CN102093959A - Production method of recombinant multifunctional cellulase - Google Patents
Production method of recombinant multifunctional cellulase Download PDFInfo
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- CN102093959A CN102093959A CN 201010585035 CN201010585035A CN102093959A CN 102093959 A CN102093959 A CN 102093959A CN 201010585035 CN201010585035 CN 201010585035 CN 201010585035 A CN201010585035 A CN 201010585035A CN 102093959 A CN102093959 A CN 102093959A
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Abstract
The invention belongs to the field of microbial fermentation engineering, and relates to a production method of recombinant multifunctional cellulose. The production method is characterized by comprising the following steps: transforming a multifunctional cellulose gene into Tremella fuciformis spores to obtain a Tremella fuciformis spore engineered strain serving as a production strain; and carrying our shake flask fermentation by utilizing sugar cane bagasse powder as an optimal culture medium under optimized culture conditions, thus obtaining the recombinant multifunctional cellulase. The sugar cane bagasse culture medium has a pH value of 5.0-5.5 and comprises the following components: sugar cane bagasse powder 17-23 g/L, yeast extract 3.1-4.9 g/L, dipotassium hydrogen phosphate 1 g/L, potassium dihydrogen phosphate 0.46 g/L and magnesium sulfate 1 g/L. The optimal culture conditions are as follows: the culture temperature is 25DEG C, the culture time is 4 days, and the rotation speed of a shaking table is 180r/min. The optimal enzyme yields are as follows: xylanase of enzyme activity 1.09*10<5>U/L, and carboxymethyl cellulase (CMCase) of enzyme activity 3.7*10<4>U/L. By utilizing the method provided by the invention, Tremella fuciformis spore engineered strain and cheap agricultural waste sugar cane bagasse can be effectively utilized to produce the recombinant multifunctional cellulose.
Description
Technical field
The invention belongs to the microbial fermentation engineering field, be specifically related to a kind of method of utilizing white fungus gemma engineering strain to produce multifunctional cellulase.Utilize the method for bagasse High-efficient Production 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 of white fungus gemma engineering strain of the present invention production 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.And the promotor of white fungus gemma engineering strain is constitutive promoter, is not subjected to the influence of other inductors, and production performance is stable.Simultaneously, the substratum that the present invention uses is agricultural wastes bagasse, 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 white fungus gemma engineering strain High-efficient Production reorganization multifunctional fibre element is provided.
The present invention is achieved through the following technical solutions above-mentioned purpose:
Invention provides a kind of production method of the multifunctional cellulase of recombinating, multifunctional cellulase gene transformation white fungus gemma obtains white fungus gemma engineering strain, as producing bacterial strain, with the bagasse powder be main raw material as substratum, shake flask fermentation production reorganization multifunctional cellulase.
The prescription of described substratum is: bagasse powder (20 order) 17-23g/L, yeast extract paste 3.1-4.9 g/L, dipotassium hydrogen phosphate 1g/L, potassium primary phosphate 0.46g/L, sal epsom 1g/L, pH value 5.0-5.5; The condition of shake flask fermentation is leavening temperature 23-25 ℃, fermentation time 3-4d, rotating speed 165-190r/min.Optimal culture condition is: 25 ℃ of culture temperature, incubation time 4d, shaking speed 180r/min.
Described white fungus gemma engineering strain bacterial classification 60-72h in age, the inoculum size of bacterial strain in substratum is 1-5%, i.e. the white fungus gemma bacterial classification of the inoculation of medium 1-5mL of every 100mL, the OD of its gemma concentration
600Value is 1.5-1.9.This white fungus gemma engineering strain is that multifunctional cellulase gene transformation white fungus gemma obtains, and its preparation method can be referring to refined refined Master's thesis (the multifunctional cellulase gene of Zhao
MfcThe research of genetic transformation white fungus gemma, Agricultural University Of South China's Master's thesis, in June, 2008).
