CN102154412A - Method for increasing sugar yield of cellulase enzymolysis through pretreatment by using ion liquid - Google Patents
Method for increasing sugar yield of cellulase enzymolysis through pretreatment by using ion liquid Download PDFInfo
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Abstract
The invention relates to a method for increasing the sugar yield of cellulase enzymolysis through pretreatment by using ion liquid. In the method, natural lignin from different sources is pretreated by using different types of ion liquids to increase the sugar yield of enzymolysis. The method comprises the following steps of: firstly, pretreating natural lignin at certain temperature under fully stirring condition by using an ion liquid; secondly, extracting regenerated cellulose from a mixed liquor and drying; and lastly, adding cellulase which can decompose the regenerated cellulose serving as a substrate into reducing sugar at certain temperature, pH and concentration to generate liquid glucose mainly containing glucose. By adopting the method, the sugar yield of lignin can be remarkably increased, the accessibility and the reaction efficiency of cellulase can be effectively increased, the liquid glucose reaches the fermentation level at the final reaction stage, and the entire technical process is simplified.
Description
Technical field
The invention belongs to the Mierocrystalline cellulose Application Areas, be specifically related to a kind of method of utilizing ionic liquid pretreatment to improve the cellulase hydrolysis sugar yield.
Background technology
Along with the rapid exhaustion of Nonrenewable energy resources resource oil, Sweet natural gas, colliery, metal mineral reserves etc., the supply and demand contradiction of the energy and resource becomes increasingly conspicuous, and the development and utilization of novel material had caused common people's concern already.Exist a large amount of available natural resourcess at occurring in nature, Mierocrystalline cellulose is exactly an example.Mierocrystalline cellulose is the abundantest renewable resources of occurring in nature, can be widely used in industrial circles such as papermaking, coating.By biotechnology natural wooden fiber's element being converted into biochemical industry products such as ethanol with high economic worth and social value, lactic acid, is the focus of present scientific circles and industrial community research.
The chemical structure of natural cellulose is each other with β-1 by D-Glucopyranose ring, the worm-like polymer that the 4-glycosidic link is formed by connecting, a large amount of oh groups has formed the huge hydrogen bond of number in the molecule, these hydrogen bonds and intermolecular Van der Waals force make Mierocrystalline cellulose form fine and close crystalline structure, seriously having hindered chemical reagent or biological enzyme contacts and effect with the effective of cellulose surface, make it have all undissolved character in water and ordinary organic solvents, be difficult to direct utilization, this is a big obstacle of Mierocrystalline cellulose Application and Development.And natural lignin is carried out pre-treatment, break its fine and close crystalline texture, can realize cellulosic regeneration and functionalization, impel crystalline cellulose to the amorphous state cellulose conversion, improve the effective active area of Mierocrystalline cellulose and enzyme, final its sugar yield that improves is an important channel that effectively utilizes the natural fiber cellulosic material.
Ionic liquid (ionic liquids, ILs) be the ionic system that under the temperature of room temperature or a little higher than room temperature, is in a liquid state, be that a kind of fusing point is near or below 100 ℃ organic salt, because its unique physics and chemical property such as air and water stability, low-steam pressure, with water easily molten and high conductivity etc. and being widely used in a lot of fields, be considered to replace in a lot of fields the green solvent of volatile organic solvent.2002, there is the investigator to find that at first chlorination 1-butyl-3-methyl-imidazoles ([C4mim] C1) ionic liquid is a kind of cellulosic fine solvent, caused and utilized the cellulosic research of ionic liquid pretreatment.Have document to select for use the ionic liquid of a series of chloros and acetoxyl that wood fibre is carried out pre-treatment, the result shows that all treated fibre crystallinities descend, and easier and enzyme reacts.But be noted that, after the pre-treatment in substrate residual ionic liquid know from experience cellulase activity had certain influence, also need use it is removed than the method for system.
