CN102994481A - Preparation method for compound enzyme system for high-efficiency degradation for lignocellulose and application thereof - Google Patents
Preparation method for compound enzyme system for high-efficiency degradation for lignocellulose and application thereof Download PDFInfo
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- CN102994481A CN102994481A CN2012105159886A CN201210515988A CN102994481A CN 102994481 A CN102994481 A CN 102994481A CN 2012105159886 A CN2012105159886 A CN 2012105159886A CN 201210515988 A CN201210515988 A CN 201210515988A CN 102994481 A CN102994481 A CN 102994481A
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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
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
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Abstract
The invention discloses a preparation method for a compound enzyme system for high-efficiency degradation for lignocellulose and an application thereof. The preparation method comprises the following steps of: step 1, activating Trichoderma reesei strains or the derivative strains thereof; step 2, preparing spore suspension from the activated Trichoderma reesei strains or the derivative strains thereof, inoculating into a seed culture medium, and culturing for 24-36 hours in a shaking manner on a shaking table with a rotational speed of 170-200 r/min and at a temperature of 28-30 DEG C; and step 3, inoculating the seed culture medium inoculated with the Trichoderma reesei strains or the derivative strains thereof into a fermentation culture medium using microcrystalline cellulose and corncobs as a carbon source in a mixing ratio of 1:4, and performing fermentation tank culture. The compound enzyme system is used for treating corncobs, gas-detonating straw and the corn straw pre-treated by NaOH at a normal temperature. Via the method disclosed by the invention, the saccharification and hydrolysis capacities of the compound enzyme system are enhanced, the preparation cost of enzymes is reduced, and the pollution to environment is low.
Description
Technical field
The present invention relates to microorganism field, relate in particular to a kind of preparation method and application thereof of prozyme system of efficient degradation lignocellulose.
Background technology
Along with the day by day exhaustion of non-renewable fossil energy, increasing research has all focused on the development and utilization of new forms of energy.Biomass energy is because its source is abundant, with low cost and recyclability and enjoy favor.Mierocrystalline cellulose is as renewable resources the abundantest on the earth, for biomass energy provides abundant source.It is reported that the Mierocrystalline cellulose total amount that the annual photosynthesis in the whole world produces can reach 10
11Ton.But Mierocrystalline cellulose is the wire high molecular polymer that is combined into Isosorbide-5-Nitrae-glycosidic link by glucose molecule.Cellulosic molecule is difficult to directly be utilized, and needs just can be utilized through after being degraded into small molecules carbohydrate material.Mierocrystalline cellulose can be directly degraded by acid, alkaline purification or by enzymatic treatment.The enzymic degradation Mierocrystalline cellulose is because the advantage of pollution-free, less energy-consumption and gradually replacing acid, alkali treatment method.Lignocellulolytic enzymes extensively is present in the natural organism, in fields such as food, brewing industry, processing of agriculture product, feed, medicine, environment protection and chemical industry boundless application prospect and application potential is arranged.Therefore, the prozyme cording of the method preparation has wide practical use.
The method of production of cellulose enzyme generally adopts liquid submerged fermentation and solid state fermentation both at home and abroad.The solid state fermentation ventilation effect is not fine, and operability is relatively poor, is not suitable for industry and amplifies.The carbon source that is used for the product enzyme under the liquid state fermentation condition has Microcrystalline Cellulose or paper pulp, although enzyme activity is higher, cost is higher, and environmental pollution is serious, is not suitable for scale operation; Also useful cheap raw material wheat bran, straw and wheat stalk, but enzyme activity is lower; Also useful acid-treated corn cob residue ferments as raw material, because there is toxic by-products furfural etc. in the residue after the acid treatment, thalli growth is had restraining effect, and therefore, enzyme activity is lower.The production method of existing production of cellulose enzyme all can not satisfy can prepare again the more full needs of enzyme system when the assurance enzyme activity is higher.
Summary of the invention
For above-mentioned technical problem, the present invention has designed and developed a kind of preparation method and application thereof of prozyme system of efficient degradation lignocellulose, purpose is to prepare the prozyme system of ligocellulose degradation of containing simultaneously cellulase and hemicellulase, reduce the production cost of enzyme, improve catalytic efficiency, reduce the pollution to environment.
