CN102604904B - Production method of glucose dehydrogenase - Google Patents
Production method of glucose dehydrogenase Download PDFInfo
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- CN102604904B CN102604904B CN 201210065718 CN201210065718A CN102604904B CN 102604904 B CN102604904 B CN 102604904B CN 201210065718 CN201210065718 CN 201210065718 CN 201210065718 A CN201210065718 A CN 201210065718A CN 102604904 B CN102604904 B CN 102604904B
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- fermented liquid
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- phosphate dehydrogenase
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Abstract
The invention relates to a production method of glucose dehydrogenase, which mainly uses flow feeding technology in microbial fermentation and genetically engineered microorganisms to ferment to produce glucose dehydrogenase. The production method specifically includes: culturing recombinant escherichia coli containing glucose dehydrogenase gene at the culture temperature of 37 DEG C so as to express glucose dehydrogenase; inoculating the recombinant escherichia coli to culture medium for fermentation, adding supplementary medium into fermented broth containing the recombinant escherichia coli during fermentation by the flow feeding technology, and increasingly the flow feeding speed in a gradient form with prolonging of the fermentation time; and adding inducer when initial growth phase of the bacterium is over, reducing the temperature from 37 DEG C to about 28 DEG C, and maintaining constant temperature until fermentation is over. By the method, fermentation conditions for fermentation process meet actual needs of the bacterium as far as possible, the density of the bacterium can be increased to a high level, the yield of glucose dehydrogenase is increased, and great industrialapplication value is achieved.
Description
Technical field
The invention belongs to bioengineering field, be specifically related to a kind of method of utilizing genetic engineering bacterium fermentative production Hexose phosphate dehydrogenase, mainly use the flow feeding technology of microbial fermentation aspect
Background technology
Hexose phosphate dehydrogenase (Glucose dehydrogenase, abbreviation GlcDH) is a member of short chain alcohol dehydrogenase enzyme family, at coenzyme NAD (P)
+
Catalysis β-D-conversion of glucose to be gluconic acid when existing.Hexose phosphate dehydrogenase is widely used in aspects such as catalytic preparation chiral alcohol, alcohol acid, amino acid, these enzymic catalytic reactions generally all need the participation of coenzyme NAD (P) H, and these coenzyme are expensive, so the regeneration of NAD (P) H is very important for subduing cost.U.S. Shi Guibao company has effectively utilized Hexose phosphate dehydrogenase and has carried out coenzyme NAD P in the large-scale production that utilizes synthetic (the s)-4-chloro-3-beta-hydroxymethyl butyrate of geotrichum candidum desaturase asymmetric reduction 4-chloro-3-carbonyl methyl-butyrate
+
The circulation of-NADPH, the productive rate of this reaction are up to 95%, and product is the chiral raw material of anticholesteremic agent.
In the prior art, Hexose phosphate dehydrogenase mainly extracts from the liver of animal, and perhaps by fermentation of bacillus, the source is restricted.And domestic Hexose phosphate dehydrogenase all mainly relies on import.Because Hexose phosphate dehydrogenase is with a wide range of applications, market demand also increases day by day, and therefore, the production method of developing a kind of Hexose phosphate dehydrogenase of Cheap highly effective is of great practical significance
Summary of the invention
Technical problem to be solved by this invention is to overcome that the source of Hexose phosphate dehydrogenase in the prior art is restricted and the problem that is difficult to meet the need of market provides a kind of method of utilizing genetic engineering bacterium fermentative production Hexose phosphate dehydrogenase.
For solving above technical problem, the present invention takes following technical scheme:
A kind of production method of Hexose phosphate dehydrogenase, the recombination bacillus coli of the Hexose phosphate dehydrogenase that is first construction expression and it is seeded in the initial medium ferments, in the fermented liquid that comprises described recombination bacillus coli, add supplemented medium by the flow feeding technology in the fermenting process, when fermentation parameter reaches setting threshold, in fermented liquid, add inductor again, and continue the fermentation certain hour to fermentation ends, from the culture of fermented liquid, obtain Hexose phosphate dehydrogenase at last; Wherein, during flow feeding, gradient increases progressively the flow acceleration of supplemented medium along with the prolongation of fermentation time, remains unchanged after adding inductor.
