CN101875926B - Method for preparing liquid cellulase - Google Patents
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- CN101875926B CN101875926B CN2009102596776A CN200910259677A CN101875926B CN 101875926 B CN101875926 B CN 101875926B CN 2009102596776 A CN2009102596776 A CN 2009102596776A CN 200910259677 A CN200910259677 A CN 200910259677A CN 101875926 B CN101875926 B CN 101875926B
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Abstract
The invention relates to a method for preparing liquid cellulase from trichoderma viride, which comprises the followings steps of: culturing trichoderma viride mycelia by using a mechanical agitator tank, and transferring to an airlift fermentation tank for culture after all glucose is consumed and the concentration of the mycelia reaches the standard. The method has the advantages of promoting the growth of trichoderma viride mycelium cells, avoiding the shearing force to the mycelia because of the increase of the agitator speed in the fermentation process when dissolved oxygen is insufficient, and preparing the cellulase with high efficiency by combining the mechanical agitator tank with the airlift fermentation tank. Compared with the conventional method for preparing the cellulase by only using the mechanical agitator tank or the airlift fermentation tank, the method for preparing the cellulase of the invention can improve the enzymatic activity of filter paper by 50 to 150 percent, and is particularly suitable for popularization and application in industrial production.
Description
Technical field
The present invention relates in technical field of biological fermentation, particularly relate to a kind of preparation method of liquid cellulase.
Background technology
Cellulase (cellulase) is meant energy degraded cellulose β-1,4 glucoside bond, makes Mierocrystalline cellulose become the general name of one group of enzyme of cellobiose and glucose, has been that synergistic polycomponent enzyme is.Cellulase has tens yuan the market sales revenue every year in China, has application widely in industry such as weaving, alcohol, feeds, but the domestic market share more than 90% is captured by external product at present, therefore carry out the cellulase fermentations Study on Technology, reducing production costs has very important practical sense.
The production of cellulase is the same with other zymin, mainly contains two kinds of solid fermentation and liquid submerged fermentations, the less investment of solid fermentation method, technology is simple, and product price is cheap, but the level of automation of solid fermentation method is low, labour intensity is big, unstable product quality.Therefore, along with the development of liquid fermenting enzymatic process and the raising of bacterial classification performance, adopting solution fermentation producd fibers enzyme element is inexorable trend.
Plain main mechanical agitator tank or the airlift fermentor of adopting of existing solution fermentation producd fibers enzyme.When using mechanical agitator tank fermentative production cellulase; in the respiratory climacteric phase of viride; dissolved oxygen sharply descends; fall below zero through regular meeting; therefore must improve dissolved oxygen, the method that improves dissolved oxygen in the fermented liquid during the fermentation mainly is the raising air flow, increases tank pressure, improves mixing speed.In order to improve the dissolved oxygen level in the Trichoderma Viride production of cellulose enzyme respiratory climacteric phase mash, can improve air flow respectively, increase tank pressure, improve mixing speed.But find in the practical application that it has been the upper limit that the raising of air flow reaches 1: 1.5, and it is limited to the raising of dissolved oxygen to increase air flow.Improve tank pressure to 0.8kg/cm
3Also be the upper limit of the pressure-bearing of common fermentation jar.Can promote to mix by stirring in the fermenting process, heat and mass transfer, dissolved oxygen level in the mash is improved also bigger, but the raising of mixing speed produces shearing action to hyphomycetic thalline, thereby influence the synthesizing of growth, form and product of bacterium.Discovery that can be clearly in the fermenting process brings up to one regularly when rotating speed, and mycelium is obviously interrupted, and occurs a large amount of mycelial fragments in the mash.Therefore, only by the way, be difficult to satisfy viride in the demand of respiratory climacteric to dissolved oxygen.
If when directly using airlift fermentor production of cellulose enzyme, owing in the fermentation liquid insoluble substances such as pure cellulose, Microcrystalline Cellulose and lime carbonate are arranged, in the fermentation initial stage, the mixing of materials effect is bad.Especially at 8～20 hours of fermentation, because the quick growth of viride mycelium, the thickness phenomenon appearred in fermentation liquid, airlift fermentor can't be finished the thorough mixing of material, anoxic condition appears in mycelia, occurs the mycelia tubercle too early, reduces the output of liquid cellulase greatly.
Therefore, be necessary to propose a kind of method of efficient production cellulase.
