CN101705215A - Xylanase/cellobiase composite enzyme and preparation method thereof - Google Patents
Xylanase/cellobiase composite enzyme and preparation method thereof Download PDFInfo
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- CN101705215A CN101705215A CN200910210153A CN200910210153A CN101705215A CN 101705215 A CN101705215 A CN 101705215A CN 200910210153 A CN200910210153 A CN 200910210153A CN 200910210153 A CN200910210153 A CN 200910210153A CN 101705215 A CN101705215 A CN 101705215A
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- wheat bran
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
The invention provides a xylanase/cellobiase composite enzyme prepared by using Aspergillus niger CGMCC 3.3147 and a preparation method thereof. The xylanase and the cellobiase can be efficiently co-produced through the Aspergillus niger, wherein the produced xylanase can reach 10,000 IU/g and the cellobiase can reach 900 IU/g through a preferable fermentation culture medium; and the prepared composite enzyme can be mixed-used directly with the cellobiase in the production of straw fermented alcohol, xylitol, citric acid and the like. The composite enzyme and the preparation method have great realistic significance for solving the problems of environment pollution, food shortage and energy crisis.
Description
Technical field
The present invention relates to biological field, specifically, relate to a kind of zytase/cellobiase prozyme and preparation method thereof.
Background technology
The content of xylan in plant cell wall is only second to Mierocrystalline cellulose, is the abundantest hemicellulose of content.Mierocrystalline cellulose, hemicellulose, xylogen account for 30～40%, 15～30%, 10～20% respectively in the cellulose raw material.Wherein xylan is the chief component of hemicellulose.Xylan is the polymkeric substance of being made up of D-wood sugar main chain (linking to each other with β-1,4 key) and L-arabinose branch (a-1,2 and a-1,3 is continuous).It is combined in the surface of cellulose micro-fibers, and interconnects, and has constituted the interconnective network of hard cell.This hard network has hindered cellulase to cellulose adsorption, thereby has influenced cellulosic degradation rate.Cellulose raw material zymamsis at present mainly is that the hexose that utilizes the cellulose degraded Mierocrystalline cellulose to obtain carries out zymamsis, and hemicellulose wherein is not fully used, and has caused the wasting of resources and serious environmental to pollute.
Zytase is the enzyme of single-minded degradation of xylan, belongs to hydrolase, xylan can be decomposed into wood oligose.Zytase is the macromole activated protein with quaternary structure, and mainly by β-1,4-D-endo-xylanase, β-1, circumscribed xylosidase of 4-D-and α-L-arabinose glycosides enzyme, α-D-amino acid aldehydic acid enzyme etc. take off the side chain enzyme and form.Zytase is by destroying the covalent cross-linking (pectinose residue replacement district) in the xylan molecule and by the joining region that hydrogen bond forms, making xylan degrading.As a kind of industrial enzymes, zytase has important use in ethanol fermentation, fodder industry and foodstuffs industry etc. and is worth.
Cellulase is a kind of multi-component prozyme, it mainly comprises endo-type dextranase (EC3.2.1.4, also claim Cx enzyme, CMC enzyme), circumscribed-type dextranase (EC3.2.1.91 also claims C1 enzyme, avicelase) and cellobiase (EC3.2.1.21 also claims beta-glucosidase).Relevant cellulosic enzymic hydrolysis mechanism is not studied clear so far yet fully, but generally believes and natural cellulose is being hydrolyzed in the process of glucose, must rely on the synergy of above-mentioned 3 kinds of components just can finish.Cellulose macromolecule at first progressively is degraded into cellobiose under the effect of C1 enzyme and Cx enzyme, cellobiase then further is hydrolyzed into cellobiose 2 glucose.Because cellobiose has had strong inhibitory effects to C1 enzyme and Cx enzyme activity, will have a strong impact on cellulosic degradation rate if lack the cellobiase component in the cellulase plural components.And lack the cellobiase component in the present known cellulase production bacterial strain, so be necessary to add the cellobiase of external source in the production process.
In the present cellulose raw material zymamsis, produce the appearance of the recombination yeast of alcohol along with assimilating five-carbon sugar (mainly being wood sugar), hemicellulose is expected to be fully used, but, be wood sugar with xylan degrading so need to add zytase because yeast can not directly utilize hemicellulose.Exploitation therefore cheap, high-effective xylanase just becomes very urgent.
The present invention is directed to the problem that the cellulose raw material zymamsis is run into, a kind of zytase/cellulase prozyme and preparation method thereof is provided.
Summary of the invention
The purpose of this invention is to provide the zytase/cellobiase prozyme that utilizes Aspergillus niger strain CGMCC3.3147 preparation.
