CN102127532A - Method for using transgenic Coprinus cinereus to efficiently express recombinant enzyme - Google Patents
Method for using transgenic Coprinus cinereus to efficiently express recombinant enzyme Download PDFInfo
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Abstract
The invention belongs to the field of microbial fermentation engineering, and relates to a preparation method of a recombinant multi-function cellulase. The method is characterized in that multi-function cellulase genes are used to carry out genetic transformation on Coprinus cinereus to obtain a transgenic Coprinus cinereus strain which is used as a producing strain, a mixture of bagasse powder and bean pulp powder is the optimal culture medium, and the recombinant multi-function cellulase is prepared by shake fermentation under the optimal culture conditions. The formula of the bagasse powder and bean pulp powder mixed culture medium comprises 27.9-30.0g/L bagasse powder, 4.3-6g/L bean pulp powder, 0.5-1g/L dipotassium hydrogen phosphate, 0.23-0.69g/L potassium dihydrogen phosphate and 0.5-1g/L magnesium sulfate, and the pH value is 6.25-7.82. The optimal culture conditions are as follows: the culture temperature is 36-37 DEG C, the culture time is 7 days, and the rotation speed of a shaking table is 210r/min. The optimal enzyme yields are as follows: the enzyme activity of Xylanase is 1.66*10<5>U/L, and the enzyme activity of CMCase is 1.7*10<4>U/L. The method provided by the invention can effectively enable the transgenic Coprinus cinereus strain to produce the recombinant multi-function cellulase from low-cost agricultural waste bagasse and bean pulp powder.
Description
Technical field
The invention belongs to the microbial fermentation engineering field, be specifically related to a kind of method of utilizing transgenosis Coprinus cinereus bacterial strain to efficiently express the reorganization multifunctional cellulase.Utilize bagasse and dregs of beans to efficiently express the method for reorganization multifunctional cellulase.
Background technology
Mierocrystalline cellulose, hemicellulose are one of the abundantest renewable resourcess of nature, are the main components of plant cell wall.The earth produces several hundred million tons plant dry matter (polysaccharose substance) by photosynthesis every year, and its main component is exactly Mierocrystalline cellulose and hemicellulose.The degraded of the zymetology of Mierocrystalline cellulose and hemicellulose is again generally acknowledge at present the most effective, the method for transformation of cleaning.Its biological degradation needs the effect of cellulase.And cellulase is an a kind of prozyme system, mainly comprises inscribe-β-1,4-D-dextranase (E.C. 3.2.1.4), circumscribed-β-1,4-D-dextranase (E.C. 3.2.9.11) and beta-glucosidase (E.C. 3.2.1.21).Cellulosic degraded needs the synergy of these enzymes just can finish, and the single enzyme component is very poor to cellulosic degradation capability.Multifunctional cellulase (MFC) has endo-beta-1,4-glucanase, circumscribed-β-1 simultaneously, and 4-dextranase and inscribe-β-1, and 3 kinds of enzymic activitys of 4-zytase can efficiently be decomposed natural cellulose.
At present the main production bacterial strain of production of cellulose enzyme have that wood is mould, mould and inulinase, production method is to obtain cellulase by solid fermentation or liquid fermenting to these filamentous funguss, because the cellulase components that these filamentous funguss produced is single, the vigor of enzyme is lower in actual applications, degraded cellulose (cellulosic as mentioned above degraded needs the synergy of plurality of enzymes just can finish) effectively, and complex manufacturing, the production cost height of cellulase at present.The multifunctional cellulase that transgenosis Coprinus cinereus bacterial strain of the present invention is expressed has endo-beta-1,4-glucanase, circumscribed-β-1 simultaneously, and 4-dextranase and inscribe-β-1,4-zytase 3 kinds of enzymic activitys, degraded celluloses efficiently.Simultaneously, the substratum that the present invention uses is agricultural wastes bagasse and dregs of beans, and production cost is low.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of method of utilizing transgenosis Coprinus cinereus bacterial strain to efficiently express reorganization multifunctional fibre element is provided.