Technical scheme provided by the invention is:
A kind of preparation method of prozyme system of efficient degradation lignocellulose may further comprise the steps:
Step 1, activation Li's Trichoderma strains or its derivative strain;
Step 2, Li's Trichoderma strains or its derivative strain after will activating are prepared into spore suspension, are inoculated in the seed culture medium and cultivate, and temperature is 28 ~ 30 ℃, and rotating speed is that 24 ~ 36h is cultivated in concussion on 170 ~ 200 rev/mins the shaking table;
Step 3, the seed culture medium that will inoculate Li's Trichoderma strains or its derivative strain are seeded to take Microcrystalline Cellulose and corn cob as carbon source and ratio of mixture is carried out fermentor cultivation in the fermention medium of 1:4.
Preferably, among the preparation method of the prozyme system of described efficient degradation lignocellulose, the seed culture medium that will inoculate Li's Trichoderma strains or its derivative strain in the described step 3 is seeded to according to 5 ~ 10% inoculum sizes and carries out fermentor cultivation in the described fermention medium.
Preferably, among the preparation method of the prozyme of described efficient degradation lignocellulose system, by mass percentage, described fermention medium comprises: carbon source 2% ~ 8%, and organic nitrogen source 0.1 ~ 5%, inorganic nitrogen-sourced concentration is 0 ~ 2%, inorganic salt 0.0001 ~ 10%.
Preferably, among the preparation method of the prozyme system of described efficient degradation lignocellulose, when carrying out fermentor cultivation by described step 3, pH regulator is 3.0 ~ 5.0, temperature is controlled to be 20 ~ 40 ℃, shaking speed is controlled at 100 ~ 250 rev/mins, carries out deep layer liquid state fermentation, and fermentation time is 60 ~ 200 hours.
Preferably, among the preparation method of the prozyme of described efficient degradation lignocellulose system, described carbon source content the best is 3% ~ 5%.
Preferably, among the preparation method of the prozyme of described efficient degradation lignocellulose system, described fermentor tank volume is 5L, and the 3L fermention medium is housed.
Utilize the application of a kind of prozyme system of aforesaid method preparation, described prozyme system is processed corn cob and gas explosion stalk and through the pretreated maize straw of NaOH normal temperature.
The preparation method of the prozyme of efficient degradation lignocellulose of the present invention system mixes carbon source as fermention medium with a small amount of Microcrystalline Cellulose with corn cob, because of corn cob wide material sources, cheap, reduced production cost.Xylanase activity is significantly higher than pure Microcrystalline Cellulose as the xylan enzyme activity of carbon source in the prozyme system that application present method obtains, and cellulase activity decreases.The prozyme system that utilizes present method preparation processed corn cob and gas explosion stalk and through the pretreated maize straw of NaOH normal temperature, found that the hydrolysis effect to corn cob is higher than Microcrystalline Cellulose as the treatment effect of carbon source, has also obtained certain raising to pretreated maize straw reducing sugar content.Raw material among the preparation method of the present invention does not need to carry out the method pre-treatment such as acid or alkali; therefore; can not produce toxic by-products in the preparation process, the prozyme of preparation ties up to the fields such as food, brewing industry, processing of agriculture product, feed, medicine, environment protection and chemical industry boundless application prospect and application potential.
Description of drawings
Fig. 1 is that prozyme of the present invention is that cellulase vigor and Xylanase activity are with fermentation time Changing Pattern figure.
Embodiment
The present invention is described in further detail below in conjunction with accompanying drawing, can implement according to this with reference to the specification sheets literal to make those skilled in the art.
The invention provides a kind of preparation method of prozyme system of efficient degradation lignocellulose, may further comprise the steps:
Step 1, activation Li's Trichoderma strains or its derivative strain;
Step 2, Li's Trichoderma strains or its derivative strain after will activating are prepared into spore suspension, are inoculated in the seed culture medium and cultivate, and temperature is 28 ~ 30 ℃, and rotating speed is that 24 ~ 36h is cultivated in concussion on 170 ~ 200 rev/mins the shaking table;
Step 3, the seed culture medium that will inoculate Li's Trichoderma strains or its derivative strain are seeded to take Microcrystalline Cellulose and corn cob as carbon source and ratio of mixture is carried out fermentor cultivation in the fermention medium of 1:4.
Among the preparation method of the prozyme system of described efficient degradation lignocellulose, the seed culture medium that will inoculate Li's Trichoderma strains or its derivative strain in the described step 3 is seeded to according to 5 ~ 10% inoculum sizes and carries out fermentor cultivation in the described fermention medium.
