CN102559641A - Method for producing beta-1,3-1,4-glucanase through submerged fermentation of recombinant Pichia pastoris liquid - Google Patents

Method for producing beta-1,3-1,4-glucanase through submerged fermentation of recombinant Pichia pastoris liquid Download PDF

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Publication number
CN102559641A
CN102559641A CN2011104485239A CN201110448523A CN102559641A CN 102559641 A CN102559641 A CN 102559641A CN 2011104485239 A CN2011104485239 A CN 2011104485239A CN 201110448523 A CN201110448523 A CN 201110448523A CN 102559641 A CN102559641 A CN 102559641A
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beta
glucanase
liquid
submerged fermentation
pichia pastoris
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刘钦松
李小刚
吴永坤
白杨
敬科举
卢英华
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SHANDONG BOAOKE BIOTECHNOLOGY CO Ltd
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SHANDONG BOAOKE BIOTECHNOLOGY CO Ltd
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Abstract

The invention relates to a method for producing beta-1,3-1,4-glucanase through submerged fermentation of recombinant Pichia pastoris liquid. In the method, recombinant Pichia pastoris is used as a strain, and yeast powder, peptone, yeast nitrogen source, biotin, glycerin, H3PO4, CaSO4.2H2O, K2SO4, MgSO4.7H2O and KOH are used as raw materials. The method comprises conducting liquid submerged fermentation to produce beta-glucanase, and filtering fermentation filtrate through a plate frame to obtain filter residue and beta-glucanase filtrate; conducting ultrafiltration and spray drying for the obtained beta-glucanase filtrate to obtain powdered beta-glucanase product; and drying the filter residue to be used as a feed additive. No waste is produced, and high-yield fermentation of the beta-1,3-1,4-glucanase is achieved. Therefore, the method of the invention is suitable for industrial production.