Cellulase is meant can hydrocellulose β-1,4 glucoside bond, generates the general name of all enzymes of glucose and cellobiose.Cellulase is not single a kind of enzyme, but multi-component prozyme system, the cellulase of Que Dinging mainly contains three kinds of component: endo-type β-1,4 dextranases and also claims Cx enzyme, CMC enzyme now.It can cut with the noncrystalline domain of form in cellulose polymer compound inside at random, and is littler than middle key to the susceptibility of terminal key.Circumscribed-type β-1,4 dextranase also claims C1 enzyme, cellobiohydrolase or avicelase.It can generate soluble cellobiose from the non reducing end cutting of cellulose chain.Cellobiase also claims beta-glucosidase, and the cell-oligosaccharide of its energy hydrolysis fiber disaccharides and short chain generates glucose, and is very fast to the hydrolysis of cellobiose and procellose, and along with the increase hydrolysis rate decline of glucose polymerization degree, the specificity of this kind of enzyme is poor.
At present, the source of the plain enzyme of industrial fibre mainly is the cellulase that aerobic filamentous fungus produces, as: wood mould (Trichoderma), aspergillus (Aspergillus), mould (Penicillium) etc.The cellulase system of different sources is formed the content difference, and is also different, wherein most widely used with the cellulase of Trichodermareesei, the mould generation of healthy and free from worry wood to the natural cellulose enzymolysis efficiency of different sources, and its advantage is that output is big, enzyme is lived height, is easy to extract and use; Shortcoming then is that the cellulase system composition of Trichoderma is undesirable, and β-glucose glycosides enzyme relatively lacks.The accumulation that this will cause cellobiose in the cellulase hydrolysis liquid is one of inefficient major reason of enzymolysis process, can produce the composition that enzyme can improve enzyme system by beta-glucosidase or many bacterium mixed culture of adding external source.
The interaction of Mierocrystalline cellulose enzyme-to-substrate is the process of a more complicated.The katalysis of enzyme is the interactional process of enzyme-to-substrate, product, and a plurality of factors such as concentration of the vigor of the speed of cellulase hydrolysis saccharification and transformation efficiency and enzyme, the character of substrate and product are closely related.Temperature is the important parameter that enzymolysis process will be considered to the highly significant that influences of enzyme mechanism.The optimal temperature of the plain enzyme effect of industrial fiber is generally at 40 ℃ ~ 60 ℃, and is different and different with the source of enzyme.But the optimum temperuture of enzyme is not the characteristic constant of enzyme, the specific enzyme, selects optimum temps need test definite in concrete technology.Another important parameters is pH, and the optimal pH of acidic cellulase is about 4.8, and neutral cellulase is decided according to the cellulase kind of selecting for use about 6.8.And other enzymatic hydrolysis condition as enzyme carrying capacity and solid-to-liquid ratio etc., can influence the efficient and the sugar yield of enzymolysis too, and then influence the technology cost.
Relevant report in recent years mainly concentrates on the condition optimizing aspect of cellulosic pretreatment process and enzymolysis process, seek more effectively preprocessing means and raising enzyme method alive, ionic liquid is a kind of novel green cellosolve of rising in recent years, for cellulosic development research important effect is arranged, and the research association that adapts to the cellulase of its cellulosic substrate of handling further promotes cellulosic application, its product glucose can be applicable to fields such as degradable material lactic acid, biofuel ethanol, has significant values.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing ionic liquid pretreatment to improve the cellulase hydrolysis sugar yield.
The ionic liquid pretreatment of utilizing that the present invention proposes improves the method for cellulase hydrolysis sugar yield, at first select for use different types of ionic liquid respectively the natural lignin of different sources to be carried out pre-treatment, obtain regenerated cellulose, under certain enzymatic hydrolysis condition, carry out enzymolysis then, and carry out sampling and testing at set intervals, observe the changing conditions of the reducing sugar yield of cellulosic substrate in whole enzymolysis process and final sugar yield, and with compare without pretreated natural lignin sugar yield, the research ionic liquid pretreatment is to the influence of xylogen sugar yield.
