CN100467609C - Method for saccharification of lignocellulose by ultrasonic synergistic catalysis of modified cellulose - Google Patents
Method for saccharification of lignocellulose by ultrasonic synergistic catalysis of modified cellulose Download PDFInfo
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- CN100467609C CN100467609C CNB2006101299177A CN200610129917A CN100467609C CN 100467609 C CN100467609 C CN 100467609C CN B2006101299177 A CNB2006101299177 A CN B2006101299177A CN 200610129917 A CN200610129917 A CN 200610129917A CN 100467609 C CN100467609 C CN 100467609C
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Abstract
The invention discloses a saccharifying method of xylon cellulose through ultrasonic coordinated modified cellulose enzyme, which comprises the following steps: grinding and predisposoing xylon in the raw material through alkaline; reacting activated methoxy carbowax and cellulose enzyme in the citrate-sodium citrate buffer solution to obtain modified cellulose enzyme; blending modified cellulose enzyme, beta-glucosidase, amylase and pectase to obtain composite liquid; adding composite enzyme into predisposed raw material according to corresponding proportion; proceeding enzyme catalytic reaction through ultrasound; filtering; decompressing; evaporating; obtaining condensed sugar liquid.
Description
Technical field
The present invention relates to a kind of method of saccharification of lignocellulose by ultrasonic synergistic catalysis of modified cellulose.The technology that belongs to the lignocellulose saccharification.
Background technology
Production of fuel ethanol technology can be divided into two processes, is respectively the saccharification of raw material and the fermenting process of sugar, and soon feedstock conversion is the process of fermentable saccharide and utilizes carbohydrate to carry out the process of production of ethanol from microbial fermentation.In whole process of production, biocatalysis technology is widely adopted.The production of traditional fuel alcoholic acid is raw material with grain, mainly uses amylase as catalyzer starch to be changed into fermentable carbohydrate, and its output is subjected to the restriction of grain resource, is difficult to satisfy for a long time energy demand.The biomass waste of lignocellulose-containing is another raw material sources of producing alcohol fuel, and it comprises abandoned biomass contained in agricultural crop straw, forestry processing waste material, bagasse and the municipal wastes etc.Third generation alcohol fuel with various lignocelluloses as raw materials for production, there is stable crystalline texture because lignocellulose has complex construction and the Mierocrystalline cellulose itself that xylogen, Mierocrystalline cellulose, hemicellulose wrap up mutually, makes lignocellulose be difficult to be decomposed into the carbohydrate that can be used for fermenting alcohol.The saccharification of lignocellulose is one of subject matter of transforming fuel alcohol.
The saccharification of lignocellulose at present mainly comprises acid hydrolysis and biological enzyme dual mode, acid hydrolysis process is because cost height, seriously polluted, abandoned gradually using, what now just be widely studied is the Mashing process that utilizes cellulase biocatalysis decomposing lignocellulose.This technology has less energy-consumption, free of contamination advantage, but because the lignocellulose complex structure is difficult to decomposition; The cellulase functioning efficiency is low, and its functioning efficiency mainly depends on the pretreatment technology of raw material; Efficiently, low-cost, the pretreatment technology of pollution remain exploitation, so thereby to reduce cost all be present lignocellulose Mashing process existing questions to the consumption that improves enzymatic efficient and reduce enzyme.Using prozyme catalysis is the effective means that improves enzyme catalysis efficient, but because the working conditions of different zymins is variant, makes that the prozyme Catalytic processes is very complicated or be difficult to bring into play fully the katalysis of enzyme.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing saccharification of lignocellulose by ultrasonic synergistic catalysis of modified cellulose.This method has catalytic efficiency height, raw material comprehensive utilization, the low characteristics of environmental pollution.
For achieving the above object, the present invention is realized by following technical proposals.A kind of method of ultrasonic synergistic modified-cellulose enzyme catalysis saccharification of cellulose reaction is characterized in that comprising following process:
1, the pre-treatment of raw material: maize straw being ground into 20～40 order particles, getting the raw material that crushes, is 1:10～20 by the grams per milliliter solid-to-liquid ratio, react 3h～8h with mass concentration 3%～10%NaOH in 25 ℃, suction filtration gets filter residue and filtrate, and filter residue is washed to the pH value and is 7～8, and is standby.With mass concentration 98% vitriol oil filtrate being transferred to the pH value is 3～6, is heated to 30～80 ℃, and precipitation 30～60min centrifugation obtains xylogen.
