CN105802937B - The preparation method of high temperature resistant lipase - Google Patents
The preparation method of high temperature resistant lipase Download PDFInfo
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- 108090001060 Lipase Proteins 0.000 title claims abstract description 56
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- 238000002360 preparation method Methods 0.000 title claims abstract description 35
- 238000000855 fermentation Methods 0.000 claims abstract description 26
- 230000004151 fermentation Effects 0.000 claims abstract description 26
- 241000894006 Bacteria Species 0.000 claims abstract description 24
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- 238000010899 nucleation Methods 0.000 claims abstract description 7
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- 238000012545 processing Methods 0.000 claims abstract description 3
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- 229940062042 oxygen 50 % Drugs 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 2
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- 125000003535 D-glucopyranosyl group Chemical group [H]OC([H])([H])[C@@]1([H])OC([H])(*)[C@]([H])(O[H])[C@@]([H])(O[H])[C@]1([H])O[H] 0.000 description 1
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- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
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- 239000000463 material Substances 0.000 description 1
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- 238000003786 synthesis reaction Methods 0.000 description 1
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- 230000014616 translation Effects 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C12N15/09—Recombinant DNA-technology
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- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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Abstract
The invention discloses a kind of preparation methods of high temperature resistant lipase, comprising the following steps: will from bacillus subtilis (Bacillus subtilis) high temperature resistant lipase gene be cloned into Pichia pastoris (Pichia pastoris) in；Pichia pastoris is activated again, the Pichia pastoris single colonie after activation processing is then inoculated in progress shaking flask culture in shaking flask；The bacterium solution of shaking flask culture is moved in seeding tank and is cultivated；The bacterium solution of seed tank culture is moved to and carries out fermented and cultured in fermentor；Lipase synergist is finally added in fermentation liquid, arrives the lipase after being adsorbed, being dried.The features such as lipase that preparation method of the present invention obtains, is resistant to higher temperature, wider pH sphere of action and tolerance good to organic solvent, expands the application field of lipase, solves the problems, such as in feed granulating lipase not heatproof.
Description
Technical field
The present invention relates to fermentation arts more particularly to a kind of preparation methods of high temperature resistant lipase.
Background technique
Lipase (triacylglycerol lipase EC3.1.1.3) is a kind of hydrolase being widely present, and can be urged
Change the fracture of triglycerides key.Diversity because of its catalysis and the superiority as a kind of biocatalyst are widely applied
In fields such as food, process hides, feed, washing, oil and fat chemicals.Lipase is applied to have in animal-breeding as feed addictive
Wide application prospect, first lipase can eliminate the lipid anti-nutritional factors in vegetality feedstuff, while can be more
Mend the deficiency of lipase in poult cub alimentary canal.But in the lipase product for having realized industrialization production, pH stability
Poor to the tolerance of acid mostly within the scope of neutral meta-alkali, the kind for being suitable for feed is seldom.
The preparation method of lipase mainly has extraction method and microbe fermentation method.The source of lipase is mainly plant, animal
And microorganism.Such as the seed of oil crops in plant, if the pancreas of higher mammal and adipose tissue are all extraction methods in animal
The source of lipase is extracted, extraction method resource is limited, complex process, low output；It is not affected by environment for microbe fermentation method, money
Source is abundant, and the producing enzyme period is short, and product is more simple and at low cost, is easily managed in production, it is easy to accomplish industrialized production, but mesh
The heat resistance of the preceding lipase using microbe fermentation method preparation is all poor.
Bacillus is the important member of yielding lipase in microorganism, and produced lipase is higher with being resistant to
The features such as temperature, wider pH sphere of action and tolerance good to organic solvent, but under field conditions (factors), the enzyme of the bacterium
Low output is difficult to meet the requirement of industrialized production using conventional breeding methods.If a kind of new use fat can be studied
The preparation method of enzyme, had not only overcome the lipase non-refractory of microbe fermentation method preparation in the prior art, but also can guarantee this
Preparation method industrialized production is necessary.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art, provide a kind of preparation of high temperature resistant lipase
Method.
