase play roles, organic matters are degraded and further converted into humus, and the harmless treatment of the compost is realized. Catalase is a stable deoxyenzyme, has the functions of efficiently decomposing hydrogen peroxide and avoiding high oxidation of cells, and is widely applied to the engineering fields of textiles, food, environmental protection and the like. The research on the fermentation of the microbial catalase started in the early 50 s of the last century, and in recent 30 years, with the continuous development and improvement of microbial screening technology, scientists obtained a large number of catalase production strains through different screening modes and studied the strains in aspects such as fermentation optimization strategies and the like. Experiments show that the expression level of catalase in pichia pastoris is closely related to the culture conditions, and the influence of the expression conditions on the expression level is very obvious.

However, the research on improving the expression level of the microorganism catalase in the prior art is only limited to the aspect of optimizing the culture conditions such as temperature, pH, inorganic salt ion concentration, shaking table rotating speed and the like, and no innovation in the aspect of fermentation process is seen.

Disclosure of Invention

The invention aims to provide a method for improving the expression quantity of catalase in pichia pastoris, which can obviously improve the expression quantity and the enzyme activity of the catalase by optimizing the fermentation process of the pichia pastoris.

In order to achieve the purpose, the invention provides the following technical scheme: a method for improving the expression level of catalase in pichia pastoris comprises the following steps:

s1, providing a pichia pastoris seed solution, a basic salt culture medium and a fermentation tank;

s2, adding the basic salt culture medium into the fermentation tank, and then performing pretreatment and parameter setting on the fermentation tank;

s3, inoculating the seed liquid into a basic salt culture medium in the fermentation tank for fed-batch fermentation, and continuously adding glycerol for fermentation culture after the glycerol in the basic salt culture medium is consumed in the fed-batch fermentation process;

s4, after the glycerol added in the step S3 is consumed, adding methanol and a culture medium to continue the fed-batch fermentation.

Further, step S2 specifically includes:

s21, correcting a pH electrode and a dissolved oxygen electrode, adding the basic salt culture medium into the fermentation tank, sealing, and placing the fermentation tank into a large sterilization pot for sterilization;

s22, after the fermentation tank in the step S21 is cooled, the fermentation tank is placed on a fermentation platform to be installed, cooling water and an air pump power supply are turned on, an air pipe is connected for ventilation, and the pressure of the fermentation tank is maintained at 0.05 Mpa; setting relevant parameters, and calibrating the slope of the dissolved oxygen electrode under the determined rotating speed and ventilation capacity, wherein the value is 100%;

and S23, after all parameters of temperature stability are correct, inoculating the pichia pastoris seed liquid into a basic salt culture medium in a fermentation tank, starting fermentation timing, and recording all parameters.

Further, before inoculating the pichia pastoris seed solution, the method further comprises the following steps: transferring the pichia pastoris seed solution into a sterilized centrifuge cup under the aseptic condition, centrifuging for 10min at the temperature of 4 ℃ and the rpm of 8000, and discarding the supernatant.

Further, step S3 specifically includes: during the fed-batch fermentation process, the strains consume the glycerol in the basic salt culture medium and enter an exponential growth phase from an adaptation phase, the dissolved oxygen in the fermentation tank is reduced, and the dissolved oxygen is increased after the glycerol in the basic salt culture medium is consumed; and continuously adding 50% (v/v) glycerol at the flow rate of 1.65mL/min for 3-9 h.

Further, step S3 further includes: in the subsequent fermentation process of adding glycerin, the dissolved oxygen is controlled by mixing pure oxygen and air, and the dissolved oxygen concentration is controlled to be 30-60% by adjusting the ventilation volume, the flow rate or the rotating speed of oxygen.

Further, step S3 further includes: and in the subsequent fermentation process of adding the glycerol, 25% (v/v) ammonia water and 30% (v/v) phosphoric acid are added to control the pH value to be 4-6.

