CN107384879B - A kind of fermentation manufacturing technique of recombinant yeast pichia pastoris bacterium production liver detoxification enzyme - Google Patents
A kind of fermentation manufacturing technique of recombinant yeast pichia pastoris bacterium production liver detoxification enzyme Download PDFInfo
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Abstract
The present invention provides a kind of fermentation manufacturing technique of recombinant yeast pichia pastoris bacterium production liver detoxification enzyme, including strain preparation, bacterial strain activation, first order seed culture, secondary seed culture, fermented and cultured and inducing expression and puts tank step.The strain is the recombinant yeast pichia pastoris bacterium of the enzyme gene containing liver detoxification.The fermented and cultured and inducing expression includes four-stage, and respectively thallus concentration stage, glycerol fed-batch fermentation stage, carbon source hungry stage and the high dissolved oxygen of high density induce feed phase.The present invention reaches 50000-56000 U/g, fermentation time 132-144h by the enzyme activity of the liver detoxification enzyme of recombinant yeast pichia pastoris bacterium fermenting and producing.
Description
Technical field
The present invention relates to a kind of fermentation manufacturing techniques of recombinant yeast pichia pastoris bacterium, and in particular to a kind of recombinant yeast pichia pastoris bacterium
The fermentation manufacturing technique for producing liver detoxification enzyme, belongs to technical field of bioengineering.
Background technique
Fermentation Engineering refers to using modern engineering technology means, is human being's production using certain specific functions of microorganism
Useful product, or directly microbe application in a kind of new technology of industrial processes.The content of Fermentation Engineering includes bacterium
The breeding of kind, sterilizing, expands culture and inoculation, fermentation process and the separating-purifying of product etc. at the preparation of culture medium.
Due to the complexity of biological respinse, from laboratory to pilot scale, from pilot scale to large-scale production process in will appear
Many problems, here it is Fermentation Engineering technique scale-up problems.The technology with purified product is separated from fermentation liquid: including solid-liquid point
From technology (centrifuge separation, be separated by filtration, the techniques such as precipitation and separation), cell wall breaking technology (ultrasound, high shear, osmotic pressure, table
Face activating agent and lywallzyme etc.), purified technology of protein (precipitation method, chromatography and ultrafiltration etc.), last there are also products
Packing processes technology (vacuum drying and the frost secretary in charge of something are dry etc.).These are all the aspects of field of fermentation engineering primary study.
The industrial fermentation production of recombination engineering bacteria is even more the key points and difficulties in Fermentation Engineering.Although engineering strain has
There is the ability of transgenosis, but how to realize its effective expression and industrial production, is still the important technical links in production process.
It carries out expanding fermentation recombinant yeast pichia pastoris bacterium using common Pichia pastoris zymotechnique, expression liver detoxification enzyme
Efficiency is very low, can not therefrom extract effective liver detoxification enzyme, and the liver detoxification enzyme enzyme activity of acquisition is very low；Therefore, why
The enzyme activity for the liver detoxification enzyme that sample raising transgenic Pichia yeast is obtained by fermenting and producing is the important topic of this field research.
Summary of the invention
In view of the deficiencies of the prior art, the object of the present invention is to provide a kind of recombinant yeast pichia pastoris bacterium to produce liver detoxification enzyme
Fermentation manufacturing technique, significantly improve the enzyme activity of liver detoxification enzyme.
To solve existing technical problem, the technical solution adopted by the present invention is that:
A kind of fermentation manufacturing technique of recombinant yeast pichia pastoris bacterium production liver detoxification enzyme, including strain prepares, bacterial strain activates,
First order seed culture, secondary seed culture, fermented and cultured and inducing expression and put tank step.
The strain is the recombinant yeast pichia pastoris bacterium of the enzyme gene containing liver detoxification.
The fermented and cultured and inducing expression includes four-stage, respectively thallus concentration stage, glycerol fed-batch fermentation
Stage, carbon source hungry stage and the high dissolved oxygen of high density induce feed phase.
