CN109486690A - A kind of preparation method improving Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield - Google Patents
A kind of preparation method improving Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield Download PDFInfo
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Abstract
A kind of preparation method improving Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield, it include: the culture of first order seed, the culture of secondary seed, high density fermentation, induce producing enzyme, the extraction of enzyme, enzyme solution spray drying, the acquisition of single-cell methanol protein, the induction producing enzyme stage, it will continue hungry culture 2-3h after the seed culture in high density fermentation stage to dissolved oxygen rebound, into methanol-polyalcohol induction period, the polyalcohol is pentaerythrite, glycerol, trimethylolethane, xylitol, one of sorbierite, polyalcohol MPC is added according to the volume ratio of 0.5-2:1 in methyl alcohol, common stream adds.Speed of agitator is constant, and constant speed stream adds methanol-polyalcohol of the PTM1 containing 12mL/L for flowing plus inducing, and gradient increases methanol-polyhydric alcohol concentration, and 1mL/L/h-6mL/L/h increases 1mL/L/h per hour, controls dissolved oxygen in 10%-20%.The present invention can be improved Pichia pastoris fermenting fat enzyme enzyme activity while increase the yield of Methanol Protein.
Description
Technical field
The present invention relates to new material application technologies, and in particular to a kind of raising Pichia pastoris fermenting fat enzyme enzyme activity and first
The preparation method of alcohol protein yield.
Background technique
Lipase is a kind of ester bond water for mainly hydrolyzing the triglycerides formed by the insoluble long chain fatty acids of glycerol and water
Enzyme is solved, the fields such as food processing, novel biomaterial, biomedicine, chiral drug resolution are widely used in.In bakery product
Have the function of strengthening tendons using lipase, improve structure of dough texture and bread.The butterfat hydrolysis of lipase can change
Kind dairy products quality, can be in enhancing cheese and milk powder flavor, cheese ripening, cream and the esterlysis modification of ice cream etc.；Lipase exists
It is mainly used for aid digestion, reducing blood lipid in medicine company, also can be used for the diseases such as clinical diagnosis piarhemia disease, pancreatitis.
Methanol Protein is the single cell protein produced using industrial methanol as raw material, referred to as second generation single cell protein.It
It is mycoprotein obtained from being grown, bred for main nutrient source with methanol as microorganism.It is single compared with native protein
The crude protein content of cell Methanol Protein will be high than fish meal and soybean, essential amino acid, minerals and Wei Sheng rich in
Element, nutritive value is high, can be used for part instead of fish meal, soybean, bone meal, meat and skimmed milk power and be applied to animal and fowl fodder or
Other chemical fields.Relative to other methods production single cell protein for, Methanol Protein have it is resourceful, production not by
The features such as weather influence, aggregate velocity are fast, quality is stablized.
China's protein feed insufficiency of supply-demand reaches several ten million tons every year, with the raising of living standards of the people and the hair of aquaculture
Exhibition, the imbalance between supply and demand of forage protein are further exacerbated by.Crude protein content in single-cell methanol protein will than fish meal and soybean
Height, essential amino acid rich in, minerals and vitamins, nutritive value is high, is one particularly significant and have development
The product of prospect.And product present in current Methanol Protein production is single, zymotechnique is outmoded, at high cost and benefit is low
Problem；For lipase as biological enzyme formulation, reaction condition is mild, and high catalytic efficiency is widely used in food, fermentation, process hides, doctor
The industry such as medicine, daily use chemicals, feed, still, the fat of output during the current production lipase of combined ferment simultaneously and Methanol Protein
Enzyme enzyme activity is low and the Methanol Protein low output of output.
Summary of the invention
(1) the technical issues of solving
In view of the deficiencies of the prior art, the object of the present invention is to provide a kind of raising Pichia pastoris fermenting fat enzyme enzyme activity and first
The preparation method of alcohol protein yield can be improved Pichia pastoris fermenting fat enzyme enzyme activity while increase the yield of Methanol Protein.
