CN110343677A - The method for improving Pichia pastoris catalase expression quantity - Google Patents
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Abstract
The present invention relates to a kind of methods for improving Pichia pastoris catalase expression quantity, comprising the following steps: S1, provides Pichia yeast seed liquor, basal salt media and fermentor；S2, the basal salt media is incorporated into the fermentor, then pretreatment and parameter setting is carried out to the fermentor；S3, it the seed liquor is accessed to carries out fed-batch cultivation in the basal salt media in the fermentor, during the fed-batch cultivation, after the glycerol in the basal salt media runs out of, continuously add glycerol and carry out fermented and cultured；After S4, the glycerol after add in step S3 run out of, adds methanol and culture medium continues fed-batch cultivation.This method can significantly improve the expression quantity and enzymatic activity of catalase by the zymotechnique of optimization Pichia pastoris.
Description
Technical field
The present invention relates to a kind of methods for improving Pichia pastoris catalase expression quantity, belong to microorganism field.
Background technique
The Biochemical processes of compost aerobic fermentation are all the enzymatic reactions carried out in enzyme presence.In the compost maturity phase,
The oxidoreducing enzyme such as catalase, dehydrogenase, polyphenol oxidase have played effect, organic matter degradation and are further converted into
Humus realizes the harmless treatment of compost.Catalase is a kind of stable deoxyenzyme, and effect is efficient-decomposition mistake
Hydrogen oxide avoids cell high oxidation, is widely used in engineering fields such as weaving, food, environmental protection.It is opened at the beginning of the fifties in last century
Begun microbial catalase fermentation research, in the past 30 years, continuous development and perfect, section with microbe to screen technology
Scholars have obtained a large amount of catalases by different screening modes and have produced bacterial strain, and it is excellent to have carried out fermentation to these bacterial strains
Change the research of strategy etc..Experiment shows that catalase expression and condition of culture in Pichia pastoris are in close relations,
Influence of the expression condition to expression quantity level is also extremely significant.
However, being limited to optimization temperature to raising microbial catalase expression quantifier elimination in prior art
The condition of culture levels such as degree, pH, inorganic ion concentration, shaking speed, have no the innovation in terms of zymotechnique.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for improving Pichia pastoris catalase expression quantity, are finished by optimizing
The zymotechnique of red yeast can significantly improve the expression quantity and enzymatic activity of catalase.
In order to achieve the above objectives, the invention provides the following technical scheme: a kind of raising Pichia pastoris hydrogen peroxide expression of enzymes
The method of amount, comprising the following steps:
S1, Pichia yeast seed liquor, basal salt media and fermentor are provided；
S2, the basal salt media is incorporated into the fermentor, then the fermentor is pre-processed and joined
Number setting；
S3, it the seed liquor is accessed to carries out fed-batch cultivation in the basal salt media in the fermentor,
During the fed-batch cultivation, after the glycerol in the basal salt media runs out of, continuously adds glycerol and carry out fermentation training
It supports；
After S4, the glycerol after add in step S3 run out of, adds methanol and culture medium continues fed-batch cultivation.
Further, step S2 is specifically included:
S21, correction pH electrode and dissolved oxygen electrode, the basal salt media is incorporated in the fermentor, will after sealing
The fermentor sterilizes as big autoclave；
S22, after the fermentor of step S21 is cooling, place it on fermentation platform and installed, open cooling water and air pump
Power supply, and connect ventilation pipe and ventilate, maintain tank to be pressed in 0.05Mpa；Relevant parameter is set again, and in determining revolving speed and
Slope calibration, numerical value 100% are carried out to the dissolved oxygen electrode under ventilatory capacity；
S23, after temperature stablize parameters it is all correct after, the Pichia yeast seed liquor is accessed in fermentor
In basal salt media, when starting zymometer, and various parameters are recorded.
Further, before being inoculated with the Pichia yeast seed liquor further include: aseptically finish red ferment for described
Female bacterium seed liquor moves into the Centrifuge Cup of sterilizing, and at 4 DEG C, 10min is centrifuged under conditions of 8000rpm, abandons supernatant.
Further, step S3 is specifically included: during the fed-batch cultivation, strain can consume the basic salt culture
Glycerol in base, and enter exponential phase of growth from the laundering period, during this, the dissolved oxygen decline in the fermentor, when described
After glycerol in basal salt media runs out of, dissolved oxygen rises；Followed by afterflow plus 50% (v/v) glycerol, flow velocity 1.65mL/
Min is 3~9h between the stream added-time.
