CN102115735A - High-density industrial fermentation method for recombinant pichia pastoris phytase - Google Patents
High-density industrial fermentation method for recombinant pichia pastoris phytase Download PDFInfo
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Abstract
The invention relates to the field of fermentation, in particular to a high-density industrial fermentation method for recombinant pichia pastoris phytase. The high-density industrial fermentation method for the recombinant pichia pastoris phytase comprises the following steps of: 1) inoculating fermentation work seed liquid to a fermentation tank; 2) supplementing and feeding glucose; 3) starving; and 4) performing methanol-induced expression on phytase. The method has the advantages that: according to the growth rule of thalli, by combining a dissolved-oxygen-stat (DO-stat) and substrate concentration feedback supplementing method, an expensive glucose electrode is not required to be used, and the supplementing speed of 50 percent glucose solution is adjusted according to the growth speed and concentration of the thalli after inoculation, so that high-density fermentation is realized, and the phytase is efficiently expressed by adjusting the supplementing speed of methanol.
Description
Technical field
The present invention relates to the fermentation field, relate to a kind of recombinant yeast pichia pastoris phytase high-density industrial fermentation method particularly.
Background technology
The pichia pastoris phaff expression system is nearly more than the 20 years a kind of eukaryotic expression systems that grow up, and is used to the manufacture order cell protein in early days, and up to 1987, people such as Cgata were used for pichia spp expression of exogenous gene (JMCregg, et al.1987) first.
Pichia pastoris is research and uses exogenous protein expression system very widely that present having above 400 kinds of albumen obtains expressing.The advantage of this system comprises: the high expression level of (1) foreign protein; (2) foreign protein excretory high-level efficiency; (3) efficient post transcriptional modificaiton, for example glycosylation; (4) can realize high-density culture.
(alcohol oxidase AOX) is key enzyme in the Pichia pastoris system to alcohol oxidase, also is first enzyme in the methanol metabolic pathway.The gene that two kinds of coding alcohol oxidases are arranged in the pichia spp: AOX1 and AOX2, wherein AOX1 is the principal element of alcohol oxidase vigor in the decision cell.The AOX promotor has been regulated the expression of alcohol oxidase at transcriptional level, this has wherein comprised dual regulating and controlling effect: AOX expression by inhibitation system when having inhibition carbon source such as glycerine, glucose, foreign gene also is in not expression status, and when being sole carbon source with methyl alcohol, the AOX gene could high expression level.Therefore the restructured Pichia pastoris in expression foreign protein is divided into three phases, glycerine batch fermentation stage, glycerine feed supplement stage and foreign gene abduction delivering stage usually.
The pasteur recombinant yeast pichia pastoris has following physiological characteristic: (1) aerobic type bacterial strain needs a large amount of oxygen supplys during cultivation.(2) have the Crabtree effect, the threshold value of effect is very low, generally about 0.1-1g/L.Substrate concn surpasses this value, and thalli growth is had inhibiting metabolic by-prods (as ethanol) just can produce thereupon, and there is very big restraining effect in (3) ethanol to the expression of thalline production, particularly foreign protein.So when expressing foreign protein, influenced by several factors, consider the factor of thalline self, also to take the external environment condition in the culturing process into account, influence as nutritive ingredient, temperature, pH, oxyty DO, particularly substrate concn, how to control the stream rate of acceleration of nutritive substance, make that nutritive substance just satisfies the needs of microorganism in the fermentor tank.
When the pichia spp high-density culture, what great majority adopted at present is that the FM21 basis salt that invitrogen company provides is made into the fermention medium that contains 4% glycerine, the less glucose of using, because glucose is the carbon source of checking property by force of AOX promotor, it checks effect and is better than glycerine.But glucose is fermentation field a kind of cultivation carbon source commonly used, and its price is compared with glycerine has very strong competitive edge.Therefore substituting glycerine with relatively inexpensive glucose selects as a kind of ideal that the growth carbon source becomes on the industrial production.Therefore will realize the optimal growth of pichia spp cell under the glucose environment, the starting point concentration of glucose and feed rate how to control glucose are key points.