The present invention is achieved through the following technical solutions above-mentioned purpose:
Invention provides a kind of transgenosis Coprinus cinereus to efficiently express the method for recombinase, multifunctional cellulase gene transformation Coprinus cinereus obtains transgenosis Coprinus cinereus bacterial strain, as producing bacterial strain, with bagasse powder and dregs of beans be main raw material as substratum, shake flask fermentation express recombinant multifunctional cellulase.
The prescription of described substratum is: bagasse powder (20 order) 27.9-30g/L, bean cake powder (40 order) 4.3-6g/L, dipotassium hydrogen phosphate 0.5-1.5g/L, potassium primary phosphate 0.23-0.69g/L, sal epsom 0.5-1.5g/L, pH value 6.25-7.82; The condition of shake flask fermentation is leavening temperature 36-37 ℃, fermentation time 5-7d.Optimal culture condition is: 37 ℃ of culture temperature, incubation time 7d, shaking speed 180-210 r/min.
Described transgenosis Coprinus cinereus bacterial strain bacterial classification 84-96h in age, the inoculum size of bacterial strain in substratum is 1-1.5%, i.e. the Coprinus cinereus bacterium liquid of the inoculation of medium 1-1.5mL of every 100mL, bacterium dry weight 0.1-0.15g.This transgenosis Coprinus cinereus bacterial strain is that multifunctional cellulase gene transformation Coprinus cinereus obtains, its preparation method can referring to Yang Pei week Ph D dissertation (the multifunctional cellulase gene (
Mfc) the clone and the expression study in Coprinus cinereus, Agricultural University Of South China's Ph D dissertation, in June, 2008).
Can adopt following preferred version to carry out fermentative production:
(1) with described transgenosis Coprinus cinereus inoculation on the Coprinus cinereus solid medium, cultivate 7d for 37 ℃, inoculation 1-5% bacterial classification in the 100mL seed culture medium, 37 ℃, 210r/min shaking culture 84-96h;
(2) preparation bagasse substratum, bagasse powder (20 order) 27.9-30g/L, bean cake powder 4.3-6g/L, dipotassium hydrogen phosphate 1g/L, potassium primary phosphate 0.46g/L, sal epsom 1g/L, pH value 6.25-7.82, be sub-packed in the 250mL triangular flask every bottled liquid measure 100mL, autoclaving;
(3) every bottle of bacterial classification as cultivating in the culture medium inoculated 1mL step (1) of step (2) in ultra-clean work, inoculation is placed on 36-37 ℃ of following shaking table and cultivates shaking speed 210r/min.
(4) cultivating 7d stops cultivating centrifugal collection fermented liquid.
Coprinus cinereus solid culture based formulas wherein is that the material of following parts by weight constitutes: 10 parts of glucose, 2 parts of l-asparagines, 0.1 part of adenine sulfate, 25 parts of Stock A damping fluids, 1 part of Stock B damping fluid, 10 parts of Stock C damping fluids, 14 parts in agar, water are supplemented to 1000 parts of gross weights.Stock A buffer formulation is that the material of following parts by weight constitutes: 20 parts of ammonium tartrates, KH
2PO
440 parts, anhydrous Na SO
411.6 part, anhydrous Na
2HPO
490 parts, water is supplemented to 1000 parts of gross weights.Stock B buffer formulation is that the material of following parts by weight constitutes: 0.04 part of VitB1, water are supplemented to 1000 parts of gross weights.Stock C buffer formulation is that the material of following parts by weight constitutes: MgCl
2H
225 parts of O, water are supplemented to 1000 parts of gross weights.The seed culture based formulas is that the material of following parts by weight constitutes: 20 parts of Microcrystalline Celluloses, and 4 parts of yeast extract pastes, 1 part of dipotassium hydrogen phosphate, 0.46 part of potassium primary phosphate, 1 part in sal epsom, 20 parts in agar, water are supplemented to 1000 parts of gross weights.
The present invention is that method by the screening of single factor, PB design has obtained optimal medium prescription and the optimal culture condition that transgenosis Coprinus cinereus bacterial strain efficiently expresses reorganization multifunctional fibre element.Not only the High-efficient Production for the reorganization multifunctional cellulase provides a kind of effective means, and provides the scientific theory foundation for the reasonable utilization of agricultural wastes.