The present invention also at the deficiency of cellulose raw material pre-treatment with zymotechnic, provides a kind of easy and simple to handle, and cost is low, the enzyme activity height, and the solid fermentation of enzyme reasonable mixture ratio of components is produced the method for zytase/cellobiase prozyme.
The present invention is a starting strain with aspergillus niger (Aspergillus niger) CGMCC3.3147, with this bacterial strain at corn cob, wheat straw, cultivate on the agriculture and forestry organic waste materials such as straw, to wheat bran in the culture medium prescription, urea, ammonium sulfate, potassium primary phosphate, sal epsom, cobalt chloride, initial pH, moisture totally 8 factors (wherein two are culture condition) is studied, select for use the Plackett-Burman of N=12 to use design, screening influences the key factor of zytase and disaccharidase output, result according to the Plackett-Burman experimental analysis utilizes the steepest hill climbing to determine the central point of center combination test, approach the peak response zone, last design centre combination experiment, simulate a dihydric phenol polynomial equation, find out the optimal medium prescription.The optimization fermentative medium formula that obtains by aforesaid method is:
Agricultural waste material 50～80%, wheat bran 15～45%, urea 1～3%, ammonium salt 0.2～1%, phosphoric acid salt 0.03～0.1%, sal epsom 0.002～0.008%, cobalt chloride 0.0001～0.001%, excess water, initial pH5.0～7.0; After add entry to adjust moisture be 60～80%.
Also comprise trace element in the described fermention medium, as sal epsom, cobalt chloride etc., the content of general sal epsom is 0.002%-0.004%; The content of cobalt chloride is generally 0.0001%-0.0005%.
Agricultural waste material of the present invention comprises the remainder behind the results farm crop seeds such as maize straw, corn cob, wheat stalk, straw;
Described ammonium salt is ammonium sulfate and/or ammonium nitrate.
Described phosphoric acid salt is dipotassium hydrogen phosphate and/or potassium primary phosphate.
Described fermention medium is the solid fermentation substratum.
The preparation method of zytase of the present invention/cellobiase prozyme is: the aspergillus niger mutagenic strain is seeded in the above-mentioned fermention medium by 1～15% inoculum size, cultivated 4～5 days at 26-40 ℃; Cultivate according to ordinary method then and produce prozyme, wherein, inoculum size preferred 10%.
With inoculation to fermention medium, the preparation method of zytase of the present invention/cellobiase prozyme also comprises the variation aspergillus niger is inserted earlier in the wheat bran substratum by 1～2% inoculum size, cultivates 5-10 days 20-30 ℃ of following condition.
Described wheat bran substratum contains agricultural waste material 50～80%, wheat bran 15～45%, urea 1～3%, ammonium salt 0.2～1%, phosphoric acid salt 0.03～0.1%, excess water.
In culturing process, wheat bran substratum and fermention medium can adopt the solid medium of identical component.
Also comprise trace element in the described wheat bran substratum, as sal epsom, cobalt chloride etc., the content of general sal epsom is 0.002%-0.004%; The content of cobalt chloride is generally 0.0001%-0.0005%.
Wherein, described ammonium salt is ammonium sulfate and/or ammonium nitrate, preferred ammonium nitrate.
Described phosphoric acid salt is dipotassium hydrogen phosphate and/or potassium primary phosphate, preferred dipotassium hydrogen phosphate.
Key point of the present invention is to utilize bacterial classification CGMCC3.3147 and optimizes culture medium prescription, makes zytase and cellobiase high yield simultaneously, and it produces the enzyme zytase can reach 10,000IU/g, and cellobiase can reach 900IU/g.
The present invention is by Aspergillus niger strain CGMCC3.3147 efficiently co-producing xylanase and cellobiase, and by preferred fermention medium, it produces enzyme zytase can reach 10,000IU/g, and cellobiase can reach 900IU/g; The prozyme of preparation can directly mix with cellulase in the productions such as being used in stalk fermentation alcohol, Xylitol, citric acid. and solve environmental pollution, food shortage and energy dilemma for the mankind and be of great immediate significance.
Description of drawings
Fig. 1 is zytase/cellobiase prozyme production process route figure.
Embodiment
For the ease of understanding the present invention, the present invention is described in further detail below in conjunction with embodiment.These embodiment just play the effect of explanation, and the present invention is not limited to these embodiment.
The bacterial strain that is adopted among the following embodiment is aspergillus niger (Aspergillus niger) CGMCC3.3147 (available from a Chinese common micro-organisms culture presevation administrative center); Employed method is the normal experiment method if no special instructions.