For solving the problems of the technologies described above, the present invention adopts following technical scheme:
For breaking the crystalline texture of natural lignin, improve the contact area and the reactable of cellulase, at first natural lignin is carried out ionic liquid pretreatment, concrete steps are:
(1) natural lignin's powder is carried out drying treatment, and its particle diameter is controlled at 80 μ m～120 μ m;
(2) with the ionic liquid be solvent, at 100～150 ℃, natural lignin's powder and ion liquid mass percent is under the condition of 5～20 ﹪, the xylogen powder is joined in the ionic liquid, impose stirring, inner cellulose was dissolved in the ionic liquid fully in 1～5 hour, formed the ionic liquid solution of natural lignin;
(3) in the ionic liquid of xylogen, add deionized water and carry out cellulosic regeneration, the volume of the deionized water that is added is equivalent to 5～20 times of xylogen ionic liquid solution volume, filtering precipitate then, carry out lyophilize after this throw out cleaned 1～5 time repeatedly, obtain regenerated cellulose;
(4) take by weighing a certain amount of regenerated cellulose then, add damping fluid, the weight ratio of regenerated cellulose and damping fluid is 1:5～1:40, and the ratio according to 5～30FPU/g substrate adds cellulase again, carries out 20～80h enzymolysis; In enzymolysis process, extract sugar every 3～12h and separate liquid, and measure the sugar yield of this moment, finish until enzyme digestion reaction, and measure final sugar yield.
Among the present invention, employed natural wooden fiber element is the common solid waste of occurring in nature in the preprocessing process, comprising: comprising: the corn stalk xylogen, in straw straw xylogen, corn cob xylogen, Cassava stalk, rice husk or the bagasse etc. any.
Among the present invention, employed ionic liquid is 1-propenyl-3-Methylimidazole villaumite [AMIM] Cl, 3-methyl-N-butyl-pyridinium tribromide salt [BMPy] Br, 1-butyl-3-Methylimidazole villaumite [BMIM] Cl, 1-ethyl-3-Methylimidazole tetrafluoro boron salt [EMIM] BF
4Or among 1-butyl-3-Methylimidazole trifluoromethyl sulfonic acid [BMIM] OTF etc. any.
Among the present invention, in enzymolysis process, employed ph value of buffer solution is consistent with the scope of application of selected cellulase, to guarantee that at utmost bringing into play enzyme lives.The cellulase system that selected cellulase is viride, Trichodermareesei, aspergillus niger, healthy and free from worry wood is mould or nipa palm aspergillus etc. originated.
Among the present invention, in the enzymolysis process, stirring velocity is 150～190r/min, carries out under a certain steady temperature in 40～60 ℃.
Beneficial effect of the present invention is: the present invention can obviously improve the sugar yield of xylogen, effectively improves the accessibility and the reaction efficiency of cellulase, but makes the final liquid glucose of reaction reach fermentation level, has simplified whole technological process.
Embodiment
Further specify the present invention below by embodiment.
Embodiment 1
The sugar yield of the straw straw fiber element that ionic liquid [AMIM] Cl is pretreated.
(1) ionic liquid [AMIM] Cl preprocessing straw straw fiber element
A) with straw straw fiber element at 100 ℃ of following drying treatment 24h, its particle diameter announced be controlled at 80 μ m～120 μ m.
B) accurately weighing 5 g straw straws and 45 g ionic liquid [AMIM] Cl put into the 100ml round-bottomed flask jointly, and in 130 ℃ of sealings, intense mechanical stirs 2h and dissolves fully to Mierocrystalline cellulose, this moment solution to be dark-brown thick.