(2) technical solution
To achieve the above object, the present invention is achieved by the following technical programs: a kind of raising Pichia pastoris fermenting fat enzyme
The preparation method of enzyme activity and Methanol Protein yield, a kind of raising Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield
Preparation method, comprising:
The culture of first order seed；
The culture of secondary seed；
High density fermentation；
Induce producing enzyme；
The extraction of enzyme；
Enzyme solution spray drying；
The acquisition of single-cell methanol protein；
The induction producing enzyme stage will continue hungry culture 2-3h after the seed culture in high density fermentation stage to dissolved oxygen rebound,
Into methanol-polyalcohol induction period, polyalcohol MPC is added according to the volume ratio of 0.5-2:1 in methyl alcohol, common stream adds.It stirs
Mix that revolving speed is constant, constant speed stream adds methanol-polyalcohol containing 12 mL/L PTM1 for flowing plus inducing, and it is more that gradient increases methanol-
First determining alcohol, 1 mL/L/h-6 mL/L/h increase by 1 mL/L/h per hour, control dissolved oxygen in 10%-20%.
Preferably, first order seed cultivation stage: Pichia pastoris is accessed on primary-seed medium, in 30 DEG C of shaking tables
Shaken cultivation 22-26 hours, shaking speed 200-300rpm, obtain level-one shake-flask seed.Wherein primary-seed medium is adopted
With YPD culture medium, YPD culture medium (g/L): yeast powder 10, peptone 20, glucose 20 (separately go out)；Solid medium contains 20
G/L agar powder.
Preferably, secondary seed cultivation stage: after the trace element solution of 4mL is added in every liter of minimal medium, then
PH value is adjusted to 5.0, level-one shake-flask seed is inoculated on the minimal medium after trace element solution is added, in 30-32
DEG C, pH5.0, ventilatory capacity 0.5-1.2v/ (v × min), speed of agitator 400-650rpm, incubation time 22-26h.Wherein
Fermentation medium weight percent (basal salts medium, BSM) (g/L): glycerol 40, K2SO418,
MgSO4·7H2O 14.9, KOH 4.13,85%H3PO426.7 mL/L, CaSO4·2H20.93,4.35 mL/L PTM1 of O is micro-
Secondary element (filtration sterilization)；
Inorganic microelement weight percent PTM1 (g/L): CuSO4·5H2O 6, KI 0.09, MnSO4·H2O 3, H3BO3
0.02, MoNa2O4·2H2O 0.2, CoCl2·6H2O 0.92, ZnCl220, FeSO4·7H2O 65, biotin 0.2,
H2SO4 5.0 mL。
Preferably, the high density fermentation stage:
A seed liquor prepares and inoculation
The recombinant bacterial strain for taking -80 DEG C of glycerol tubes to save, crosses on YPD solid plate, is placed in 30 DEG C of constant incubator cultures
2-3 days to growing single colonie.Picking single colonie is inoculated in 500 mL triangle shake bottles of the fluid nutrient medium of YPD containing 50mL, sets
In 30 DEG C, 220 rpm are cultivated for 24 hours.
High density fed-batch fermentation executes on the 3-L fermentor containing 1200 mL BSM culture mediums, and fermentation parameter is as follows:
30 DEG C of temperature, pH 5.5,2.0 vvm of ventilatory capacity and initial speed 200 rpm.
B batch culture and glycerol feeding
The 150 mL seed liquors of 16 h are cultivated by access fermentation tank culture medium, the automatic ammonium hydroxide that adds controls pH 5.5.
Revolving speed is 100 rpm to 500 rpm per hour.It cultivates (17-27h) after 16 h, 50% (v/v) is carried out in a manner of exponential fed-batch
Glycerol (containing 12 mL L -1PTM1) feed supplement, adjusts 4.0 vvm of ventilatory capacity and 850 rpm of revolving speed.Feed rate is preceding 6 h
The feed rate of the mL/h/L of respectively 20.25,24.3,28.8,34.2,40.8 and 48.6, subsequent 4 h are 45 mL/h/L.