Among the preparation method of the prozyme system of described efficient degradation lignocellulose, by mass percentage, described fermention medium comprises: carbon source 2% ~ 8%, and organic nitrogen source 0.1 ~ 5%, inorganic nitrogen-sourced concentration is 0 ~ 2%, inorganic salt 0.0001 ~ 10%.
Among the preparation method of the prozyme system of described efficient degradation lignocellulose, when carrying out fermentor cultivation by described step 3, pH regulator is 3.0 ~ 5.0, temperature is controlled to be 20 ~ 40 ℃, shaking speed is controlled at 100 ~ 250 rev/mins, carries out deep layer liquid state fermentation, and fermentation time is 60 ~ 200 hours.
Among the preparation method of the prozyme system of described efficient degradation lignocellulose, described carbon source content the best is 3% ~ 5%.
Among the preparation method of the prozyme system of described efficient degradation lignocellulose, described fermentor tank volume is 5L, and the 3L fermention medium is housed.
Utilize the application of a kind of prozyme system of aforesaid method preparation, described prozyme system is processed corn cob and gas explosion stalk and through the pretreated maize straw of NaOH normal temperature.
Experiment material
Buy in Chinese industrial microbial strains preservation center (CICC), CICC13052 with Trichodermareesei (Trichoderma reesei Rut C-30) in this experiment.
Seed culture medium comprises Microcrystalline Cellulose 2%, corn steep liquor 1.5%, and glucose 1%, regulating pH is 4.5.
Fermention medium of the present invention with Microcrystalline Cellulose and corn cob as carbon source, wherein corn cob meal is pulverized through pulverizer and is formed, corn steep liquor is as organic nitrogen source, ammonium sulfate is as inorganic nitrogen-sourced, potassium primary phosphate, sal epsom, calcium carbonate are as the inorganic salt composition, and glycerine is as the carbon source at strain fermentation initial stage.Therefore, the fermention medium of determining comprises: corn steep liquor 1.0 ~ 2.5%, (NH
4)
2SO4 0.2 ~ 0.8%, KH
2PO
40 ~ 0.6%, MgSO
47H
2O 0 ~ 0.1%, CaCO
30 ~ 0.25%, glycerine 0 ~ 0.25%, regulating pH is 4.0 ~ 5.0.Wherein, organic nitrogen source can also be yeast powder, peptone; Inorganic nitrogen-sourcedly can also be ammonium chloride, ammonium nitrate.
Draw the typical curve of glucose and xylose, utilize filter paper enzyme activity and Xylanase activity in the prozyme system that standard curve determination the present invention obtains.
1) drafting of glucose typical curve:
Preparation 10mg/ml glucose mother liquid is diluted to 2.0,3.3,5.0 and the glucose solution sample of 6.7mg/ml.The glucose solution sample of getting 0.5ml is added to the 0.05M of 1ml, in the citric acid solution of pH4.8, adds the DNS reaction solution of 3ml again, mixing, and boiling water bath heating 5min is cooled to room temperature.Use spectrophotometric determination under wavelength 540nm, to measure absorbancy OD.Take OD540 as ordinate zou, corresponding glucose concn is X-coordinate, the drawing standard curve.
2) drafting of wood sugar typical curve
Preparation 0.01M(=0.15g/100ml damping fluid) xylose mother liquid is diluted to the xylose solution sample of 10,5,3.33 and 2 μ mol/ml.Get in the xylan substrate solution that 0.2ml xylose solution sample is added to 1.8ml, add the 3mlDNS reaction solution, mixing, boiling water bath heating 5min, cooling is also centrifugal, uses spectrophotometer to measure supernatant liquor absorbancy OD under wavelength 540nm.Take OD540 as ordinate zou, corresponding xylose concentration is X-coordinate, the drawing standard curve.
3) filter paper enzyme activity is measured
In vitro add 1.0ml 0.05M, pH 4.8 sodium citrate buffer solutions, the dilution enzyme liquid of interpolation 0.5ml is heated to 50 ℃, adds a filter paper band and mixes, and 50 ℃ of water-bath 60min add 3.0mlDNS reaction solution mixing termination reaction immediately.Boiling water bath heating 5min is transferred to cooling bath, cooling.Get the mixed solution behind the 0.45ml color reaction, add 2.0ml deionized water or distilled water, fully mixing.Under 540nm, read absorbancy, read the glucose concn of sample by typical curve.