2, cellulase modification: the preparation mass concentration is 6%～15% cyanuric chloride benzole soln, and 2～20 ℃ of recrystallizations twice obtain the cyanuric chloride of purifying.Mass ratio by mono methoxy polyethylene glycol 4000 and 4A molecular sieve and anhydrous sodium carbonate is 1: (2～6): (4～10) add mono methoxy polyethylene glycol 4000,4A molecular sieve and anhydrous sodium carbonate in the solvent benzol, after the stirring at normal temperature 30～60 minutes, filter, remove 4A molecular sieve and yellow soda ash and obtain filtrate, mol ratio according to polymethoxy ethylene glycol 4000 and the cyanuric chloride of purifying is that 2～2.5: 1 cyanuric chloride with purifying joins in the filtrate then, normal temperature condition reaction 20～50 hours down.Solution through concentrating, filters again, and cryodrying obtains the activatory mono methoxy polyethylene glycol.Is that 1: 2～6 to join pH be in 5.0～7.0 citric acid-sodium citrate buffer with cellulase and activation mono methoxy polyethylene glycol according to mass ratio, and controlled temperature is 0 ℃～8 ℃, reacts 1～5 hour.Adopting molecular weight cut-off is that 5000～10000 ultra-filtration membrane is isolated the modified-cellulose enzyme of getting continuously with mono methoxy polyethylene glycol glue, and frozen drying obtains the modified-cellulose enzyme.
3, the activation of enzyme: by the modified-cellulose enzyme and the mass ratio of β-glucuroide, amylase and polygalacturonase is after 10:1:5:5 mixes, and activates 5～15min in 40～60 ℃ of water, obtains prozyme liquid.
4, the saccharification of raw cellulose: mass ratio 1:8～10 by particulate material and water in the filter residue that step 1 obtains add entry, adjust the temperature to 45～55 ℃, are 5～6 with mass concentration 98% vitriol oil pH value.Press the mixed enzyme solution after 0.5～1% of maize straw weight adds activation, utilize sonic oscillation to promote the enzymatic hydrolysis reaction, sonic oscillation power is 80～800w, and oscillation frequency is 20～40kHz, and the reaction times is 3～8h.It is standby that suction filtration gets liquid glucose.
5, concentrating of liquid glucose: 40 ℃～70 ℃ of the liquid glucoses of step 4, it is 25～35% liquid glucoses that reduction vaporization obtains mass concentration, is used for the zymotechnique of alcohol fuel.
The invention has the advantages that: whole Mashing process has been avoided High Temperature High Pressure and acid treatment process, and equipment cost reduces, and energy consumption is relatively low, pollutes little; The modified-cellulose enzyme strengthens the adaptive faculty of environment, can add and reach higher activity simultaneously with other auxiliary enzymes preparations; Sonic oscillation can effectively improve the efficient of enzymic catalytic reaction, reduces the reaction times; To the raw material comprehensive utilization, can obtain lignin product; Final saccharification liquid concentration is higher, and does not contain the deleterious toxic component of fermented yeast.
Embodiment
Embodiment one: maize straw is ground into 40 order particles, gets the raw material that 10g crushes, add 8%NaOH150ml in 25 ℃ of reaction 8h, suction filtration gets filter residue and filtrate.With 6.5ml mass concentration 98% vitriol oil filtrate pH value is adjusted into 3,50 ℃ of temperature, precipitation 30min centrifugation obtains purified xylogen 0.92g, and extraction yield is 84.4%, and product purity is 82%.It is 8 that filter residue is washed to the pH value, adds 80mlH again in filter residue
2O adjusts temperature to 50 ℃, with 1.2ml concentration 2mol/L sulfuric acid the pH value is transferred to 6.5.
The 20.0g cyanuric chloride is dissolved in the 300mL benzene, behind twice in 20 ℃ of recrystallizations, obtains the cyanuric chloride 17.9g of purifying.The 4A molecular sieve that in the there-necked flask of 1000mL, adds 5.0g, the 10.0g anhydrous sodium carbonate, 500mL benzene and 20.0g mono methoxy polyethylene glycol 4000, stirring at normal temperature is after 30 minutes, and B filters, and removes 4A molecular sieve and yellow soda ash; Filtrate collection adds the cyanuric chloride 0.4613g of purifying then in there-necked flask, normal temperature condition reacted 36 hours down.Solution is through concentrating, and B filters, and is-14 ℃ of following cryodryings in temperature then, obtains activatory mono methoxy polyethylene glycol 4000.The activation mono methoxy polyethylene glycol 4000 and the pH that add 4.0g in the flask of 500mL are 6.8 citric acid-sodium citrate buffer 100mL, attemperation is 4 ℃, activation mono methoxy polyethylene glycol 4000 fully adds the 1.0g cellulases in the dissolving back, react that to utilize molecular weight cut-off after 2 hours be that 5000 polysulphones hyperfiltration membrane separation obtains the modified-cellulose enzyme.