In order to solve the above technical problems, technical solution proposed by the present invention are as follows:
A kind of preparation method of high temperature resistant lipase, comprising the following steps:
(1) it will be cloned into from the high temperature resistant lipase gene of bacillus subtilis (Bacillus subtilis) complete
In red yeast (Pichia pastoris)；
(2) to through step (1), treated, and Pichia pastoris is activated, then by the Pichia pastoris after activation processing
Single colonie is inoculated in progress shaking flask culture in shaking flask；It is activated using the culture of YPD slant medium until growing single colonie；
(3) bacterium solution of shaking flask culture is moved in seeding tank and is cultivated；
(4) bacterium solution of seed tank culture is moved to and carries out fermented and cultured in fermentor；
(5) lipase synergist is added in the fermentation liquid that step (4) obtains, arrives the rouge after being adsorbed, being dried
Fat enzyme.
Above-mentioned preparation method, it is preferred that the fermented and cultured include the thalline quantity multiplicative stage, the thallus hungry stage and
Methanol solution induction period.
Above-mentioned preparation method, it is preferred that the operating procedure of the thalline quantity multiplicative stage includes: by seed tank culture
Bacterium solution be seeded to fermentor after, control cultivation temperature is 30 DEG C ± 0.5 DEG C, and adjusts pH to 5.0 ± 0.1；Control fermentation initial stage
Revolving speed in fermentor is 100-120rpm, ventilation quantity 1:(1.8-2.2) vvm；As fermentation thalli increases, adjust revolving speed and
Ventilation quantity controls the dissolved oxygen in fermentor and is greater than 25%；After culture 14-16 hours, when the dissolved oxygen in fermentor is gone back up to rapidly
When 95% or more, addition concentration is 30-35% Glucose Liquid, control in fermentation tank culture medium sugared concentration always 0.5% with
Under, stop mending sugar when thallus weight in wet base reaches 160 ± 10g/L.
Above-mentioned preparation method, it is preferred that after the thallus hungry stage is Glucose Liquid feed supplement, in fermentor
Dissolved oxygen from 25% rapidly rise to 95% or more when, maintain starvation 10-15 minutes.
Above-mentioned preparation method, it is preferred that when bacterium solution pH value when observation starvation has the tendency that rising, start stream and add
Methanol solution induction: first added with 3.0 ± 0.2ml/L/h speed (mending 3.0 ± 0.2ml of methanol in fermentation liquid per liter per hour) stream
Methanol solution 2 hours, the dissolved oxygen in fermentor was controlled 50% or more；The flow acceleration of methanol solution, control are slowly improved again
Dissolved oxygen in fermentor is 10% or more, and wherein the flow acceleration maximum of solution is no more than 10.0 ± 0.2ml/L/h.
Above-mentioned preparation method, it is preferred that methanol solution induction time terminates fermentation after reaching 110-120h.
Above-mentioned preparation method, it is preferred that the seed tank culture is divided into first class seed pot culture and the training of secondary seed tank
It supports；The first class seed pot incubation refers to that the bacterium solution that will be cultivated in shaking flask moves to the first class seed pot using YPD as culture medium
Middle culture, inoculum concentration 1.5-2%, mixing speed 200-300rpm；Cultivation temperature is 30 DEG C ± 0.5 DEG C, ventilation quantity 1:
(1-1.2)vvm；Incubation time is 20-24 hours；The secondary seed tank culture refer to the thallus by first class seed pot culture with
10% inoculum concentration culture transferring is cultivated to using BSM to expand in the secondary seed tank of culture medium, and secondary seed tank culture is constant temperature training
It supports, cultivation temperature is 30 DEG C ± 0.5 DEG C, mixing speed 180-200rpm, and incubation time is 15-18 hours, ventilation quantity 1:(1-
1.2)vvm；The pH of bacterium solution is 5.0 ± 0.1 in the secondary seed tank incubation.