Description

The recombinant yeast pichia pastoris liquid submerged fermentation is produced β-1,3-1,4-LSD method
Technical field
The invention provides a kind of recombinant yeast pichia pastoris liquid submerged fermentation and produce β-1,3-1, the method for 4-LSD belongs to the fermentation engineering field.
Background technology
Along with the continuous development of China industry, beta-glucanase at food, brewage, the demand in the industry such as weaving constantly increases; Because beta-glucanase has vital role to the beta-glucan in barley, the rye, thus be mainly used in brewing industry in early days abroad, and in the 1970s and 1980s in this field widespread use.China's brewing industry development is swift and violent; China's beer ultimate production occupied the first in the world in continuous 4 years; In the production of beer, states such as the U.S., Japan and Denmark have all adopted beta-glucanase as one of main zymin of brewing industry; Add beta-glucanase during beer saccharification and can decompose beta-glucan gel in the Fructus Hordei Germinatus, reduce wheat juice viscosity, improve wheat juice filtering velocity and yield, help improving beer flavor, be held in the non-biostability of sampling wine; The test of Jiangshan of Zhejiang Province brew-house is thought, when using the Fructus Hordei Germinatus of solubilizing poorly, adds beta-glucanase, and wort filtration speed can accelerate 33%, and wheat juice amount increases by 1.8%, has improved the yield and the plant factor of raw material, has also improved beer quality; In Fructus Hordei Germinatus is made, add high temperature resistant beta-glucanase and can significantly reduce beta-glucan content in the finished product Fructus Hordei Germinatus, make the beta-glucan content of finished product Fructus Hordei Germinatus reach acceptability limit, improved index relevant in the Fructus Hordei Germinatus simultaneously with beta-glucan.
Beta-glucanase has become the key areas that biotechnology is used as the development research of fodder enzyme preparation in fodder industry; Barley is one of main cereal of China, and output is only second to wheat, paddy and corn, occupies the 4th; In recent years, along with mass-producing, modernization that animal and fowl fodder is produced, the grain trough especially consumption of barley feed constantly increases, and this trend makes the demand of beta-glucanase also continue to increase; External is the main successful Application of compound enzymic preparation in barley type feed with the beta-glucanase, has opened up an important channel with higher economic worth for solving grain trough resources such as barley; At present, China has also applied the compound enzymic preparation that contains beta-glucanase in feed; In the pig feed that with barley is main energy, add beta-glucanase, its capacity usage ratio can improve 13%, and protein utilization improves 21%; Yu Dongyou is in " barley type diet adds beta-glucanase to INFLUENCE OF YUCCA EXTRACT " literary composition; 180 piglets are shown with middle swine rearing test-results: compare with control group; Add compound enzymic preparation in the diet and make piglet and middle daily gain in pigs improve 20.66% and 11.56% respectively, the material anharmonic ratio has reduced by 8.87% and 13.61% respectively; The Economic and Efficiency Analysis result shows that the unit bodies weightening finish feed cost of enzyme-added group of piglet and middle pig has descended 8.44% and 13.26% respectively than not enzyme-added group; Reduced the pollution of movement simultaneously to environment; Add in barley diet that beta-glucanase is enzyme-added can to significantly improve IDGF level in the duckling blood; Contain the alternative ordinary maize type of adding beta-glucanase feed in 50% diet laying hen, egg laying performance is significantly improved; In containing the barley meal feed, add the beta-glucanase carp of feeding; The result shows; Add beta-glucanase in the feed that contains barley (35%), can improve the rate of body weight gain and reduction feed coefficient of carp, improve the digestibility of bait crude protein, crude fat and dry-matter.
Beta-glucanase also has extremely important application in sugar-refining industry; According to reported in literature,, VISOSE formation will cause approximately 7 tons of sucrose of per 10,000 tons of sugarcane Direct Loss because of consuming sucrose; Because the existence of VISOSE causes the sugar-refining industry extraction yield to reduce, residual sucrose amount increases in the last molasses, and the loss that causes is 3-5 a times that sucrose inversion becomes the VISOSE loss; The method of sugar refinery control VISOSE that has is to add sterilant, and not only cost increases, and does not meet the requirement of green food; Part sugar refinery uses LSD to handle the VISOSE of frying in shallow oil in the juice, has reduced sugar loss, has obtained better effects.