Embodiment 1
The aspergillus niger CGMCC3.3147 strain inclined plane that selection is grown fine, the picking slant pore inserts in the wheat bran bottle of the substratum that contains 20% wheat bran and 70% maize straw gently, tampon and bandage kraft paper beyond the Great Wall, firmly shaking up the wheat bran bottle is uniformly dispersed spore in substratum, in moving to the wheat bran bottle between cultivation, put 27 ℃ and cultivate down.Cultivate the 10th day spore and cover with fully that to move into refrigerating chamber behind the whole substratum standby.
The composition of solid fermentation substratum is 20% wheat bran, 70% maize straw, urea 1%, ammonium sulfate 0.2%, potassium primary phosphate 0.03%, sal epsom 0.002%, cobalt chloride 0.0001%, excess water, initial pH5.6.
Stir moisture 65% after adding water.Solid medium is packed in the tray into 121 ℃ of autoclavings 60 minutes.Sterilized water is poured in the wheat bran bottle, after stirring, be poured on the tray.Solid medium and wheat bran bacterial classification are mixed thoroughly, paved, and cover lid, was cultivated 10 days by 27 ℃.
Cultured prozyme added to pour into behind 1～2 times of mixing of water carry out enzymolysis in the pretreating reacting container.
Embodiment 2
The aspergillus niger CGMCC3.3147 strain inclined plane that selection is grown fine, the picking slant pore inserts and contains in the wheat bran bottle of 20% wheat bran and 70% wheat stalk substratum gently, tampon and bandage kraft paper beyond the Great Wall, firmly shaking up the wheat bran bottle is uniformly dispersed spore in substratum, in moving to the wheat bran bottle between cultivation, put 28 ℃ and cultivate down.Cultivate the 7th day spore and cover with fully that to move into refrigerating chamber behind the whole substratum standby.
The solid culture based component is that 20% wheat bran, 70% wheat stalk, urea 1.5%, ammonium sulfate 0.2%, potassium primary phosphate 0.04%, sal epsom 0.003%, cobalt chloride 0.0002%, surplus are water, initial pH5.6.Stir moisture 70% after adding water.
Solid medium is packed in the tray into 121 ℃ of autoclavings 60 minutes.Sterilized water is poured in the wheat bran bottle, after stirring, be poured on the tray.Solid medium and wheat bran bacterial classification are mixed thoroughly, paved, and cover lid, was cultivated 7 days by 28 ℃.
Embodiment 3
The aspergillus niger CGMCC3.3147 strain inclined plane that selection is grown fine, the picking slant pore inserts and contains in the wheat bran bottle of 30% wheat bran and 60% straw substratum gently, tampon and bandage kraft paper beyond the Great Wall, firmly shaking up the wheat bran bottle is uniformly dispersed spore in substratum, in moving to the wheat bran bottle between cultivation, put 27 ℃ and cultivate down.Cultivate the 10th day spore and cover with fully that to move into refrigerating chamber behind the whole substratum standby.
The solid culture based component is that 30% wheat bran, 60% straw, urea 2.0%, ammonium sulfate 0.1%, potassium primary phosphate 0.04%, sal epsom 0.003%, cobalt chloride 0.0005%, surplus are water, initial pH6.0.
Stir moisture 75% after adding water.Solid medium is packed in the tray into 121 ℃ of autoclavings 60 minutes.Sterilized water is poured in the wheat bran bottle, after stirring, be poured on the tray.Solid medium and wheat bran bacterial classification are mixed thoroughly, paved, and cover lid, was cultivated 10 days by 27 ℃.
Embodiment 4
The aspergillus niger CGMCC3.3147 strain inclined plane that selection is grown fine, the picking slant pore inserts and contains in the wheat bran bottle of 15% wheat bran and 80% corn cob substratum gently, tampon and bandage kraft paper beyond the Great Wall, firmly shaking up the wheat bran bottle is uniformly dispersed spore in substratum, in moving to the wheat bran bottle between cultivation, put 29 ℃ and cultivate down.Cultivate the 8th day spore and cover with fully that to move into refrigerating chamber behind the whole substratum standby.
The solid culture based component is that 15% wheat bran, 80% corn cob, urea 1.5%, ammonium sulfate 0.2%, potassium primary phosphate 0.04%, sal epsom 0.003%, cobalt chloride 0.0005%, surplus are water, initial pH6.0.
Stir moisture 75% after adding water.Solid medium is packed in the tray into 121 ℃ of autoclavings 60 minutes.Sterilized water is poured in the wheat bran bottle, after stirring, be poured on the tray.Solid medium and wheat bran bacterial classification are mixed thoroughly, paved, and cover lid, was cultivated 8 days by 29 ℃.
Claims (9)
1. the preparation method of zytase/cellobiase prozyme comprises: Aspergillus niger strain CGMCC3.3147 is connected in the fermention medium by 1～15% inoculum size, cultivated 4～5 days at 26～40 ℃, produce out prozyme.