Further, step S3 further include: in the subsequent fermentations of stream glycerol adding, dissolved oxygen uses pure oxygen instead and air is mixed
Control is closed, and the flow by adjusting ventilatory capacity, oxygen or revolving speed control oxyty in 30%-60%.
Further, step S3 further include: pass through stream plus 25% (v/v) ammonium hydroxide in the subsequent fermentations of stream glycerol adding
With 30% (v/v) phosphoric acid control pH 4~6.
Further, the specific steps of step S4 are as follows: after stream plus 50% (v/v) glycerol, rise again to dissolved oxygen
When, the mixed liquor of stream plus 0.1%~1% (v/v) pure methanol and fed-batch medium, flow velocity 0.55mL/min.
It further, further include using Ni2+Column chromatography isolates and purifies catalase: in extraction step S4
The recombination crude enzyme liquid of culture medium, to Ni2+Column is chromatographed after being activated, then is purified to catalase.
Further, extract the recombination crude enzyme liquid the following steps are included:
(1) after the completion of step S4,5min is centrifuged at 4 DEG C, under conditions of 5000g and collects thallus；
(2) thallus sterilizing ddH2Washing is resuspended in O, is centrifuged 5min at 4 DEG C, under conditions of 5000g and collects thallus；
(3) thallus is resuspended with combination buffer, thallus is crushed using sonioation method, obtains ultrasonication liquid；
(4) by the ultrasonication liquid at 4 DEG C, it is centrifuged 20min under conditions of 30000g, collects supernatant.
Further, to the Ni2+The activation processing of column the following steps are included:
(1) it after sufficiently suspension column packing saves liquid, is fitted into and is stood into chromatographic column；
(2) setting column flow rate is 10CV/h, and when 20% ethyl alcohol when save in column flow to column top, ddH is added2O is carried out
Wash column；
(3) to ddH in column2When O flow to column top, charge buffer liquid is added and carries out washing column；
(4) when the charge buffer liquid stream to column top, combination buffer is added and washes column, and column packing is stored in knot
It closes in buffer.
Compared with prior art, the beneficial effects of the present invention are raising Pichia pastoris catalase tables of the invention
Up to amount method under the conditions of fermentation tank culture, with fed-batch fermentation technique logarithmic phase addition glycerol improve Pichia pastoris
Bacterium number amount, addition methanol improve catalase expression quantity and enzymatic activity.Therefore this method in terms of zymotechnique by being created
Newly, it improves Pichia pastoris catalase expression quantity and enzyme activity is horizontal, for realizing that it is particularly significant that compost hazard-free processing has
Meaning.
The above description is only an overview of the technical scheme of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, with presently preferred embodiments of the present invention, detailed description is as follows below.
Specific embodiment
With reference to embodiment, the embodiment of the present invention is furthur described in detail.Following embodiment is used for
Illustrate the present invention, but is not intended to limit the scope of the invention.
The method of raising Pichia pastoris catalase expression quantity of the invention, comprising the following steps:
S1, Pichia yeast seed liquor, basal salt media and fermentor are provided；
S2, the basal salt media is incorporated into the fermentor, then the fermentor is pre-processed and joined
Number setting；
S3, it the seed liquor is accessed to carries out fed-batch cultivation in the basal salt media in the fermentor,
During the fed-batch cultivation, after the glycerol in the basal salt media runs out of, continuously adds glycerol and carry out fermentation training
It supports；
After S4, the glycerol after add in step S3 run out of, adds methanol and culture medium continues fed-batch cultivation.
In the present invention, step S2 is specifically included:
S21, correction pH electrode and dissolved oxygen electrode, the basal salt media is incorporated in the fermentor, will after sealing
The fermentor sterilizes as big autoclave；
S22, after the fermentor of step S21 is cooling, place it on fermentation platform and installed, open cooling water and air pump
Power supply, and connect ventilation pipe and ventilate, maintain tank to be pressed in 0.05Mpa；Relevant parameter is set again, and in determining revolving speed and
Slope calibration, numerical value 100% are carried out to the dissolved oxygen electrode under ventilatory capacity；
S23, after temperature stablize parameters it is all correct after, the Pichia yeast seed liquor is accessed in fermentor
In basal salt media, when starting zymometer, and various parameters are recorded.