The flow feeding technology is a kind of traditional method that is used for improving cell concentration in the fermenting process.This method can be controlled the concentration of nutritive ingredient in when fermentation nutrient solution, therefore uses more satisfactory in cell growth or product form the technological process of the limited sensitivity of concentration of substrate.
According to the present invention, described fermentation parameter can be the cell optical density(OD) of fermented liquid, dissolved oxygen in the fermented liquid or the carbon source total concn in the fermented liquid etc.
If without specified otherwise, the cell optical density(OD) described in the present invention all refers to OD
600
Value.OD
600
It is the standard method of following the trail of microorganism growth in the liquid culture.OD is optical density(optical density(OD)) abbreviation, the optical density(OD) that sponges of expression detected material is the proper noun in the detection method.Light is by detected material, and the capacity volume variance of front and back namely is the energy that detected material sponges, and under the specific wavelength, becomes quantitative relationship with a kind of concentration of detected material with absorbed energy.OD
600
What refer to is exactly that certain solution is at the light absorption value at 600nm wavelength place.Light absorption value is proportional to the concentration of the extinction material in the solution, and correspondingly the transmitance with sample is inversely proportional to.
The preferred a kind of production method of the present invention may further comprise the steps:
(1), the preparatory stage: make up genetic engineering bacterium, preparation seed culture medium, initial medium and supplemented medium; Genetic engineering bacterium is activated in described seed culture medium, and the seed liquor that will obtain after will activating then is inoculated in the initial medium ferments, by volume, and seed liquor/initial medium during inoculation=1% ~ 4%; Described genetic engineering bacterium is the recombination bacillus coli of having expressed Hexose phosphate dehydrogenase;
(2), fermenting process: the different steps that the present invention carries out according to fermentation, cell are divided into six stages to the spending rate difference of nutritive ingredient in the nutrient solution with fermenting process, and with the cell optical density(OD) (OD in the nutrient solution
600
) divide this six stages, be followed successively by fs 0 ~ 4, subordinate phase 4 ~ 8, phase III 8 ~ 12, quadravalence section 12 ~ 16, five-stage 16 ~ 20 and the 6th stage OD
600
＞20;
Cell optical density(OD) (OD in fermented liquid
600
) along with the carrying out of fermentation reaches successively at 4,8,12,16,20 o'clock, in fermented liquid, replenish supplemented medium by the flow feeding technology, and the flow acceleration of supplemented medium corresponds to 0 ~ 10g/l/h, 10 ~ 20g/l/h, 20 ~ 30g/l/h, 30 ~ 40g/l/h, 40 ~ 50g/l/h successively in batches; As cell optical density(OD) (OD
600
)＞20 o'clock add inductor in fermented liquid, and the adding quality of inductor in fermented liquid account for 0.01% ~ 0.2% of fermented liquid total amount; Under the constant situation of the flow acceleration that keeps supplemented medium, continue fermentation 16 ~ 20 hours then;
The first five of fermenting process stage is the thalli growth stage, it is lower that the flow acceleration of supplemented medium keeps, between 0 ~ 40 g/l/h, and the flow acceleration of supplemented medium should along with the fermentation carrying out accelerate gradually, to adapt to thalli growth to the increase in demand of nutritive substance in the nutrient solution; The 6th stage of fermenting process is the abduction delivering stage, and the flow acceleration of supplemented medium should keep comparatively fast, between 40 ~ 50 g/l/h.
(3), after step (2) finishes, adopt ordinary method from the culture of step (2) gained fermented liquid, to obtain Hexose phosphate dehydrogenase, through subsequent disposal, finish whole process of production.
Preferably, described initial medium comprises peptone 6 ~ 15g/L, yeast powder 5 ~ 10g/L, glycerine 6 ~ 18g/L, sal epsom 1 ~ 4g/L, phosphoric acid salt 4 ~ 8g/L and sodium-chlor 5 ~ 12g/L; Described supplemented medium comprises peptone, yeast powder and glycerine, and peptone by mass: yeast powder: glycerine=2:1:14 ~ 15.
Preferably, in the fermenting process, dissolved oxygen of fermentation liquid maintains between 30% ~ 40%, and fermented liquid pH value is controlled between 6.8 ~ 7.3 (available ammoniacal liquor adjusting).