In addition, beta-glucanase also has many researchs reports in the control of plant harmful fungoid, hydrolysed leaven cell walls, application aspect modified and solubilized; Beta-glucanase also can be used for playing tangible tooth-cleaning action in the toothpaste, is applied in the washing composition to strengthen the soil removability of cleaning product, also is widely used aspect many in food, papermaking, wellfracturing etc. simultaneously.
A large amount of research work of external beta-glucanase start from the sixties in 20th century; Feed and brewing industry have been widely used in by the end of the eighties; Some zymin companies such as Denmark Novo, Dutch Gist, the numerous and confused product of releasing oneself such as U.S. Miles and Finland Cultor company; The commercial enzyme preparation of these zymin companies has plenty of simple beta-glucan zymin, and what have then is mixed type zymin (except that beta-glucanase, also containing AMS, beta-amylase and proteolytic enzyme etc.); China is since the just research of the beta-glucanase eighties in 20th century, though do a lot of work so far, the situation of import zymin monopolization home market is not broken yet.
Summary of the invention
The present invention is in order to provide a kind of recombinant yeast pichia pastoris liquid submerged fermentation to produce β-1,3-1, the method for 4-LSD.
Technical scheme of the present invention is following:
A kind of working method of beta-glucanase utilizes pichia spp as bacterial classification, with yeast powder, peptone, yeast nitrogen, vitamin H, glycerine, H 3PO 4, CaSO 42H 2O, K 2SO 4, MgSO 47H 2O, KOH are raw material, carry out deep liquid time fermentative prodn beta-glucanase, and ferment filtrate obtains filter residue and beta-glucanase liquid through Plate Filtration; Resulting beta-glucanase liquid is obtaining powdery beta-glucan enzyme product through ultrafiltration and spraying drying; The gained filter residue can be used as fodder additives, does not produce any waste; Specifically comprise the steps:
1, strain preparation and enlarged culturing
1.1 pichia spp test tube slant bacterial classification is changed over to (pH6.6～6.8 in the 3L triangular flask 600mL first order seed substratum that are contained in that prepared under aseptic condition; Sterilising conditions: 115 ℃ of sterilization 30min), it is 12～14 subsequent use that 250rpm, 30 ± 1 ℃ cultivate.
1.2 bacterial classification is carried out enlarged culturing with the 100L seeding tank
With the yeast powder 480g of technical grade, peptone 900g, 1M phosphoric acid buffer (pH=6.0) 6L, yeast nitrogen (no amino acid) 900g, vitamin H 24mg, glycerine 600g drop into respectively in the 100L seeding tank, with the tap water constant volume to 60L; Stir; Directly sterilize with steam then, 115 degree insulations 30 minutes are cooled to 30 degree again, insert the 600mL first order seed bacterium liquid of 1.1.1 gained set by step; Under the condition of air quantity 0.6: 1,30 ± 1 ℃ of temperature, pressure tank 0.06～0.08MPa, cultivated 30 hours, the pichia spp bacterial classification that gets enlarged culturing is subsequent use.
2, produce beta-glucanase with the 1000L ferment tank
With the glycerine of technical grade, 30kg; H 3PO4,18L; CaSO 42H 2O, 600g; K 2SO 4, 9.6kg; MgSO 47H 2O, 7.2kg; KOH, 1.8kg drop into respectively in the 1000L seeding tank, with the tap water constant volume to 600L; Stir; Directly sterilize with steam then, 115 degree insulations 30 minutes are cooled to 30 degree again, and the sterilization back is by 28%NH 4OH adjusts pH to 5.0, adds the trace element (CuSO of 2.61L again 45H 2O, 0.6g/L; KI, 0.08g/L; MnSO 4H 2O, 0.3g/L; Na 2MoO 42H 2O, 0.2g/L; Biotin, 0.2g/L; 98% dense H 2SO 4, 5mL/L), insert 60L first order seed nutrient solution; Under the condition of air quantity 1: 0.6～0.8,30 ± 1 ℃ of temperature, pressure tank 0.06v～0.08MPa, cultivate; Mend glycerine after for some time, to about weight in wet base length to 200, begin to use methanol induction, induced 120 hours; Obtain fermented liquid; Utilize flame filter press that fermented liquid is carried out solid-liquid separation, obtain filter residue and beta-glucanase clear liquid, so-called air quantity ratio is one cubic metre of fermented liquid and the ratio of the needed air input of PM.
3, the β that step 2 is obtained-1,3-1, it is that 1000 daltonian ultra-filtration membranes filter and concentrate crude enzyme liquid that 4-LSD clear liquid adopts molecular weight; Adopt centrifugal spray-drying tower to carry out spraying drying then; 140～150 ℃ of inlet temperatures export 50～60 ℃, obtain powdery β-1,3-1,4-LSD embedding product.