Further, the specific step of step S4 is: after the feeding of 50% (v/v) glycerol is finished, when the dissolved oxygen rises again, the mixed solution of 0.1-1% (v/v) pure methanol and the feeding culture medium is fed at the flow rate of 0.55 mL/min.

Further, the method also comprises adopting Ni₂₊And (3) separating and purifying catalase by column chromatography: extracting the recombinant crude enzyme solution of the medium in step S4 for Ni₂₊Activating the column, performing chromatography, and purifying catalase.

Further, the step of extracting the recombinant crude enzyme solution comprises the following steps:

(1) after the step S4 is finished, centrifuging for 5min at 4 ℃ and 5000g to collect thalli;

(2) sterilization ddH for thallus₂O heavy suspension washing, centrifuging for 5min at 4 ℃ under the condition of 5000g, and collecting thalli;

(3) resuspending the thallus by using a binding buffer solution, and crushing the thallus by adopting an ultrasonic crushing method to obtain an ultrasonic crushing solution;

(4) and centrifuging the ultrasonication solution at 4 ℃ and 30000g for 20min, and collecting supernatant.

Further, to the Ni₂₊The activation treatment of the column comprises the following steps:

(1) filling the suspension column filler preservation solution into a chromatographic column and standing;

(2) setting the flow rate of the column at 10CV/h, and adding ddH when 20% ethanol stored in the column flows to the top of the column₂O, washing the column;

(3) ddH in column₂When the O flows to the top of the column, adding a charge buffer solution to wash the column;

(4) and when the charge buffer flows to the top of the column, adding a binding buffer solution to wash the column, and storing the column packing in the binding buffer solution.

Compared with the prior art, the invention has the beneficial effects that: according to the method for improving the expression quantity of the pichia pastoris catalase, under the condition of fermentation tank culture, glycerol is added in a logarithmic phase by using a fed-batch fermentation process to improve the quantity of the pichia pastoris, and methanol is added to improve the expression quantity and the enzyme activity of the catalase. Therefore, the method improves the expression level and enzyme activity level of the catalase in the pichia pastoris by innovating the fermentation process, and has very important significance for realizing the harmless treatment of the compost.

The foregoing is a summary of the present invention, and in order to provide a clear understanding of the technical means of the present invention and to be implemented in accordance with the present specification, the following is a detailed description of the preferred embodiments of the present invention.

Detailed Description

The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The method for improving the expression level of the catalase in the pichia pastoris comprises the following steps:

s1, providing a pichia pastoris seed solution, a basic salt culture medium and a fermentation tank;

s2, adding the basic salt culture medium into the fermentation tank, and then performing pretreatment and parameter setting on the fermentation tank;

s3, inoculating the seed liquid into a basic salt culture medium in the fermentation tank for fed-batch fermentation, and continuously adding glycerol for fermentation culture after the glycerol in the basic salt culture medium is consumed in the fed-batch fermentation process;

s4, after the glycerol added in the step S3 is consumed, adding methanol and a culture medium to continue the fed-batch fermentation.

In the present invention, step S2 specifically includes:

s21, correcting a pH electrode and a dissolved oxygen electrode, adding the basic salt culture medium into the fermentation tank, sealing, and placing the fermentation tank into a large sterilization pot for sterilization;

s22, after the fermentation tank in the step S21 is cooled, the fermentation tank is placed on a fermentation platform to be installed, cooling water and an air pump power supply are turned on, an air pipe is connected for ventilation, and the pressure of the fermentation tank is maintained at 0.05 Mpa; setting relevant parameters, and calibrating the slope of the dissolved oxygen electrode under the determined rotating speed and ventilation capacity, wherein the value is 100%;

and S23, after all parameters of temperature stability are correct, inoculating the pichia pastoris seed liquid into a basic salt culture medium in a fermentation tank, starting fermentation timing, and recording all parameters.

Step S2 further includes, prior to inoculating the pichia pastoris seed solution: transferring the pichia pastoris seed solution into a sterilized centrifuge cup under the aseptic condition, centrifuging for 10min at the temperature of 4 ℃ and the rpm of 8000, and discarding the supernatant.