Summary of the invention
Therefore, the purpose of this invention is to provide a kind of recombinant yeast pichia pastoris phytase high-density industrial fermentation method, thereby can pass through high density fermentation, efficiently express the production phytase.
According to recombinant yeast pichia pastoris phytase high-density industrial fermentation method of the present invention, may further comprise the steps:
1) the work seed liquor of will fermenting is inoculated in fermentor tank;
2) glucose feed supplement stream adds the stage:
In 0-6 hour, add glucose, 6-9 hour with the velocity flow of 40mL/h, add glucose with 80mL/h stream, 9-12 hour, add glucose with 140mL/h stream, 12-14 hour, add glucose with 220mL/h stream, 14-16 hour, add glucose with 320mL/h stream, 16-17 hour, add glucose with 500mL/h stream, 17 hours-mending sugar finishes, add with 600mL/h stream, the thalline weight in wet base reaches and stops to mend sugar behind the 180g/L, and the concentration of wherein said glucose is 50%, contain PTM
112mL/L;
3) the hungry stage:
After stopping to add glucose, do not add any carbon source, hungry 10-15 minute, observe DO and pH value, when DO begins to rise, when pH also begins to rise, enter induction period;
4) the methanol induction Expressing Recombinant Phytase stage:
Added methyl alcohol 2 hours with the 3.0mL/h velocity flow, 3.4mL/h velocity flow added methyl alcohol 2 hours, 3.8mL/h velocity flow added methyl alcohol 2 hours, 4.2mL/h velocity flow added methyl alcohol 2 hours, 4.6mL/h velocity flow added methyl alcohol 12 hours, 5.6mL/h velocity flow added methyl alcohol 24 hours, 6.6mL/h velocity flow added methyl alcohol 24 hours, 7.6mL/h velocity flow added methyl alcohol 24 hours, 8.6mL/h velocity flow added methyl alcohol 24 hours, the 9.0mL/h velocity flow added methyl alcohol 24 hours, and the 8.6mL/h velocity flow added methyl alcohol 24 hours, wherein, described methyl alcohol contains PTM
112mL/L.
Therefore, the method according to this invention comprises three phases: glucose feed supplement stream adds stage, hungry stage and foreign gene abduction delivering stage.Glucose feed supplement stream adds the stage, and thalline adds 50% according to given pace to medium flow from the inoculation beginning and contains PTM
1The glucose feed supplement liquid of 12ml/L, thalline utilizes carbon source for growth, along with thalli growth, adjusts the flow acceleration of glucose according to thalli growth speed, the concentration of sugar stops to mend sugar all the time below 0.5% in the control substratum after the thalline weight in wet base reaches about 180g/L.After stopping to mend sugar, can influence next step abduction delivering because of residual glucose or metabolic intermediate ethanol, kept starvation 15-30 minute, observation DO (dissolved oxygen) value rises near 100%, when the pH value has the trend of rising, beginning to add the methyl alcohol that contains 12ml/L PTM induces, thalline is carbon source abduction delivering phytase with methyl alcohol, methanol concentration must be controlled, the too high meeting of methanol concentration produces toxic action to pichia spp, concentration is low excessively, does not then have effectively to induce effect, after inducing beginning, because the change that thalline can not conform at once, methyl alcohol can't be fully used, so early stage, the methyl alcohol flow can not be too high, add methyl alcohol with the 3.0mL/h velocity flow and (contains PTM
112mL/L) 2 hours,, slowly increase the methyl alcohol flow again along with thalline progressively adapts to the methyl alcohol environment.
Advantage of the present invention is according to the thalli growth rule, in conjunction with DO-stat and concentration of substrate feedback feed supplement method, do not need to use expensive glucose electrode, from inoculating the back just according to thalli growth speed and concentration, adjust 50% glucose solution feed supplement speed, thereby the realization high density fermentation by adjusting the feed supplement speed of methyl alcohol, is realized efficiently expressing of phytase again.
