CN1036792A - The manufacture method of high active cellulase - Google Patents
The manufacture method of high active cellulase Download PDFInfo
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- CN1036792A CN1036792A CN 88105339 CN88105339A CN1036792A CN 1036792 A CN1036792 A CN 1036792A CN 88105339 CN88105339 CN 88105339 CN 88105339 A CN88105339 A CN 88105339A CN 1036792 A CN1036792 A CN 1036792A
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- fermentation
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- substratum
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Abstract
A kind of manufacture method of high active cellulase, utilize the fermentation residue that left behind in the process of natural cellulose class fermentation of materials production of cellulose enzyme, multiple be used for again replacing the natural cellulose class material in the substratum and use as carbon source, carry out the fermentative production cellulase, can not only obtain having more highly active cellulase, can save fermentation raw material simultaneously again and solve the problem of environmental pollution that brings owing to fermentation residue.
Description
The invention belongs to the production technique of microorganism.
At present, the production of cellulase the natural cellulose class materials that cellulase is had an inducing action of adopting carry out as the solid fermentating mode of carbon source more.These natural cellulose substances such as corn stalk powder, wheat straw powder, straw powder, sorghum straw powder or the like, after fermentation, carry over as a large amount of residues.Because these residues are by microbiological degradation, sharp exhausted, be difficult to the refuse that decomposes again and utilizes so become, will not cause the serious environmental pollution as if not handling.Japan once speciallyyed permit (clear 58-42759) Suzuki and repaiied people's such as Tibetan invention: utilize the residue of cellulase hydrolysis natural cellulose to use as the carbon source of substratum, produce cellulase.Though utilized residue like this, and can shorten fermentation period, can not improve enzyme and live.And more crucial is, they come the hydrolysis natural cellulose with cellulase earlier, and this needs a large amount of enzymes, so production cost is quite high, is difficult to application at present.And because the cellulase that utilizes the natural cellulose residue after the hydrolysis to produce, its enzyme is lived and is not significantly improved, and has also influenced applying of this technology.
The present invention is intended to overcome the deficiency of prior art, utilize the fermentation residue that left behind in the process of natural cellulose class fermentation of materials production of cellulose enzyme, the multiple natural cellulose class material that is used for again replacing in the substratum, produce cellulase, it can be turned waste into wealth, and fermentation residue is utilized again, overcome the pollution problem that brings thus, can save fermentation raw material exponentially again, reduce production costs, can also produce active higher cellulase simultaneously.
Fungal cellulase is an inducible enzyme, the mould low molecular weight carbohydrate that can utilize glucose etc. easily to be absorbed, to utilize well of wood, but the cellulase activity of producing is very low.In order to produce high active cellulase, usually inductive substance-the natural cellulose of cellulase is used as the carbon source of substratum.Although natural cellulose class material is difficult to decompose, to utilize, natural cellulose class material has stronger inducing action to cellulase.It is generally acknowledged that utilize the content of xylogen high more, cellulosic degree of crystallinity is high more, difficult more by the natural cellulose material of microbiological degradation, utilization, the activity of the cellulase of being produced is also high more.Use the natural cellulose material and do carbon source, it is mould to cultivate wood, and during the production of cellulose enzyme, microorganism is in growth and development process, lower the monose that easily is absorbed in the natural cellulose, utilizes, low molecule oligosaccharides and degree of crystallinity, the Mierocrystalline cellulose that utilizes that easily is decomposed preferentially decomposes utilization.Thereby, just contain Mierocrystalline cellulose and the xylogen that more difficulty is decomposed in the fermentation residue that utilizes that is not decomposed that the fermentation back is left.If utilize these to be difficult to the fermentation residue that is utilized by microbiological degradation under normal conditions, replace the natural cellulose class material in the substratum, when carrying out the fermentative production cellulase, just can obtain having more highly active cellulase.Can save simultaneously fermentation raw material again and solve the problem of environmental pollution that brings owing to fermentation residue.