4) Xylanase activity is measured
Add the dilution enzyme liquid of the substrate solution (use 0.05M, the citrate buffer solution dissolving of pH5.3 prepares 1% xylan suspension) of 1.8ml and 0.2ml extremely in vitro, 50 ℃ of water-bath 5min, the DNS reaction solution of interpolation 3.0ml, mixing.Boiling water bath heating 5min shifts sample to cooling bath, then the unreacted substrate of centrifugation.Supernatant liquor reads the absorbancy of sample in the 540nm place, according to the wood sugar typical curve, the absorbancy of measuring the sample of pipe is converted to the concentration of wood sugar.
5) application of prozyme system
In the 50ml shaking flask, add 5 ~ 15% pre-treatment or untreated substrate, the loading capacity of enzyme is 5 ~ 10FPU/g dry bottom thing, be 50 ℃ in temperature, when shaking speed is 150 rev/mins, enzymolysis 48 ~ 96h, the 1ml that takes a sample respectively, the centrifuging and taking supernatant liquor is measured the total reducing sugars in the supernatant liquor.
Embodiment one
Li's Trichoderma strains or its derivative strain after the activation on the PDA slant medium are prepared into spore suspension, are inoculated in the fresh seed culture medium according to the inoculum size of the first per-cent and cultivate.Microcrystalline Cellulose mixes with different ratios with corn cob, get 1.5g different mixing proportion raw material and add pack into the triangular flask of 250ml of 50ml fermentation culture based sols, in 120 ℃ of sterilization 15min, the seed culture based sols of inoculation Li's Trichoderma strains or its derivative strain after the cooling, in 28 ~ 30 ℃, 150 ~ 220 rev/mins fermented 5 ~ 7 days, and the centrifuging and taking supernatant liquor is measured cellulase and Xylanase activity.The result is as shown in table 1, and when Microcrystalline Cellulose and corn cob mixed with the ratio of 2:8, cellulase activity was pure Microcrystalline Cellulose 94.6% during as carbon source, and Xylanase activity is during as carbon source 10 times of pure Microcrystalline Cellulose.Therefore, selecting the best proportioning of Microcrystalline Cellulose and corn cob is the experiment that 1:4 carries out following embodiment.
Table 1 corn cob Substitute For Partial Microcrystalline Cellulose is on impact and the hydrolysis effect of the fermentation of prozyme system
Embodiment two
According to the embodiment among the embodiment one, Li's Trichoderma strains after the activation and derivative strain thereof are inoculated in the seed culture medium and cultivate, then access contains 3L fermentation culture based sols and the 5l fermentation cylinder for fermentation of packing into of the mixing raw material of 150g/L, fermentation ends is measured cellulase activity and Xylanase activity, the results are shown in Figure 1, cellulase activity reaches 9.7IU/ml behind 96h, Xylanase activity reaches 3000IU/ml at 132h.
Embodiment three
The crude enzyme liquid of preparation among the embodiment two is processed corn cob, gas explosion stalk, do not add the beta-glucosidase crude enzyme liquid, concentration of substrate is 5%, the loading capacity of enzyme is 5 ~ 10IU/g substrate, hydrolyzation system is the pH4.8 of 20ml, and the citric acid solution of 0.05M, hydrolysis time are 48h.As shown in table 2, wherein, enzyme liquid 1 is that Microcrystalline Cellulose and corn cob are as the fermention medium of carbon source, enzyme liquid 2 is that Microcrystalline Cellulose is as the fermention medium of carbon source, the hydrolysis effect of 1 pair of corn cob of enzyme liquid is 33g/L, be 1.59 times of enzyme liquid 2 hydrolysis effects, the gas explosion stalk during as substrate the hydrolysis effect difference of two kinds of enzyme liquid less.
The activity ratio of the different proportionings of table 2 with different substrate hydrolysis test-results
Embodiment four
Carry out prozyme liquid and the beta-glucosidase crude enzyme liquid of preparation among the embodiment two composite, process the pretreated maize straw of 2%NaOH normal temperature, the loading capacity of enzyme is 5 ~ 10IU/g substrate, concentration of substrate is 5%, hydrolysis temperature is 50 ℃, hydrolyzation system is the citric acid buffer system of pH4.5 ~ 5.5, and hydrolysis time is 48 ~ 96h, and hydrolysis finishes to measure concentration of reduced sugar.The prozyme system of the method preparation is 49.4g/L to pretreated maize straw reducing sugar content, and pure Microcrystalline Cellulose is 40g/L as the prozyme system of the zymotechnique of carbon source to pretreated maize straw reducing sugar content, and the former has improved 23.5% than the latter.
