CN102041249A - Preparation of cellulase by trichoderma reesei - Google Patents
Preparation of cellulase by trichoderma reesei Download PDFInfo
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- CN102041249A CN102041249A CN2009101533549A CN200910153354A CN102041249A CN 102041249 A CN102041249 A CN 102041249A CN 2009101533549 A CN2009101533549 A CN 2009101533549A CN 200910153354 A CN200910153354 A CN 200910153354A CN 102041249 A CN102041249 A CN 102041249A
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Abstract
The title of the invention is preparation of cellulase by trichoderma reesei. The invention provides a novel method for producing cellulose, which comprises the following steps: preparing a suspension of trichoderma reesei, placing the suspension on a shaking table with a constant temperature of 30 DEG C, incubating for 48 h at a shaking speed of 180 r/min, pretreating corn stalks, and preparing a liquid fermentation medium for substrates by a modified Msndels nutrient salt; inoculating the liquid seeds in an inoculation amount of 10% into the fermentation medium, culturing for 24 h in a thermostatic shaking incubator at a temperature of 32 DEG C and a rotating speed of 150 r/min, decreasing the temperature to 28 DEG C and further culturing for 36 h; centrifuging the fermented liquid at 3000 r/min for 15 min to obtain a supernatant of crude enzyme solution, adding ammonium sulfate with a concentration of 60% (W/V) into the supernatant, allowing the solution to stand overnight, adding a proper amount of filtering agents, suction-filtering to obtain enzyme slurry, vacuum drying for 1 d at a temperature of 10 DEG C, and pulverizing to enzyme powder. The invention has a simple operation process and a high yield, and is applicable to large-scale industrialized production.
Description
One, technical field
The invention provides a kind of new cellulase preparation method, relate to the Production by Microorganism Fermentation cellulase.
Two, technical background
Cellulase (cellulase) refers to the general name of the glucogenic class of enzymes of degraded cellulose, and it is not single enzyme of planting, and is mainly to come from fungi and bacterium but play synergistic polycomponent enzyme.Wherein 3 kinds of main enzyme components are inscribe β-1,4-dextranase (Cx enzyme, CMC enzyme); Exoglucanase (also claiming avicelase); β one glucuroide.
Cellulase is at feed, and there is enormous and latent market aspects such as food, oil recovery, medicine, chemical industry, weaving.In ox, sheep, swine rations, add a certain amount of cellulase, can significantly improve the utilization ratio of feed, increase the weight that increases day by day, shorten fattening period, reduce feedstuff-meat ratio, can improve product hair rate of sheep and the milk yield of milk cow simultaneously.In fruit press, pollen beverage, add cellulase, can improve the extraction yield of juice, promote the juice clarification; Be that raw material makes soy sauce and adds cellulase in the process protein and carbohydrate in the cell are discharged with the soybean, so both can improve soy sauce concentration, improve quality of sauce, can shorten the production cycle again, improve productive rate; In oil recovery process, in order to increase oil production rate, the existing normal pressing filtering liquid of band pressure that adopts is pressed in the stratum, to keep subterranean fracture, keeps subsurface pressure.A kind of enzyme agent made from cellulase is suitable emulsion splitter.This emulsion splitter specificity is strong, has no side effect, to stratum and environmental nonpollution.In pulp and paper industry, the defibrination performance that cellulase can improve deinking efficiency, improve paper pulp and water filtering performance, be modified into paper physicals, improve into the tensile index of paper, the flexibility of tissue paper.
The a large amount of grain fermentative production of ethanol of domestic for a long time alcohol production enterprise's consumption, along with progressively promoting the use of of alcohol fuel, huge ethanol demand is remarkable day by day to the threat that the grain security of China forms, and therefore selecting the grain of fermenting alcohol to substitute becomes crucial problem.Utilize fermenting crops straws to produce the focus that alcohol fuel has become people's research in recent years, yet the cellulase preparation cost accounts for biological 50%～60% of the total cost that transforms of Mierocrystalline cellulose during with stalk fibre system ethanol, be about 50 times of stalk, cause the cellulose ethanol cost high.The cellulase of the deep liquid fermentation process production of generally adopting at present, its production cost still has certain distance apart from the saccharification of practical application cellulase hydrolysis.This research has been carried out further research to its optimum condition of enzyme production, in the hope of finding the most adaptable method of mutagenic strain cellulase-producing, for the industrial mass production cellulase provides reliable theoretical foundation on the basis that obtains the mutagenesis cellulase producing bacteria.The present invention utilizes Trichodermareesei fermentative production cellulase, and its production technique is simple, and productive rate is big, is suitable for scale operation.