Summary of the invention
The present invention seeks to the deficiency at the existing fermentation technique of cellulase, provide a kind of easy and simple to handle, cost is low, and the viride that utilizes that enzyme activity is stable prepares the method for cellulase in a large number.
Trichoderma Viride production of cellulose enzyme process mainly is divided into three phases: one, viride utilizes the carbon source of easily utilizing in the substratum, and somatic cells increases fast, and mycelia is long, spread growth, respiratory intensity is obvious, and dissolved oxygen consumption is very fast, does not secrete this moment or the plain enzyme of a small amount of eccrine fiber; Two, viride utilizes in the substratum difficulty to utilize carbon source, somatic cells concentration kept stable, and mycelia is long, and uneven grain is arranged in the cell, the plain enzymes of a large amount of eccrine fibers this moment; Three, carbon source in the substratum and nitrogenous source are consumed substantially, and tubercle appears in the mycelium chap, senesce with dead, and the plain enzyme speed of eccrine fiber obviously descends.
For guaranteeing that viride can just be necessary that according to its fermenting characteristic each parameter in the control fermenting process promotes the course of fermentation of viride, so proposes the method for preparing cellulase of the present invention with its maximum capacity production liquid cellulase.
The method for preparing cellulase of the present invention is to utilize mechanical agitator tank and airlift fermentor to unite the cultivation viride.
Specifically, the method for preparing cellulase of the present invention comprises the steps:
(1) viride is inoculated in the mechanical agitator tank that fermention medium is housed heat insulating culture earlier;
(2) in fermented liquid in the mechanical agitator tank glucose consumption to content less than 0.01%, and the mycelium 0.8g/100ml that weighs changes airlift fermentor over to and carries out fermentation culture, whole fermentation period is 90～100 hours.
The parameter control of mechanical agitator tank also can be selected according to the needs of concrete bacterial classification, and general may command mixing speed is 100～260r/min, and air quantity is 1: 0.2～0.5vvm, and tank pressure is 0.02～0.05Mpa; Preferred mixing speed is 150～230r/min, and air quantity is 1: 0.3～0.4vvm, and tank pressure is 0.03Mpa.
The parameter control of airlift fermentor also can be selected according to the needs of concrete bacterial classification, and general may command jar face pressure reduction is 0.025～0.045Mpa, and ventilation is 0.7～1.0vvm, and tank pressure is 0.04～0.06Mpa; Preferred jar of face pressure reduction is 0.04Mpa, and ventilating is that 0.8vvm, tank pressure are 0.05Mpa.
The liquid amount of fermention medium described in the mechanical agitator tank is a mechanical agitator tank volumetrical 70%～90%.
The inoculum size of viride is 5%～12% of a fermention medium cumulative volume.
Culture temperature is 28-32 ℃ in the fermenting process, preferred 30 ℃.
In the method for the present invention, step 1) and 2) between, also comprise step 1 ') survey glucose content, survey mycelia body weight, microscopy every sampling in 3～5 hours between incubation period;
Step 2) afterwards, also comprise step 2 ') survey cellulase content every sampling in 6～10 hours between incubation period.
Wherein, the measuring method of cellulase is: use 0.1g filter paper as substrate, add the 0.9mLpH4.8 citrate buffer solution, add the thick enzyme centrifuged supernatant of 0.1mL again, 50 ℃ of water bath with thermostatic control shaking table reaction 30min, with 3,5-dinitrosalicylic acid method is measured the reducing sugar that generates, with glucose as a standard sugar generates the required enzyme amount of 1 μ mol reducing sugar with per minute and is defined as a cellulose enzyme unit of activity (IU).The mash centrifuging and taking supernatant liquor that liquid fermenting is good is crude enzyme liquid.Citrate buffer solution is that 21g Citric Acid, usp, Anhydrous Powder, 7.8g sodium hydroxide adding distil water are settled to 2L, and pH is 4.8.
The glucose assays method is: adopt the DNS method, with glucose as a standard.
The measuring method of mycelia body weight is: get the 100ml fermented liquid, 5000 rev/mins centrifugal 10 minutes, claim weight in wet base to be mycelium weight in wet base among every 100ml to the solid under centrifugal.
Described viride can be selected the bacterial strain of this area preparing liquid cellulase commonly used, and preferred deposit number is the viride of CICC13052, according to above-mentioned condition, utilizes the output of the cellulase of this bacterial strain preparation can reach 13～36IU/ml.