Preferably, it induces the producing enzyme stage: will continue after the fermentation liquid culture in high density fermentation stage to dissolved oxygen rebound hungry
Cultivate 2-3h.Into methanol-polyalcohol induction period, polyalcohol MPC, common stream is added according to the volume ratio of 1:1 in methyl alcohol
Add.Revolving speed is constant, and constant speed stream adds methanol-polyalcohol containing 12 mL/L PTM1 for flowing plus inducing, and it is more that gradient increases methanol-
First determining alcohol, 1 mL/L/h-6 mL/L/h increase by 1 mL/L/h per hour, control dissolved oxygen in 10%-20%.
Preferably, the extraction stage of enzyme:
A, it is separated using the fermentation liquid that tab (s) centrifuge collects the induction producing enzyme stage, tab (s) centrifuge speed
12000rpm, liquid inlet volume 500L/h collect supernatant and heavy phase respectively；
B, heavy phase aggravates 1 times of phase volume of clear water, stirs evenly, is centrifuged again, collects supernatant and heavy phase respectively,
Supernatant merges with the supernatant of above-mentioned a collection step；Ultrafiltration membrane concentration through molecular cut off 5000Ku, obtains lipase
Concentrate is collected into enzyme solution holding vessel, adds fructus hordei germinatus aleuron, fructus hordei germinatus aleuron dosage=lipase concentrate total enzyme activity/meter
Draw solid enzymatic activity；Wherein, lipase concentrate total enzyme activity refers to that the enzymatic activity of unit volume lipase concentrate and lipase are dense
The product of contracting liquid total volume, plan solid enzymatic activity, which refers to, prepares the solid enzymatic activity that production obtains, and stirs evenly, is transported to spraying
It is dried in drying machine, heavy phase is spare.
Preferably, enzyme solution spray-drying stage: adjustable spraying dryer entrance mouth temperature to 135~155 DEG C, leaving air temp
To 60-75 DEG C, adjusting feed rate is 500L/h, and after spray drying, collecting dry powder is lipase.
Preferably, the acquisition stage of single-cell methanol protein: heavy phase, that is, single-cell methanol protein of b collection step is hanged
Clear water is added in liquid, and thallus weight in wet base is 700-750g/L in suspension, is sent into spray dryer, adjustable spraying dryer entrance mouth temperature
For degree to 135~155 DEG C, leaving air temp to 60-75 DEG C, adjusting feed rate is 400-500L/h, is spray-dried, spraying dry
After dry, the dry powder of collection is single-cell methanol protein.
(3) beneficial effect
Compared with prior art, the present invention provides a kind of raising Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield
Preparation method, have it is following the utility model has the advantages that
When adding methanol while polyalcohol MPC is added during inducing producing enzyme in the present invention, can be by biomass from original
300g/L is improved to 500g/L, improves 67%；By the lipase activity in fermentation liquid from 1.5 × 104U/ml improves to 2 ×
104U/ml improves 33%.
Specific embodiment
Technical solution of the present invention is clearly and completely described below with reference to embodiment, it is clear that described embodiment
Only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1
A kind of preparation method improving Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield, comprising:
The culture of first order seed；
The culture of secondary seed；
High density fermentation；
Induce producing enzyme；
The extraction of enzyme；
Enzyme solution spray drying；
The acquisition of single-cell methanol protein；
Wherein the preferred embodiment of the present embodiment is in the induction producing enzyme stage by the fermentation liquid culture in high density fermentation stage to dissolved oxygen
Continue hungry culture 2h after rebound.Into methanol-polyalcohol induction period, which is pentaerythrite, glycerol, trihydroxy methyl
One of ethane, xylitol, sorbierite, are added polyalcohol MPC according to the volume ratio of 1:1 in methyl alcohol, and common stream adds.Fermentation
Liquid speed of agitator is constant, and constant speed stream adds methanol-polyalcohol of the PTM1 containing 12mL/L for flowing plus inducing, and it is more that gradient increases methanol-
First determining alcohol, 2mL/L/h(increase by 1 mL/L/h per hour), dissolved oxygen is controlled 13%.