Three, summary of the invention
1. utilize Trichodermareesei to prepare cellulase, its feature comprises following step:
(1) Trichodermareesei bacterial classification seed selection
Trichodermareesei (Triehoderma reesel Rut C 1) is seeded on the test tube slant of potato, glucose, agar, cultivates after 7 days preservation below 4 ℃ in refrigerator in 30 ℃ of thermostat containers.
(2) spore suspension preparation
To cultivate ripe inclined-plane washing, the vibration that is covered with spore with sterilized water, with the Hematocyte Counter counting, being diluted to spore count is 10
7～10
8Individual/mL.
(3) shake a bottle liquid seeds preparation
Inoculum size by 2% inserts spore suspension and is equipped with in the 2L triangular flask of 500mL nutrient solution, places 30 ℃ of constant temperature shaking tables then, 180r/min shaking culture 48h.
(4) configuration of nutrition saline solution
With MandelsShi nutritive salt is that the basis adds NaNO again
34.0g/l it is standby to be configured to a kind of new nutritive salt.
(5) maize straw pre-treatment
Earlier maize straw is carried out pre-treatment.With 0.75% rare H
2SO
4, (150 ℃) heating 50min on glycerol bath (with 30mL small steel cylinder dress maize peel, over dry material 5g), steel cylinder takes out back cooling (termination reaction) rapidly from glycerol bath, and hot water wash 3 times is expected air-dry standby.
(6) configuration liquid fermentation medium
MsndelsShi nutritive salt with improvement is substrate configuration liquid fermentation medium, adds the maize straw 80g/L that handles well, peptone 6g/L, and maltose 10g/L adds a certain amount of KH again
2PO
4/ K
2HPO
4Buffered soln is regulated PH in about 5.8, joins pack into the triangle of 1L of 200ml and shakes bottle.
(7) fermentation culture
Inoculum size with 10% will be cultivated sophisticated triangular flask liquid seeds and be inserted liquid fermentation medium, put into the isothermal vibration incubator and cultivate 24h under 32 ℃, 150r/min rotating speed, and downward modulation temperature to 28 ℃ continues to cultivate 36h.
(8) cellulase extracts
With the centrifugal 10min of 5000r/min, supernatant liquor is a crude enzyme liquid with fermented liquid, adds concentration ammonium sulfate to saturation ratio 20% in supernatant, gets supernatant again, adds ammonium sulfate to saturation ratio 60%, gets the Na that precipitation is dissolved in pH 6.0
2HPO
4-NaH
2PO
4In the damping fluid, obtain enzyme liquid, dialysis desalting also concentrates, and adds suitable filtering medium suction filtration and gets enzyme mud, places 10 ℃ of following vacuum-drying 1d, grinds to form the enzyme powder at last.
Operating procedure of the present invention is simple, and raw material sources are extensive, and production cost is low, and is pollution-free, is suitable for large-scale industrial production, is a kind of method of ideal production of cellulose enzyme.
Claims (5)
1. utilize Trichodermareesei to prepare cellulase, its feature comprises following step:
(1) Trichodermareesei bacterial classification seed selection
Trichodermareesei (Triehoderma reesel Rut C 1) is seeded on the test tube slant of potato, glucose, agar, cultivates after 7 days preservation below 4 ℃ in refrigerator in 30 ℃ of thermostat containers.
(2) spore suspension preparation
To cultivate ripe inclined-plane washing, the vibration that is covered with spore with sterilized water, with the Hematocyte Counter counting, being diluted to spore count is 10
7～10
8Individual/mL.
(3) shake a bottle liquid seeds preparation
Inoculum size by 2% inserts spore suspension and is equipped with in the 2L triangular flask of 500mL nutrient solution, places 30 ℃ of constant temperature shaking tables then, 180r/min shaking culture 48h.
(4) configuration of nutrition saline solution
With MsndelsShi nutritive salt is that the basis adds NaNO again
34.0g/l it is standby to be configured to a kind of new nutritive salt.
(5) maize straw pre-treatment
Earlier maize straw is carried out pre-treatment.With 0.75% rare H
2SO
4, (150 ℃) heating 50min on glycerol bath (with 30mL small steel cylinder dress maize peel, over dry material 5g), steel cylinder takes out back cooling (termination reaction) rapidly from glycerol bath, and hot water wash 3 times is expected air-dry standby.
