CN101654669B - Method for producing cellulase in high yield - Google Patents
Method for producing cellulase in high yield Download PDFInfo
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- CN101654669B CN101654669B CN2009100919689A CN200910091968A CN101654669B CN 101654669 B CN101654669 B CN 101654669B CN 2009100919689 A CN2009100919689 A CN 2009100919689A CN 200910091968 A CN200910091968 A CN 200910091968A CN 101654669 B CN101654669 B CN 101654669B
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Abstract
The invention relates to a method for producing cellulase in high yield. Cellulase liquid fermentation thallus has big demand of oxygen, the invention improves fermentation level of cellulase by controlling the concentration of fermentation thallus, thereby increasing the yield of cellulase. Experiments indicate that the fermentation level of cellulase can be improved from 15 FPAIU/ml to 33 FPAIU/ml when the concentration of fermentation thallus is 20 g/L by controlling amounts of dissolved oxygen and supplementary materials. The invention can obviously promote the fermentation level of cellulose, thereby increasing the yield of cellulase; and the method has a great referential significance to other types of high oxygen-consumption fermentations.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of method of high cellulase-producing.
Background technology
Mierocrystalline cellulose is the abundantest glucide of occurring in nature content, is the one type of reproducible resource and the energy.How the utilization but Mierocrystalline cellulose is difficult to be decomposed is that pendulum in people in front a difficult problem for the resource that can directly utilize with the energy with cellulose conversion.Cellulosic degraded has two approach: chemical method and biological process, and low, the contaminate environment of poor specificity, purity of chemical method, and can overcome these shortcomings with biological enzyme, therefore, cellulase is once finding just to have received the attention of countries in the world scientific circles.
Cellulase (cellulase) is meant ability degraded cellulose β-1,4 glucoside bond, makes Mierocrystalline cellulose become the general name of one group of enzyme of cellobiose and glucose; Be that synergistic polycomponent enzyme is, its main ingredient is inscribe β-1,4 a LSD (EC3.2.1.4; Also claim the Cr enzyme), (EC3.2.1.91 also claims the Cl enzyme to exoglucanase; Avicelase) and β one glucuroide (EC3.2.1.21 also claims CB enzyme or cellose).Preceding two kinds of main dissolving cellulos of enzyme, a kind of enzyme in back is converted into glucose with cellobiose, when the active ratio of these three kinds of staples is suitable, just can accomplish cellulosic degraded, can be used for producing fuel alcohol, food and various chemical.
In addition, the discovered in recent years cellulase is with a wide range of applications at numerous industrial circles, like food, feed, medicine, weaving, washing composition and paper industry etc.As previously mentioned, the cellulose degraded Mierocrystalline cellulose is compared tool with chemical method and is had great advantage, but the cellulase consumption is big; Cost is high, has limited it equally and has used, and therefore must screen suitable bacterial strain; Optimization for fermentation technology to be improving the output of cellulase, thereby reduces cost.
Cellulase is had the broad research in more than 40 year abroad, the representative bacterial strain of cellulase producing bacteria has that wood is mould, mould and aspergillus etc., and wherein Trichodermareesei is considered to one of best cellulase producing bacteria; China has also carried out extensive studies to Study on Cellulase with producing; But adopt solid fermentating mode mostly; Though solid fermentation has many superior parts; But still have fermentation level unstable, be inappropriate for drawbacks such as scale operation, therefore highly significant the research of cellulase liquid submerged fermentation.
The present invention has proposed to improve fermentation level, the method for high cellulase-producing through each factor in the control fermenting process through the preparation process of research liquid cellulase.
Summary of the invention
The method that the purpose of this invention is to provide a kind of high cellulase-producing.
The present invention improves the cellulase fermentations level through the zymogenic cell concentration of control, thereby improves the output of cellulase.
The liquid zymophyte body of cellulase is higher to the demand of oxygen, causes fermenting process oxygen under-supply when cell concentration is too high, and enzyme is produced in influence.If cell concentration is low excessively, product enzyme speed is slower, and fermentation period is long, and thalline is aging, does not reach the ideal effect equally.So the cell concentration of control fermented liquid is to solve above contradiction, improves the effective way of fermentation level.