4, dextranase activity is measured
Adopt the DNS method, an enzyme activity unit (U) is defined as under 50 ℃, pH 6.0 conditions, and PM catalytic substrate (lichenstarch) hydrolysis produces the enzyme amount of 1 μ mol glucose.Catalytic substrate (lichenstarch) hydrolysis produces the enzyme amount of 1mol glucose; Lichenstarch as substrate is the glycogen of lichens Cetraria islandica (Iceland's lichens); Its structure is similar with the cereal beta-glucan; Because structure is simple relatively, lichenstarch is often as measuring β-1,3-1, the substrate of 4-dextranase activity; This polymkeric substance that constitutes (mainly being linear structure) by glucose unit contains 30% β-1,3 key and 70% β-1,4 key.
Concrete operations are following:
Add 100 μ L crude enzyme liquid (supernatant or diluent) and 0.4mL, 50 ℃ of water bath heat preservation 10min in the 0.5mL lichenstarch reaction solution of 50 ℃ of preheatings; Add 1mL DNS reaction solution and 3mL water then, put into boiling water at once and boil 5min with stopped reaction; Put into the water condensation after the taking-up immediately, sentencing the pipe that adds substrate and inactivator liquid at 540nm is that reference solution is measured absorbancy.
The calculating of enzyme activity:
Configuration glucose standardized solution is made the glucose typical curve according to the method described above, draws the relation of glucose concn and absorbancy.
Through the glucose concn that typical curve is found, can calculate enzyme activity (U mL -1).
r＝(CGlc?V?F)/(v?t)
R-speed of response [μ mol (mL.min) -1], enzymic activity [U mL -1]
CGlc-glucose concn [μ mol mL -1]
V-measures volume (1.0mL)
V-sample volume and 70% β-1,4 key (0.1mL)
The t-reaction times (10min)
F-crude enzyme liquid extension rate (-)
Description of drawings
Accompanying drawing is a fermentation culture accompanying drawing of the present invention.
5, embodiment:
Below in more detail technical essential of the present invention is made an explanation through instance, for understanding the present invention better foundation is provided.
Embodiment:
1), under aseptic condition, changes pichia spp test tube slant bacterial classification over to prepared (pH6.6～6.8 in the 3L triangular flask 600mL first order seed substratum that are contained in; Sterilising conditions: 115 ℃ of sterilization 30min), it is 12～14 subsequent use that 250rpm, 30 ± 1 ℃ cultivate.
2) with the 100L seeding tank bacterial classification is carried out enlarged culturing
With the yeast powder 480g of technical grade, peptone 900g, 1M phosphoric acid buffer (pH=6.0) 6L, yeast nitrogen (no amino acid) 900g, vitamin H 24mg, glycerine 600g drop into respectively in the 100L seeding tank, with the tap water constant volume to 60L; Stir; Directly sterilize with steam then, 115 degree insulations 30 minutes are cooled to 30 degree again, insert the 600mL first order seed bacterium liquid of 1.1.1 gained set by step; Under the condition of air quantity 0.6: 1,30 ± 1 ℃ of temperature, pressure tank 0.06～0.08MPa, cultivated 30 hours, the pichia spp bacterial classification that gets enlarged culturing is subsequent use.
3), produce beta-glucanase with the 1000L ferment tank
With the glycerine of technical grade, 30kg; H 3PO 4, 18L; CaSO 42H 2O, 600g; K 2SO 4, 9.6kg; MgSO 47H 2O, 7.2kg; KOH, 1.8kg drop into respectively in the 1000L seeding tank, with the tap water constant volume to 600L; Stir; Directly sterilize with steam then, 115 degree insulations 30 minutes are cooled to 30 degree again, and the sterilization back is by 28%NH 4OH adjusts pH to 5.0, adds the trace element (CuSO of 2.61L again 45H 2O, 0.6g/L; KI, 0.08g/L; MnSO 4H 2O, 0.3g/L; Na 2MoO 42H 2O, 0.2g/L; Biotin, 0.2g/L; 98% dense H 2SO 4, 5mL/L), insert 60L first order seed nutrient solution; Under the condition of air quantity 1: 0.6～0.8,30 ± 1 ℃ of temperature, pressure tank 0.06v～0.08MPa, cultivate, mend glycerine after for some time, to about weight in wet base length to 200, begin to use methanol induction; To induce 120 hours, and to obtain fermented liquid, the foam that produces in order eliminating in the fermenting process; The skimmer that in 100L fermentor tank and 1000L fermentor tank, adds silicone oil; Utilize flame filter press that fermented liquid is carried out solid-liquid separation, obtain filter residue and beta-glucanase clear liquid, record β-1; 3-1, the enzyme activity of 4-LSD are 53189.32U/mL.
4) β that step 2 is obtained-1,3-1, it is that 1000 daltonian ultra-filtration membranes filter and concentrate crude enzyme liquid that 4-LSD clear liquid adopts molecular weight; Adopt centrifugal spray-drying tower to carry out spraying drying then; 140～150 ℃ of inlet temperatures export 50～60 ℃, obtain powdery β-1,3-1,4-LSD embedding product.