Above-mentioned preparation method, it is preferred that the culture medium that the shaking flask cultivation stage uses for YPD culture medium, culture
Temperature is 30 DEG C ± 0.5 DEG C, and the time of culture is 24-32 hours, the revolving speed 200-220rpm of incubation；
Above-mentioned preparation method, it is preferred that in the step (5), the lipase synergist is mainly by polyvinylamine, ring
Dextrin and cornstarch composition；Wherein the quality of the polyvinylamine and fermentating liquid volume ratio are 0.1% ± 0.02%；Ring paste
The addition of essence and the ratio of fermentating liquid volume are 1% ± 0.1%；The ratio unit is Kg/L.
Above-mentioned preparation method, it is preferred that in the step (5), dry process is using spray drying, dry temperature
No more than 100 DEG C；Finally obtained lipase product moisture is no more than 10%.
Pichia pastoris yeast is an eukaryon efficient expression system of rising in recent years, has the advantages that many uniquenesses,
It has rapidly developed as one of the main expression system for being widely used in recombinant protein production in molecular biology field.It has with
Lower advantage: (1), can be with accurate expression purpose foreign protein with the promoter of accuracy controlling；(2) expression efficiency is high, expression
Foreign protein can account for summary table reach albumen 90% or more, be conducive to isolating and purifying for destination protein；(3) in simple synthesis culture
High Density Cultivation can be achieved in base.
The present invention clones the gene of producing bacillus subtilis thermostable lipase, constructs and produces high temperature resistant lipase
Genetic engineering bacterium；Then it will be expressed in Pichia pastoris from the high temperature resistant lipase gene of bacillus, realize resistance to height
The industrialized production of warm lipase.
Cyclodextrin (Cyclodextrin, abbreviation CD) is a series of general name of cyclic oligosaccharides, usually contains 6~12 D-
Glucopyranose units.Each D ()-glucopyranose for constituting cyclodextrin molecular is chair conformation.Each glucose unit is equal
Cyclization is combined with Isosorbide-5-Nitrae-glycosidic bond.Since the glycosidic bond of connection glucose unit cannot rotate freely, cyclodextrin is not cylindric
Molecule but the annulus tapered slightly.The primary hydroxyl of its cyclodextrin defines the osculum of taper, and its secondary hydroxyl defines cone
The big mouth of shape.The present invention special construction hydrophilic and inner cavity (Cavity) hydrophobic using the outer rim (Rim) of cyclodextrin, in fermentation liquid
Middle addition cyclodextrin can play a protective role to zymoprotein；Polyvinylamine is additionally added in fermentation liquid simultaneously, it and cyclodextrin
Between there is synergic catalytic effect, the synergistic effect of two kinds of substances can further improve heat resistance, the stability of lipase.
Compared with the prior art, the advantages of the present invention are as follows:
(1) gene source for the high temperature resistant lipase that the present invention obtains is resistant to higher temperature in bacillus subtilis, compared with
The features such as wide pH sphere of action and tolerance good to organic solvent, the application field of lipase is expanded, solves feeding
Material granulation in lipase not heatproof the problem of.
(2) lipase preparation method of the invention, is added to the synergist of lipase, further increases the heat-resisting of lipase
Property, stability.
(3) thallus during preparation method of the present invention in lipase fermentation liquid can be used as animal and fowl fodder, be a kind of fine
Single-cell protein feed；Thus preparation method of the invention realizes no pollution substantially, zero-emission, and the rate of recovery is high.
(4) the high temperature resistant lipase that the present invention obtains, 85 DEG C of heat preservation 5min enzymatic activitys retain 50% or more, are better than existing city
The commercial enzyme preparation sold can satisfy the requirement of feed high temperature granulating.
Specific embodiment
To facilitate the understanding of the present invention, present invention work more comprehensively, is meticulously described below in conjunction with preferred embodiment,
But the protection scope of the present invention is not limited to the following specific embodiments.
Unless otherwise defined, all technical terms used hereinafter and the normally understood meaning of those skilled in the art
It is identical.Technical term used herein is intended merely to the purpose of description specific embodiment, is not intended to the limitation present invention
Protection scope.
Except there is a special instruction, the various reagents used in the present invention, raw material be can commodity commercially or
Person can the product as made from well known method.
Used culture medium and composition of raw materials in following embodiments are shown in Table 1.