C) stop to stir, and the cellulose ionic liquid solution of gained washed three times repeatedly with 5 times of deionized waters to its volume, obtain white sediment at last, i.e. regenerated cellulose, after the filtration that product lyophilize 48 h grind into powders are stand-by, and utilize vacuum rotary steam to reclaim ionic liquid.
(2) utilize Richter scale wood enzyme that regenerated cellulose is carried out enzymolysis
A) accurate weighing 3 g regeneration straw rod fibers element places the 250ml Erlenmeyer flask stand-by.Add pH=4.8 damping fluid 60g again, after stirring, add total enzyme according to the ratio of 15FPU/g substrate and live to the Richter scale wood enzyme of 45FPU, put into 50 ℃ after the sealing, rotating speed is to carry out enzymolysis in 170 rev/mins the constant temperature oscillator.
B), get the 1mL enzymolysis solution in centrifuge tube, with the centrifugal 5min of the rotating speed of 5000rpm every 3h, get supernatant liquor 0.5mL, add 0.5mL buffered soln and 3mL DNS solution (autogamy), in boiling water, carry out common thermal response 10min, be settled to 25ml capping plug mixing after the cooling rapidly.With the same condition solution that does not add cellulase be blank sample with the spectrophotometer zero clearing, with 10 mm cuvettes, measure absorbancy at spectrophotometer wavelength 540nm place, get final product in the substitution glucose typical curve glucose content.In the reaction later stage, because increasing, sugar yield slows down, sampling interval time extends to 6 h, 12 h, 18 h.
C) enzyme digestion reaction is finished behind the 72h, stopped reaction, and measure final sugar yield, its result data is as shown in the table.
| Time/h | 3 | 6 | 9 | 12 | 24 | 36 | 54 | 72 |
| Sugar yield/% | 12.86 | 15.47 | 16.03 | 17.02 | 17.35 | 18.14 | 18.52 | 18.98 |
Embodiment 2
The sugar yield of the straw straw fiber element that ionic liquid [BMPy] Br is pretreated
(1) ionic liquid [BMPy] Br preprocessing straw straw fiber element
A) with straw straw fiber element at 100 ℃ of following drying treatment 24h, its particle diameter announced be controlled at 80 μ m～120 μ m.
B) accurately weighing 5 g straw straws and 45 g ionic liquid [BMPy] Br put into the 100ml round-bottomed flask jointly, and in 130 ℃ of sealings, intense mechanical stirs 2h and dissolves fully to Mierocrystalline cellulose, this moment solution to be dark-brown thick.
C) stop to stir, and the cellulose ionic liquid solution of gained washed three times repeatedly with 5 times of deionized waters to its volume, obtain white sediment at last, i.e. regenerated cellulose, after the filtration that product lyophilize 48 h grind into powders are stand-by, and utilize vacuum rotary steam to reclaim ionic liquid.
(2) utilize Richter scale wood enzyme that regenerated cellulose is carried out enzymolysis
A) accurate weighing 3 g regeneration straw rod fibers element places the 250ml Erlenmeyer flask stand-by.Add pH=4.8 damping fluid 60g again, after stirring, add total enzyme according to the ratio of 15FPU/g substrate and live to the Richter scale wood enzyme of 45FPU, put into 50 ℃ after the sealing, rotating speed is to carry out enzymolysis in 170 rev/mins the constant temperature oscillator.
B), get the 1mL enzymolysis solution in centrifuge tube, with the centrifugal 5min of the rotating speed of 5000rpm every 3h, get supernatant liquor 0.5mL, add 0.5mL buffered soln and 3mL DNS solution (autogamy), in boiling water, carry out common thermal response 10min, be settled to 25ml capping plug mixing after the cooling rapidly.With the same condition solution that does not add cellulase be blank sample with the spectrophotometer zero clearing, with 10 mm cuvettes, measure absorbancy at spectrophotometer wavelength 540nm place, get final product in the substitution glucose typical curve glucose content.In the reaction later stage, because increasing, sugar yield slows down, sampling interval time extends to 6 h, 12 h, 18 h.