In the present invention, step S3 specifically includes: during the fed-batch fermentation process, the strains consume the glycerol in the basic salt culture medium and enter an exponential growth phase from an adaptation phase, the dissolved oxygen in the fermentation tank is reduced, and the dissolved oxygen is increased after the glycerol in the basic salt culture medium is consumed; and continuously adding 50% (v/v) glycerol at the flow rate of 1.65mL/min for 3-9 h, preferably 6 h. Step S3 further includes: in the subsequent fermentation process of adding glycerin, the dissolved oxygen is controlled by mixing pure oxygen and air, and the dissolved oxygen concentration is controlled to be 30-60% by adjusting the ventilation volume, the flow rate or the rotating speed of oxygen. Step S3 further includes: and in the subsequent fermentation process of adding the glycerol, 25% (v/v) ammonia water and 30% (v/v) phosphoric acid are added to control the pH to be 4-6, preferably 5.

In the present invention, the specific steps of step S4 are: after the addition of 50% (v/v) glycerol, when the dissolved oxygen rises again, 0.1-1% (v/v) of the mixture of pure methanol and the addition medium is added, preferably 0.5% (v/v) of the mixture of pure methanol and the addition medium, at a flow rate of 0.55 mL/min.

In the present invention, the use of Ni is also included₂₊And (3) separating and purifying catalase by column chromatography: extracting the recombinant crude enzyme solution of the medium in step S4 for Ni₂₊Activating the column, performing chromatography, and purifying catalase.

The method for extracting the recombinant crude enzyme solution comprises the following steps:

(1) after the step S4 is finished, centrifuging for 5min at 4 ℃ and 5000g to collect thalli;

(2) sterilization ddH for thallus₂O heavy suspension washing, centrifuging for 5min at 4 ℃ under the condition of 5000g, and collecting thalli;

(3) resuspending the thallus with a binding buffer (1 × binding buffer), and crushing the thallus by an ultrasonic crushing method to obtain an ultrasonic crushing liquid;

(4) and centrifuging the ultrasonication solution at 4 ℃ and 30000g for 20min, and collecting supernatant.

To the Ni₂₊The activation treatment of the column comprises the following steps:

(1) after fully suspending the column packing preservation solution, 2mL of the suspension is put into a chromatographic column and stands still (the column volume CV is 1.5 mL);

(2) setting the flow rate of the column at 10CV/h, and adding 3CV ddH when 20% ethanol stored in the column flows to the top of the column₂O, washing the column;

(3) ddH in column₂When the O flows to the top of the column, adding a charge buffer solution (5CV 1 × charge buffer) to wash the column;

(4) when the charge buffer flows to the top of the column, binding buffer (5CV 1 × binding buffer) is added to wash the column, and the column packing is stored in the binding buffer.

The purification of the catalase comprises the following steps:

the column was washed with a binding buffer (10CV 1 × binding buffer), a washing buffer (10CV 1 × wash buffer), an eluent (3CV1 × elute buffer) and a membrane regeneration solution (3CV1 × strip buffer), respectively, by loading the supernatant with ultrasound, and collecting one tube per 1mL, and storing at 4 ℃ for future use.

The pichia pastoris seed liquid can be obtained by the following method:

activation of cryopreserved strains

Taking out the strain from a refrigerator at-80 ℃, performing water bath sterilization at 50 ℃, inoculating the strain to a YPD culture medium, and culturing for three days.

Preparation of seed liquid

Preparing a seed solution: a single colony of Pichia pastoris was picked and inoculated into BMGY seed medium (500ml), shake-cultured at 28 ℃ and 200rpm for 24 h. The seed solution was aseptically transferred to a sterilized centrifuge cup, centrifuged at 8000rpm at 4 ℃ for 10min, the supernatant was discarded, the cells were resuspended in a basal salt medium, and inoculated into a 5L fermenter containing 10% (200ml) inoculum size.