The thallus concentration stage；PH 6.5~7.0 is controlled by way of feeding back Feeding ammonia water, air vent ratio is
0.6vvm, revolving speed are 120~160 r/min；Fermentation temperature is 25 DEG C ± 0.5；Tank pressure is 0.05~0.07MPa, and DO value is 25%
~35%, 12~14 h are cultivated, this stage, thallus weight in wet base was up to 250g/L or more.
The glycerol fed-batch fermentation stage；Stream plus 30% glycerol into fermentor, glycerol feeding speed are 17~20
mL·(h·L)-1, control air vent amount is adjusted to 1.4~1.6vvm, control 25 DEG C ± 0.5 of fermentation temperature, tank pressure 0.05~
0.07MPa, DO value are 25%~35%, and the glycerol feed supplement time controls 22~26 h, and thallus OD600 reaches 1.8~2.0.
The carbon source hungry stage；30 DEG C ± 0.5 of temperature are controlled in fermentor, air vent amount is 0.8~1.2vvm,
Tank pressure is 0.05~0.07MPa, carries out 1~1.5h of hungry culture.
The high dissolved oxygen of the high density induces feed phase: stream plus methanol and 50% sorbitol solution 1:1 mixed liquor；Fermentation
Temperature control is 24~29 DEG C, and control pH is 5.5~7.1, and air vent amount is 0.8~1.2vvm, tank pressure for 0.05~
0.07MPa, DO value are 25%~35%.
The fermented and cultured and expression induction step: the active constituent content of the fermentation medium of use are as follows: yeast extract
10~30g/L, 6.7~20g/L of peptone, 3.3~10ug/L of biotin, 23.3~70mL/L of glycerol, K2HPO45~15g/L,
0~8 mL/L of brewer's wort.
The fermented and cultured and expression induction step: the active constituent content of the fermentation medium of use are as follows: yeast extract
20g/L, peptone 15g/L, biotin 5ug/L, K2HPO415g/L, 8 mL/L of brewer's wort, 40 g/L of glycerol.
The first order seed culture: picking activates recombinant yeast pichia pastoris bacterium, is inoculated in culture medium, culture to culture solution
Thallus is collected behind OD600=0.6~0.8；Thallus is fermented again, fermentation temperature is 25 DEG C ± 0.5；Tank pressure is 0.06MPa；Training
It supports to culture solution OD600=1.5~1.8；
The secondary seed culture: inoculative proportion 1%, temperature control at 24~26 DEG C, fermentation liquid PH is 6.5~
7.0, dissolved oxygen content control is 25%~35%.
By adopting the above-described technical solution, having the technical effect that of reaching of the present invention
The present invention reaches 50000-56000 U/ by the enzyme activity of the liver detoxification enzyme of recombinant yeast pichia pastoris bacterium fermenting and producing
G, fermentation time 132-144h.
Specific embodiment
A kind of fermentation manufacturing technique of the recombinant yeast pichia pastoris bacterium of embodiment 1 production liver detoxification enzyme
Step 1, strain prepare
The strain used belongs to transgenic engineered bacteria, the recombination for the recombinant yeast pichia pastoris bacterium of the enzyme gene containing liver detoxification
The deposit number of Pichia yeast is CCTCC M2017278；The deposit date is on May 22nd, 2017, depositary institution was in CCTCC-
State's Type Tissue Collection, address are Wuhan, China, and classification naming is Pichia pastoris X33.
Step 2, bacterial strain activation
The activation: bacterial strain is crossed culture three times.
Scribing line culture for the first time: taking recombinant yeast pichia pastoris bacterium 0.8~1.2g of freeze-dried powder, is dissolved in the training of 1mL YPD liquid asepsis
It supports in base, takes bacterium solution to cross on YPD plating medium, whole sterile working；Scribed culture medium is placed in 25 DEG C of constant temperature trainings
It supports in case and cultivates 48h；
Second of scribing line culture: it from picking single colonie on the plate of primary scribing line culture, is trained again in the YPD plate of blank
Support culture of crossing on base, whole sterile working；
Scribed culture medium is placed in 25 DEG C of constant incubators and cultivates 48h；
Third time scribing line culture: from picking single colonie on the plate of second of scribing line culture, again in the YPD plate of blank
It crosses and cultivates on culture medium, whole sterile working；
Scribed culture medium is placed in 25 DEG C of constant incubators and cultivates 48h.