Step S2 is before being inoculated with the Pichia yeast seed liquor further include: aseptically by the Pichia yeast
Seed liquor moves into the Centrifuge Cup of sterilizing, and at 4 DEG C, 10min is centrifuged under conditions of 8000rpm, abandons supernatant.
In the present invention, step S3 is specifically included: during the fed-batch cultivation, strain can consume the basic salt culture
Glycerol in base, and enter exponential phase of growth from the laundering period, during this, the dissolved oxygen decline in the fermentor, when described
After glycerol in basal salt media runs out of, dissolved oxygen rises；Followed by afterflow plus 50% (v/v) glycerol, flow velocity 1.65mL/
Min is 3~9h, preferably 6h between the stream added-time.Step S3 further include: in the subsequent fermentations of stream glycerol adding, dissolved oxygen is used instead
Pure oxygen and air mixing control, and the flow by adjusting ventilatory capacity, oxygen or revolving speed control oxyty in 30%-60%.
Step S3 further include: pass through stream plus 25% (v/v) ammonium hydroxide and 30% (v/v) phosphoric acid control in the subsequent fermentations of stream glycerol adding
PH processed is 4~6, preferably 5.
In the present invention, the specific steps of step S4 are as follows: after stream plus 50% (v/v) glycerol, rise again to dissolved oxygen
When, the mixed liquor of stream plus 0.1%~1% (v/v) pure methanol and fed-batch medium, preferably 0.5% (v/v) pure methanol and stream add
The mixed liquor of culture medium, flow velocity 0.55mL/min.
It further include using Ni in the present invention2+Column chromatography isolates and purifies catalase: in extraction step S4
The recombination crude enzyme liquid of culture medium, to Ni2+Column is chromatographed after being activated, then is purified to catalase.
Extract the recombination crude enzyme liquid the following steps are included:
(1) after the completion of step S4,5min is centrifuged at 4 DEG C, under conditions of 5000g and collects thallus；
(2) thallus sterilizing ddH2Washing is resuspended in O, is centrifuged 5min at 4 DEG C, under conditions of 5000g and collects thallus；
(3) thallus is resuspended with combination buffer (1 × binding buffer), thallus is crushed using sonioation method, is obtained
To ultrasonication liquid；
(4) by the ultrasonication liquid at 4 DEG C, it is centrifuged 20min under conditions of 30000g, collects supernatant.
To the Ni2+The activation processing of column the following steps are included:
(1) it after sufficiently suspension column packing saves liquid, takes 2mL to be fitted into chromatographic column and stands (column volume CV:1.5mL)；
(2) setting column flow rate is 10CV/h, and when 20% ethyl alcohol when save in column flow to column top, 3CV ddH is added2O
It carries out washing column；
(3) to ddH in column2When O flow to column top, charge buffer liquid (1 × charge of 5CV buffer) is added and is washed
Column；
(4) it when the charge buffer liquid stream to column top, is added combination buffer (1 × binding of 5CV buffer)
Column is washed, and column packing is stored in combination buffer.
The catalase is purified the following steps are included:
With ultrasonic supernatant loading, combination buffer (1 × binding of 10CV buffer), washing buffer are used respectively
(1 × wash of 10CV buffer), eluent (1 × elute of 3CV buffer) and membrane regeneration liquor (3CV1 × strip
Buffer chromatographic column) is washed, every 1mL collects a pipe, and 4 DEG C save backup.
Pichia yeast seed liquor of the invention can also obtain by the following method:
Freeze the activation of bacterial strain
Bacterial strain is taken out, 50 DEG C of water-bath solution bacterium from -80 DEG C of refrigerators, by strain inoculated to YPD culture medium, cultivates three
It.
The preparation of seed liquor
Make seed liquor: picking Pichia pastoris single colonie is seeded in seed culture medium BMGY (500ml), and 28 DEG C,
200rpm shaking table culture is for 24 hours.Aseptically seed liquor is moved into the Centrifuge Cup of sterilizing, at 4 DEG C, the condition of 8000rpm
Lower centrifugation 10min abandons supernatant, and thallus is resuspended with basal salt media, is inoculated in fermentor containing 5L by 10% (200ml) inoculum concentration
In.