C) enzyme digestion reaction is finished behind the 72h, stopped reaction, and measure final sugar yield, its result data is as shown in the table.
| Time/h | 3 | 6 | 9 | 12 | 24 | 36 | 54 | 72 |
| Sugar yield/% | 7.73 | 10.33 | 10.85 | 12.12 | 13.06 | 14.39 | 15.28 | 15.96 |
Comparative Examples 1
The sugar yield of natural straw straw fiber element
(1) with straw straw fiber element at 100 ℃ of following drying treatment 24h, its particle diameter announced be controlled at 80 μ m～120 μ m.
(2) accurate weighing 3 g grass stalk Mierocrystalline celluloses place the 250ml Erlenmeyer flask stand-by.Add pH=4.8 damping fluid 60g again, after stirring, add total enzyme according to the ratio of 15FPU/g substrate and live to the Richter scale wood enzyme of 45FPU, put into 50 ℃ after the sealing, rotating speed is to carry out enzymolysis in 170 rev/mins the constant temperature oscillator.
B), get the 1mL enzymolysis solution in centrifuge tube, with the centrifugal 5min of the rotating speed of 5000rpm every 3h, get supernatant liquor 0.5mL, add 0.5mL buffered soln and 3mL DNS solution (autogamy), in boiling water, carry out common thermal response 10min, be settled to 25ml capping plug mixing after the cooling rapidly.With the same condition solution that does not add cellulase be blank sample with the spectrophotometer zero clearing, with 10 mm cuvettes, measure absorbancy at spectrophotometer wavelength 540nm place, get final product in the substitution glucose typical curve glucose content.In the reaction later stage, because increasing, sugar yield slows down, sampling interval time extends to 6 h, 12 h, 18 h.
(3) enzyme digestion reaction is finished behind the 72h, stopped reaction, and measure final sugar yield, its result data is as shown in the table.
| Time/h | 3 | 6 | 9 | 12 | 24 | 36 | 54 | 72 |
| Sugar yield/% | 5.12 | 6.04 | 6.66 | 7.30 | 8.02 | 8.40 | 8.72 | 8.86 |
By comparing with Comparative Examples 1, embodiment 1 and 2 sugar yield all are greatly increased, especially [AMIM] Cl ionic liquid, final sugar yield has been improved more than two times, the proof ionic liquid pretreatment has active influence for the sugar yield that improves natural lignin, and for natural straw straw xylogen, it is more obvious that glyoxaline ion liquid improves degree than pyridines.No matter and for embodiment or Comparative Examples, the Changing Pattern of sugar yield is consistent, main output of sugar concentrates on preceding 24 h of reaction.
Embodiment 3
The cellulosic sugar yield of corn stalk that ionic liquid [AMIM] Cl is pretreated
(1) ionic liquid [AMIM] Cl pre-treatment corn stalk Mierocrystalline cellulose
A) with the corn stalk Mierocrystalline cellulose at 100 ℃ of following drying treatment 24h, its particle diameter announced be controlled at 80 μ m～120 μ m.
B) accurately weighing 5 g corn stalks and 45 g ionic liquid [AMIM] Cl put into the 100ml round-bottomed flask jointly, and in 130 ℃ of sealings, intense mechanical stirs 2h and dissolves fully to Mierocrystalline cellulose, this moment solution to be dark-brown thick.
C) stop to stir, and the cellulose ionic liquid solution of gained washed three times repeatedly with 5 times of deionized waters to its volume, obtain white sediment at last, i.e. regenerated cellulose, after the filtration that product lyophilize 48 h grind into powders are stand-by, and utilize vacuum rotary steam to reclaim ionic liquid.