The YPD culture medium, effective component include: yeast extract, peptone and glucose, yeast extract: peptone: grape
The mass ratio of sugar is 1:2:2.
The preparation of the YPD fluid nutrient medium the following steps are included:
(1) 10g yeast extract and 20g peptone is taken to be dissolved in 900mL purified water, 121 DEG C of sterilizing 20min；
(2) glucose 20g is taken to be dissolved in 100mL purified water, 115 DEG C of sterilizing 15min；
(3) solution that step (1) and step (2) obtain is mixed to get YPD fluid nutrient medium.
The preparation method of the YPD plating medium: it is identical as the preparation method of YPD fluid nutrient medium, only in step (1)
Solution in be added 2g agar；And the mixed solution inverted plate for obtaining step (3).
Step 3, first order seed culture
The recombinant yeast pichia pastoris bacterium single colonie that picking third time scribing line culture obtains is seeded in the sterile YPD liquid training of 10mL
It supports in base, cultivates in 250rpm, the isothermal vibration incubator of 25 DEG C of conditions to culture solution OD600=0.6~0.8；
Above-mentioned culture solution is centrifuged 5min in 3000rpm, collects thallus；
The thallus of collection is resuspended in the 50L seeding tank of the sterile YPD fluid nutrient medium containing 25L, ventilation ratio 0.6vvm,
Revolving speed is 260 r/min；Fermentation temperature is 25 DEG C ± 0.5；Tank pressure is 0.06MPa；It cultivates to culture solution OD600=1.5~1.8,
Thallus weight in wet base reaches 240 ± 10g/L, obtains first order seed culture solution.
Step 4, secondary seed culture
Using the fermentor of volume 5T as secondary seed tank, it is packed into secondary seed medium 2.5T；Temperature control is 25 DEG C,
PH is adjusted with ammonium hydroxide and is controlled in 6.5-7.0, and dissolved oxygen content control is 25%~35%；
First order seed culture solution 25L is inoculated with into secondary seed medium；I.e. inoculative proportion is 1%(percentage by volume).It connects
After kind, 6~8h is cultivated, secondary seed culture solution is transferred to fermentor when culture to OD600 is 2.3 or so.
The secondary seed medium: effective component and content are as follows: yeast powder 10g/L, peptone 20g/L, glycerol 80mL/
L。
Step 5, fermented and cultured and inducing expression
Use capacity for the fermentor of 30T, be packed into fermentation medium amount be 15T, by 15%(percentage by volume) inoculation
Ratio is inoculated with secondary seed culture solution.
The fermentation medium: the content of effective component are as follows: yeast extract 30g/L, peptone 20g/L, biotin 10ug/L,
Glycerol 70mL/L, K2HPO4 15g/L。
After the completion of inoculation, fermented and cultured and inducing expression, including four-stage are carried out；
First stage thallus concentration stage: PH 6.5-7.0, air vent ratio are controlled by way of feeding back Feeding ammonia water
For 0.6vvm, revolving speed is 150 r/min；Fermentation temperature is 25 DEG C ± 0.5；Tank pressure is 0.06MPa, and DO value is 25%~35%, training
12 h are supported, this stage, thallus weight in wet base was up to 250g/L or more.
Fermentation parameter is recorded every 4 h, when DO value rises very rapidly up to 100%, thallus concentration stage terminates.
Second stage is the glycerol fed-batch fermentation stage: being enriched with biomass further, stream plus 30% glycerol into fermentor
(percentage is percentage by volume), glycerol feeding speed is 18 mL (hL)-1, control air vent amount and be adjusted to
1.5vvm, revolving speed are 150 r/min, control 25 DEG C ± 0.5 of fermentation temperature, and tank presses 0.06MPa, and DO value is 25%~35%, glycerol
The feed supplement time controls 24 h, and thallus OD600 reaches 1.8~2.0, and thallus weight in wet base can reach 350g/L or more.