Although embodiment of the present invention are open as above, but it is not restricted to listed utilization in specification sheets and the embodiment, it can be applied to various suitable the field of the invention fully, for those skilled in the art, can easily realize other modification, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the legend of describing.
Claims (7)
1. the preparation method of the prozyme of efficient degradation lignocellulose system is characterized in that, may further comprise the steps:
Step 1, activation Li's Trichoderma strains or its derivative strain;
Step 2, Li's Trichoderma strains or its derivative strain after will activating are prepared into spore suspension, are inoculated in the seed culture medium and cultivate, and temperature is 28 ~ 30 ℃, and rotating speed is that 24 ~ 36h is cultivated in concussion on 170 ~ 200 rev/mins the shaking table;
Step 3, the seed culture medium that will inoculate Li's Trichoderma strains or its derivative strain are seeded to take Microcrystalline Cellulose and corn cob as carbon source and ratio of mixture is carried out fermentor cultivation in the fermention medium of 1:4.
2. the preparation method of the prozyme of efficient degradation lignocellulose as claimed in claim 1 system, it is characterized in that the seed culture medium that will inoculate Li's Trichoderma strains or its derivative strain in the described step 3 is seeded to according to 5 ~ 10% inoculum sizes and carries out fermentor cultivation in the described fermention medium.
3. the preparation method of the prozyme of efficient degradation lignocellulose as claimed in claim 1 system is characterized in that, by mass percentage, described fermention medium comprises: carbon source 2% ~ 8%, organic nitrogen source 0.1 ~ 5%, inorganic nitrogen-sourced concentration are 0 ~ 2%, inorganic salt 0.0001 ~ 10%.
4. the preparation method of the prozyme of efficient degradation lignocellulose as claimed in claim 3 system, it is characterized in that, when carrying out fermentor cultivation by described step 3, pH regulator is 3.0 ~ 5.0, temperature is controlled to be 20 ~ 40 ℃, shaking speed is controlled at 100 ~ 250 rev/mins, carries out deep layer liquid state fermentation, and fermentation time is 60 ~ 200 hours.
5. the preparation method of the prozyme of efficient degradation lignocellulose as claimed in claim 4 system is characterized in that described carbon source content the best is 3% ~ 5%.
6. the preparation method of the prozyme of efficient degradation lignocellulose as claimed in claim 1 system is characterized in that described fermentor tank volume is 5L, and the 3L fermention medium is housed.
7. an application that utilizes the prozyme system of method claimed in claim 1 preparation is characterized in that, described prozyme system is processed corn cob and gas explosion stalk and through the pretreated maize straw of NaOH normal temperature.
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| CN103497942A (en) * | 2013-09-25 | 2014-01-08 | 中国科学院微生物研究所 | Myceliophthora thermophila ferment as well as preparation method and application thereof |
| CN105505901A (en) * | 2014-09-26 | 2016-04-20 | 中国科学院天津工业生物技术研究所 | Composition for inducing fungi to realize high-yield production of cellulase and application method thereof |
| CN105695437A (en) * | 2016-01-27 | 2016-06-22 | 江苏康维生物有限公司 | Method for preparing low-beta-glucosidase cellulase additive |
| CN108707595A (en) * | 2018-07-03 | 2018-10-26 | 华东理工大学 | A method of improving Cellulase-producing Fungi yield |
| CN115975819A (en) * | 2022-11-21 | 2023-04-18 | 福建师范大学 | Furfural-tolerant trichoderma reesei mutant strain and application thereof |
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| CN103497942B (en) * | 2013-09-25 | 2016-03-23 | 中国科学院微生物研究所 | Fermented product of a kind of thermophilic fungus destroyed wire and its preparation method and application |
| CN105505901A (en) * | 2014-09-26 | 2016-04-20 | 中国科学院天津工业生物技术研究所 | Composition for inducing fungi to realize high-yield production of cellulase and application method thereof |
| CN105695437A (en) * | 2016-01-27 | 2016-06-22 | 江苏康维生物有限公司 | Method for preparing low-beta-glucosidase cellulase additive |
| CN108707595A (en) * | 2018-07-03 | 2018-10-26 | 华东理工大学 | A method of improving Cellulase-producing Fungi yield |
| CN108707595B (en) * | 2018-07-03 | 2021-07-27 | 华东理工大学 | Method for improving yield of cellulase produced by fungi |
| CN115975819A (en) * | 2022-11-21 | 2023-04-18 | 福建师范大学 | Furfural-tolerant trichoderma reesei mutant strain and application thereof |
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