Embodiment
Be embodiments of the invention below, the recombinant yeast pichia pastoris among the embodiment, genotype is Mut
+
More than test used various culture medium prescriptions referring to table 1
Embodiment 1
1, work seed culture
Single bacterium colony of picking is cultivated about 28 hours to the dense 60g/L that reaches of bacterium to first order seed substratum (the bottled 50mlYPD of 250ml triangle) from the YPD streak plate, obtains the first order seed nutrient solution; Again above-mentioned substratum is cultivated 26 hours to the dense 64g/L of bacterium with 1% inoculum size access secondary seed medium (the bottled 100mlYPD of 500ml triangle), promptly get the work seed liquor of fermenting.
2, prepare before the inoculation
Demarcate the pH meter probe with pH6.86 and pH4.00, and to demarcate DO (dissolved oxygen) electrode with saturated sodium sulphite anhydrous 99.3 be 1%, the good fermentation tank culture medium 15L of weighing expects that (BSM) pours in the fermentor tank then, 121 ℃ of autoclavings 30 minutes.
3, inoculation
Being cooled to after 30 ℃ with aseptic method is the PTM of 4.4mL/L with concentration
166ml adds in the substratum of the poison that disappeared, adjusts pH to 4.6 with 25% ammoniacal liquor again, at 0.05MPa, and 2.5m
3/ h, it is 100% that the 600rpm state is proofreaied and correct the DO electrode down.Be inoculated in the 50L full automatic control fermentor tank with 6% the inoculum size work seed liquor of will fermenting then, the initial mixing speed in inoculation back is 200rpm, air flow quantity 2.0m
3/ h, tank pressure 0.05MPa, it is 4.6 that whole fermentation process is controlled pH automatically.
4, glucose stream adds the stage
After being seeded to substratum, thalline has individual adaptive process to new environment, this process thalli growth is slow, the glucose that needs seldom, the oxygen that consumes is also few, and along with thalli growth enters logarithmic phase, the carbon source of consumption and oxygen also increase thereupon, pH is controlled at 4.6 automatically in whole process, and adjusting rotating speed, air flow, glucose flow acceleration are kept DO (dissolved oxygen) more than 30%.In 0-6 hour, add 50% glucose with the velocity flow of 40mL/h and (contain PTM
112ml/L), 6-9 hour, add 50% glucose with 80mL/h stream and (contain PTM
112ml/L), 9-12 hour, add 50% glucose with 140mL/h stream and (contain PTM
112ml/L), 12-14 hour, add 50% glucose with 220mL/h stream and (contain PTM
112mL/L), 14-16 hour, add 50% glucose with 320mL/h stream and (contain PTM
112mL/L), 16-17 hour, add 50% glucose with 500mL/h stream and (contain PTM
112mL/L), 17 hours-mending sugar finishes, and add 50% glucose with 600mL/h stream and (contain PTM
112mL/L), the thalline weight in wet base reaches 180g/L.The fermentation process contrast of method of the present invention and prior art sees Table 2.
Table 2 glucose stream adds the technology comparison sheet
Bend sharp effect (claiming glucose effect Crabtree effect again) according to Ke Lebo, promptly under the dextrose culture-medium of high density and aerobic conditions, find that the cell growth is suppressed and generates the alcoholic acid phenomenon during culturing cell, pichia spp produces ethanol in culturing process, thalli growth is suppressed, bigger harm be after the utilization of inducing competitive inhibition methyl alcohol in the process, cause induce bad in addition the failure.Therefore in the present invention, the flow acceleration according to the different growth phase adjustment glucose of thalline gives an amount of carbon source, thereby does not cause the excess accumulation of glucose, prevents to produce Ke Lebo and bends sharp effect, uses identical glucose can obtain more thalline.