The phase III carbon source hungry stage: after glycerol feed phase, 30 DEG C ± 0.5 of temperature are controlled in fermentor, is turned
180 r/min of speed, air vent amount are 1 vvm, and tank pressure is 0.06MPa, carry out hungry culture 1h.
The high dissolved oxygen of fourth stage high density induces feed phase: after starvation culture, stream plus methanol and 50% sorbierite
Solution 1:1(volume ratio) mixed liquor；
Fermentation temperature control is 25 DEG C, and control pH is 6.5-7.0, and 200 r/min of speed of agitator, air vent amount is 1
Vvm, tank pressure are 0.06MPa, and DO value is 25%~35%.
Initial methanol and sorbierite mixing acceleration of liquid are 15 mL (hL)-1, flow the added-time between maintain 4 h；
Then flow acceleration control is at 35 mL (hL)-1, 2 h of time maintenance；
Then methanol content is detected every 2 h, controls flow acceleration, maintain methanol content in 2%-3%, pure oxygen ventilatory capacity is
0.5vvm, control DO value are 50%；The total 80h of the fermentation time of fourth stage.
Step 6 puts tank
After fermented and cultured, tank is put, obtains liver detoxification enzyme fermentation liquid.
A kind of fermentation manufacturing technique of the recombinant yeast pichia pastoris bacterium of embodiment 2 production liver detoxification enzyme
Fermenting and producing is carried out according to the method for embodiment 1, is only changed the step and is fermented in 5 fermented and cultureds and expression induction step
The concentration of culture medium carries out embodiment 2；
The fermentation medium concentration that embodiment 2 uses is the 2/3 of 1 concentration of embodiment；That is the effective component of fermentation medium
Content are as follows: yeast extract 20g/L, peptone 13.3g/L, biotin 6.7ug/L, glycerol 46.7mL/L, K2HPO4 10g/L。
A kind of fermentation manufacturing technique of the recombinant yeast pichia pastoris bacterium of embodiment 3 production liver detoxification enzyme
Fermenting and producing is carried out according to the method for embodiment 1, is only changed the step and is fermented in 5 fermented and cultureds and expression induction step
The concentration of culture medium carries out embodiment 3；
The fermentation medium concentration that embodiment 3 uses is the 1/3 of 1 concentration of embodiment；That is the effective component of fermentation medium
Content are as follows: yeast extract 10g/L, peptone 6.7g/L, biotin 3.3ug/L, glycerol 23.3mL/L, K2HPO4 5g/L。
It is tested, the enzyme activity of the liver detoxification enzyme of detection embodiment 1-3 production, concrete outcome is shown in Table 1；
Table 1
As it can be seen that embodiment 2 is preferred embodiment.
A kind of fermentation manufacturing technique of the recombinant yeast pichia pastoris bacterium of embodiment 4 production liver detoxification enzyme
Fermenting and producing is carried out according to the method for embodiment 1, changes the step fourth order in 5 fermented and cultureds and expression induction step
The fermentation temperature and pH of section (the high dissolved oxygen of high density induces feed phase) carry out embodiment 4-7；
The fermentation temperature and pH that embodiment 4-7 is used are shown in Table 2；
Table 2
The fermenting and producing of embodiment 4-7 the results are shown in Table 3；
Table 3
Seen from table 3, embodiment 5 is preferred embodiment, i.e. fourth stage in step 5 fermented and cultured and expression induction step
The fermentation temperature of (the high dissolved oxygen of high density induces feed phase) is preferably 28 DEG C, and pH is preferably 6.0 ± 0.1.