Described fermention medium is that Trichoderma Viride production liquid cellulase each component content of substratum commonly used is Microcrystalline Cellulose 3～5%, corn steep liquor 1～2%, glucose 1～2%, urea 0.1～0.2%, ammonium sulfate 0.1～0.2% and dipotassium hydrogen phosphate 0.05～0.1%, contains Microcrystalline Cellulose 4%, corn steep liquor 2%, glucose 2%, urea 0.2%, ammonium sulfate 0.1% and dipotassium hydrogen phosphate 0.05% in the preferred fermention medium.
Described step 2) also comprise in: fermenting, stream adds fresh fermention medium after 70-80 hour.
The composition of described fresh fermention medium can be identical or different with fermention medium, the fresh culture of preferred following composition: Microcrystalline Cellulose 3-5%, corn steep liquor 1-3%, bean cake powder 1-2%, ammonium sulfate 0.1-1%, dipotassium hydrogen phosphate 0.1-0.5%; The more preferably fresh culture of following composition: Microcrystalline Cellulose 5%, corn steep liquor 2%, bean cake powder 1%, ammonium sulfate 0.5%, dipotassium hydrogen phosphate 0.2%.
The viride seed is inoculated in the mechanical agitator tank of the substratum that fibre-bearing inductor Microcrystalline Cellulose and nutritive salt are housed.When glucose consumption finishes, bacterium is dense reach standard after, change airlift fermentor over to and carry out fermentation culture.
In the described method, before step 1), further comprising the steps of:
Slant culture: under aseptic technique, the viride original seed is inserted in the PDA test tube slant substratum, cultivated 5～7 days for 30 ℃;
Seed culture: under aseptic condition, slant strains is inserted in the triangular flask, putting vibrates on the bottle swingging machine 28～32 ℃ cultivated 20～24 hours; The seeding tank of then shake-flask seed being transferred continues 28～32 ℃ and cultivated 20～24 hours; The seed culture based component is a glucose 1～2%, corn steep liquor 1～2%, urea 0.1～0.2%, ammonium sulfate 0.1～0.2%, potassium primary phosphate 0.1～0.2%, sal epsom 0.05～0.1%; Triangular flask is identical with the seed tank culture based component.
Preferred seed culture based component is a glucose 1%, corn steep liquor 1%, urea 0.1%, ammonium sulfate 0.2%, potassium primary phosphate 0.1%, sal epsom 0.05%.
The method of utilizing viride to produce liquid cellulase of the present invention, by mechanical agitator tank and airlift fermentor coupling, both can promote the growth of viride somatic cells, avoided simultaneously when dissolved oxygen is not enough, improving in the fermenting process mixing speed again to mycelial shearing force, can high efficiency preparation cellulase, with only with mechanical agitator tank or only compare with airlift fermentor production of cellulose enzyme, the production of cellulose enzyme can make filter paper enzyme activity improve 50～150% in this way, is particularly suitable for applying in industrial production.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.Specialize as nothing, the viride that adopts among the embodiment is available from Chinese industrial microbial strains preservation administrative center, and culture presevation is numbered CICC13052.
Embodiment 1
Example only adopts the mechanical agitating fermentation tank preparing liquid cellulase in contrast.
Is 35m with viride (CICC13052) at liquid amount
350m
3Fermentative production cellulase in the mechanical agitating fermentation tank, mixing speed are 150r/min, and ventilation is 1: 0.5vvm, tank pressure are 0.05Mpa.Wherein contain Microcrystalline Cellulose 3%, corn steep liquor 1%, glucose 1%, urea 0.1%, ammonium sulfate 0.1% and dipotassium hydrogen phosphate 0.05% in the fermention medium.
Temperature is controlled at 30 ℃ in the fermenting process, and fermentation period is 94 hours.
Ferment per 8 hours test samples once, and the fermented liquid of at every turn emitting all will be measured cellulase activity and residual glucose immediately.
The result shows, uses the filter paper enzyme activity of mechanical agitator tank cellulase-producing to be up to 15.42IU/ml.The fermentation final volume is 33m
3
Embodiment 2
Example only adopts the airlift fermentor preparing liquid cellulase in contrast.
Is 35m with viride at liquid amount
350m
3Fermentative production cellulase in the airlift fermentor, jar face pressure reduction is 0.04Mpa, ventilating is that 0.8vvm, tank pressure are 0.05Mpa.Wherein contain Microcrystalline Cellulose 3%, corn steep liquor 1%, glucose 1%, urea 0.1%, ammonium sulfate 0.1% and dipotassium hydrogen phosphate 0.05% in the fermention medium.