According to the present invention, it is constant to have carried out flow velocity respectively, changes stream and adds time and stream to add the time constant, changes the control experiment of flow velocity, description of test, and thalline weight in wet base in the present invention is the highest, concrete experimental result sees Table 3:
Table 3 different in flow rate and stream add the contrast experiment of time
5, the hungry stage
After stopping to add glucose, do not add any carbon source, hungry 10-15 minute, observe DO and pH value, when DO rises rapidly, when pH is also on the rise, enter induction period.
6, the methanol induction Expressing Recombinant Phytase stage
Thalline is carbon source abduction delivering phytase with methyl alcohol, methanol concentration must be controlled, the too high meeting of methanol concentration produces toxic action to pichia spp, concentration is low excessively, does not then have effectively to induce effect, after inducing beginning, because the change that thalline can not conform at once, methyl alcohol can't be fully used, so early stage, the methyl alcohol flow can not be too high, add methyl alcohol with the 3.0mL/h velocity flow and (contains PTM
112mL/L) 2 hours, along with thalline progressively adapts to the methyl alcohol environment, slowly increase the methyl alcohol flow again, the 3.4mL/h velocity flow adds methyl alcohol and (contains PTM
112mL/L) 2 hours, the 3.8mL/h velocity flow added methyl alcohol and (contains PTM
112mL/L) 2 hours, the 4.2mL/h velocity flow added methyl alcohol and (contains PTM
112mL/L) 2 hours, the 4.6mL/h velocity flow added methyl alcohol and (contains PTM
112mL/L) 12 hours, the 5.6mL/h velocity flow added methyl alcohol and (contains PTM
112mL/L) 24 hours, the 6.6mL/h velocity flow added methyl alcohol and (contains PTM
112mL/L) 24 hours, the 7.6mL/h velocity flow added methyl alcohol and (contains PTM
112mL/L) 24 hours, the 8.6mL/h velocity flow added methyl alcohol and (contains PTM
112mL/L) 24 hours, the 9.0mL/h velocity flow added methyl alcohol and (contains PTM
112mL/L) 24 hours, the 8.6mL/h velocity flow added methyl alcohol and (contains PTM
112mL/L) 24 hours.Fermentation of the present invention and art methods contrast see Table 4.
Table 4, methyl alcohol stream add the technology comparison sheet
Use the stream of glucose among the present invention to add the thalline of technology cultivation because non-existent alcoholic acid accumulation in substratum, after inducing beginning, can comparatively fast finish the carbon source conversion between glucose-methyl alcohol, compare with existing technology, rapid and the amount of the inducing increase of methyl alcohol utilization, rate of growth is also fast so enzyme is lived.
Methanol induction method in according to the present invention has carried out respectively that the amount of inducing is constant in earlier stage, changes induction time, and induction time is constant, changes the control experiment of the amount of inducing, and its weight in wet base and enzyme are lived does not all have height among the present invention, and concrete outcome sees Table 5
Different induction times of table 5 induction period and the amount of inducing contrast experiment
Claims (1)
1. a recombinant yeast pichia pastoris phytase high-density industrial fermentation method is characterized in that, said method comprising the steps of:
1) the work seed liquor of will fermenting is inoculated in fermentor tank;
2) glucose feed supplement stream adds the stage:
In 0-6 hour, add glucose, 6-9 hour with the velocity flow of 40mL/h, add glucose with 80mL/h stream, 9-12 hour, add glucose with 140mL/h stream, 12-14 hour, add glucose with 220mL/h stream, 14-16 hour, add glucose with 320mL/h stream, 16-17 hour, add glucose with 500mL/h stream, 17 hours-mending sugar finishes, add with 600mL/h stream, the thalline weight in wet base reaches 180g/L, and the concentration of wherein said glucose is 50%, contain PTM
112mL/L;
3) the hungry stage:
After stopping to add glucose, do not add any carbon source, hungry 10-15 minute, observe DO and pH value, when DO begins to rise, when pH also begins to rise, enter induction period;
4) the methanol induction Expressing Recombinant Phytase stage:
Added methyl alcohol 2 hours with the 3.0mL/h velocity flow, 3.4mL/h velocity flow added methyl alcohol 2 hours, 3.8mL/h velocity flow added methyl alcohol 2 hours, 4.2mL/h velocity flow added methyl alcohol 2 hours, 4.6mL/h velocity flow added methyl alcohol 12 hours, 5.6mL/h velocity flow added methyl alcohol 24 hours, 6.6mL/h velocity flow added methyl alcohol 24 hours, 7.6mL/h velocity flow added methyl alcohol 24 hours, 8.6mL/h velocity flow added methyl alcohol 24 hours, the 9.0mL/h velocity flow added methyl alcohol 24 hours, and the 8.6mL/h velocity flow added methyl alcohol 24 hours, wherein, described methyl alcohol contains PTM
112mL/L.