Preferably, described inductor is sec.-propyl-β-D-sulfo-galactopyranoside (IPTG).
Preferably, before the interpolation inductor, leavening temperature is controlled at 37 ℃; Leavening temperature control behind the interpolation inductor is at 28 ℃.Be the first five stage (thalli growth stage) of fermenting process, temperature should be controlled at 37 ℃, is beneficial to thalline breeding growth, and the 6th stage of fermenting process (abduction delivering stage), leavening temperature should be controlled at 28 ℃, is beneficial to the generation of inducing of Hexose phosphate dehydrogenase.
Preferably, in the step (1), described seed culture medium comprises peptone 10g/L, yeast powder 5g/L and NaCl 10g/L, and the pH value is 7.0, needs high-temperature sterilization before using.
Because the enforcement of above technical scheme, the present invention compared with prior art has following advantage:
1, the present invention utilizes the method for genetic engineering bacterium fermentation to obtain Hexose phosphate dehydrogenase, adopting the technology of flow feeding according to the different steps of fermenting process is the fermented liquid material that supplements the nutrients gradually, fermentation condition in the fermenting process is adapted with the thalline actual needs as far as possible, be conducive to the growth of thalline and efficiently expressing of foreign protein greatly.
2, the present invention can effectively avoid because the excessive substrate that causes of certain stage substratum of fermentation suppresses, and can effectively control the speed of growth of abduction delivering stage thalline.
When 3, producing Hexose phosphate dehydrogenase with the inventive method, the ratio of the expression amount of Hexose phosphate dehydrogenase in total protein can reach 6.3g/L up to 47.2%.
Embodiment
The present invention will be further described in detail below in conjunction with specific embodiment, but be not limited to these embodiment.
The structure of the genetic engineering bacterium among the present invention---recombination bacillus coli is finished [seeing " molecular cloning " (Science Press, second edition, 2002)] according to conventional molecular biosciences operation.The BIOTECH-10L fermentor tank that the fermentor tank that this experiment is used is produced as Shanghai Baoxing Biology Equipment Engineering Co., Ltd.
Each substratum composed as follows:
Seed culture medium:
Peptone 10 g/L, yeast powder 5 g/L, NaCl 10 g/L regulate pH=7.0, sterilize 20 minutes for 121 ℃.
Initial medium:
Peptone 12g/L, yeast powder 6 g/L, glycerine 18 g/L, sal epsom 2.3 g/L, potassium primary phosphate 1.7 g/L, dipotassium hydrogen phosphate 2.6 g/L, sodium-chlor 10 g/L regulate pH=7.2, add defoamer 0.5 g/L, sterilize 30 minutes for 121 ℃.Initial loading liquid measure 5L.
Supplemented medium:
Peptone is 60 g/L, and yeast powder is 30 g/L, and glycerine is 432 g/L, sterilizes 30 minutes for 121 ℃.
Below pass through the contrast of several different fermentations production methods of Hexose phosphate dehydrogenase, production method of the present invention is described further.
Embodiment 1
Present embodiment provides the method that does not adopt flow feeding technology malaga in next life glucocorticoid dehydrogenase in a kind of fermenting process, and detailed process is as follows:
The recombination bacillus coli seed that the glycerine pipe is preserved after 15 hours, inserts fermentor tank by 2% inoculum size through the seed culture medium activation.The fermentor tank parameter arranges: 37 ℃ of temperature, and initial air flow 5L/min, initial stirring velocity 200rpm, initial dissolved oxygen (DO) is 100%.By regulating mixing speed and air flow control fermenting process dissolved oxygen level between 30%-40%, control pH=7.2 ± 0.1 with ammoniacal liquor.Fermented about 3 hours, and worked as OD
600
Reach at 4 o'clock, begin to be cooled to 28 ℃, and add the IPTG account for fermented liquid total amount 0.03%, continue to cultivate and went up to 100% to dissolved oxygen in 12 hours, the substratum nutritive ingredient exhausts secondary fermentation and finishes.
Adopt this zymotechnique, fermented at the end in 15 hours, cell concentration is (with cell optical density(OD) OD
600
Expression) reach 15, Hexose phosphate dehydrogenase output can reach 1.6g/L.