Compared with prior art, the present invention has following beneficial effect:
1. method of the present invention render transgenic Coprinus cinereus bacterial strain effectively utilizes cheap agricultural wastes bagasse and dregs of beans to produce the reorganization multifunctional cellulase.
2. the reorganization multifunctional cellulase that the inventive method produced, the molecular weight of its enzyme size and natural from Fushou spiral shell gastric juice isolating multifunctional cellulase big or small consistent, be 45.8KDa, the activity of enzyme is also with natural consistent.Reorganization multifunctional cellulase than prokaryotic expression system and yeast expression system expression all has significant advantage on zymologic property, the reorganization multifunctional cellulase activity of prokaryotic expression system is low, and be that intracellular enzyme is difficult for purifying, the reorganization multifunctional cellulase enzyme that yeast expression system is expressed is because by excessive glycosylation, so the activity of enzyme is also not as good as natural multifunctional cellulase.
Description of drawings
Fig. 1 is transgenosis Coprinus cinereus bacterial strain is cultivated the reorganization multifunctional cellulase that produces under different carbon sources a enzymic activity.
Fig. 2 is transgenosis Coprinus cinereus bacterial strain is cultivated the reorganization multifunctional cellulase that produces under different nitrogen sources a enzymic activity.
Fig. 3 bagasse addition is to the Xylanase of reorganization multifunctional cellulase and the influence of CMCase vigor.
Fig. 4 bean cake powder addition is to the Xylanase of reorganization multifunctional cellulase and the influence of CMCase vigor.
Fig. 5 ferments initial pH value to the Xylanase of reorganization multifunctional cellulase and the influence of CMCase vigor.
Fig. 6 inoculum size is to the Xylanase of reorganization multifunctional cellulase and the influence of CMCase vigor.
Fig. 7 sal epsom addition is to the Xylanase of reorganization multifunctional cellulase and the influence of CMCase vigor.
Fig. 8 potassium primary phosphate addition is to the Xylanase of reorganization multifunctional cellulase and the influence of CMCase vigor.
Fig. 9 dipotassium hydrogen phosphate addition is to the Xylanase of reorganization multifunctional cellulase and the influence of CMCase vigor.
Zytase (Xylanase) the vigor result of the Plackett-Burman experimental design of Figure 10 N=12 and MFC.
Endoglucanase (CMCase) the vigor result of the Plackett-Burman experimental design of Figure 11 N=12 and MFC.
Embodiment
Below in conjunction with embodiment, technical solution of the present invention is described further.
The preparation substratum:
Seed culture medium: Microcrystalline Cellulose 20g, yeast extract paste 4g, dipotassium hydrogen phosphate 1g, potassium primary phosphate 0.46g, sal epsom 1g, distilled water is settled to 1000mL, and is standby behind the autoclaving.
Coprinus cinereus solid medium: glucose 10 g, l-asparagine 2 g, adenine sulfate 0.1 g, Stock A damping fluid 25 mL, Stock B damping fluid 1 mL, Stock C damping fluid 10 mL, agar 14 g, distilled water is settled to 1000mL, and is standby behind the autoclaving.
Stock A damping fluid: ammonium tartrate 20 g, KH
2PO
440 g, anhydrous Na SO
411.6 g, anhydrous Na
2HPO
490 g, distilled water is settled to 1000mL, adds under several chloroform room temperatures to preserve.
Stock B damping fluid: VitB1 0.04 g, distilled water is settled to 1000mL, 4 ℃ of preservations behind the filtration sterilization.
Stock C damping fluid: MgCl
2H
2O 25 g, distilled water is settled to 1000mL, autoclaving, room temperature
The following preservation.
The enzyme substratum is produced on the basis: carbon source 20g, nitrogenous source 4g, dipotassium hydrogen phosphate 1g, potassium primary phosphate 0.46g, sal epsom 1g, and regulating initial pH is 6.25, distilled water is settled to the 1000mL high-temperature sterilization.
The contrast of the multiple production method of reorganization multifunctional cellulase:
(1) make up transgenosis Coprinus cinereus bacterial strain, its construction process is referring to following paper: the multifunctional cellulase gene (
Mfc) the clone and the expression study in Coprinus cinereus, Agricultural University Of South China's Ph D dissertation, in June, 2008.Make up and obtain transgenosis Coprinus cinereus bacterial strain TCles11, be inoculated in the Coprinus cinereus solid medium and cultivate 7d for dull and stereotyped 37 ℃, 1-5% is in the 100mL seed culture medium in inoculation, and 37 ℃, 210r/min shaking culture 4d.