The method of high cellulase-producing of the present invention is to play fermentation ends when fermenting to certain cell concentration, and the control cell concentration keeps stable, and the cell concentration of said fermentation strain is controlled at 10～30g/L.
The cell concentration of control fermentation strain is at 10～30g/L.Preferably cell concentration is controlled at 20g/L.Wherein, cell concentration comes in the dry cell weight in every liter of fermented liquid.
Said fermentation strain is a Trichodermareesei.
For Trichodermareesei fermentative prepn cellulase, saidly be meant 18-48 hour when fermenting to certain cell concentration; Preferred 24-48 hour.
The control of said cell concentration realizes through control dissolved oxygen concentration and control feed supplement amount.
Said dissolved oxygen concentration is controlled at 20-30%; Said feed supplement amount is controlled at 50-100ml/h.
Wherein, The component of said feed supplement is identical or slightly different with the component of the initial fermention medium that uses; As can be according to the common practise of this area, in fermention medium, adding an amount of generation to cellulase has components such as the sophorose of inducing action, lactose, chest nut powder as feed supplement.
Among the present invention, the vigor filter paper enzyme activity iu of cellulase, method for expressing is: a filter paper enzyme activity iu (FPAIU/ml) is defined as: PM generates the enzyme amount of 1.0umol glucose (in reducing sugar) in the enzyme digestion reaction.
The present invention can obviously improve the cellulase fermentations level, thereby improves the output of cellulase.Experiment showed, through the control dissolved oxygen in 20-30% and feed supplement amount and to keep cell concentration at 20g/L at 50-100ml/h, the cellulase fermentations level is brought up to 33FPAIU/ml by 15FPAIU/ml.In addition, the method for control fermented liquid cell concentration provided by the present invention has the certain experiences meaning to other high oxygen consumption fermentations.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
Embodiment 1
1) slant culture: be inoculated in respectively on the solid medium under Trichodermareesei (available from the Chinese Academy of Sciences microbial strains storehouse) aseptic condition, cultivated 7 days under 28 ℃ of conditions;
2) seed culture: be inoculated in liquid nutrient medium respectively under the bacterial classification aseptic condition with the step 1) cultivation, 28 ℃, the 200r/min shaking table was cultivated 48 hours;
3) fermentation culture: by the volume ratio of liquid nutrient medium is 5% inoculum size, and seed is inoculated in the fermentor tank, and under 28 ℃ of conditions, stirring velocity is 300r/min, cultivates 7 days.
4) feed supplement is cultivated: ferment after 24 hours, with the feed supplement of 50-100ml/h speed, the control dissolved oxygen is at 20-30%.
Wherein the culture medium prescription of step 1) use is: inclined-plane prescription (g/L): potato 100; Glucose 10 grams; Agar 20.
Step 2 wherein) culture medium prescription that uses is:
Seed prescription (g/L): yeast powder 10; Sal epsom 2; Potassium primary phosphate 15; Glucose 20; Steeping water 10.
Trace element (ppm): ferrous sulfate 3; Copper sulfate 1.3; Boric acid 0.6; Manganous sulfate 3.1; Zinc sulfate 9.
Wherein the culture medium prescription of step 3) use is:
Fermentating formula (g/L): yeast powder 5; Sal epsom 0.6; Potassium primary phosphate 5; Glucose 10; Steeping water 18; Ammonium sulfate 3; Filter paper powder 50; Tween 1.
Trace element (ppm): ferrous sulfate 5; Copper sulfate 2; Boric acid 1; Manganous sulfate 5; Zinc sulfate 10.
Wherein the culture medium prescription of step 4) use is:
Feed supplement prescription (g/L): yeast powder 4; Sal epsom 0.6; Potassium primary phosphate 5; Lactose 5; Steeping water 10; Ammonium sulfate 3; Filter paper powder 30; Sophorose 3; Tween 1.
Trace element (ppm): ferrous sulfate 5; Copper sulfate 2; Boric acid 1; Manganous sulfate 5; Zinc sulfate 10.