The lipase activity power result that it is obtained is as follows: during inducing producing enzyme, when adding methanol while being added
Biomass can be improved from original 300g/L to 500g/L, improve 67% by polyalcohol MPC；By the lipase activity in fermentation liquid
Power is from 1.5 × 104U/ml is improved to 2 × 104U/ml improves 33%.
Methanol Protein yield improves to 30g/L from original 18g/L, improves 67%.
Embodiment 2
A kind of preparation method improving Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield, comprising:
The culture of first order seed；
The culture of secondary seed；
High density fermentation；
Induce producing enzyme；
The extraction of enzyme；
Enzyme solution spray drying；
The acquisition of single-cell methanol protein；
Wherein the preferred embodiment of the present embodiment is in the induction producing enzyme stage by the fermentation liquid culture in high density fermentation stage to dissolved oxygen
Continue hungry culture 2h after rebound.Into methanol-polyalcohol induction period, which is pentaerythrite, glycerol, trihydroxy methyl
One of ethane, xylitol, sorbierite, are added polyalcohol MPC according to the volume ratio of 1:1 in methyl alcohol, and common stream adds.Fermentation
Liquid speed of agitator is constant, and constant speed stream adds methanol-polyalcohol of the PTM1 containing 12mL/L for flowing plus inducing, and it is more that gradient increases methanol-
First determining alcohol, 3mL/L/h(increase 1mL/L/h per hour), dissolved oxygen is controlled 15%.
The lipase activity power result that it is obtained is as follows: during inducing producing enzyme, when adding methanol while being added
Biomass can be improved from original 310g/L to 510g/L, improve 64.5% by polyalcohol MPC；By the lipase in fermentation liquid
Vigor is from 1.6 × 104U/ml is improved to 2.1 × 104U/ml improves 31.25%.
Methanol Protein yield improves to 30.6g/L from original 18.6g/L, improves 64.5%.
Embodiment 3
A kind of preparation method improving Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield, comprising:
The culture of first order seed；
The culture of secondary seed；
High density fermentation；
Induce producing enzyme；
The extraction of enzyme；
Enzyme solution spray drying；
The acquisition of single-cell methanol protein；
Wherein the preferred embodiment of the present embodiment is in the induction producing enzyme stage by the fermentation liquid culture in high density fermentation stage to dissolved oxygen
Continue hungry culture 2h after rebound.Into methanol-polyalcohol induction period, which is pentaerythrite, glycerol, trihydroxy methyl
One of ethane, xylitol, sorbierite, are added polyalcohol MPC according to the volume ratio of 1:1 in methyl alcohol, and common stream adds.Fermentation
Liquid speed of agitator is constant, and constant speed stream adds methanol-polyalcohol of the PTM1 containing 12mL/L for flowing plus inducing, and it is more that gradient increases methanol-
First determining alcohol, 4mL/L/h(increase by 1 mL/L/h per hour), dissolved oxygen is controlled 17%.
The lipase activity power result that it is obtained is as follows: during inducing producing enzyme, when adding methanol while being added
Biomass can be improved from original 320g/L to 520g/L, improve 62.5% by polyalcohol MPC；By the lipase in fermentation liquid
Vigor is from 1.5 × 104U/ml is improved to 2 × 104U/ml improves 33%.
Methanol Protein yield improves to 30.6g/L from original 18.6g/L, improves 64.5%.
Embodiment 4
A kind of preparation method improving Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield, comprising:
The culture of first order seed；
The culture of secondary seed；
High density fermentation；
Induce producing enzyme；
The extraction of enzyme；
Enzyme solution spray drying；
The acquisition of single-cell methanol protein；
Wherein the preferred embodiment of the present embodiment is in the induction producing enzyme stage by the fermentation liquid culture in high density fermentation stage to dissolved oxygen
Continue hungry culture 3h after rebound.Into methanol-polyalcohol induction period, which is pentaerythrite, glycerol, trihydroxy methyl
One of ethane, xylitol, sorbierite, are added polyalcohol MPC according to the volume ratio of 1:1 in methyl alcohol, and common stream adds.Fermentation
Liquid speed of agitator is constant, and constant speed stream adds methanol-polyalcohol of the PTM1 containing 12mL/L for flowing plus inducing, and it is more that gradient increases methanol-
First determining alcohol, 4mL/L/h(increase by 1 mL/L/h per hour), dissolved oxygen is controlled 19%.