Example one culture method for increasing Pichia pastoris bacterial yield by supplementing glycerol

Inoculating the seed culture solution into a fermentation tank containing 5L with an inoculation amount of 10% (200mL), allowing the inoculated seed culture solution to enter an exponential growth phase after a period of adaptation, wherein 50% glycerol is added by flowing through a feeding pipe at a flow rate of about 1.65mL/min, and feeding time is set to be 3h, 6h and 9h, and a control is set. The dissolved oxygen in the fermentation tank is controlled by mixing pure oxygen and air, the air and the oxygen are respectively provided with a flow meter, the dissolved oxygen concentration is controlled to be 30-60% by adjusting the ventilation quantity, the flow rate or the rotating speed of the oxygen, and the pH is controlled to be about 5 by feeding 25% ammonia water and 30% phosphoric acid in the period. The fermentation broth was taken after the end of fermentation to determine the amount of yeast, and the results are shown in Table 1.

TABLE 1 Effect of different glycerol additions on the number of bacteria

As can be seen from the above table, after the yeast in the fermentation tank is supplemented with the nutrition of the medium by adding 50% glycerol at the logarithmic phase, the viable count of the yeast is increased compared with the control group, which indicates that the glycerol supplemented fermentation process is adopted to contribute to the increase of the bacterial count, and when the supplementation time is 6 hours, the yeast count is 4.7X10⁹cfu/ml。

Example II expression method for improving enzyme activity of pichia pastoris catalase by inducing methanol with different concentrations

After the glycerol feeding is finished, when the dissolved oxygen amount in the fermentation tank rises again, the mixed liquid of the pure methanol and the basic culture medium is added by using a feeding tube, the methanol concentration in the culture medium is set to be 0.1%, 0.5% and 1% in the feeding process, the flow rate is about 0.55mL/min, and meanwhile, the control is set. After the fermentation was completed, the fermentation supernatant was collected, and the catalase activity was measured, and the results are shown in Table 2.

TABLE 2 Catalase Activity with addition of different methanol concentrations

Methanol concentration (%)	Enzyme activity (u/ml)
		0.1	15321
0.5	20267
		1	16967
ck	2790

As can be seen from the above table, after glycerol is supplemented, methanol is continuously supplemented, the catalase activity is improved compared with that of a control group, and methanol with the concentration of 0.5% is the optimal supplement concentration, which indicates that the use of the 0.5% methanol supplement fermentation process is beneficial to the improvement of the catalase activity, and the highest enzyme activity is 20267 u/ml.

EXAMPLE III method for increasing catalase expression level in 0.5% methanol under different induction times

After the glycerol feeding is finished, when the dissolved oxygen in the fermentation tank rises again, adding a mixed solution of 0.5 percent pure methanol and a basic culture medium by using a feeding tube, wherein the flow rate is about 0.55mL/min, and the feeding time is set to be 48h, 72h and 96 h. After the fermentation was completed, the fermentation supernatant was collected, and the catalase activity was measured, and the results are shown in Table 3.

TABLE 30.5% Catalase Activity with methanol at different Induction times

As can be seen from the table above, after adding 50% glycerol for 6h, 0.5% methanol is continuously added, the induction time is 72h, and the highest enzyme activity is 20348 u/ml.

In summary, the following steps: according to the method for improving the expression quantity of the pichia pastoris catalase, under the condition of fermentation tank culture, glycerol is added in a logarithmic phase by using a fed-batch fermentation process to improve the quantity of the pichia pastoris, and methanol is added to improve the expression quantity and the enzyme activity of the catalase. Therefore, the method improves the expression level and enzyme activity level of the catalase in the pichia pastoris by innovating the fermentation process, and has very important significance for realizing the harmless treatment of the compost.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (4)