Embodiment 2
Present embodiment provides the method that adopts constant speed flow feeding technology malaga in next life glucocorticoid dehydrogenase in a kind of fermenting process, and detailed process is as follows:
The recombination bacillus coli seed that the glycerine pipe is preserved after 15 hours, inserts fermentor tank by 2% inoculum size through the seed culture medium activation.The fermentor tank parameter arranges: 37 ℃ of temperature, and initial air flow 5L/min, initial stirring velocity 200rpm, initial dissolved oxygen (DO) is 100%.By regulating mixing speed and air flow control fermenting process dissolved oxygen level between 30%-40%, control pH=7.2 ± 0.1 with ammoniacal liquor.Fermented about 3 hours, and worked as OD
600
Reach at 4 o'clock, beginning is added supplemented medium with the flow acceleration of 40 constant g/l/h in fermented liquid.About 14 hours, OD
600
Reach at 20 o'clock, begin to be cooled to 28 ℃, and add the IPTG that accounts for fermented liquid total amount 0.03%, keep the feed supplement speed of 40 g/l/h to continue to cultivate secondary fermentation in 14 hours end.It is 600rpm that fermenting process stirs maximum speed, and the air flow maximum is to 20L/min, and dissolved oxygen can be controlled between 30%-40% always.
Adopt this zymotechnique, fermented at the end in 28 hours, cell concentration is (with cell optical density(OD) OD
600
Expression) reach 68, Hexose phosphate dehydrogenase output can reach 4.8g/L.
Embodiment 3
Present embodiment provides the method that adopts gradient flow feeding technology malaga in next life glucocorticoid dehydrogenase of the present invention in a kind of fermenting process, and detailed process is as follows:
The recombination bacillus coli seed that the glycerine pipe is preserved after 15 hours, inserts fermentor tank by 2% inoculum size through the seed culture medium activation.The fermentor tank parameter arranges: 37 ℃ of temperature, and initial air flow 5L/min, initial stirring velocity 200rpm, initial dissolved oxygen (DO) is 100%.By regulating mixing speed and air flow control fermenting process dissolved oxygen level between 30%-40%, control pH=7.2 ± 0.1 with ammoniacal liquor.Fermented about 3 hours, and worked as OD
600
Reach at 4 o'clock, beginning is added supplemented medium with the flow acceleration of 8 g/l/h in fermented liquid; About 5 hours, work as OD
600
Reach at 8 o'clock, adjusting feed supplement speed is 16g/l/h; About 7 hours, work as OD
600
Reach at 12 o'clock, adjusting feed supplement speed is 24 g/l/h; 8.5 about hour, work as OD
600
Reach at 16 o'clock, adjusting feed supplement speed is 32 g/l/h; About 10 hours, work as OD
600
Reach at 20 o'clock, adjusting feed supplement speed is 40 g/l/h, begins to be cooled to 28 ℃ simultaneously, and adds the IPTG that accounts for fermented liquid total amount 0.03%, keeps the feed supplement speed of 40 g/l/h to continue to cultivate secondary fermentation in 18 hours end.It is 600rpm that fermenting process stirs maximum speed, and the air flow maximum is to 20L/min, and dissolved oxygen can be controlled between 30%-40% always.
Adopt this zymotechnique, fermented at the end in 28 hours, cell concentration is (with cell optical density(OD) OD
600
Expression) reach 95, Hexose phosphate dehydrogenase output can reach 6.3g/L.