Temperature is controlled at 30 ℃ in the fermenting process, and fermentation period is 92 hours.
Ferment per 8 hours test samples once, and the fermented liquid of at every turn emitting all will be measured cellulase activity and residual sugar immediately.
The result shows, uses the Microcrystalline Cellulose filter paper enzyme activity to be up to 13.24IU/ml.The fermentation final volume is 33m
3
Embodiment 3
(culture presevation numbering CICC13052) is 35m at liquid amount with viride
350m
3Heat insulating culture in the mechanical agitating fermentation tank, mixing speed are 150r/min, and ventilation is 1: 0.5vvm, tank pressure are 0.05Mpa.Wherein contain Microcrystalline Cellulose 3%, corn steep liquor 1%, glucose 1%, urea 0.1%, ammonium sulfate 0.1% and dipotassium hydrogen phosphate 0.05% in the fermention medium.
Ferment per 4 hours test samples once.
When glucose consumption to content less than 0.01%, mycelium weighs and changes airlift fermentor over to behind the 0.8g/100ml and carry out fermentation culture, jar face pressure reduction is 0.04Mpa, ventilating is that 0.8vvm, tank pressure are 0.05Mpa.Ferment per 8 hours test samples once, and the fermented liquid of at every turn emitting all will be measured cellulase activity immediately.
Temperature is controlled at 30 ℃ in the fermenting process, and whole fermentation period is 94 hours.
The result shows, uses mechanical agitator tank and airlift fermentor coupled fermentation, and the cellulase filter paper enzyme activity is up to 25.30IU/ml.The fermentation final volume is 33m
3
Embodiment 4
(culture presevation numbering CICC13052) is 30m at liquid amount with viride
350m
3Heat insulating culture in the mechanical agitating fermentation tank, mixing speed are 220r/min, and ventilation is 1: 0.3vvm, tank pressure are 0.02Mpa.Wherein contain Microcrystalline Cellulose 4%, corn steep liquor 2%, glucose 2%, urea 0.2%, ammonium sulfate 0.1% and dipotassium hydrogen phosphate 0.05% in the fermention medium.
Ferment per 4 hours test samples once.
When glucose consumption to content less than 0.01%, mycelium weighs and changes airlift fermentor over to behind the 0.8g/100ml and carry out fermentation culture, jar face pressure reduction is 0.025Mpa, ventilating is that 0.96vvm, tank pressure are 0.04Mpa.
Ferment per 8 hours test samples once, and the fermented liquid of at every turn emitting all will be measured cellulase activity immediately.Fermenting, stream adds fresh culture after 70 hours.Composition is Microcrystalline Cellulose 5%, corn steep liquor 2%, bean cake powder 1%, ammonium sulfate 0.5%, dipotassium hydrogen phosphate 0.2% in the substratum that stream adds.The fermented liquid of at every turn emitting all will be measured cellulase activity and residual sugar immediately.
Temperature is controlled at 30 ℃ in the fermenting process, and whole fermentation period is 96 hours.
The result shows, uses mechanical agitator tank and airlift fermentor coupling stream to add the fermentative production cellulase, and filter paper enzyme activity is up to 35.21IU/ml.The fermentation final volume is 33m3.
Embodiment 5
(culture presevation numbering CICC13052) is 30m at liquid amount with viride
350m
3Heat insulating culture in the mechanical agitating fermentation tank, mixing speed are 260r/min, and ventilation is 1: 0.2vvm, tank pressure are 0.05Mpa.Wherein contain Microcrystalline Cellulose 4%, corn steep liquor 2%, glucose 2%, urea 0.2%, ammonium sulfate 0.1% and dipotassium hydrogen phosphate 0.05% in the fermention medium.
Ferment per 5 hours test samples once.
When glucose consumption to content less than 0.01%, mycelium weighs and changes airlift fermentor over to behind the 0.8g/100ml and carry out fermentation culture, jar face pressure reduction is 0.045Mpa, ventilating is that 0.7vvm, tank pressure are 0.06Mpa.