The lipase activity power result that it is obtained is as follows: during inducing producing enzyme, when adding methanol while being added
Biomass can be improved from original 300g/L to 500g/L, improve 67% by polyalcohol MPC；By the lipase activity in fermentation liquid
Power is from 1.5 × 104U/ml is improved to 2 × 104U/ml improves 33%.
Methanol Protein yield improves to 30g/L from original 18g/L, improves 67%.
Embodiment 5
A kind of preparation method improving Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield, comprising:
The culture of first order seed；
The culture of secondary seed；
High density fermentation；
Induce producing enzyme；
The extraction of enzyme；
Enzyme solution spray drying；
The acquisition of single-cell methanol protein；
Wherein the preferred embodiment of the present embodiment is in the induction producing enzyme stage by the fermentation liquid culture in high density fermentation stage to dissolved oxygen
Continue hungry culture 3h after rebound.Into methanol-polyalcohol induction period, which is pentaerythrite, glycerol, trihydroxy methyl
One of ethane, xylitol, sorbierite, are added polyalcohol MPC according to the volume ratio of 0.5:1 in methyl alcohol, and common stream adds.Hair
Zymotic fluid speed of agitator is constant, and constant speed stream adds methanol-polyalcohol of the PTM1 containing 12mL/L for flowing plus inducing, and gradient increases methanol-
Polyhydric alcohol concentration, 4mL/L/h(increase by 1 mL/L/h per hour), dissolved oxygen is controlled 13%.
The lipase activity power result that it is obtained is as follows: during inducing producing enzyme, when adding methanol while being added
Biomass can be improved from original 300g/L to 450g/L, improve 50% by polyalcohol MPC；By the lipase activity in fermentation liquid
Power is from 1.5 × 104U/ml is improved to 1.7 × 104U/ml improves 13%.
Methanol Protein yield improves to 27g/L from original 18g/L, improves 50%.
Embodiment 6
A kind of preparation method improving Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield, comprising:
The culture of first order seed；
The culture of secondary seed；
High density fermentation；
Induce producing enzyme；
The extraction of enzyme；
Enzyme solution spray drying；
The acquisition of single-cell methanol protein；
Wherein the preferred embodiment of the present embodiment is in the induction producing enzyme stage by the fermentation liquid culture in high density fermentation stage to dissolved oxygen
Continue hungry culture 3h after rebound.Into methanol-polyalcohol induction period, which is pentaerythrite, glycerol, trihydroxy methyl
One of ethane, xylitol, sorbierite, are added polyalcohol MPC according to the volume ratio of 0.5:1 in methyl alcohol, and common stream adds.Hair
Zymotic fluid speed of agitator is constant, and constant speed stream adds methanol-polyalcohol of the PTM1 containing 12mL/L for flowing plus inducing, and gradient increases methanol-
Polyhydric alcohol concentration, 4mL/L/h(increase by 1 mL/L/h per hour), dissolved oxygen is controlled 15%.
The lipase activity power result that it is obtained is as follows: during inducing producing enzyme, when adding methanol while being added
Biomass can be improved from original 300g/L to 450g/L, improve 50% by polyalcohol MPC；By the lipase activity in fermentation liquid
Power is from 1.5 × 104U/ml is improved to 1.7 × 104U/ml improves 13%.
Methanol Protein yield improves to 27g/L from original 18g/L, improves 50%.