Can adopt following preferred version to carry out fermentative production:
(1) described white fungus gemma engineering strain is inoculated on the PDSA solid medium, cultivates 4～5d for 25 ℃, picking list colony inoculation in 100mL PDSB liquid nutrient medium, 25 ℃, 180r/min shaking culture 72h;
(2) preparation bagasse substratum, bagasse powder (20 order) 17-23g/L, yeast extract paste 3.1-4.9 g/L, dipotassium hydrogen phosphate 1g/L, potassium primary phosphate 0.46g/L, sal epsom 1g/L, pH value 5.0-5.5, 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 25 ℃ of following shaking tables and cultivate shaking speed 165-190r/min.
(4) stop cultivating centrifugal collection fermented liquid behind the cultivation 4d.
PDSA solid culture based formulas wherein is that the material of following parts by weight constitutes: 200 parts of potatos, 20 parts of glucose, 3 parts of dipotassium hydrogen phosphates, 1.5 parts in sal epsom, 20 parts of glucose, 20 parts in agar, water is supplemented to 1000 parts of gross weights.PDSA liquid culture based formulas is that the material of following parts by weight constitutes: 200 parts of potatos, 20 parts of glucose, 3 parts of dipotassium hydrogen phosphates, 1.5 parts in sal epsom, 20 parts of glucose, water is supplemented to 1000 parts of gross weights.
The present invention is optimal medium prescription and the optimal culture condition that has obtained white fungus gemma engineering strain High-efficient Production reorganization multifunctional fibre element by the method for single factor screening, PB design and response surface experiment.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 can make white fungus gemma engineering strain utilize cheap agricultural wastes bagasse production reorganization multifunctional cellulase effectively.
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 48.6KDa, 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 white fungus gemma engineering strain is cultivated the reorganization multifunctional cellulase that produces under different nitrogen sources a enzymic activity;
Fig. 2 is white fungus gemma engineering strain is cultivated the reorganization multifunctional cellulase that produces under different carbon sources a enzymic activity;
Fig. 3 yeast extract paste is to the interaction of the Xylanase vigor of reorganization multifunctional cellulase;
Fig. 4 rotating speed is to the interaction of the Xylanase vigor of reorganization multifunctional cellulase;
Fig. 5 yeast extract paste is to the interaction of the CMCase vigor of reorganization multifunctional cellulase;
Fig. 6 rotating speed is to the interaction of the CMCase vigor of reorganization multifunctional cellulase;
Zytase (Xylanase) the vigor result of the Plackett-Burman experimental design of Fig. 7 N=12 and MFC;
Endoglucanase (CMCase) the vigor result of the Plackett-Burman experimental design of Fig. 8 N=12 and MFC;
Design of Fig. 9 response surface and result;
The polynary secondary model and the variable analysis of zytase (Xylanase) vigor of Figure 10 response surface design MFC;
The polynary secondary model and the variable analysis of endoglucanase (CMCase) vigor of Figure 11 response surface design MFC.
Embodiment
Below in conjunction with embodiment, technical solution of the present invention is described further.
The preparation substratum:
The PDSA substratum: potato (peeling) 200g, glucose 20g, dipotassium hydrogen phosphate 3g, sal epsom 1.5g, glucose 20g, agar 20g, distilled water is settled to 1000mL, and is standby behind the autoclaving.
The PDSB substratum: do not add agar in the PDSA substratum, standby behind the autoclaving.
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 5.5, distilled water is settled to 1000ml, high-temperature sterilization.
The contrast of the multiple production method of reorganization multifunctional cellulase:
(1) according to refined refined Master's thesis (the multifunctional cellulase gene of Zhao
MfcThe research of genetic transformation white fungus gemma, Agricultural University Of South China's Master's thesis, in June, 2008) method, make up and obtain white fungus gemma engineering strain ycLes3, be inoculated in PDSA and cultivate 4～5d for dull and stereotyped 25 ℃, picking list colony inoculation is in 100mL PDSB substratum, and 25 ℃, 180 r/min shaking culture 3d.
(2) produce on the basis of enzyme substratum on the basis, with glucose is sole carbon source, ammonium sulfate, primary ammonium phosphate, peptone, yeast extract paste, extractum carnis, Semen Maydis powder, wheat bran, analysis for soybean powder are respectively the 25 ℃ of 180rpm shaking culture of bacterium liquid among the above-mentioned PDSB of nitrogenous source inoculation 1mL, get the fermented liquid of cultivating 96h and measure its enzymic activity, filter out optimum nitrogen source.