Embodiment one improves the cultural method of Pichia pastoris bacterium amount by supplement glycerol
Seed culture fluid is inoculated in fermentor containing 5L by 10% (200ml) inoculum concentration, by a Duan Shiying after inoculation
Enter exponential phase of growth after phase, 50% glycerol of addition is flowed by feed supplement pipe at this time, flow velocity is about 1.65mL/min, feed supplement time
3h, 6h, 9h tri- processing are set as, while control is set.Dissolved oxygen uses pure oxygen and air mixing control, air and oxygen instead in fermentor
Gas has flowmeter respectively, in subsequent fermentations, makes oxyty control by the flow or revolving speed that adjust ventilatory capacity, oxygen
In 30%-60%, pH is by stream plus the control of 25% ammonium hydroxide and 30% phosphoric acid 5 or so during this period.Hair is being taken after fermentation
Zymotic fluid measures saccharomycete quantity, as a result such as table 1.
Influence of the different glycerol additive amounts of table 1 to bacterium number amount
As can be seen from the above table, saccharomycete in the logarithm culture period stage adds 50% glycerol supplementing culture medium in fermentor
After nutrition, saccharomycete number of viable has increase compared with control group, illustrates to facilitate mentioning for bacterium amount using glycerol fed-batch fermentation technique
Rise, and the feed supplement time be 6h when, saccharomycete quantity be 4.7X109cfu/ml。
The expression of two various concentration methanol induction of embodiment raising Pichia pastoris catalase enzyme activity
After glycerol feed supplement, when dissolved oxygen amount rises again in fermentor, pure methanol and basis are added with feed supplement pipe
The mixed liquor of culture medium, during feed supplement, methanol concentration is set as 0.1%, 0.5% in culture medium, and 1% 3 processing, flow velocity is about
For 0.55mL/min, while control is set.Fermented supernatant fluid is collected after fermentation, measures its catalase enzyme activity, as a result
Such as table 2.
Table 2 adds catalase enzyme activity under the conditions 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 adding glycerol, continue to add methanol, catalase enzyme activity is compared with control group compared to equal
It is improved, and the methanol that concentration is 0.5% is best feed supplement concentration, illustrates to facilitate using 0.5% methanol feeding zymotechnique
The promotion of hydrogen peroxide enzyme activity, highest enzyme activity are 20267u/ml.
The method of catalase expression quantity is improved under 3 0.5% methanol difference induction time of embodiment
After glycerol feed supplement, when dissolved oxygen amount rises again in fermentor, with feed supplement pipe add 0.5% pure methanol with
The mixed liquor of basal medium, flow velocity are about 0.55mL/min, and the feed supplement time is set as 48h, 72h, 96h tri- processing.Wait ferment
After collect fermented supernatant fluid, its catalase enzyme activity is measured, as a result such as table 3.
Catalase enzyme activity under 3 0.5% methanol difference induction time of table
As can be seen from the above table, after adding 50% glycerol 6h, continue to add 0.5% methanol and induction time is 72h, most
High enzyme activity is 20348u/ml.
In summary: the method for raising Pichia pastoris catalase expression quantity of the invention is in fermentation tank culture condition
Under, Pichia yeast quantity is improved in logarithmic phase addition glycerol with fed-batch fermentation technique, addition methanol improves peroxidating
Hydrogenase expression amount and enzymatic activity.Therefore this method improves Pichia pastoris catalase by being innovated in terms of zymotechnique
Expression quantity and enzyme activity are horizontal, for realizing that compost hazard-free processing has a very important significance.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of method for improving Pichia pastoris catalase expression quantity, which comprises the following steps:
S1, Pichia yeast seed liquor, basal salt media and fermentor are provided；
S2, the basal salt media is incorporated into the fermentor, then to the fermentor carry out pretreatment and parameter set
It sets；
S3, it the seed liquor is accessed to carries out fed-batch cultivation in the basal salt media in the fermentor, described
During fed-batch cultivation, after the glycerol in the basal salt media runs out of, continuously adds glycerol and carry out fermented and cultured；
After S4, the glycerol after add in step S3 run out of, adds methanol and culture medium continues fed-batch cultivation.
2. improving the method for Pichia pastoris catalase expression quantity as described in claim 1, which is characterized in that step S2 tool
Body includes:
S21, correction pH electrode and dissolved oxygen electrode, the basal salt media is incorporated in the fermentor, will be described after sealing
Fermentor sterilizes as big autoclave；
S22, after the fermentor of step S21 is cooling, place it on fermentation platform and installed, open cooling water and air pump electricity
Source, and connect ventilation pipe and ventilate, maintain tank to be pressed in 0.05Mpa；Relevant parameter is set again, and in determining revolving speed and is led to
Slope calibration, numerical value 100% are carried out to the dissolved oxygen electrode under tolerance；
S23, after temperature stablize parameters it is all correct after, the Pichia yeast seed liquor is accessed into the basis in fermentor
In salt culture medium, when starting zymometer, and various parameters are recorded.