1 culture medium of table and composition of raw materials
Embodiment 1:
A kind of preparation method of high temperature resistant lipase of the invention, comprising the following steps:
(1) gene cloning procedures: Pichia pastoris will be cloned into from the high temperature resistant lipase gene of bacillus subtilis
In, cloning procedure is conventional technical process；
(2) seed preparatory phase: the Pichia pastoris obtained after step (1) is grown through YPD slant medium culture finish it is red
Then saccharomycete single colonie is inoculated in shaking flask (loading amount 100mLYPD culture medium in 500mL shaking flask) and is cultivated, control shaking flask
Revolving speed 220rpm, 30 DEG C of constant-temperature table culture 26h or so, obtain shake-flask seed, weight in wet base 56g/L.
(3) level-one 200L seed tank culture: being placed with 100L YPD culture medium in seeding tank, shakes step (1) is cultured
Bottle seed be inoculated into first class seed pot with 2% inoculum concentration, control mixing speed be 200rpm, 30 DEG C of constant temperature, air quantity 6m3/h
(1:1vvm) cultivates 24 hours, obtains the bacterium solution of weight in wet base 70g/L.
(4) second level 2000L seed tank culture: having 1000L BSM culture medium in seeding tank, first class seed pot is cultured
Bacterium solution culture transferring is to secondary seed tank, inoculum concentration 10%, controls mixing speed 180rpm, and 30 DEG C of constant temperature, air quantity 60m3/ h (1:
1vvm), it cultivates 18 hours, entire incubation controls pH5.0 with 25% ammonium hydroxide, finally obtains the bacterium solution of weight in wet base 100g/L.
(5) fermentation stage:
The bacterium solution that step (4) obtains is seeded in 20 tons of fermentors with 10% inoculum concentration (has 10 tons of BSM trainings in fermentor
Support base), 30 DEG C of cultivation temperature are controlled, with 25% ammonium hydroxide tune pH to 5.0, initially controls revolving speed 100rpm, ventilation quantity 1200m3/h
(1:2vvm) adjusts revolving speed and ventilation quantity control dissolved oxygen is greater than 25% as thallus increases.Culture 16 hours, dissolved oxygen returns rapidly
Stream plus 30% Glucose Liquid after rising control in culture medium sugared concentration always below 0.5%.
Stop mending sugar when thallus weight in wet base reaches 160g/L, after dissolved oxygen rises rapidly, maintain starvation 15 minutes, observation
Dissolved oxygen rises to close to 100%, when pH value has the tendency that rising, then starts stream plus methanol induction.
Methanol solution (containing PTM112ml/L) is first added 2 hours with 3.0ml/L/h speed stream, control dissolved oxygen 50% or more,
As thallus gradually adapts to methanol environment, then it is slowly increased methanol flow, max-flow adds methanol speed to be 10.0ml/L/h, control
10% or more dissolved oxygen.The entire methanol induction time is 120h, and the final thallus vigor detected in fermentation liquid is 12280U/mL (state
The measurement of mark method), then terminate to ferment, putting tank volume is 11500L.
(6) 11.5kg polyvinylamine, 115kg cyclodextrin are added in the fermentation liquid for obtaining step (5), are stirred
Afterwards, it then adds 870kg cornstarch and is stirred evenly as carrier, be spray-dried in the case where being no more than 100 DEG C, control moisture exists
10% hereinafter, obtain lipase finished product 2564kg.
(7) heat resistance measures:
1 gram of finished product lipase is weighed, is accurate to 0.0002 gram, it is (commercially available after 220000 times of dilution according to National Standard Method of Determination
Sample dilutes 100000 times), it 40 DEG C, reacts 15 minutes, measurement enzyme activity is 44062.4U/g, then 1.2ml the sample solution to be tested is taken to fill respectively
It in 1.5ml centrifuge tube, places it in kickboard, is respectively put into 65 DEG C, 75 DEG C, 85 DEG C, 95 DEG C of temperature water baths, at once timing 5
Minute, heat treatment, which finishes to take out at once, is put into tap water that cooling is spare, the enzyme activity initial using product as compareing (100%),
Opposite enzyme activity is calculated, 85 DEG C of heat preservation 5min enzymatic activitys retain 55%, the results are shown in Table 2:
2 lipase temperature tolerance results of comparison table of table
Embodiment 2:
A kind of preparation method of high temperature resistant lipase of the invention, comprising the following steps:
(1) gene cloning procedures: Pichia pastoris will be cloned into from the high temperature resistant lipase gene of bacillus subtilis
In, cloning procedure is conventional technical process；
(2) seed preparatory phase: the Pichia pastoris obtained after step (1) is grown through YPD slant medium culture finish it is red
Then saccharomycete single colonie is inoculated in shaking flask (loading amount 100mLYPD culture medium in 500mL shaking flask) and is cultivated, control shaking flask
Revolving speed 200rpm, 30 DEG C of constant-temperature table culture 31h or so, obtain shake-flask seed, weight in wet base 59g/L.