Claims (5)

1. a recombinant yeast pichia pastoris liquid submerged fermentation is produced β-1,3-1, and 4-LSD method, this method utilizes pichia spp as bacterial classification, with yeast powder, peptone, yeast nitrogen, vitamin H, glycerine, H 3PO 4, CaSO 42H 2O, K 2SO 4, MgSO 47H 2O, KOH are raw material, carry out liquid submerged fermentation and produce beta-glucanase, and ferment filtrate obtains filter residue and beta-glucanase liquid through Plate Filtration; Resulting beta-glucanase liquid is obtaining powdery beta-glucan enzyme product through ultrafiltration and spraying drying; It is characterized in that the gained filter residue can be used as fodder additives, does not produce any waste.
2. according to claim 1; A kind of recombinant yeast pichia pastoris liquid submerged fermentation is produced β-1; 3-1; 4-LSD method is characterized in that: pichia spp test tube slant bacterial classification is changed over to (pH6.6～6.8 in the 3L triangular flask 600mL first order seed substratum that are contained in that prepared under aseptic condition; Sterilising conditions: 115 ℃ of sterilization 30min), 250rpm, 30 ± 1 ℃ cultivate 12～14 hours subsequent use.
3. according to claim 1, a kind of recombinant yeast pichia pastoris liquid submerged fermentation is produced β-1,3-1; 4-LSD method is characterized in that: with the yeast powder 480g of technical grade, peptone 900g; 1M phosphoric acid buffer (pH=6.0) 6L, yeast nitrogen (nothing, amino acid) 900g, vitamin H 24mg; Glycerine 600g drops into respectively in the 100L seeding tank, with the tap water constant volume to 60L; Stir; Directly sterilize with steam then, 115 degree insulations 30 minutes are cooled to 30 degree again, insert the 600mL first order seed bacterium liquid of 1.1.1 gained set by step; Under the condition of air quantity 0.6: 1,30 ± 1 ℃ of temperature, pressure tank 0.06～0.08MPa, cultivated 30 hours, the pichia spp bacterial classification that gets enlarged culturing is subsequent use.
4. according to claim 1, a kind of recombinant yeast pichia pastoris liquid submerged fermentation is produced β-1,3-1, and 4-LSD method is characterized in that: with the glycerine of technical grade, 30kg; H 3PO 4, 18L; CaSO 42H 2O, 600g; K 2SO 4, 9.6kg; MgSO 47H 2O, 7.2kg; KOH, 1.8kg drop into respectively in the 1000L seeding tank, with the tap water constant volume to 600L; Stir; Directly sterilize with steam then, 115 degree insulations 30 minutes are cooled to 30 degree again, and the sterilization back is by 28%NH 4OH adjusts pH to 5.0, adds the trace element of 2.61L again, inserts 60L first order seed nutrient solution; Under the condition of air quantity 1: 0.6～0.8,30 ± 1 ℃ of temperature, pressure tank 0.06v～0.08MPa, cultivate; Mend glycerine after for some time, to about weight in wet base length to 200, begin to use methanol induction, induced 120 hours; Obtain fermented liquid; Utilize flame filter press that fermented liquid is carried out solid-liquid separation, obtain filter residue and beta-glucanase clear liquid, record the enzyme 51723u/L alive of beta-glucanase clear liquid.
5. according to claim 1, a kind of recombinant yeast pichia pastoris liquid submerged fermentation is produced β-1,3-1, and 4-LSD method is characterized in that: trace element is CuSO 45H 2O, 0.6g/L; KI, 0.08g/L; MnSO 4H 2O, 0.3g/L; Na 2MoO 42H2O, 0.2g/L; Biotin, 0.2g/L; 98% dense H 2SO 4, 5mL/L.
CN2011104485239A 2011-12-29 2011-12-29 Method for producing beta-1,3-1,4-glucanase through submerged fermentation of recombinant Pichia pastoris liquid Pending CN102559641A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104328099A (en) * 2014-11-13 2015-02-04 河南天冠纤维乙醇有限公司 Method for producing beta-glucanase by using recombined pichia pastoris
CN106820091A (en) * 2016-12-13 2017-06-13 安徽翠鸟生物技术有限公司 A kind of preparation method of food fermentation enzyme powder
CN113969271A (en) * 2021-12-06 2022-01-25 厦门大学 Foam separation method of beta-glucanase in fermentation liquor

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104328099A (en) * 2014-11-13 2015-02-04 河南天冠纤维乙醇有限公司 Method for producing beta-glucanase by using recombined pichia pastoris
CN106820091A (en) * 2016-12-13 2017-06-13 安徽翠鸟生物技术有限公司 A kind of preparation method of food fermentation enzyme powder
CN113969271A (en) * 2021-12-06 2022-01-25 厦门大学 Foam separation method of beta-glucanase in fermentation liquor
CN113969271B (en) * 2021-12-06 2023-12-22 厦门大学 Foam separation method of beta-glucanase in fermentation liquor

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Application publication date: 20120711