Ferment per 10 hours test samples once, and the fermented liquid of at every turn emitting all will be measured cellulase activity immediately.Fermenting, stream adds fresh culture after 75 hours.Composition is Microcrystalline Cellulose 5%, corn steep liquor 2%, bean cake powder 1%, ammonium sulfate 0.5%, dipotassium hydrogen phosphate 0.2% in the substratum that stream adds.The fermented liquid of at every turn emitting all will be measured cellulase activity and residual sugar immediately.
Temperature is controlled at 30 ℃ in the fermenting process, and whole fermentation period is 100 hours.
The result shows, uses mechanical agitator tank and airlift fermentor coupling stream to add the fermentative production cellulase, and filter paper enzyme activity is up to 34.253U/ml.The fermentation final volume is 33m
3
Claims (9)
1. a method of utilizing viride to prepare cellulase is characterized in that, is to utilize mechanical agitator tank and airlift fermentor to unite the cultivation viride, comprises the steps:
(1) viride is inoculated in the mechanical agitator tank that fermention medium is housed heat insulating culture earlier;
(2) in fermented liquid in the mechanical agitator tank glucose consumption to content less than 0.01%, and mycelium weighs more than the 0.8g/100ml, changes airlift fermentor over to and carries out fermentation culture, whole fermentation period is 90～100 hours.
2. the method for claim 1 is characterized in that, the mixing speed of described mechanical agitator tank is 100～260r/min, and air quantity is 1: 0.2～0.5vvm, and tank pressure is 0.02～0.05Mpa.
3. method as claimed in claim 2 is characterized in that, the mixing speed of described mechanical agitator tank is 150～230r/min, and air quantity is 1: 0.3～0.4vvm, and tank pressure is 0.03Mpa.
4. the method for claim 1 is characterized in that, the jar face pressure reduction of described airlift fermentor is 0.025～0.045Mpa, and ventilation is 0.7～1.0vvm, and tank pressure is 0.04～0.06Mpa.
5. method as claimed in claim 4 is characterized in that, the jar face pressure reduction of described airlift fermentor is 0.04Mpa, and ventilating is that 0.8vvm, tank pressure are 0.05Mpa.
6. the method for claim 1 is characterized in that, described step 2) in also comprise: fermenting, stream adds fermention medium after 70～80 hours.
7. the method for claim 1 is characterized in that, step 1) and 2) between, also comprise step 1 ') survey glucose content, survey mycelia body weight, microscopy every sampling in 3～5 hours between incubation period; Step 2) afterwards, also comprise step 2 ') survey cellulase content every sampling in 6～10 hours between incubation period.
8. the method for claim 1 is characterized in that, and is before step 1), further comprising the steps of:
A) slant culture: under aseptic technique, the viride original seed is inserted in the PDA test tube slant substratum, cultivated 5～7 days for 30 ℃;
B) seed culture: under aseptic condition, slant strains is inserted in the triangular flask, putting vibrates on the bottle swingging machine 28～32 ℃ cultivated 20～24 hours; The seeding tank of then shake-flask seed being transferred continues 28～32 ℃ and cultivated 20～24 hours; The seed culture based component is a glucose 1～2%, corn steep liquor 1～2%, urea 0.1～0.2%, ammonium sulfate 0.1～0.2%, potassium primary phosphate 0.1～0.2%, sal epsom 0.05～0.1%; Triangular flask is identical with the seed tank culture based component.
9. the method for claim 1 is characterized in that, described viride is the viride of culture presevation numbering CICC13052.
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| CN106489536B (en) * | 2016-12-09 | 2018-04-27 | 天津农学院 | A kind of brown mushroom liquid spawn high density production method |
| CN106497998B (en) * | 2016-12-09 | 2018-10-02 | 天津农学院 | A kind of control method of brown mushroom polysaccharide deep layer liquid state fermentation |
| CN108330120B (en) * | 2018-02-01 | 2021-08-03 | 湖南鸿鹰生物科技有限公司 | Fermentation method for producing complex enzyme and co-producing vitamin B12 by using Neurospora sitophila |
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| CN1560222A (en) * | 2004-03-04 | 2005-01-05 | 江南大学 | Coupling biologic reactor |
| CN101182503A (en) * | 2007-11-23 | 2008-05-21 | 河南天冠企业集团有限公司 | Production process of acidic liquid cellulase |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1560222A (en) * | 2004-03-04 | 2005-01-05 | 江南大学 | Coupling biologic reactor |
| CN101182503A (en) * | 2007-11-23 | 2008-05-21 | 河南天冠企业集团有限公司 | Production process of acidic liquid cellulase |
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