Embodiment 7
A kind of preparation method improving Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield, comprising:
The culture of first order seed；
The culture of secondary seed；
High density fermentation；
Induce producing enzyme；
The extraction of enzyme；
Enzyme solution spray drying；
The acquisition of single-cell methanol protein；
Wherein the preferred embodiment of the present embodiment is in the induction producing enzyme stage by the fermentation liquid culture in high density fermentation stage to dissolved oxygen
Continue hungry culture 3h after rebound.Into methanol-polyalcohol induction period, which is pentaerythrite, glycerol, trihydroxy methyl
One of ethane, xylitol, sorbierite, are added polyalcohol MPC according to the volume ratio of 2:1 in methyl alcohol, and common stream adds.Fermentation
Liquid speed of agitator is constant, and constant speed stream adds methanol-polyalcohol of the PTM1 containing 12mL/L for flowing plus inducing, and it is more that gradient increases methanol-
First determining alcohol, 4mL/L/h(increase by 1 mL/L/h per hour), dissolved oxygen is controlled 17%.
The lipase activity power result that it is obtained is as follows: during inducing producing enzyme, when adding methanol while being added
Biomass can be improved from original 300g/L to 480g/L, improve 60% by polyalcohol MPC；By the lipase activity in fermentation liquid
Power is from 1.5 × 104U/ml is improved to 1.55 × 104U/ml improves 3%.
Methanol Protein yield improves to 28.8g/L from original 18g/L, improves 60%.
Embodiment 8
A kind of preparation method improving Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield, comprising:
The culture of first order seed；
The culture of secondary seed；
High density fermentation；
Induce producing enzyme；
The extraction of enzyme；
Enzyme solution spray drying；
The acquisition of single-cell methanol protein；
Wherein the preferred embodiment of the present embodiment is in the induction producing enzyme stage by the fermentation liquid culture in high density fermentation stage to dissolved oxygen
Continue hungry culture 3h after rebound.Into methanol-polyalcohol induction period, which is pentaerythrite, glycerol, trihydroxy methyl
One of ethane, xylitol, sorbierite, are added polyalcohol MPC according to the volume ratio of 2:1 in methyl alcohol, and common stream adds.Fermentation
Liquid speed of agitator is constant, and constant speed stream adds methanol-polyalcohol of the PTM1 containing 12mL/L for flowing plus inducing, and it is more that gradient increases methanol-
First determining alcohol, 4mL/L/h(increase by 1 mL/L/h per hour), dissolved oxygen is controlled 19%.
The lipase activity power result that it is obtained is as follows: during inducing producing enzyme, when adding methanol while being added
Biomass can be improved from original 300g/L to 480g/L, improve 60% by polyalcohol MPC；By the lipase activity in fermentation liquid
Power is from 1.5 × 104U/ml is improved to 1.55 × 104U/ml improves 3%.
Methanol Protein yield improves to 28.8g/L from original 18g/L, improves 60%.
In conclusion add ratio and control relevant parameter can for change methanol-polyalcohol MPC stream that the present embodiment is enumerated
The yield for significantly improving fermenting fat enzyme enzyme activity and Methanol Protein, reaches the purpose of the present invention.
Although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, it is possible to understand that
These embodiments can be carried out with a variety of variations, modification, replacement without departing from the principles and spirit of the present invention and become
Type, the scope of the present invention is defined by the appended.
Claims (8)
1. a kind of preparation method for improving Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield, comprising:
The culture of first order seed；
The culture of secondary seed；
High density fermentation；
Induce producing enzyme；
The extraction of enzyme；
Enzyme solution spray drying；
The acquisition of single-cell methanol protein；
It is characterized in that, the induction producing enzyme stage, will continue hungry after the seed culture in high density fermentation stage to dissolved oxygen rebound
Culture 2-3h is starved, into methanol-polyalcohol induction period, which is pentaerythrite, glycerol, trimethylolethane, xylose
One of alcohol, sorbierite, are added polyalcohol MPC according to the volume ratio of 0.5-2:1 in methyl alcohol, and common stream adds.Speed of agitator
Constant, constant speed stream adds methanol-polyalcohol containing 12 mL/L PTM1 for flowing plus inducing, and it is polynary pure and strong that gradient increases methanol-
Degree, 1 mL/L/h-6 mL/L/h increase by 1 mL/L/h per hour, control dissolved oxygen in 10%-20%.