The fermentation culture process comprises: under 28 ℃ of conditions, stirring velocity is 300r/min, and air flow is 1: 1; Fermented about 18 hours; Begin when cell concentration reaches 10g/L with the feed supplement of 50-100ml/h speed, the control dissolved oxygen is kept cell concentration 10g/L at 20-30%; Cultivated fermentation level 15FPAIU/ml 7 days.
Embodiment 2
1) slant culture: be inoculated in respectively on the solid medium under Trichodermareesei (available from the Chinese Academy of Sciences microbial strains storehouse) aseptic condition, cultivated 7 days under 28 ℃ of conditions;
2) seed culture: be inoculated in liquid nutrient medium respectively under the bacterial classification aseptic condition with the step 1) cultivation, 28 ℃, the 200r/min shaking table was cultivated 48 hours;
3) fermentation culture: by the volume ratio of liquid nutrient medium is 5% inoculum size, and seed is inoculated in the fermentor tank, and under 28 ℃ of conditions, stirring velocity is 300r/min, cultivates 7 days.
4) feed supplement is cultivated: ferment about 24 hours with the feed supplement of 50-100ml/h speed, the control dissolved oxygen is at 20-30%.
Wherein the culture medium prescription of step 1) use is:
Inclined-plane prescription (g/L): potato 100; Glucose 10 grams; Agar 20.
Step 2 wherein) culture medium prescription that uses is:
Seed prescription (g/L): yeast powder 10; Sal epsom 2; Potassium primary phosphate 15; Glucose 20; Steeping water 10.
Trace element (ppm): ferrous sulfate 3; Copper sulfate 1.3; Boric acid 0.6; Manganous sulfate 3.1; Zinc sulfate 9.
Wherein the culture medium prescription of step 3) use is:
Fermentating formula (g/L): yeast powder 5; Sal epsom 0.6; Potassium primary phosphate 5; Glucose 10; Steeping water 18; Ammonium sulfate 3; Filter paper powder 50; Tween 1.
Trace element (ppm): ferrous sulfate 5; Copper sulfate 2; Boric acid 1; Manganous sulfate 5; Zinc sulfate 10.
Wherein the culture medium prescription of step 4) use is:
Feed supplement prescription (g/L): yeast powder 4; Sal epsom 0.6; Potassium primary phosphate 5; Lactose 5; Steeping water 10; Ammonium sulfate 3; Filter paper powder 30; Sophorose 3; Tween 1.
Trace element (ppm): ferrous sulfate 5; Copper sulfate 2; Boric acid 1; Manganous sulfate 5; Zinc sulfate 10.
The fermentation culture process comprises: under 28 ℃ of conditions, stirring velocity is 300r/min, and air flow is 1: 1; Fermented about 24 hours; Begin when cell concentration reaches 20g/L with the feed supplement of 50-100ml/h speed, the control dissolved oxygen is kept cell concentration 20g/L at 20-30%; Cultivated fermentation level 33FPAIU/ml 7 days.
Embodiment 3
1) slant culture: be inoculated in respectively on the solid medium under Trichodermareesei (available from the Chinese Academy of Sciences microbial strains storehouse) aseptic condition, cultivated 7 days under 28 ℃ of conditions;
2) seed culture: be inoculated in liquid nutrient medium respectively under the bacterial classification aseptic condition with the step 1) cultivation, 28 ℃, the 200r/min shaking table was cultivated 48 hours;
3) fermentation culture: by the volume ratio of liquid nutrient medium is 5% inoculum size, and seed is inoculated in the fermentor tank, and under 28 ℃ of conditions, stirring velocity is 300r/min, cultivates 7 days.
4) feed supplement is cultivated: fermented about 48 hours, with the feed supplement of 50-100ml/h speed, the control dissolved oxygen is at 20-30%.
Wherein the culture medium prescription of step 1) use is:
Inclined-plane prescription (g/L): potato 100; Glucose 10 grams; Agar 20.
Step 2 wherein) culture medium prescription that uses is:
Seed prescription (g/L): yeast powder 10; Sal epsom 2; Potassium primary phosphate 15; Glucose 20; Steeping water 10.