The bacterial classification that the present invention uses has wood mould (Trichoderma T and Trichoderma S), aspergillus niger (Aspergillus niger P
1), and Penicillium notatum (Penicillium Ju) (above bacterial strain be this school preserve bacterial classification).
The natural cellulose class material that the present invention uses has usefulness pulverizer powder such as corn stalk, wheat straw, straw, sorghum straw to become chaff, also has the MandelsShi nutritive medium in addition, and it is composed as follows:
（NH
4）
2SO
4～1.4g
KH
2PO
4～2.0g
MgSO
4·7H
2o～0.3g
Urea～0.3g
CaCl
2～0.3g
Trace element: FeSO
47H
2O～5mg
MnSO
4·H
2O～1.56mg
ZnSO
4～1.4mg
CoCl
2～2.0mg
Water 1l
The residue of the natural cellulose class material after fermentation, the analytical results of its composition is an example with the corn stalk powder, lists in the subordinate list 1.By table 1 as seen, along with the increase of the fermentation times of natural cellulose class material, because low molecule carbohydrate and the labile cellulosic utilization of part make it content of cellulose and reduced, the total reducing sugar amount has also reduced.And the content of xylogen has increased, and Protein content also increases to some extent because of the increase of tropina.
Accompanying drawing is a process flow sheet of the present invention.Wherein, 1～natural cellulose class material, 2～nutritive medium 3～sterilization rotary spherical digester 4～proving room 5～extractor 6～water 7～enzyme liquid
As shown in the figure, natural cellulose class material 1 Ensure Liquid liquid 2 is made fermention medium, after inserting in the rotary spherical digester 3 sterilization, cooling, inoculation, be transported to fermentation in the proving room 4 again, fermentation finishes, and delivers in the extractor 5, adds water 6 and carries out suction filtration or squeezing, enzyme liquid 7 is separated from fermented product, and obtained fresh fermentation residue, these fermentation residues are directly delivered in the sterilization rotary spherical digester 3 (perhaps that it is air-dry standby), by controlling 50～75% water content, and after adding the nutritive medium 2 identical, use the technological process below repeating again as the carbon source of substratum with former substratum.
The measuring method of cellulase activity, with 20mg Xinhua filter paper is substrate, the acetate buffer solution 0.9ml that adds pH4.8 0.1M, add the enzyme liquid 0.1ml that diluted through suitable again, 40 ℃ of reactions 30 minutes, with 3,5-dinitrosalicylic acid method (DNS method) is measured the reducing sugar that generates, with glucose as a standard sugar.Enzymic activity represents with international unit, and promptly to produce the glucose of 1 μ mol be an international unit IU to per minute.
Be a most preferred embodiment of the present invention below.
Corn stalk meal (25 order) 100g that takes pulverizer powder one-tenth adds Mandels nutrition and crosses 200ml, pack in the triangular flask of 5l, in 120 ℃ of sterilizations 15 minutes, behind the inoculation wood mould (Trichoderma T), in 28 ℃ of fermentations 3 days, fermentation finishes, add water 1000ml, place 2～3 hours (often stirring) under the room temperature, filter with nylon cloth then, it is stand-by to leach enzyme liquid, and obtains the 81.2g fermentation residue.Again one time fermentation residue 50g is added Mandels nutritive medium 100ml, pack into the triangular flask of 1l, sterilization, inoculation and culturing process above repeating, add the flooding enzyme then, again after filtration, enzyme and residue are separated, promptly obtained Secondary Fermentation residue 45.4g, move in circles successively, can obtain fermentation residue, the 4th fermentation residue for the third time ...
In order to compare the activity of the cellulase of producing by corn stalk powder and fermentation residue, take respectively method for preparing once, secondary, three fermentation residues and each 10g of former corn stalk powder, add Mandels nutritive medium 20ml, pack in the triangular flask of 250ml, by above-mentioned similarity condition sterilization, inoculation (Trichoderma T), in 28 ℃ of cultivations 4 days, record activity and the results are shown in the subordinate list 2.