(2) produce on the basis of enzyme substratum on the basis, with the yeast extract paste is only nitrogen source, Pericarpium Musae powder (60 order), buckwheat flour (60 order), Semen Maydis powder (60 order), bagasse (20 order), Microcrystalline Cellulose, oat, glucose, sucrose, maltose, wood chip are respectively the 37 ℃ of 210rpm shaking culture of bacterium liquid in the above-mentioned seed culture medium of carbon source inoculation 1mL, get the fermented liquid of cultivating 7d and measure its enzymic activity, filter out optimum carbon source.
(3) produce on the basis of enzyme substratum on the basis, with the carbon source that filters out is sole carbon source, peptone, extractum carnis, yeast extract paste, urea, ammonium sulfate, primary ammonium phosphate, bean cake powder (40 order), analysis for soybean powder (40 order), wheat bran (40 order), Semen Maydis powder (60 order) are respectively the 37 ℃ of 210rpm shaking culture of bacterium liquid in the above-mentioned seed culture medium of nitrogenous source inoculation 1mL, get the fermented liquid of cultivating 7d, adopt the DNS method to measure its enzymic activity, determine the suitableeest nitrogenous source.
(4) be sole carbon source with the above-mentioned bagasse that filters out, bean cake powder is an only nitrogen source, the bagasse powder (20 order) that in liquid fermentation medium, adds 10-30g/L, the bean cake powder (40 order) that adds 2-6g/L, the dipotassium hydrogen phosphate that adds 0.5-1.5g/L, the potassium primary phosphate that adds 0.23-0.69g/L, the sal epsom that adds 0.5-1.5g/L, regulate the initial pH4.5-8.0 of fermention medium, inoculation 0.5-2.5mL bacterial classification, 37 ℃ of 210rpm shaking culture are got the fermented liquid of cultivating 7d, adopt the DNS method to measure its enzymic activity.
(5) utilization Design-Expert 7.1.3 software, selecting 11 factors, experiment number for use is 12 Plackett-Burman design, investigate carbon source, nitrogenous source, leavening temperature, inoculum size, initial pH to producing the significance of enzyme influence, remaining 6 null terms are done errot analysis.Each factor is got 2 levels, and " low-level " and " high level " all decides according to the experiment situation.With Xylanase vigor and CMCase vigor is response value, adopts Design-Expert 7.1.3 software to set up analysis of variance model, and selecting the factor of P＜0.05 is the main effect factor.
The above results is shown in Fig. 1-9, Figure 10-11.
The difference that influences that different carbon source shown in Figure 1 is produced the reorganization multifunctional cellulase to the TCles11 bacterial strain is wherein remarkable with the effect of bagasse.Therefore determine that bagasse produces the optimum carbon source of reorganization multifunctional cellulase for the TCles11 strain fermentation.As shown in Figure 2, inorganic nitrogen-sourced ammonium sulfate, urea and primary ammonium phosphate are not obvious to the influence that the TCles11 bacterial strain produces the reorganization multifunctional cellulase, and the influence of organic nitrogen source bean cake powder, peptone, yeast extract paste, extractum carnis, Semen Maydis powder, wheat bran, analysis for soybean powder is obvious, wherein the effect of bean cake powder is the most remarkable, determines that therefore bean cake powder produces the optimum nitrogen source of reorganization multifunctional cellulase as the TCles11 strain fermentation.Not obvious by dipotassium hydrogen phosphate shown in Fig. 3-9, potassium primary phosphate and sal epsom to the influence that the TCles11 bacterial strain produces the reorganization multifunctional cellulase, and the influence of bagasse, dregs of beans, initial pH value, inoculum size is obvious.The result of comprehensive Figure 10, Figure 11 shows that bagasse, initial pH value, inoculum size are for influencing the main effect factor that the TCles11 bacterial strain produces the reorganization multifunctional cellulase.