Trace element (ppm): ferrous sulfate 3; Copper sulfate 1.3; Boric acid 0.6; Manganous sulfate 3.1; Zinc sulfate 9.
Wherein the culture medium prescription of step 3) use is:
Fermentating formula (g/L): yeast powder 5; Sal epsom 0.6; Potassium primary phosphate 5; Glucose 10; Steeping water 18; Ammonium sulfate 3; Filter paper powder 50; Tween 1.
Trace element (ppm): ferrous sulfate 5; Copper sulfate 2; Boric acid 1; Manganous sulfate 5; Zinc sulfate 10.
Wherein the culture medium prescription of step 4) use is:
Feed supplement prescription (g/L): yeast powder 4; Sal epsom 0.6; Potassium primary phosphate 5; Lactose 5; Steeping water 10; Ammonium sulfate 3; Filter paper powder 30; Sophorose 3; Tween 1.
Trace element (ppm): ferrous sulfate 5; Copper sulfate 2; Boric acid 1; Manganous sulfate 5; Zinc sulfate 10.
The fermentation culture process comprises: under 28 ℃ of conditions, stirring velocity is 300r/min, and air flow is 1: 1; Fermented about 24 hours; Begin when cell concentration reaches 30g/L with the feed supplement of 50-100ml/h speed, the control dissolved oxygen is kept cell concentration 30g/L at 20-30%; Cultivated fermentation level 28FPAIU/ml 7 days.
Claims (7)
1. method that improves yield of cellulase; It is characterized in that; When Trichodermareesei ferments to certain cell concentration, play fermentation ends, the control cell concentration keeps stable, and the cell concentration of said fermentation strain is controlled at 10～30g/L; Improve the cellulose fermentation level, thereby improve the output of cellulase.
2. the method for claim 1 is characterized in that, said ferment to the time of certain cell concentration be 18-48 hour.
3. the method for claim 1 is characterized in that, the cell concentration of fermentation strain is controlled at 20g/L.
4. method as claimed in claim 2 is characterized in that, saidly is meant 24～48 hours when fermenting to certain cell concentration.
5. like the arbitrary described method of claim 1-4, it is characterized in that the control of said cell concentration realizes through control dissolved oxygen concentration and control feed supplement amount.
6. method as claimed in claim 5 is characterized in that said dissolved oxygen concentration is controlled at 20-30%; Said feed supplement amount is controlled at 50-100ml/h.
7. any application of described method in preparing liquid cellulase of claim 1-6.
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| CN102952791B (en) * | 2012-11-12 | 2014-12-03 | 熊鹏 | Method for replenishing nitrogen source culture fungus in batch to produce cellulase |
| CN112795491B (en) * | 2021-01-29 | 2023-03-17 | 武汉新华扬生物股份有限公司 | Fermentation method for producing high-activity acidic cellulase by trichoderma reesei |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250485A (en) * | 2008-04-16 | 2008-08-27 | 中国石油化工股份有限公司 | Trichoderma reesei cultivation method for improving yield of cellulase |
| CN101407797A (en) * | 2008-10-23 | 2009-04-15 | 肇东市日成酶制剂有限公司 | Method for producing cellulose, xylanase, glucanase and pectic enzyme by submerged fermentation of Trichoderma reesei |
| CN101418289A (en) * | 2008-11-28 | 2009-04-29 | 天津科技大学 | Trichoderma reesei liquid submerged fermentation cellulase and enzymatic beating process thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250485A (en) * | 2008-04-16 | 2008-08-27 | 中国石油化工股份有限公司 | Trichoderma reesei cultivation method for improving yield of cellulase |
| CN101407797A (en) * | 2008-10-23 | 2009-04-15 | 肇东市日成酶制剂有限公司 | Method for producing cellulose, xylanase, glucanase and pectic enzyme by submerged fermentation of Trichoderma reesei |
| CN101418289A (en) * | 2008-11-28 | 2009-04-29 | 天津科技大学 | Trichoderma reesei liquid submerged fermentation cellulase and enzymatic beating process thereof |
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