2. preparation method as claimed in claim 1, it is characterized in that, described fermention medium contains following component in siccative: agricultural waste material 50～80%, wheat bran 15～45%, urea 1～3%, ammonium salt 0.2～1%, phosphoric acid salt 0.03～0.1%, sal epsom 0.002～0.008%, cobalt chloride 0.0001～0.001%, excess water, initial pH5.0～7.0; After add entry to adjust moisture be 60～80%.
3. preparation method as claimed in claim 2 is characterized in that, described fermention medium is a solid medium.
4. preparation method as claimed in claim 2 is characterized in that described agricultural waste material is selected from one or more in maize straw, corn cob, wheat stalk and the straw.
5. preparation method as claimed in claim 2 is characterized in that, described ammonium salt is ammonium sulfate and/or ammonium nitrate; Described phosphoric acid salt is dipotassium hydrogen phosphate and/or potassium primary phosphate.
6. preparation method as claimed in claim 1 is characterized in that, with inoculation to fermention medium, also comprise Aspergillus niger strain CGMCC3.3147 inserted in the wheat bran substratum by 1～2% inoculum size earlier, cultivated 5～10 days 20～30 ℃ of following conditions.
7. preparation method as claimed in claim 6, it is characterized in that, described wheat bran substratum contains agricultural waste material 50～70%, wheat bran 15～45%, urea 1～3%, ammonium salt 0.2～1%, phosphoric acid salt 0.03～0.1%, excess water, wherein said agricultural waste material are to be selected from maize straw, wheat stalk, corn cob and the straw one or more.
8. zytase/cellobiase prozyme as the arbitrary described preparation method preparation of claim 1～7.
9. the zytase as claimed in claim 8/application of cellobiase prozyme in stalk fermentation alcohol, Xylitol, citric acid production.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910210153.8A CN101705215B (en) | 2009-10-29 | 2009-10-29 | Xylanase/cellobiase composite enzyme and preparation method thereof |
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| CN200910210153.8A CN101705215B (en) | 2009-10-29 | 2009-10-29 | Xylanase/cellobiase composite enzyme and preparation method thereof |
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| CN101705215A true CN101705215A (en) | 2010-05-12 |
| CN101705215B CN101705215B (en) | 2015-06-17 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102392004A (en) * | 2011-10-27 | 2012-03-28 | 安徽丰原发酵技术工程研究有限公司 | Composite enzyme of cellulase, xylanase and cellobiase, as well as preparation method thereof |
| CN102839132A (en) * | 2012-09-21 | 2012-12-26 | 中粮生物化学（安徽）股份有限公司 | Aspergillus niger moldy bran culture medium and preparation method thereof, and Aspergillus niger moldy bran culture method |
| CN103233049A (en) * | 2013-05-10 | 2013-08-07 | 天津科技大学 | Method for fermentation production of xylitol using aureobasidium pullulans mutant strain |
| US10759727B2 (en) | 2016-02-19 | 2020-09-01 | Intercontinental Great Brands Llc | Processes to create multiple value streams from biomass sources |
-
2009
- 2009-10-29 CN CN200910210153.8A patent/CN101705215B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| MUNISWARAN等: "PRODUCTION OF CELLULASES FROM COCONUT COIR PITH IN SOLID-STATE FERMENTATION", 《JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY》 * |
| 黄林等: "玉米芯酶解糖化条件研究", 《中国酿造》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102392004A (en) * | 2011-10-27 | 2012-03-28 | 安徽丰原发酵技术工程研究有限公司 | Composite enzyme of cellulase, xylanase and cellobiase, as well as preparation method thereof |
| CN102839132A (en) * | 2012-09-21 | 2012-12-26 | 中粮生物化学（安徽）股份有限公司 | Aspergillus niger moldy bran culture medium and preparation method thereof, and Aspergillus niger moldy bran culture method |
| CN103233049A (en) * | 2013-05-10 | 2013-08-07 | 天津科技大学 | Method for fermentation production of xylitol using aureobasidium pullulans mutant strain |
| CN103233049B (en) * | 2013-05-10 | 2014-12-10 | 天津科技大学 | Method for fermentation production of xylitol using aureobasidium pullulans mutant strain |
| US10759727B2 (en) | 2016-02-19 | 2020-09-01 | Intercontinental Great Brands Llc | Processes to create multiple value streams from biomass sources |
| US11840500B2 (en) | 2016-02-19 | 2023-12-12 | Intercontinental Great Brands Llc | Processes to create multiple value streams from biomass sources |
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| CN101705215B (en) | 2015-06-17 |
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