Get modified-cellulose enzyme 50mg, β-glucuroide 5mg, amylase 25mg, polygalacturonase 25mg respectively, in 20ml, 50 ℃ of water, activate 15min.Add the enzyme liquid after activating then in solution, carry out enzymic catalytic reaction in ultrasonic field, sonic oscillation power is 80w, and oscillation frequency is 20kHz, reaction 8h.Suction filtration gets filtrate, and it is 35.5% liquid glucose 12ml that 55 ℃ of reduction vaporizations of filtrate obtain concentration of reduced sugar, and this liquid glucose is used for the zymotechnique of alcohol fuel.The degradation rate that records lignocellulose is 67.2%, conversion coefficient 44.3%.
Embodiment two: maize straw is ground into 20 order particles, gets the raw material that 10g crushes, add 5%NaOH150ml in 25 ℃ of reaction 3h, suction filtration gets filter residue and filtrate.With 4.2ml mass concentration 98% vitriol oil filtrate pH value is adjusted into 3,50 ℃ of temperature, precipitation 30min centrifugation obtains purified xylogen 0.73g, and extraction yield is 67%, and product purity is 79%.It is 8 that filter residue is washed to the pH value, adds 80mlH again in filter residue
2O adjusts temperature to 50 ℃, with 1.1ml concentration 2mol/L sulfuric acid the pH value is transferred to 6.5.
The 10.0g cyanuric chloride is dissolved in the 150mL benzene, and 12 ℃ of recrystallizations twice obtain the cyanuric chloride 8.7g of purifying.The 4A molecular sieve that in the there-necked flask of 1000mL, adds 2.5g, the 8.0g anhydrous sodium carbonate, 400mL benzene and 10.0g mono methoxy polyethylene glycol 4000, stirring at normal temperature is after 40 minutes, and B filters, and removes 4A molecular sieve and yellow soda ash; Filtrate collection adds the cyanuric chloride 0.2306g of purifying then in there-necked flask, normal temperature condition reacted 38 hours down.Solution is through concentrating, and B filters, and temperature is-14 ℃ of following cryodryings, obtains activatory mono methoxy polyethylene glycol 4000.Adding 8.0g activation mono methoxy polyethylene glycol 4000 and pH are 7.0 citric acid-sodium citrate buffer 200mL in the flask of 500mL, attemperation is 4 ℃, activation mono methoxy polyethylene glycol 4000 fully adds the 2.0g cellulases in the dissolving back, and react after 2 hours with molecular weight cut-off is that 4000 polysulphones hyperfiltration membrane separation obtains the modified-cellulose enzyme.
Distinguish extracting cellulose enzyme 50mg, β-glucuroide 5mg, amylase 25mg, polygalacturonase 25mg, in 20ml, 50 ℃ of water, activate 10min.Add the enzyme liquid after activating then in solution, carry out enzymic catalytic reaction in ultrasonic field, sonic oscillation power is 300w, and oscillation frequency is 40kHz, reaction 4h.Press filtration gets filtrate, and filtrate is heated, and to obtain concentration of reduced sugar be 30.2% liquid glucose 12ml to reduction vaporization, can be used for the zymotechnique of alcohol fuel.The degradation rate that records lignocellulose is 54.2%, conversion coefficient 36%.
Embodiment three: maize straw is ground into 40 order particles, gets the raw material that 10g crushes, add 3%NaOH120ml in 25 ℃ of reaction 3h, suction filtration gets filter residue and filtrate.With 3.6ml mass concentration 98% vitriol oil filtrate pH value is adjusted into 6,30 ℃ of temperature, precipitation 30min centrifugation obtains purified xylogen 0.66g, and extraction yield is 60.6%, and product purity is 72%.It is 8 that filter residue is washed to the pH value, adds 80mlH again in filter residue
2O adjusts temperature to 50 ℃, and the pH value is 6.5.