1. A method for improving the expression level of catalase in pichia pastoris is characterized by comprising the following steps:

s1, providing a pichia pastoris seed solution, a basic salt culture medium and a fermentation tank;

s2, adding the basic salt culture medium into the fermentation tank, and then performing pretreatment and parameter setting on the fermentation tank;

step S2 specifically includes:

s21, correcting a pH electrode and a dissolved oxygen electrode, adding the basic salt culture medium into the fermentation tank, sealing, and placing the fermentation tank into a large sterilization pot for sterilization;

s22, after the fermentation tank in the step S21 is cooled, the fermentation tank is placed on a fermentation platform to be installed, cooling water and an air pump power supply are turned on, an air pipe is connected for ventilation, and the pressure of the fermentation tank is maintained at 0.05 Mpa; setting relevant parameters, and calibrating the slope of the dissolved oxygen electrode under the determined rotating speed and ventilation capacity, wherein the value is 100%;

s23, after all parameters of temperature stability are correct, the pichia pastoris seed liquid is inoculated into a basic salt culture medium in a fermentation tank, fermentation timing is started, and all parameters are recorded; before inoculating the pichia pastoris seed solution, the method also comprises the following steps: transferring the pichia pastoris seed solution into a sterilized centrifuge cup under the aseptic condition, centrifuging for 10min at the temperature of 4 ℃ and the rpm of 8000, and removing the supernatant;

s3, inoculating the seed liquid into a basic salt culture medium in the fermentation tank for fed-batch fermentation, and continuously feeding glycerol for fermentation culture after the glycerol in the basic salt culture medium is consumed in the fed-batch fermentation process; in the subsequent fermentation process of adding glycerol, the dissolved oxygen is controlled by mixing pure oxygen and air, and the dissolved oxygen concentration is controlled to be 30-60% by adjusting the ventilation capacity, the flow rate or the rotating speed of oxygen; and controlling the pH value to be 4-6 by adding 25% (v/v) ammonia water and 30% (v/v) phosphoric acid in a flowing manner;

step S3 specifically includes: when the glycerol in the basic salt culture medium is completely consumed, the dissolved oxygen rises; continuously adding 50% (v/v) glycerol at the flow rate of 1.65mL/min for 6-9 h;

s4, after the glycerol added in the step S3 is consumed, after the feeding of 50% (v/v) glycerol is finished, and when the dissolved oxygen rises again, feeding a mixed solution of 0.1-1% (v/v) pure methanol and a feeding culture medium at a flow rate of 0.55mL/min for 48-96 h.

2. The method for increasing the expression level of catalase in pichia pastoris according to claim 1, further comprising using Ni₂₊And (3) separating and purifying catalase by column chromatography: extracting the recombinant crude enzyme solution of the medium in step S4 for Ni²⁺Activating the column, performing chromatography, and purifying catalase.

3. The method for improving the expression level of pichia pastoris catalase as claimed in claim 2, wherein the extraction of the recombinant crude enzyme solution comprises the following steps:

(1) after the step S4 is finished, centrifuging for 5min at 4 ℃ and 5000g to collect thalli;

(2) sterilization ddH for thallus₂O heavy suspension washing, centrifuging for 5min at 4 ℃ under the condition of 5000g, and collecting thalli;

(3) resuspending the thallus by using a binding buffer solution, and crushing the thallus by adopting an ultrasonic crushing method to obtain an ultrasonic crushing solution;

(4) and centrifuging the ultrasonication solution at 4 ℃ and 30000g for 20min, and collecting supernatant.

4. The method for increasing the expression level of catalase in pichia pastoris according to claim 2, wherein Ni is added to the pichia pastoris^{2 +}The activation treatment of the column comprises the following steps:

(1) filling the suspension column filler preservation solution into a chromatographic column and standing;

(2) setting the column flow rate at 10CV/h, and adding ddH when 20% ethanol stored in the column flows to the top of the column₂O, washing the column;

(3) ddH in column₂When the O flows to the top of the column, adding a charge buffer solution to wash the column;

(4) and when the charge buffer flows to the top of the column, adding a binding buffer solution to wash the column, and storing the column packing in the binding buffer solution.