(3) level-one 200L seed tank culture: being placed with 100L YPD culture medium in seeding tank, shakes step (1) is cultured
Bottle seed be inoculated into first class seed pot with 2% inoculum concentration, control mixing speed be 260rpm, 30 DEG C of constant temperature, air quantity 7m3/h
(1:1.16vvm) cultivates 21 hours, obtains the bacterium solution of weight in wet base 80g/L.
(4) second level 2000L seed tank culture: being placed with 1000L BSM culture medium in seeding tank, first class seed pot culture is good
Strain culture transferring to secondary seed tank, inoculum concentration 10% controls mixing speed 200rpm, 30 DEG C of constant temperature, air quantity 70m3/ h (1:
1.16vvm), it cultivates 15 hours, entire incubation controls pH5.0 with 25% ammonium hydroxide, finally obtains the bacterium solution of weight in wet base 116g/L.
(5) fermentation stage:
Bacterium solution that step (3) obtains is seeded in 20 tons of fermentors with 10% inoculum concentration to (fermentor there are 10 tons of BSM cultures
Base), 30 DEG C of cultivation temperature are controlled, with 25% ammonium hydroxide tune pH to 5.0, initially controls revolving speed 110rpm, ventilation quantity 1300m3/ h (1:
2.16vvm), as thallus increases, revolving speed and ventilation quantity are adjusted, control dissolved oxygen is greater than 25%.Culture 14 hours, dissolved oxygen returns rapidly
Stream plus 30% Glucose Liquid after rising control in culture medium sugared concentration always below 0.5%.
Stop mending sugar when thallus weight in wet base reaches 160g/L, after dissolved oxygen rises rapidly, maintain starvation 12 minutes, observation
Dissolved oxygen rises to 98%, when pH value has the tendency that rising, then starts stream plus methanol induction.
Methanol solution (containing PTM112ml/L) is first added 2 hours with 3.0ml/L/h speed stream, control dissolved oxygen 50% or more,
As thallus gradually adapts to methanol environment, then it is slowly increased methanol flow, max-flow adds methanol speed to be 10.0ml/L/h, control
10% or more dissolved oxygen.The entire methanol induction time is 110h, and the final thallus vigor detected in fermentation liquid is 13459U/mL (state
The measurement of mark method), then terminate to ferment, putting tank volume is 11000L.
(6) 11kg polyvinylamine, 110kg cyclodextrin are added in the fermentation liquid for obtaining step (5), are stirred
Afterwards, it then adds 830kg cornstarch and is stirred evenly as carrier, be spray-dried in the case where being no more than 100 DEG C, control moisture exists
10% hereinafter, obtain lipase finished product 2493kg.