(3) produce on the basis of enzyme substratum on the basis, with the nitrogenous source that filters out is only nitrogen source, glucose, sucrose, maltose, Pericarpium Musae powder, Microcrystalline Cellulose, bagasse, processing bagasse, corn cob meal, Semen Maydis powder are respectively the 25 ℃ of 180rpm shaking culture of bacterium liquid among the above-mentioned PDSB of carbon source inoculation 1mL, get the fermented liquid of cultivating 96h, adopt the DNS method to measure its enzymic activity, determine the suitableeest carbon source.
(4) 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, rotating speed, inoculum size, kind age, fermentation time, initial pH to producing the significance of enzyme influence, remaining 3 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.
(5) according to the Box-Bohnken principle of design, by the factor that the Plackett-Burman design is determined, each factor is got 3 levels, experimentizes with (1,0,1) coding.With zytase (Xylanase) vigor and endoglucanase (CMCase) vigor is response value, by Design-Expert 7.1.3 software data are carried out the quadratic regression match, obtain polynary quadratic regression equation, analyze the main effect and the interaction of each factor, analysis of experimental data is obtained the partial regression coefficient estimated value of polynary each variable of quadratic regression equation, thereby the quadratic response face of foundation empirical model, draw response surface stereoscopic analysis figure according to regression equation, draw response surface canonical parse result, thereby seek optimum level, and then determine the best enzyme culture system that produces.
The above results is shown in Fig. 1-4, Fig. 7-5.
As shown in Figure 1, inorganic nitrogen-sourced ammonium sulfate and primary ammonium phosphate are not obvious to the influence that the ycLes3 bacterial strain produces the reorganization multifunctional cellulase, and the influence of organic nitrogen source peptone, yeast extract paste, extractum carnis, Semen Maydis powder, wheat bran, analysis for soybean powder is obvious, wherein the effect of yeast extract paste is the most remarkable, determines that therefore yeast extract paste produces the optimum nitrogen source of reorganization multifunctional cellulase as the ycLes3 strain fermentation.The difference that influences that different carbon source shown in Figure 2 is produced the reorganization multifunctional cellulase to the ycLes3 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 ycLes3 strain fermentation.The result of synthesizing map 7, Fig. 8 shows that bagasse, yeast extract paste, rotating speed and fermentation time are for influencing the main effect factor that the reorganization multifunctional cellulase is produced in the ycLes3 fermentation.The result of Figure 10 and Figure 11 shows, it is fine to test selected model highly significant and model-fitting degree, and data residual error (Residual) causes that by error immediately Model Selection is fit to.Fig. 3-Fig. 6 is for to draw response surface stereoscopic analysis figure and level line thereof by Design Expert7.1.3 software according to regression equation, and the shape response surface stereographic map of the response curved surface of investigation institute match is as shown below.Utilize this picture group to analyze and to estimate to any 2 kinds of factor interactions in the fermentation condition of influence reorganization multifunctional cellulase vigor, and definite best level of factor scope, the strong and weak size of isocontour shape reflection interaction, the significant interaction between oval expression two factors.As seen from the figure, the production of the low-level or high-level multifunctional cellulase that all is unfavorable for recombinating of factor has only when bagasse, yeast extract paste, fermentation time, rotating speed are controlled between the certain limit, and enzyme activity is just the highest.
(1) with the ycLes3 inoculation of embodiment 1 preparation on the PDSA flat board, cultivate 4d for 25 ℃, picking list colony inoculation in 100mL PDSB liquid nutrient medium, 25 ℃, 180r/min shaking culture 72h;
(2) preparation bagasse substratum, bagasse powder 20.31g/L, yeast extract paste 4.11 g/L, dipotassium hydrogen phosphate 1g/L, potassium primary phosphate 0.46g/L, sal epsom 1g/L regulates pH value 5.5; 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 25 ℃ of following shaking tables and cultivate, and shaking speed is 180r/min.
(4) stop cultivating centrifugal collection fermented liquid behind the cultivation 4d.