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Cited By (8)
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| CN102517223A (en) * | 2011-12-28 | 2012-06-27 | 温州市龙泰轻工机械有限公司 | High-density fermentation process and equipment for methanol type pichia pastoris |
| CN105420320A (en) * | 2015-12-28 | 2016-03-23 | 山东鲁抗医药股份有限公司 | Collagen phased mixing, induction and fermentation method |
| CN105861464A (en) * | 2016-05-24 | 2016-08-17 | 谢飞 | Recombined hansenula polymorpha fermentation technology |
| CN107586765A (en) * | 2017-10-20 | 2018-01-16 | 山东奥博生物科技有限公司 | A kind of industrial fermentation process of phytase |
| CN108396017A (en) * | 2017-10-20 | 2018-08-14 | 山东奥博生物科技有限公司 | A kind of industrial fermentation process of mannase |
| CN105219749B (en) * | 2015-11-04 | 2019-08-27 | 广东溢多利生物科技股份有限公司 | Optimize the phytic acid enzyme mutant improved and its encoding gene and application |
| CN111088304A (en) * | 2019-12-27 | 2020-05-01 | 深圳康泰生物制品股份有限公司 | Fermentation induction process for expressing recombinant enterovirus 71 type virus-like particles, enterovirus 71 type vaccine and preparation method thereof |
| CN113667709A (en) * | 2021-08-28 | 2021-11-19 | 西安德诺海思医疗科技有限公司 | Fermentation method of recombinant humanized collagen |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102517223A (en) * | 2011-12-28 | 2012-06-27 | 温州市龙泰轻工机械有限公司 | High-density fermentation process and equipment for methanol type pichia pastoris |
| CN105219749B (en) * | 2015-11-04 | 2019-08-27 | 广东溢多利生物科技股份有限公司 | Optimize the phytic acid enzyme mutant improved and its encoding gene and application |
| CN105420320A (en) * | 2015-12-28 | 2016-03-23 | 山东鲁抗医药股份有限公司 | Collagen phased mixing, induction and fermentation method |
| CN105420320B (en) * | 2015-12-28 | 2019-04-09 | 山东鲁抗医药股份有限公司 | The induction fermentation method of mixing stage by stage of collagen |
| CN105861464A (en) * | 2016-05-24 | 2016-08-17 | 谢飞 | Recombined hansenula polymorpha fermentation technology |
| CN107586765A (en) * | 2017-10-20 | 2018-01-16 | 山东奥博生物科技有限公司 | A kind of industrial fermentation process of phytase |
| CN108396017A (en) * | 2017-10-20 | 2018-08-14 | 山东奥博生物科技有限公司 | A kind of industrial fermentation process of mannase |
| CN111088304A (en) * | 2019-12-27 | 2020-05-01 | 深圳康泰生物制品股份有限公司 | Fermentation induction process for expressing recombinant enterovirus 71 type virus-like particles, enterovirus 71 type vaccine and preparation method thereof |
| CN113667709A (en) * | 2021-08-28 | 2021-11-19 | 西安德诺海思医疗科技有限公司 | Fermentation method of recombinant humanized collagen |
| CN113667709B (en) * | 2021-08-28 | 2023-11-21 | 西安德诺海思医疗科技有限公司 | Fermentation method of recombinant humanized collagen |
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