(7) heat resistance measures:
1 gram of finished product lipase is weighed, is accurate to 0.0002 gram, it is (commercially available after 240000 times of dilution according to National Standard Method of Determination
Sample dilutes 100000 times), it 40 DEG C, reacts 15 minutes, measurement enzyme activity is 48696.4U/g, then 1.2ml the sample solution to be tested is taken to fill respectively
1.5ml centrifuge tube, places it in kickboard, is respectively put into 65 DEG C, 75 DEG C, 85 DEG C, 95 DEG C of temperature water baths, and timing at once 5 divides
Clock, heat treatment, which finishes to take out at once, is put into tap water cooling spare, initial using product enzyme activity as control (100%), meter
Opposite enzyme activity is calculated, 85 DEG C of heat preservation 5min enzymatic activitys retain 54%, the results are shown in Table 3:
3 lipase temperature tolerance results of comparison table of table
Claims (8)
1. a kind of preparation method of high temperature resistant lipase, which comprises the following steps:
(1) will from bacillus subtilis (Bacillus subtilis) high temperature resistant lipase gene be cloned into and finish red ferment
Female (Pichia pastoris) in；
(2) to through step (1), treated, and Pichia pastoris is activated, then by the Pichia pastoris single bacterium after activation processing
It falls and is inoculated in progress shaking flask culture in shaking flask；
(3) bacterium solution of shaking flask culture is moved in seeding tank and is cultivated；
(4) bacterium solution of seed tank culture is moved to and carries out fermented and cultured in fermentor；
(5) lipase synergist is added in the fermentation liquid that step (4) obtains, arrives the fat after being adsorbed, being dried
Enzyme；The lipase synergist is made of polyvinylamine, cyclodextrin and cornstarch；The wherein quality and hair of the polyvinylamine
Zymotic fluid volume ratio is 0.1%；The addition of cyclodextrin and the ratio of fermentating liquid volume are 1%；The ratio unit is Kg/L；It is dry
Process using spray drying, dry temperature be no more than 100 DEG C；Finally obtained lipase product moisture is no more than 10%.
2. preparation method as described in claim 1, which is characterized in that the fermented and cultured include the thalline quantity multiplicative stage,
Thallus starvation stage and methanol solution induction period.
3. preparation method as claimed in claim 2, which is characterized in that the operating procedure packet of the thalline quantity multiplicative stage
Include: after the bacterium solution of seed tank culture is seeded to fermentor, control cultivation temperature be 30 DEG C ± 0.5 DEG C, and adjust pH to 5.0 ±
0.1；Revolving speed in control fermentation primary fermentation tank is 100-120rpm, ventilation quantity 1:(1.8-2.2) vvm；With zymophyte
Body increases, and the dissolved oxygen adjusted in revolving speed and ventilation quantity control fermentor is greater than 25%；After culture 14-16 hours, when in fermentor
When dissolved oxygen goes back up to rapidly 95% or more, addition concentration is 30-35% Glucose Liquid, controls concentration sugared in fermentation tank culture medium and begins
Eventually 0.5% hereinafter, stopping mending sugar when thallus weight in wet base reaches 160 ± 10g/L.
4. preparation method as claimed in claim 2, which is characterized in that the thallus hungry stage refers to that Glucose Liquid feed supplement is complete
Bi Hou is maintained starvation 10-15 minutes when the dissolved oxygen in fermentor rapidly rises to 95% or more from 25%.
5. preparation method as claimed in claim 2, which is characterized in that bacterium solution pH value when observation starvation has becoming for rising
When gesture, starts stream plus methanol solution induction: methanol solution first being added 2 hours with 3.0 ± 0.2ml/L/h speed stream, controls fermentor
In dissolved oxygen 50% or more；The flow acceleration for slowly improving methanol solution again, controls the dissolved oxygen in fermentor 10% or more,
The flow acceleration maximum of middle solution is no more than 10.0 ± 0.2ml/L/h.
6. preparation method as claimed in claim 5, which is characterized in that methanol solution induction time terminates after reaching 110-120h
Fermentation.
7. preparation method as described in claim 1, which is characterized in that the seed tank culture be divided into first class seed pot culture and
Secondary seed tank culture；The first class seed pot incubation refers to that the bacterium solution that will be cultivated in shaking flask is moved to using YPD as culture medium
First class seed pot in cultivate, inoculum concentration 1.5-2%, mixing speed 200-300rpm；Cultivation temperature is 30 DEG C ± 0.5 DEG C,
Ventilation quantity is 1:(1-1.2) vvm；Incubation time is 20-24 hours；The secondary seed tank culture, which refers to, trains first class seed pot
Feeding thallus is cultivated using 10% inoculum concentration culture transferring to using BSM to expand in the secondary seed tank of culture medium, secondary seed tank culture
For constant temperature incubation, cultivation temperature is 30 DEG C ± 0.5 DEG C, mixing speed 180-200rpm, and incubation time is 15-18 hours, ventilation
Amount is 1:(1-1.2) vvm；The pH of bacterium solution is 5.0 ± 0.1 in the secondary seed tank incubation.
