CN104946701A - Method for improving cellulase hydrolysis sugar field - Google Patents

Method for improving cellulase hydrolysis sugar field Download PDF

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CN104946701A
CN104946701A CN201410120195.3A CN201410120195A CN104946701A CN 104946701 A CN104946701 A CN 104946701A CN 201410120195 A CN201410120195 A CN 201410120195A CN 104946701 A CN104946701 A CN 104946701A
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cellulose
ionic compound
solvent
switching mode
carbonatoms
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谢海波
赵宗保
沈宏伟
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Dalian Institute of Chemical Physics of CAS
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Dalian Institute of Chemical Physics of CAS
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Abstract

The invention provides a method for improving cellulase hydrolysis sugar field. The method comprises the following steps: (1) taking cellulose as a raw material; (2) dissolving the cellulose in a CO2 switch-type ionic compound or a mixed solvent consisting of the CO2 switch-type ionic compound and an organic solvent, thereby obtaining a cellulose-containing solution; (3) adding an anti-solvent into the cellulose solution, regenerating, filtering and separating the dissolved cellulose, thereby obtaining the pretreated cellulose material; and (4) taking a certain amount of regenerated cellulose, adding a buffer solution and cellulase for performing enzymolysis, thereby obtaining the glucose solution. The CO2 switch-type ionic compound or the mixed solvent consisting of the CO2 switch-type ionic compound and the organic solvent serves as a solvent, the cellulose is subjected to dissolving pretreatment, and the method has the advantages that the process is simple, the solvent cost is low, the operation is convenient, the solvent can be recycled, the cellulase hydrolysis speed is high, the sugar yield is high and the like.