More than the present invention has been done detailed description; its purpose is to allow the personage that is familiar with this art can understand content of the present invention and is implemented; can not limit protection scope of the present invention with this; the equivalence that all spirit according to the present invention are done changes or modifies, and all should be encompassed in protection scope of the present invention
Claims (2)
1. the production method of a Hexose phosphate dehydrogenase, it is characterized in that: described production method is that the recombination bacillus coli of first construction expression Hexose phosphate dehydrogenase and it is seeded in the initial medium ferments, in the fermented liquid that comprises described recombination bacillus coli, add supplemented medium by the flow feeding technology in the fermenting process, when fermentation parameter reaches setting threshold, in fermented liquid, add inductor again, and continue the fermentation certain hour to fermentation ends, from the culture of fermented liquid, obtain Hexose phosphate dehydrogenase at last; Wherein, during flow feeding, gradient increases progressively the flow acceleration of supplemented medium along with the prolongation of fermentation time, remains unchanged after adding inductor;
Described production method may further comprise the steps:
(1), the preparatory stage: make up genetic engineering bacterium, preparation seed culture medium, initial medium and supplemented medium; Genetic engineering bacterium is activated in described seed culture medium, and the seed liquor that will obtain after will activating then is inoculated in the initial medium ferments, by volume, and seed liquor/initial medium during inoculation=1% ~ 4%; Described genetic engineering bacterium is for expressing the recombination bacillus coli of Hexose phosphate dehydrogenase;
Described seed culture medium comprises peptone 10g/L, yeast powder 5g/L and NaCl 10g/L, and the pH value is 7.0;
Described initial medium comprises peptone 6 ~ 15g/L, yeast powder 5 ~ 10g/L, glycerine 6 ~ 18g/L, sal epsom 1 ~ 4g/L, phosphoric acid salt 4 ~ 8g/L and sodium-chlor 5 ~ 12g/L; Described supplemented medium comprises peptone, yeast powder and glycerine, and peptone by mass: yeast powder: glycerine=2:1:14 ~ 15;
(2), fermenting process: when the cell optical density(OD) in the fermented liquid reaches 4,8,12,16,20 successively along with the carrying out of fermenting, in fermented liquid, replenish supplemented medium by the flow feeding technology, and the flow acceleration of supplemented medium corresponds to 0 ~ 10g/L/h, 10 ~ 20g/L/h, 20 ~ 30g/L/h, 30 ~ 40g/L/h, 40 ~ 50g/L/h successively in batches; When cell optical density(OD)＞20, in fermented liquid, add inductor, and the adding quality of inductor in fermented liquid accounts for 0.01% ~ 0.2% of fermented liquid total amount; Under the constant situation of the flow acceleration that keeps supplemented medium, continue fermentation 16 ~ 20 hours then;
In the fermenting process, dissolved oxygen of fermentation liquid maintains between 30% ~ 40%, and fermented liquid pH value is controlled between 6.8 ~ 7.3;
Before adding inductor, leavening temperature is controlled at 37 ℃; Leavening temperature control behind the interpolation inductor is at 28 ℃;
(3), from the culture of step (2) gained fermented liquid, obtain Hexose phosphate dehydrogenase.
2. the production method of Hexose phosphate dehydrogenase according to claim 1, it is characterized in that: described inductor is sec.-propyl-β-D-sulfo-galactopyranoside.
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|---|---|---|---|
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| CN108486027A (en) * | 2018-04-04 | 2018-09-04 | 江南大学 | A kind of FAD is the production of the glucose dehydrogenase of prothetic group, purification process |
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| CN102936585B (en) * | 2012-12-10 | 2014-11-26 | 江南大学 | Method for producing glucose oxidase via fermentation |
| CN108949662A (en) * | 2018-08-10 | 2018-12-07 | 固安鼎泰海规生物科技有限公司 | A kind of Escherichia coli fermentation culture medium for gymnoplasm grain pSFVK1-NMM production |
| CN110093327A (en) * | 2019-06-11 | 2019-08-06 | 安徽国星生物化学有限公司 | A kind of L-glufosinate-ammonium produces the fermentation process of double reduced form complex enzymes |
| CN110777100B (en) * | 2019-11-29 | 2022-08-26 | 宁波酶赛生物工程有限公司 | Escherichia coli fermentation method |
| CN114107355B (en) * | 2021-12-03 | 2024-04-23 | 浙江清肽生物科技有限公司 | Fermentation method of engineering bacteria for efficiently expressing glucose dehydrogenase and application of fermentation method |
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| AR005035A1 (en) * | 1995-12-11 | 1999-04-07 | Merck Patent Ges Mit Beschränkter Haftung | PROCEDURE TO PREPARE RECOMBINANT PROTEINS IN E. COLI, BY FERMENTATION WITH GREAT CONCENTRATION OF CELLS. |
| CN101613672A (en) * | 2009-08-04 | 2009-12-30 | 南京工业大学 | A kind of recombination bacillus coli and construction process thereof of asymmetric conversion preparation (S)-4-chloro-ethyl 3-hydroxybutanoate |
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