8. preparation method as described in claim 1, which is characterized in that the culture medium that the shaking flask culture uses is YPD culture
Base, the temperature of culture are 30 DEG C ± 0.5 DEG C, and the time of culture is 24-32 hours, the revolving speed 200-220rpm of incubation.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004067705A3 (en) * | 2003-01-30 | 2004-12-02 | Council Scient Ind Res | Stable lipase variants |
| CN101601441A (en) * | 2009-07-07 | 2009-12-16 | 广东溢多利生物科技股份有限公司 | Soluble fodder enzyme and manufacture method thereof |
| JP4631025B2 (en) * | 2004-12-22 | 2011-02-16 | 国立大学法人神戸大学 | P. pastoris and method for producing the same |
| CN101988054A (en) * | 2009-08-04 | 2011-03-23 | 上海源耀生物科技有限公司 | Method for preparing cyclodextrin lysozyme inclusion complex, prepared feed additive and application of inclusion complex in compound feed |
| CN102978181A (en) * | 2012-11-29 | 2013-03-20 | 青岛蔚蓝生物集团有限公司 | Lipase and engineering strain of recombinant expression thereof |
| CN103074315A (en) * | 2012-11-01 | 2013-05-01 | 广东溢多利生物科技股份有限公司 | Lipase LIP, gene and application thereof |
| CN103289967A (en) * | 2013-05-07 | 2013-09-11 | 沅江普济生物科技有限公司 | Extraction method for extracting superoxide dismutase from shenzhou grass |
| CN104073510A (en) * | 2013-03-27 | 2014-10-01 | 清华大学 | Method of preparing heatproof lipase and special-purpose expression vector thereof |
| CN104762278A (en) * | 2015-04-23 | 2015-07-08 | 焦作健康元生物制品有限公司 | Method for fermenting lipase |
| CN105132301A (en) * | 2015-10-16 | 2015-12-09 | 义马煤业集团煤生化高科技工程有限公司 | Pichia pastoris for producing methanol protein and lipase at same time and application thereof |
-
2016
- 2016-05-26 CN CN201610361673.9A patent/CN105802937B/en not_active Expired - Fee Related
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004067705A3 (en) * | 2003-01-30 | 2004-12-02 | Council Scient Ind Res | Stable lipase variants |
| JP4631025B2 (en) * | 2004-12-22 | 2011-02-16 | 国立大学法人神戸大学 | P. pastoris and method for producing the same |
| CN101601441A (en) * | 2009-07-07 | 2009-12-16 | 广东溢多利生物科技股份有限公司 | Soluble fodder enzyme and manufacture method thereof |
| CN101988054A (en) * | 2009-08-04 | 2011-03-23 | 上海源耀生物科技有限公司 | Method for preparing cyclodextrin lysozyme inclusion complex, prepared feed additive and application of inclusion complex in compound feed |
| CN103074315A (en) * | 2012-11-01 | 2013-05-01 | 广东溢多利生物科技股份有限公司 | Lipase LIP, gene and application thereof |
| CN102978181A (en) * | 2012-11-29 | 2013-03-20 | 青岛蔚蓝生物集团有限公司 | Lipase and engineering strain of recombinant expression thereof |
| CN104073510A (en) * | 2013-03-27 | 2014-10-01 | 清华大学 | Method of preparing heatproof lipase and special-purpose expression vector thereof |
| CN103289967A (en) * | 2013-05-07 | 2013-09-11 | 沅江普济生物科技有限公司 | Extraction method for extracting superoxide dismutase from shenzhou grass |
| CN104762278A (en) * | 2015-04-23 | 2015-07-08 | 焦作健康元生物制品有限公司 | Method for fermenting lipase |
| CN105132301A (en) * | 2015-10-16 | 2015-12-09 | 义马煤业集团煤生化高科技工程有限公司 | Pichia pastoris for producing methanol protein and lipase at same time and application thereof |
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