Claims (3)
1, a kind of manufacture method of high active cellulase, adopt cellulose substances as the carbon source in the substratum, technological process through sterilization, inoculation, fermentation, suction filtration, produce cellulase, it is characterized in that, the fermentation residue of natural cellulose class material with after enzyme liquid separates, is used as the carbon source in the substratum again again, repeatedly repeat the process of fermenting, produce high active cellulase.
2, the manufacturing processed of high active cellulase according to claim 1, it is characterized in that, the carbon source that above-mentioned fermentation residue is done in the substratum is again used when fermenting once more, the water content that needs control 50～75% with add the MandelsShi nutritive medium identical with former substratum.
3, the manufacture method of high active cellulase according to claim 1 is characterized in that, the carbon source that above-mentioned fermentation residue is done in the substratum is again used when repeatedly repeating to ferment, and needs once more the technological process through sterilization, inoculation, fermentation, suction filtration.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 88105339 CN1036792A (en) | 1988-09-23 | 1988-09-23 | The manufacture method of high active cellulase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 88105339 CN1036792A (en) | 1988-09-23 | 1988-09-23 | The manufacture method of high active cellulase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1036792A true CN1036792A (en) | 1989-11-01 |
Family
ID=4833542
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 88105339 Pending CN1036792A (en) | 1988-09-23 | 1988-09-23 | The manufacture method of high active cellulase |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1049684C (en) * | 1996-11-13 | 2000-02-23 | 山东大学 | Preparing method of high active cellulase |
| CN1298846C (en) * | 2005-01-18 | 2007-02-07 | 清华大学 | Process for producing cellulase by semicontinuous solid fermentation-extraction coupling |
| CN1298847C (en) * | 2005-01-18 | 2007-02-07 | 清华大学 | Process for electric field enhanced solid fermentation preparation of cellulase |
| CN1302105C (en) * | 2005-02-28 | 2007-02-28 | 国家粮食局科学研究院 | High-activity cellulase and its preparation method |
| CN104024399A (en) * | 2011-10-14 | 2014-09-03 | Ifp新能源公司 | Method for the continuous production of cellulases by a filamentous fungus using a carbon substrate obtained from an acid pretreatment |
| US9200296B2 (en) | 2006-05-26 | 2015-12-01 | Amyris Inc. | Production of isoprenoids |
-
1988
- 1988-09-23 CN CN 88105339 patent/CN1036792A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1049684C (en) * | 1996-11-13 | 2000-02-23 | 山东大学 | Preparing method of high active cellulase |
| CN1298846C (en) * | 2005-01-18 | 2007-02-07 | 清华大学 | Process for producing cellulase by semicontinuous solid fermentation-extraction coupling |
| CN1298847C (en) * | 2005-01-18 | 2007-02-07 | 清华大学 | Process for electric field enhanced solid fermentation preparation of cellulase |
| CN1302105C (en) * | 2005-02-28 | 2007-02-28 | 国家粮食局科学研究院 | High-activity cellulase and its preparation method |
| US9200296B2 (en) | 2006-05-26 | 2015-12-01 | Amyris Inc. | Production of isoprenoids |
| US10106822B2 (en) | 2006-05-26 | 2018-10-23 | Amyris, Inc. | Production of isoprenoids |
| CN104024399A (en) * | 2011-10-14 | 2014-09-03 | Ifp新能源公司 | Method for the continuous production of cellulases by a filamentous fungus using a carbon substrate obtained from an acid pretreatment |
| CN104024399B (en) * | 2011-10-14 | 2016-10-19 | Ifp新能源公司 | Use the method that the carbon substrate obtained from low-kappa number is produced cellulase continuously by filamentous fungi |
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