A kind of fermentation manufacturing technique of the recombinant yeast pichia pastoris bacterium of embodiment 8 production liver detoxification enzyme
Fermenting and producing is carried out according to the method for embodiment 1, changes the step fourth order in 5 fermented and cultureds and expression induction step
Section (the high dissolved oxygen of high density induces feed phase) are as follows:
After starvation culture, stream plus the stream containing ammonium citrate add methanol and 50% sorbitol solution 1:1(V/V) mixing
Liquid, the content of ammonium citrate are 10mmol/L；
Fermentation temperature control is 25 DEG C, and control pH is 6.5-7.0, and 200 r/min of speed of agitator, air vent amount is 1
Vvm, tank pressure are 0.06MPa, and DO value is 25%~35%.
Initial methanol and sorbierite mixing acceleration of liquid are respectively 15 mL (hL)-1, flow the added-time between maintain 4 h；
Then flow acceleration control is at 35 mL (hL)-1, 2 h of time maintenance；
Then methanol content is detected every 2 h, controls flow acceleration, maintain methanol content in 2%-3%, pure oxygen ventilatory capacity is
0.5vvm, control DO value are 50%；The total 80h of the fermentation time of fourth stage.
The enzyme activity of the liver detoxification enzyme of the present embodiment production is 56000 U/g, fermentation time 132h.
A kind of fermentation manufacturing technique of the recombinant yeast pichia pastoris bacterium of embodiment 9 production liver detoxification enzyme
Fermenting and producing is carried out according to the method for embodiment 1, is changed the step and is used in 5 fermented and cultureds and expression induction step
Fermentation medium are as follows: yeast extract 20g/L, peptone 15g/L, biotin 5ug/L, K2HPO415g/L, it is 8 mL/L of brewer's wort, sweet
40 g/L of oil.
The enzyme activity of the liver detoxification enzyme of the present embodiment production is 55800 U/g, fermentation time 132h.
Although the method for embodiment 9 is little with enzyme activity, the fermentation time gap of embodiment 8, cost reduces 10-12%.
The method for detecting liver detoxification enzyme enzyme activity are as follows:
1, Preparatory work of experiment
(1) reagent: liver detoxification enzyme, AFB1 standard items (1mg), 99.5% methanol, pure water.
(2) equipment: microplate reader, liquid-transfering gun (5mL, 1mL, 200 μ L, 10 μ L), 4mL centrifuge tube, thermostat water bath.
(3) preparation of reagents:
0.02mg/mL AFB1(20ppm): 1mgAFB1 is completely dissolved in 50mL purified water；
200ng/mL AFB1(200ppb): take 1mL 20ppmAFB1 to be dissolved in 99mL purified water；
0.04g/mL enzyme solution: precise 4g enzyme powder is completely dissolved in 100mL purified water, then 5000rpm centrifugation 10
Min takes supernatant；
PH5 phosphate buffer: accurately weighing 8.34 g potassium dihydrogen phosphates, and 1.34 g dipotassium hydrogen phosphates are dissolved in 1000mL
In purified water；
Adlerika: it accurately weighs 0.5g magnesium sulfate and is completely dissolved in 500mL purified water.
Enzyme activity definition: 1g liver detoxification enzyme degrades 1pptAFB1 per minute as an enzyme-activity unit.
Experimental procedure
(1) reaction system
(2) reaction process:
It rises again: each substance in reaction system table in addition to substrate A FB1 being added in 4mL centrifuge tube, same to AFB1
(200ppb) one arises from water-bath 5min at 30 DEG C；
Enzyme reaction: the AFB1 after being separately added into 500 μ L preheating into the centrifuge tube of 2,3, No. 4 experimental groups, in No. 1 blank group
AFB1 is first not added.Then each experimental group is placed in 30 DEG C of water-bath 30min；
It terminates reaction: terminating reaction to each experimental group addition 2mL100% methanol, while adding 500 μ to No. 1 blank group
LAFB1 substrate, then 8000rpm is centrifuged 5min, takes supernatant to terminate reaction solution；
Dilution: it takes 1mL to terminate reaction solution and is dissolved in 17mL sample diluting liquid.(purpose: sample is diluted to kit detection
Within the scope of, that is, it is less than 2ppb).