3. improving the method for Pichia pastoris catalase expression quantity as claimed in claim 2, which is characterized in that in inoculation institute
Before stating Pichia yeast seed liquor further include: the Pichia yeast seed liquor is aseptically moved into the Centrifuge Cup of sterilizing
In, at 4 DEG C, it is centrifuged 10min under conditions of 8000rpm, abandons supernatant.
4. improving the method for Pichia pastoris catalase expression quantity as claimed in claim 2, which is characterized in that step S3 tool
Body includes: during the fed-batch cultivation, and strain can consume the glycerol in the basal salt media, and enter from the laundering period
Exponential phase of growth, during this, dissolved oxygen decline in the fermentor, when the glycerol in the basal salt media runs out of
Afterwards, dissolved oxygen rises；Followed by afterflow plus 50% (v/v) glycerol, flow velocity 1.65mL/min is 3~9h between the stream added-time.
5. improving the method for Pichia pastoris catalase expression quantity as claimed in claim 4, which is characterized in that step S3 is also
Include: stream glycerol adding subsequent fermentations in, dissolved oxygen use instead pure oxygen and air mixing control, and by adjust ventilatory capacity,
Flow or revolving speed the control oxyty of oxygen are in 30%-60%.
6. improving the method for Pichia pastoris catalase expression quantity as claimed in claim 4, which is characterized in that step S3 is also
It include: that pH is controlled 4 by stream plus 25% (v/v) ammonium hydroxide and 30% (v/v) phosphoric acid in the subsequent fermentations of stream glycerol adding
~6.
7. improving the method for Pichia pastoris catalase expression quantity as claimed in claim 4, which is characterized in that step S4's
Specific steps are as follows: after stream plus 50% (v/v) glycerol, when dissolved oxygen rises again, stream plus 0.1%~1% (v/v) pure first
The mixed liquor of pure and mild fed-batch medium, flow velocity 0.55mL/min.
8. improving the method for Pichia pastoris catalase expression quantity as described in claim 1, which is characterized in that further include adopting
Use Ni2+Column chromatography isolates and purifies catalase: the recombination crude enzyme liquid of the culture medium in extraction step S4, to Ni2+
Column is chromatographed after being activated, then is purified to catalase.
9. improving the method for Pichia pastoris catalase expression quantity as claimed in claim 8, which is characterized in that described in extraction
Recombinate crude enzyme liquid the following steps are included:
(1) after the completion of step S4,5min is centrifuged at 4 DEG C, under conditions of 5000g and collects thallus；
(2) thallus sterilizing ddH2Washing is resuspended in O, is centrifuged 5min at 4 DEG C, under conditions of 5000g and collects thallus；
(3) thallus is resuspended with combination buffer, thallus is crushed using sonioation method, obtains ultrasonication liquid；
(4) by the ultrasonication liquid at 4 DEG C, it is centrifuged 20min under conditions of 30000g, collects supernatant.
10. improving the method for Pichia pastoris catalase expression quantity as claimed in claim 8, which is characterized in that described
Ni2+The activation processing of column the following steps are included:
(1) it after sufficiently suspension column packing saves liquid, is fitted into and is stood into chromatographic column；
(2) setting column flow rate is 10CV/h, and when 20% ethyl alcohol when save in column flow to column top, ddH is added2O carries out washing column；
(3) to ddH in column2When O flow to column top, charge buffer liquid is added and carries out washing column；
(4) when the charge buffer liquid stream to column top, combination buffer is added and washes column, and column packing is stored in conjunction with slow
In fliud flushing.
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| CN113564216A (en) * | 2021-07-20 | 2021-10-29 | 蓝科医美科学技术(吉林)有限公司 | Method for obtaining tridecapeptide through yeast fermentation and application thereof |
| CN113564216B (en) * | 2021-07-20 | 2023-07-25 | 蓝科医美科学技术(吉林)有限公司 | Method for obtaining tridecapeptide through yeast fermentation and application thereof |
| CN114164221A (en) * | 2022-01-17 | 2022-03-11 | 李宪臻 | Pichia pastoris gene engineering bacterium for heterologous expression of recombinant catalase and application |
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