2. a kind of preparation for improving Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield according to claim 1
Method, which is characterized in that
First order seed cultivation stage: Pichia pastoris is accessed on primary-seed medium, the shaken cultivation 22- in 30 DEG C of shaking tables
26 hours, shaking speed 200-300rpm, obtain level-one shake-flask seed.
3. a kind of preparation for improving Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield according to claim 1
Method, which is characterized in that
Secondary seed cultivation stage: it after the trace element solution of 4mL is added in every liter of minimal medium, then adjusts pH value and arrives
5.0, level-one shake-flask seed is inoculated on the minimal medium after trace element solution is added, at 30-32 DEG C, pH5.0, is led to
Tolerance 0.5-1.2v/ (v × min), speed of agitator 400-650rpm, incubation time 22-26h.
4. a kind of preparation for improving Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield according to claim 1
Method, which is characterized in that
The high density fermentation stage:
A seed liquor prepares and inoculation
The recombinant bacterial strain for taking -80 DEG C of glycerol tubes to save, crosses on YPD solid plate, is placed in 30 DEG C of constant incubator cultures
2-3 days to growing single colonie.Picking single colonie is inoculated in 500 mL triangle shake bottles of the fluid nutrient medium of YPD containing 50mL, sets
In 30 DEG C, 220 rpm are cultivated for 24 hours.
High density fed-batch fermentation executes on the 3-L fermentor containing 1200 mL BSM culture mediums, and fermentation parameter is as follows:
30 DEG C of temperature, pH 5.5,2.0 vvm of ventilatory capacity and initial speed 200 rpm.
B batch culture and glycerol feeding
The 150 mL seed liquors of 16 h are cultivated by access fermentation tank culture medium, the automatic ammonium hydroxide that adds controls pH 5.5.
Revolving speed is 100 rpm to 500 rpm per hour.It cultivates (17-27h) after 16 h, 50% (v/v) is carried out in a manner of exponential fed-batch
Glycerol (containing 12 mL/L PTM1) feed supplement, adjusts 4.0 vvm of ventilatory capacity and 850 rpm of revolving speed.Feed rate is preceding 6 h
The feed rate of the mL/h/L of respectively 20.25,24.3,28.8,34.2,40.8 and 48.6, subsequent 4 h are 45 mL/h/
L。
5. a kind of preparation for improving Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield according to claim 1
Method, which is characterized in that
It induces the producing enzyme stage: hungry culture 2-3h will be continued after the fermentation liquid culture in high density fermentation stage to dissolved oxygen rebound.Into
Enter methanol-polyalcohol induction period, polyalcohol MPC is added according to the volume ratio of 1:1 in methyl alcohol, common stream adds.Revolving speed is constant,
Constant speed stream adds methanol-polyalcohol containing 12 mL/L PTM1 for flowing plus inducing, gradient increase methanol-polyhydric alcohol concentration, and 1
ML/L/h-6 mL/L/h increases by 1 mL/L/h per hour, controls dissolved oxygen in 10%-20%.
6. a kind of preparation for improving Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield according to claim 1
Method, which is characterized in that
The extraction stage of enzyme:
A, it is separated using the fermentation liquid that tab (s) centrifuge collects the induction producing enzyme stage, tab (s) centrifuge speed
12000rpm, liquid inlet volume 500L/h collect supernatant and heavy phase respectively；
B, heavy phase aggravates 1 times of phase volume of clear water, stirs evenly, is centrifuged again, collects supernatant and heavy phase respectively,
Supernatant merges with the supernatant of above-mentioned a collection step；Ultrafiltration membrane concentration through molecular cut off 5000Ku, obtains lipase
Concentrate is collected into enzyme solution holding vessel, adds fructus hordei germinatus aleuron, fructus hordei germinatus aleuron dosage=lipase concentrate total enzyme activity/meter
Draw solid enzymatic activity；Wherein, lipase concentrate total enzyme activity refers to that the enzymatic activity of unit volume lipase concentrate and lipase are dense
The product of contracting liquid total volume, plan solid enzymatic activity, which refers to, prepares the solid enzymatic activity that production obtains, and stirs evenly, is transported to spraying
It is dried in drying machine, heavy phase is spare.