(2) utilize Richter scale wood enzyme that regenerated cellulose is carried out enzymolysis
A) accurate weighing 3 g regeneration cornstalk fiber element places the 250ml Erlenmeyer flask stand-by.Add pH=4.8 damping fluid 60g again, after stirring, add total enzyme according to the ratio of 15FPU/g substrate and live to the Richter scale wood enzyme of 45FPU, put into 50 ℃ after the sealing, rotating speed is to carry out enzymolysis in 170 rev/mins the constant temperature oscillator.
B), get the 1mL enzymolysis solution in centrifuge tube, with the centrifugal 5min of the rotating speed of 5000rpm every 3h, get supernatant liquor 0.5mL, add 0.5mL buffered soln and 3mL DNS solution (autogamy), in boiling water, carry out common thermal response 10min, be settled to 25ml capping plug mixing after the cooling rapidly.With the same condition solution that does not add cellulase be blank sample with the spectrophotometer zero clearing, with 10 mm cuvettes, measure absorbancy at spectrophotometer wavelength 540nm place, get final product in the substitution glucose typical curve glucose content.In the reaction later stage, because increasing, sugar yield slows down, sampling interval time extends to 6 h, 12 h, 18 h.
C) enzyme digestion reaction is finished behind the 72h, stopped reaction, and measure final sugar yield, its result data is as shown in the table.
| Time/h | 3 | 6 | 9 | 12 | 24 | 36 | 54 | 72 |
| Sugar yield/% | 12.83 | 14.75 | 15.14 | 16.13 | 17.32 | 17.61 | 17.89 | 18.14 |
Embodiment 4
The cellulosic sugar yield of corn stalk that ionic liquid [BMPy] Br is pretreated
(1) ionic liquid [BMPy] Br pre-treatment corn stalk Mierocrystalline cellulose
A) with the corn stalk Mierocrystalline cellulose at 100 ℃ of following drying treatment 24h, its particle diameter announced be controlled at 80 μ m～120 μ m.
B) accurately weighing 5 g corn stalks and 45 g ionic liquid [BMPy] Br put into the 100ml round-bottomed flask jointly, and in 130 ℃ of sealings, intense mechanical stirs 2h and dissolves fully to Mierocrystalline cellulose, this moment solution to be dark-brown thick.
C) stop to stir, and the cellulose ionic liquid solution of gained washed three times repeatedly with 5 times of deionized waters to its volume, obtain white sediment at last, i.e. regenerated cellulose, after the filtration that product lyophilize 48 h grind into powders are stand-by, and utilize vacuum rotary steam to reclaim ionic liquid.
(2) utilize Richter scale wood enzyme that regenerated cellulose is carried out enzymolysis
A) accurate weighing 3 g regeneration cornstalk fiber element places the 250ml Erlenmeyer flask stand-by.Add pH=4.8 damping fluid 60g again, after stirring, add total enzyme according to the ratio of 15FPU/g substrate and live to the Richter scale wood enzyme of 45FPU, put into 50 ℃ after the sealing, rotating speed is to carry out enzymolysis in 170 rev/mins the constant temperature oscillator.
B), get the 1mL enzymolysis solution in centrifuge tube, with the centrifugal 5min of the rotating speed of 5000rpm every 3h, get supernatant liquor 0.5mL, add 0.5mL buffered soln and 3mL DNS solution (autogamy), in boiling water, carry out common thermal response 10min, be settled to 25ml capping plug mixing after the cooling rapidly.With the same condition solution that does not add cellulase be blank sample with the spectrophotometer zero clearing, with 10 mm cuvettes, measure absorbancy at spectrophotometer wavelength 540nm place, get final product in the substitution glucose typical curve glucose content.In the reaction later stage, because increasing, sugar yield slows down, sampling interval time extends to 6 h, 12 h, 18 h.