(1) the TCles11 inoculation that embodiment 1 is prepared is cultivated 7d for 37 ℃ on Coprinus cinereus solid medium flat board, and 1-5% is in the 100mL seed culture medium in inoculation, and 37 ℃, 210r/min shaking culture 96h;
(2) preparation bagasse and bean cake powder interworking substratum, bagasse powder 30g/L, bean cake powder 6 g/L, dipotassium hydrogen phosphate 1g/L, potassium primary phosphate 0.46g/L, sal epsom 1g/L regulates pH value 6.25; Be sub-packed in the 250mL triangular flask every bottled liquid measure 100mL, autoclaving;
(3) bacterial classification of cultivating in every bottle graft kind 1mL step (1) in ultra-clean work, inoculation are placed on 37 ℃ of following shaking tables and cultivate, and shaking speed is 210r/min.
(4) cultivating 7d stops cultivating centrifugal collection fermented liquid.
Coprinus cinereus solid culture based formulas wherein is that the material of following parts by weight constitutes: 10 parts of glucose, 2 parts of l-asparagines, 0.1 part of adenine sulfate, 25 parts of Stock A damping fluids, 1 part of Stock B damping fluid, 10 parts of Stock C damping fluids, 14 parts in agar, water are supplemented to 1000 parts of gross weights.Stock A buffer formulation is that the material of following parts by weight constitutes: 20 parts of ammonium tartrates, KH
2PO
440 parts, anhydrous Na SO
411.6 part, anhydrous Na
2HPO
490 parts, water is supplemented to 1000 parts of gross weights.Stock B buffer formulation is that the material of following parts by weight constitutes: 0.04 part of VitB1, water are supplemented to 1000 parts of gross weights.Stock C buffer formulation is that the material of following parts by weight constitutes: MgCl
2H
225 parts of O, water are supplemented to 1000 parts of gross weights.The seed culture based formulas is that the material of following parts by weight constitutes: 20 parts of Microcrystalline Celluloses, and 4 parts of yeast extract pastes, 1 part of dipotassium hydrogen phosphate, 0.46 part of potassium primary phosphate, 1 part in sal epsom, 20 parts in agar, water are supplemented to 1000 parts of gross weights.
After collecting fermented liquid, adopt the DNS method to measure its enzymic activity, detected result shows zytase (Xylanase) enzyme alive 1.66 * 10
5U/L, the work of endoglucanase (CMCase) enzyme is 1.7 * 10
4U/L.
Claims (10)
1. a transgenosis Coprinus cinereus efficiently expresses the method for recombinase, it is characterized in that: multifunctional cellulase gene transformation Coprinus cinereus obtains transgenosis Coprinus cinereus TCles11, as producing bacterial strain, with bagasse powder and bean cake powder is that main raw material is as substratum, described substratum contains bagasse powder 27.9-30g/L and bean cake powder 4.3-6 g/L, shake flask fermentation express recombinant multifunctional cellulase.
2. the method for claim 1 is characterized in that the prescription of described substratum is: bagasse powder 27.9-30g/L, bean cake powder 4.3-6 g/L, dipotassium hydrogen phosphate 0.5-1.5g/L, potassium primary phosphate 0.23-0.69g/L, sal epsom 0.5-1.5g/L, pH6.25-7.82.
3. the method for claim 1 is characterized in that the condition of described shake flask fermentation is leavening temperature 36-37 ℃, fermentation time 5-7d, rotating speed 180-210r/min.
4. the method for claim 1 is characterized in that described bacterial strain bacterial classification age is 84-96h, inoculum size 1-1.5%.
5. the method for claim 1 is characterized in that may further comprise the steps:
(1) transgenosis Coprinus cinereus inoculation is cultivated on the Coprinus cinereus solid medium becomes the inoculation bacterial classification;
(2) preparation bagasse substratum: bagasse powder 27.9-30g/L, bean cake powder 4.3-6g/L, dipotassium hydrogen phosphate 0.5-1.5g/L, potassium primary phosphate 0.23-0.69g/L, sal epsom 0.5-1.5g/L, pH6.25-7.82 regulates pH value 6.25-7.82;
(3) under gnotobasis, inoculate, the bacterial classification inoculation that step (1) is obtained to the bagasse substratum, inoculum size 1-1.5%, inoculation is placed on 36-37 ℃ and cultivates down;
(4) cultivating 5-7d stops cultivating centrifugal collection fermented liquid.