(6) configuration liquid fermentation medium
MsndelsShi nutritive salt with improvement is substrate configuration liquid fermentation medium, adds the maize straw 80g/L that handles well, peptone 6g/L, and maltose 10g/L adds a certain amount of KH again
2PO
4/ K
2HPO
4Buffered soln is regulated PH in about 5.8, joins pack into the triangle of 1L of 200ml and shakes bottle.
(7) fermentation culture
Inoculum size with 10% will be cultivated sophisticated triangular flask liquid seeds and be inserted liquid fermentation medium, put into the isothermal vibration incubator and cultivate 24h under 32 ℃, 150r/min rotating speed, and downward modulation temperature to 28 ℃ continues to cultivate 36h.
(8) cellulase extracts
With the centrifugal 10min of 5000r/min, supernatant liquor is a crude enzyme liquid with fermented liquid, adds concentration ammonium sulfate to saturation ratio 20% in supernatant, gets supernatant again, adds ammonium sulfate to saturation ratio 60%, gets the Na that precipitation is dissolved in pH 6.0
2HPO
4-NaH
2PO
4In the damping fluid, obtain enzyme liquid, dialysis desalting also concentrates, and adds suitable filtering medium suction filtration and gets enzyme mud, places 10 ℃ of following vacuum-drying 1d, grinds to form the enzyme powder at last.
2. tell as claim (1), it is characterized in that used test tube slant substratum is the PDA slant medium.
3. tell as claim (4), it is characterized in that used nutrition saline solution is improved Msndels nutritive salt.
4. tell as claim (5), it is characterized in that used product enzyme substrates is a maize straw.
5. tell as claim (8), it is the highest to it is characterized in that extracting the cellulase extraction yield with 20%60% concentration sulphur ammonium earlier, can reach 99.8%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101533549A CN102041249A (en) | 2009-10-19 | 2009-10-19 | Preparation of cellulase by trichoderma reesei |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101533549A CN102041249A (en) | 2009-10-19 | 2009-10-19 | Preparation of cellulase by trichoderma reesei |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102041249A true CN102041249A (en) | 2011-05-04 |
Family
ID=43907797
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|---|---|---|---|
| CN2009101533549A Pending CN102041249A (en) | 2009-10-19 | 2009-10-19 | Preparation of cellulase by trichoderma reesei |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102229920A (en) * | 2011-07-21 | 2011-11-02 | 天津工业生物技术研究所 | Method for improving submerged fermentation level of trichoderma reesei cellulase liquid |
| CN103509770A (en) * | 2012-06-26 | 2014-01-15 | 湖南鸿鹰祥生物工程股份有限公司 | Method for producing cellulase through fermentation by adopting corn cob as main raw material |
| CN103509771A (en) * | 2012-06-26 | 2014-01-15 | 湖南鸿鹰祥生物工程股份有限公司 | Method for producing cellulase through solid state fermentation by adopting rice straws as main raw material |
| CN110396530A (en) * | 2018-04-25 | 2019-11-01 | 卢松 | A method of improving production amount of threonine and yield |
-
2009
- 2009-10-19 CN CN2009101533549A patent/CN102041249A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102229920A (en) * | 2011-07-21 | 2011-11-02 | 天津工业生物技术研究所 | Method for improving submerged fermentation level of trichoderma reesei cellulase liquid |
| CN102229920B (en) * | 2011-07-21 | 2013-03-13 | 天津工业生物技术研究所 | Method for improving submerged fermentation level of trichoderma reesei cellulase liquid |
| CN103509770A (en) * | 2012-06-26 | 2014-01-15 | 湖南鸿鹰祥生物工程股份有限公司 | Method for producing cellulase through fermentation by adopting corn cob as main raw material |
| CN103509771A (en) * | 2012-06-26 | 2014-01-15 | 湖南鸿鹰祥生物工程股份有限公司 | Method for producing cellulase through solid state fermentation by adopting rice straws as main raw material |
| CN103509771B (en) * | 2012-06-26 | 2017-05-17 | 湖南鸿鹰祥生物工程股份有限公司 | Method for producing cellulase through solid state fermentation by adopting rice straws as main raw material |
| CN110396530A (en) * | 2018-04-25 | 2019-11-01 | 卢松 | A method of improving production amount of threonine and yield |
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Application publication date: 20110504 |