C) enzyme digestion reaction is finished behind the 72h, stopped reaction, and measure final sugar yield, its result data is as shown in the table.
| Time/h | 3 | 6 | 9 | 12 | 24 | 36 | 54 | 72 |
| Sugar yield/% | 9.80 | 11.87 | 14.33 | 16.08 | 16.80 | 17.35 | 17.81 | 18.06 |
Comparative Examples 2
The sugar yield of natural corn straw fiber element
(1) with the corn stalk Mierocrystalline cellulose at 100 ℃ of following drying treatment 24h, its particle diameter announced be controlled at 80 μ m～120 μ m.
(2) accurate weighing 3 g cornstalk fiber elements place the 250ml Erlenmeyer flask stand-by.Add pH=4.8 damping fluid 60g again, after stirring, add total enzyme according to the ratio of 15FPU/g substrate and live to the Richter scale wood enzyme of 45FPU, put into 50 ℃ after the sealing, rotating speed is to carry out enzymolysis in 170 rev/mins the constant temperature oscillator.
B), get the 1mL enzymolysis solution in centrifuge tube, with the centrifugal 5min of the rotating speed of 5000rpm every 3h, get supernatant liquor 0.5mL, add 0.5mL buffered soln and 3mL DNS solution (autogamy), in boiling water, carry out common thermal response 10min, be settled to 25ml capping plug mixing after the cooling rapidly.With the same condition solution that does not add cellulase be blank sample with the spectrophotometer zero clearing, with 10 mm cuvettes, measure absorbancy at spectrophotometer wavelength 540nm place, get final product in the substitution glucose typical curve glucose content.In the reaction later stage, because increasing, sugar yield slows down, sampling interval time extends to 6 h, 12 h, 18 h.
(3) enzyme digestion reaction is finished behind the 72h, stopped reaction, and measure final sugar yield, its result data is as shown in the table.
| Time/h | 3 | 6 | 9 | 12 | 24 | 36 | 54 | 72 |
| Sugar yield/% | 6.86 | 8.08 | 8.71 | 9.53 | 10.34 | 10.68 | 10.76 | 11.04 |
By comparing with Comparative Examples 2, embodiment 3 and 4 sugar yield all are greatly increased, the degree that ionic liquid [AMIM] Cl pre-treatment improves is a little more than ionic liquid [BMPy] Br pre-treatment, but difference of them is little, two kinds of ionic liquid pretreatment have active influence for the sugar yield that improves natural corn straw xylogen, no matter and for embodiment or Comparative Examples, the Changing Pattern of sugar yield is consistent, main output of sugar concentrates on preceding 24 h of reaction.
Above-described embodiment has only expressed several embodiment of the present invention, and it describes concrete and detailed, but can not be interpreted as the restriction to scope of patent protection of the present invention.Be to be noted that correlative study of this area or technician also can make all kinds of distortion and improvement under the prerequisite of the present invention's design, these all belong to the protection domain of patent of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (6)
1. method of utilizing ionic liquid pretreatment to improve the cellulase sugar yield is characterized in that concrete steps are as follows:
(1) natural lignin's powder is carried out drying treatment, and its particle diameter is controlled at 80 μ m～120 μ m;
(2) with the ionic liquid be solvent, at 100～150 ℃, natural lignin's powder and ion liquid mass percent is under the condition of 5～20 ﹪, the xylogen powder is joined in the ionic liquid, stir, inner cellulose was dissolved in the ionic liquid fully in 1～5 hour, formed the ionic liquid solution of natural lignin;
(3) in the ionic liquid of xylogen, add deionized water and carry out cellulosic regeneration, the volume of the deionized water that is added is equivalent to 5～20 times of xylogen ionic liquid solution volume, filtering precipitate then, carry out lyophilize after this throw out cleaned 1～5 time repeatedly, obtain regenerated cellulose, grind into powder is stand-by;
(4) take by weighing a certain amount of regenerated cellulose, add damping fluid, the weight ratio of regenerated cellulose and damping fluid is 1:5～1:40, and the ratio according to 5～30FPU/g substrate adds cellulase again, carries out 20～80h enzymolysis;
(5) in enzymolysis process, extract sugar every 3～12h and separate liquid, and measure the sugar yield of this moment, finish until enzyme digestion reaction, and measure final sugar yield.