6. method as claimed in claim 5 is characterized in that described transgenosis Coprinus cinereus bacterial strain is to cultivate 7d at 37 ℃, and inoculation 1-5% inoculation bacterial classification is in the 100mL seed culture medium, and 37 ℃, 210r/min shaking culture 84-96h becomes the inoculation bacterial classification.
7. method as claimed in claim 5 is characterized in that the described cultivation of step (4) is to carry out under the condition of rotating speed 210r/min.
8. method as claimed in claim 5, it is characterized in that the described Coprinus cinereus solid culture of step (1) based formulas is the material formation of following parts by weight: 10 parts of glucose, 2 parts of l-asparagines, 0.1 part of adenine sulfate, 25 parts of Stock A damping fluids, 1 part of Stock B damping fluid, 10 parts of Stock C damping fluids, 14 parts in agar, water are supplemented to 1000 parts of gross weights.
9. method as claimed in claim 5, it is characterized in that the material that the described seed culture based formulas of step (1) is following parts by weight constitutes: 20 parts of Microcrystalline Celluloses, 4 parts of yeast extract pastes, 1 part of dipotassium hydrogen phosphate, 0.46 part of potassium primary phosphate, 1 part in sal epsom, 20 parts in agar, water are supplemented to 1000 parts of gross weights.
10. as right 8 described methods, it is characterized in that the material that described Stock A buffer formulation is following parts by weight constitutes: 20 parts of ammonium tartrates, KH
2PO
440 parts, anhydrous Na SO
411.6 part, anhydrous Na
2HPO
490 parts, water is supplemented to 1000 parts of gross weights; Stock B buffer formulation is that the material of following parts by weight constitutes: part, water is supplemented to 1000 parts of gross weights; Stock C buffer formulation is that the material of following parts by weight constitutes: MgCl
2H
225 parts of O, water are supplemented to 1000 parts of gross weights.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104312988A (en) * | 2014-10-15 | 2015-01-28 | 广西大学 | Bagasse fluid medium as well as preparation method and application thereof |
| CN106701804A (en) * | 2016-12-14 | 2017-05-24 | 四川省农业科学院土壤肥料研究所 | Multifunctional cellulase gene Mfcsg applicable to auricularia polytricha expression and application of multifunctional cellulase gene Mfcsg |
| CN107455548A (en) * | 2017-09-07 | 2017-12-12 | 中国林业科学研究院林产化学工业研究所 | A kind of method that edible mushroom solid state fermentation Soybean Meal prepares feeding level protein additive |
-
2010
- 2010-12-30 CN CN 201010613503 patent/CN102127532B/en not_active Expired - Fee Related
Non-Patent Citations (3)
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| 《中国食品学报》 20080430 杨培周等 福寿螺(Ampullaria crossean)mfc基因的分子克隆和序列分析 , * |
| 《华南农业大学博士论文》 20080630 杨培周 多功能纤维素酶基因（mfc）的克隆及其在灰盖鬼伞中的表达研究 , * |
| 《第二届全国食用菌中青年专家学术交流会论文集》 20081231 王艺红等 多功能纤维素酶基因_mfc_遗传转化草菇的研究 , * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104312988A (en) * | 2014-10-15 | 2015-01-28 | 广西大学 | Bagasse fluid medium as well as preparation method and application thereof |
| CN106701804A (en) * | 2016-12-14 | 2017-05-24 | 四川省农业科学院土壤肥料研究所 | Multifunctional cellulase gene Mfcsg applicable to auricularia polytricha expression and application of multifunctional cellulase gene Mfcsg |
| CN106701804B (en) * | 2016-12-14 | 2019-12-03 | 四川省农业科学院土壤肥料研究所 | It is suitable for multifunctional cellulase gene M fcsg and its application of Uricularia polytricha expression |
| CN107455548A (en) * | 2017-09-07 | 2017-12-12 | 中国林业科学研究院林产化学工业研究所 | A kind of method that edible mushroom solid state fermentation Soybean Meal prepares feeding level protein additive |
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