The 20.0g cyanuric chloride is dissolved in the 300mL benzene, and 3 ℃ of recrystallizations twice obtain the cyanuric chloride 18.3g of purifying.The 4A molecular sieve that in the there-necked flask of 1000mL, adds 8.0g, the 12.0g anhydrous sodium carbonate, 600mL benzene and 30.0g mono methoxy polyethylene glycol 4000, stirring at normal temperature is after 60 minutes, and B filters, and removes 4A molecular sieve and yellow soda ash; Filtrate collection adds the cyanuric chloride 0.5535g of purifying then in there-necked flask, normal temperature condition reacted 40 hours down.Solution is through concentrating, and B filters, and temperature is-14.2 ℃ of following cryodryings, obtains activatory mono methoxy polyethylene glycol 4000.Adding 8.0g activation mono methoxy polyethylene glycol 4000 and pH are 6.8 citric acid-sodium citrate buffer 200mL in the flask of 500mL, attemperation is 4 ℃, activation mono methoxy polyethylene glycol 4000 fully adds the 2.0g cellulases in the dissolving back, react that to utilize molecular weight cut-off after 2 hours be that 4000 polysulphones hyperfiltration membrane separation obtains the modified-cellulose enzyme.
Distinguish extracting cellulose enzyme 100mg, β-glucuroide 10mg, amylase 50mg, polygalacturonase 50mg, in 20ml50 ℃ of water, activate 5min.Add the enzyme liquid after activating then in solution, carry out enzymic catalytic reaction in ultrasonic field, sonic oscillation power is 500w, and oscillation frequency is 20kHz, reaction 3h.Press filtration gets filtrate, and filtrate is heated, and to obtain concentration of reduced sugar be 28% liquid glucose 15ml to reduction vaporization, can be used for the zymotechnique of alcohol fuel.The degradation rate that records lignocellulose is 56.3%, conversion coefficient 42%.
Claims (1)
1, a kind of method of saccharification of lignocellulose by ultrasonic synergistic catalysis of modified cellulose is characterized in that comprising following process:
1), the pre-treatment of raw material: maize straw is ground into 20～40 order particles, get the raw material that crushes, by the grams per milliliter solid-to-liquid ratio is 1: 10～20, in 25 ℃ of reactions 3～8 hours, suction filtration got filter residue and filtrate with mass concentration 3%～10%NaOH, and it is 7～8 that filter residue is washed to the pH value, standby, with mass concentration 98% vitriol oil filtrate being transferred to the pH value is 3～6, is heated to 30～80 ℃, precipitates centrifugation in 30～60 minutes and obtains xylogen;
2), the cellulase modification: the preparation mass concentration is 6%～15% cyanuric chloride benzole soln, twice in recrystallization, obtain the cyanuric chloride of purifying, by the mass ratio of mono methoxy polyethylene glycol 4000 and 4A molecular sieve and anhydrous sodium carbonate is 1: 2～6: 4～10 with mono methoxy polyethylene glycol 4000,4A molecular sieve and anhydrous sodium carbonate add in the solvent benzol, after the stirring at normal temperature 30～60 minutes, filter, remove 4A molecular sieve and yellow soda ash and obtain filtrate, mol ratio according to polymethoxy ethylene glycol 4000 and the cyanuric chloride of purifying is that 2～2.5: 1 cyanuric chloride with purifying joins in the filtrate then, normal temperature condition reacted 20～50 hours down, solution is again through concentrating, filter, cryodrying, obtain the activatory mono methoxy polyethylene glycol, is that 1: 2～6 to join pH be in 5.0～7.0 citric acid-sodium citrate buffer with cellulase and activation mono methoxy polyethylene glycol according to mass ratio, controlled temperature is 0 ℃～8 ℃, reacted 1～5 hour, adopting molecular weight cut-off is that 5000～10000 ultra-filtration membrane is isolated the modified-cellulose enzyme of getting continuously with mono methoxy polyethylene glycol glue, and frozen drying obtains the modified-cellulose enzyme;
3), the activation of enzyme: is 10: 1: 5 by the modified-cellulose enzyme with the mass ratio of β-glucuroide, amylase and polygalacturonase: 5 mix after, in 40～60 ℃ of water, activate 5～15 minutes, obtain prozyme liquid;
4), the saccharification of raw cellulose: mass ratio 1:8～10 by particulate material and water in the filter residue that step 1) obtains add entry, adjust the temperature to 50 ℃, regulating the pH value with mass concentration 98% vitriol oil is 6.5, press the mixed enzyme solution after 0.5% of maize straw weight adds activation, utilize sonic oscillation to promote the enzymatic hydrolysis reaction, sonic oscillation power is 80～800w, and oscillation frequency is 20～40kHz, reaction times is 3～8 hours, and it is standby that suction filtration gets liquid glucose;
5), concentrating of liquid glucose: 40 ℃～70 ℃ of the liquid glucoses of step 4), it is 25～35% liquid glucoses that reduction vaporization obtains mass concentration, is used for the zymotechnique of alcohol fuel.
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