2. the method for utilizing ionic liquid pretreatment to improve the cellulase sugar yield according to claim 1, it is characterized in that: natural wooden fiber described in the step (1) is plain to be the corn stalk xylogen, in straw straw xylogen, corn cob xylogen, Cassava stalk, rice husk or the bagasse any.
3. the method for utilizing ionic liquid pretreatment to improve the cellulase sugar yield according to claim 1 is characterized in that: ionic liquid described in the step (2) be in 1-propenyl-3-Methylimidazole villaumite, 3-methyl-N-butyl-pyridinium tribromide salt, 1-butyl-3-Methylimidazole villaumite, 1-ethyl-3-Methylimidazole tetrafluoro boron salt or the 1-butyl-3-Methylimidazole trifluoromethyl sulfonic acid any.
4. the method for utilizing ionic liquid pretreatment to improve the cellulase sugar yield according to claim 1, it is characterized in that: the pH value of described damping fluid is consistent with the scope of application of selected cellulase, to guarantee that at utmost bringing into play enzyme lives.
5. the method for utilizing ionic liquid pretreatment to improve the cellulase sugar yield according to claim 1 is characterized in that: cellulase described in the step (4) is viride, Trichodermareesei, aspergillus niger, healthy and free from worry wood is mould or nipa palm aspergillus in any.
6. the method for utilizing ionic liquid pretreatment to improve the cellulase sugar yield according to claim 1, it is characterized in that: in the enzymolysis process, stirring velocity is 150～190r/min described in the step (5), carries out under a certain steady temperature in 40～60 ℃.
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| CN105385722A (en) * | 2014-09-09 | 2016-03-09 | 中国科学院大连化学物理研究所 | Method for pre-processing lignocellulose biomass to increase sugar field of lignocellulose biomass |
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| CN105647813A (en) * | 2014-12-05 | 2016-06-08 | 中国石油化工股份有限公司 | Trichoderma viride strain, and applications thereof |
| CN105647813B (en) * | 2014-12-05 | 2019-05-21 | 中国石油化工股份有限公司 | One plant of Trichoderma viride and its application |
| CN104862345B (en) * | 2015-06-18 | 2018-10-19 | 南京林业大学 | A kind of poison-removing method of lignocellulose raw material prehydrolysis liquid glucose |
| CN104862345A (en) * | 2015-06-18 | 2015-08-26 | 南京林业大学 | Detoxification method for wood fiber raw material pre-hydrolysis sugar liquid |
| CN107418987A (en) * | 2017-08-31 | 2017-12-01 | 齐鲁工业大学 | A kind of processing method for improving wood-cellulose enzymatic saccharification |
| CN107505343A (en) * | 2017-10-19 | 2017-12-22 | 中南大学 | A kind of method that rapid evaluation lignocellulosic enzymolysis produces sugared ability |
| CN109161566A (en) * | 2018-09-29 | 2019-01-08 | 南京工业大学 | Method for producing butyric acid by using whole components of corncobs |
| CN109161566B (en) * | 2018-09-29 | 2021-07-20 | 南京工业大学 | Method for producing butyric acid by using whole components of corncobs |
| CN110066840A (en) * | 2019-03-08 | 2019-07-30 | 华南农业大学 | A kind of method that hydro-thermal-microwave coupled ion liquid two-step method lignocellulosic material improves enzymolysis efficiency |
| CN113174382A (en) * | 2021-04-16 | 2021-07-27 | 山东大学 | Cellulase system suitable for improving corn fiber sugar conversion rate in saccharification liquid preparation and application thereof |
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