PDSA solid culture based formulas wherein is that the material of following parts by weight constitutes: 200 parts of potatos, 20 parts of glucose, 3 parts of dipotassium hydrogen phosphates, 1.5 parts in sal epsom, 20 parts of glucose, 20 parts in agar, water is supplemented to 1000 parts of gross weights.PDSA liquid culture based formulas is that the material of following parts by weight constitutes: 200 parts of potatos, 20 parts of glucose, 3 parts of dipotassium hydrogen phosphates, 1.5 parts in sal epsom, 20 parts of glucose, water is 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 1.09 * 10
5U/L, the work of endoglucanase (CMCase) enzyme is 3.7 * 10
4U/L.
Claims (9)
1. the preparation method of the multifunctional cellulase of recombinating, it is characterized in that: multifunctional cellulase gene transformation white fungus gemma obtains white fungus gemma engineering strain, as producing bacterial strain, with the bagasse powder be main raw material as substratum, shake flask fermentation production reorganization multifunctional cellulase.
2. preparation method as claimed in claim 1 is characterized in that the prescription of described substratum is: bagasse powder 17-23g/L, yeast extract paste 3.1-4.9 g/L, dipotassium hydrogen phosphate 1g/L, potassium primary phosphate 0.46g/L, sal epsom 1g/L, pH value 5.0-5.5.
3. preparation method as claimed in claim 1 is characterized in that the condition of described shake flask fermentation is leavening temperature 23-25 ℃, fermentation time 3-4d, rotating speed 165-190r/min.
4. preparation method as claimed in claim 1 is characterized in that the condition of described shake flask fermentation is 25 ℃ of culture temperature, incubation time 4d, rotating speed 180r/min.
5. preparation method as claimed in claim 1 is characterized in that described bacterial strain bacterial classification age is 60 ~ 72h, inoculum size 1-5%.
6. preparation method as claimed in claim 1 is characterized in that may further comprise the steps:
(1) with the ycLes3 inoculation on the PDSA solid medium, cultivate 4～5d for 25 ℃, picking list colony inoculation in 100mL PDSB liquid nutrient medium, 25 ℃, 180r/min shaking culture 72h;
(2) preparation bagasse substratum: bagasse powder 17-23g/L, yeast extract paste 3.1-4.9 g/L, dipotassium hydrogen phosphate 1g/L, potassium primary phosphate 0.46g/L, sal epsom 1g/L regulates pH value 5.0-5.5;
(3) in Bechtop, inoculate, inoculum size 1-5%, inoculation is placed on 25 ℃ of following shaking tables and cultivates shaking speed 165-190r/min.
7.(4) stop cultivating centrifugal collection fermented liquid after cultivating 4d.
8. preparation method as claimed in claim 6, it is characterized in that the described PDSA solid culture of step (1) based formulas is the material formation of following parts by weight: 200 parts of potatos, 20 parts of glucose, 3 parts of dipotassium hydrogen phosphates, 1.5 parts in sal epsom, 20 parts of glucose, 20 parts in agar, water is supplemented to 1000 parts of gross weights.
9. preparation method as claimed in claim 6, it is characterized in that the described PDSA liquid culture of step (1) based formulas is the material formation of following parts by weight: 200 parts of potatos, 20 parts of glucose, 3 parts of dipotassium hydrogen phosphates, 1.5 parts in sal epsom, 20 parts of glucose, water is supplemented to 1000 parts of gross weights.
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Cited By (2)
| 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 |
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| CN101117621A (en) * | 2007-07-10 | 2008-02-06 | 钟冬季 | Novel method for white fungus cultivation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101117621A (en) * | 2007-07-10 | 2008-02-06 | 钟冬季 | Novel method for white fungus cultivation |
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| 《中国农业科学》 20081231 郭丽琼等 高效银耳芽孢遗传转化体系的建立 3728-3734 1-9 第41卷, 第11期 2 * |
| 《食品与发酵工业》 20101130 郑兰娟等 转基因草菇高产多功能纤维素酶的培养条件 26-29 1 第36卷, 第11期 2 * |
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 |
| 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 |
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