5. measuring the light absorption value of each experimental group using microplate reader.
It is as shown in the table to the testing result of aflatoxin B1 standard items:
Standard curve is obtained according to the testing result of standard items.
The enzyme activity of measuring samples is calculated according to standard curve using the light absorption value of microplate reader measurement measuring samples.
Unless specifically indicated with unit commonly used in the art, ratio of the present invention is mass ratio, the percentage,
It is mass percent.
Liver detoxification enzyme of the present invention, alternatively referred to as Aflatoxin-detofizyme.
Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention
Within protection scope.
Claims (2)
1. a kind of fermentation manufacturing technique of recombinant yeast pichia pastoris bacterium production Aflatoxin-detofizyme, it is characterised in that: including bacterium
It plants preparation, bacterial strain activation, first order seed culture, secondary seed culture, fermented and cultured and inducing expression and puts tank step；
The deposit number of the recombinant yeast pichia pastoris bacterium is CCTCC M2017278；
The first order seed culture: picking activates recombinant yeast pichia pastoris bacterium, is inoculated in culture medium, culture to culture solution
Thallus is collected behind OD600=0.6~0.8；Thallus is fermented again, fermentation temperature is 25 DEG C ± 0.5 DEG C, and tank pressure is 0.06MPa；
It cultivates to culture solution OD600=1.5~1.8；
The secondary seed culture: inoculative proportion 1%, at 24~26 DEG C, fermentation liquid PH is 6.5~7.0, molten for temperature control
Control for Oxygen Content is 25%~35%；
The fermented and cultured and inducing expression includes four-stage, respectively thallus concentration stage, the glycerol fed-batch fermentation stage,
Carbon source starvation stage and the high dissolved oxygen of high density induce feed phase；
The fermented and cultured and expression induction step: the active constituent content of the fermentation medium of use are as follows: yeast extract 10~
30g/L, 6.7~20g/L of peptone, 3.3~10ug/L of biotin, 23.3~70mL/L of glycerol, K2HPO45~15g/L, malt
0~8 mL/L of juice；
The thallus concentration stage: controlling PH 6.5~7.0 by way of feeding back Feeding ammonia water, and air vent ratio is
0.6vvm, revolving speed are 120~160 r/min；Fermentation temperature is 25 DEG C ± 0.5 DEG C；Tank pressure is 0.05~0.07MPa, and DO value is
25~35%, 12~14 h are cultivated, this stage thallus weight in wet base reaches 250g/L or more；
The glycerol fed-batch fermentation stage: control air vent amount is adjusted to 1.4~1.6vvm, and 25 DEG C of fermentation temperature of control ±
0.5 DEG C, tank presses 0.05~0.07MPa, and DO value is 25~35%, and stream plus 30% glycerol into fermentor, glycerol feeding speed are 17
~20 mL (hL)-1, the control of glycerol feed supplement time 22~26 h, thallus OD600 reach 1.8~2.0；
The carbon source hungry stage: 30 DEG C ± 0.5 DEG C of temperature in control fermentor, air vent amount is 0.8~1.2vvm, tank
Pressure is 0.05~0.07MPa, carries out 1~1.5h of hungry culture；
The high dissolved oxygen of the high density induces feed phase: stream plus methanol and 50% sorbitol solution 1:1 mixed liquor；Fermentation temperature
Control is 24~29 DEG C, and control pH is 5.5~7.1, and air vent amount is 0.8~1.2vvm, and tank pressure is 0.05~0.07MPa,
DO value is 25~35%.
2. a kind of fermenting and producing work of recombinant yeast pichia pastoris bacterium production Aflatoxin-detofizyme according to claim 1
Skill, it is characterised in that: the fermented and cultured and expression induction step: the active constituent content of the fermentation medium of use are as follows:
Yeast extract 20g/L, peptone 15g/L, biotin 5ug/L, K2HPO415g/L, 8 mL/L of brewer's wort, 40 g/L of glycerol.
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