7. a kind of preparation for improving Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield according to claim 1
Method, which is characterized in that
Enzyme solution spray-drying stage: adjustable spraying dryer entrance mouth temperature to 135~155 DEG C, leaving air temp to 60-75 DEG C,
Adjusting feed rate is 500L/h, and after spray drying, collecting dry powder is lipase.
8. a kind of preparation for improving Pichia pastoris fermenting fat enzyme enzyme activity and Methanol Protein yield according to claim 1
Method, which is characterized in that
The acquisition stage of single-cell methanol protein: by the heavy phase of b collection step, that is, single-cell methanol protein suspension, being added clear water,
Thallus weight in wet base is 700-750g/L in suspension, is sent into spray dryer, adjustable spraying dryer entrance mouth temperature to 135~155
DEG C, leaving air temp to 60-75 DEG C, adjusting feed rate be 400-500L/h, be spray-dried, after spray drying, receive
The dry powder of collection is single-cell methanol protein.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110129289A (en) * | 2019-05-24 | 2019-08-16 | 义马煤业集团煤生化高科技工程有限公司 | The production method of enzyme preparation in a kind of Methanol Protein fermentation liquid |
| 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 |
| CN112725201A (en) * | 2021-01-19 | 2021-04-30 | 武汉新华扬生物股份有限公司 | Liquid submerged fermentation method of pichia pastoris producing acid protease |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040115657A1 (en) * | 2000-09-01 | 2004-06-17 | Mattheos Koffas | Methanotrophic carbon metabolism pathway genes and enzymes |
| CN103045494A (en) * | 2013-01-05 | 2013-04-17 | 义马煤业集团煤生化高科技工程有限公司 | Pichia pastoris for efficiently converting methanol to produce single cell protein and application of pichia pastoris |
| 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 |
| CN105176853A (en) * | 2015-10-16 | 2015-12-23 | 义马煤业集团煤生化高科技工程有限公司 | Pichia pastoris for simultaneously producing methanol protein and xylanase and application of pichia pastoris |
| CN106520587A (en) * | 2016-10-18 | 2017-03-22 | 江南大学 | Recombinant strain producing alkaline polygalacturonate lyase and application thereof |
-
2018
- 2018-12-13 CN CN201811521497.6A patent/CN109486690A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040115657A1 (en) * | 2000-09-01 | 2004-06-17 | Mattheos Koffas | Methanotrophic carbon metabolism pathway genes and enzymes |
| CN103045494A (en) * | 2013-01-05 | 2013-04-17 | 义马煤业集团煤生化高科技工程有限公司 | Pichia pastoris for efficiently converting methanol to produce single cell protein and application of pichia pastoris |
| 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 |
| CN105176853A (en) * | 2015-10-16 | 2015-12-23 | 义马煤业集团煤生化高科技工程有限公司 | Pichia pastoris for simultaneously producing methanol protein and xylanase and application of pichia pastoris |
| CN106520587A (en) * | 2016-10-18 | 2017-03-22 | 江南大学 | Recombinant strain producing alkaline polygalacturonate lyase and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| CATHERINE B MATTHEWS ET AL.: "Development of a general defined medium for Pichia pastoris", 《BIOTECHNOL BIOENG》 * |
| 曹明等: "毕赤酵母甲醇利用及甲醇蛋白发酵条件优化", 《河南科学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110129289A (en) * | 2019-05-24 | 2019-08-16 | 义马煤业集团煤生化高科技工程有限公司 | The production method of enzyme preparation in a kind of Methanol Protein fermentation liquid |
| 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 |
| CN112725201A (en) * | 2021-01-19 | 2021-04-30 | 武汉新华扬生物股份有限公司 | Liquid submerged fermentation method of pichia pastoris producing acid protease |
| CN112725201B (en) * | 2021-01-19 | 2023-05-12 | 武汉新华扬生物股份有限公司 | Liquid submerged fermentation method of pichia pastoris for producing acid protease |
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