Description

A kind of method improving cellulase hydrolysis sugar yield
Technical field
The present invention relates to one utilizes Mierocrystalline cellulose to prepare, by utilizing CO for raw material 2in switching mode ionic compound or CO 2the mixed solvent pretreatment of fiber element that switching mode ionic compound and organic solvent form, by the method for enzyme-squash techniqued glucose, belongs to biomass economy and biochemical product field.
Background technology
Along with the worsening shortages of the Nonrenewable resources such as coal, oil, Sweet natural gas, natural cellulose is subject to people as the renewable resources that occurring in nature enriches the most and more and more pays close attention to.Because Mierocrystalline cellulose is a kind of polysaccharide that occurring in nature distribution is the widest, content is maximum, it is the main component of composition plant cell wall.The Mierocrystalline cellulose of a large amount of high-quality is contained in cotton, flax, taro fiber crops and jute portion.In wood, content of cellulose is about 50%, and the content of cellulose of cotton is higher, reaches 90%.Mierocrystalline cellulose in timber then normal and hemicellulose and xylogen exists jointly.Mierocrystalline cellulose is a kind of polysaccharide of complexity, has 8000 to 10000 glucosyl residues to be formed by connecting by β-Isosorbide-5-Nitrae-glycosidic link.By carrying out chemical catalysis hydrolysis to Mierocrystalline cellulose and enzymolysis can prepare important industrial chemicals glucose.Be that raw material can prepare a series of derived energy chemical product, as bio-ethanol, microbial oil, butanols, 5 hydroxymethyl furfural etc. by chemical catalysis method and bioconversion method with glucose.And traditionally, glucose is mainly from starch.Along with being that raw material prepares the energy, Chemicals technical development with glucose, new requirement is proposed to the supply of glucose.And traditional be that glucose prepared by raw material with starch because starch is food resource, its source is subject to the restriction of resource and policy, and therefore, how by utilizing cellulosic material, Efficient preparation of glucose receives general concern.
And natural cellulose is highly crystalline polymkeric substance, due to its intermolecular hydrogen bond action strong with intramolecule, it is made to be insoluble to traditional organic solvent, lower to the response of chemistry, biological catalyst.In order to obtain high cellulose hydrolysis efficiency, pre-treatment must be carried out to Mierocrystalline cellulose, to change its crystalline structure and physical aspect, the accessibility of raising corresponding chemical catalyzer, enzyme and catalytic efficiency.Recently based on ion liquid dissolving Mierocrystalline cellulose, ligno-cellulosic materials, realize Mierocrystalline cellulose (Biotechnology and Bioengineering, 2011,108,511-520), dissolving pre-treatment (the Green Chemistry of lignocellulose, 2012,14,1202-1210), thus improve its enzymolysis efficiency, due to its high efficiency, obtain and pay close attention to widely.Chinese patent 201110008782.X protects a kind of method utilizing ionic liquid pretreatment to improve cellulase hydrolysis product sugar.Turning over patent 201210144959.3 and protect a kind of pretreatment process improving lignocellulolyticenzymes percent hydrolysis, is also utilize ionic liquid for solvent.Realize its pre-treatment by ion liquid dissolving Mierocrystalline cellulose, lignocellulose, can significantly improve its enzymolysis efficiency, its ionic liquid liquid used is all traditional imidazolium ionic liquid.And traditional imidazolium ionic liquid is expensive, recovery, purifying, recycle cost are high, greatly limit the economic performance of this process.
In order to realize cellulosic dissolving pre-treatment, its exploitation of dissolving new system is the key of technological breakthrough.Chinese patent 201210374955.4 protects a kind of method of cellulose solution dissolving cellulos and regenerated cellulose, and its principal character utilizes cheapness, the CO of easily preparation 2switch ionic compound or CO 2the mixed solvent system that switch ionic compound and organic solvent form dissolves processing of cellulose.As a kind of new cellulose dissolution system, for cellulosic homogeneous chemistry modification and dissolve pre-treatment and provide a good platform.And dissolve the cellulose dissolution pre-treatment of processing platform based on this, then prepare glucose by zymolysis technique and also there is no pertinent literature and patent report.Patent of the present invention is based on this platform, and the cellulose dissolution pre-treatment of exploitation, improves its enzymolysis efficiency, efficiently prepares cellulose base glucose.Based on the significant application value of glucose, this dissolving preconditioning technique has cheapness, easily prepares, and the feature such as easily to recycle, has good prospects for commercial application.
Summary of the invention
In order to solve prior art Problems existing, the invention provides one and utilizing CO 2in switching mode ionic compound or CO 2the mixed solvent that switching mode ionic compound and organic solvent form is cellosolve, realizes cellulosic dissolving pre-treatment, thus improves its subsequent enzymatic hydrolysis efficiency.This method solving current production of cellulose, to dissolve the solvent cost that pretreated method exists high, purifies and recycle high in cost of production problem,
The present invention utilizes Mierocrystalline cellulose for raw material, utilizes CO 2in switching mode ionic compound or CO 2the mixed solvent that switching mode ionic compound and organic solvent form is cellosolve, realizes cellulosic dissolving pre-treatment, thus improves its subsequent enzymatic hydrolysis efficiency, and it carries out in accordance with the following steps:
1. be raw material with Mierocrystalline cellulose;
2. can with CO 2one or more raw materials of compound in reaction formation switching mode ionic compound (Switchable ionic compounds) and Mierocrystalline cellulose are mixed to form predissolve system;
Or can with CO 2reaction forms one or more raw materials of compound in switching mode ionic compound and Mierocrystalline cellulose and is mixed to form predissolve system to the organic solvent that Mierocrystalline cellulose has a good swelling action;
Or by CO 2one or more and Mierocrystalline cellulose in switching mode ionic compound (Switchable ionic compounds) are mixed to form predissolve system;
Or by CO 2one or more and Mierocrystalline cellulose in switching mode ionic compound and predissolve system is mixed to form to the organic solvent that Mierocrystalline cellulose has a good swelling action;
3. pass into CO toward system 2, by the heating of this mixed system, stirring and dissolving, release CO 2after pressure, obtain cellulose solution;
4. in cellulose solution, add anti-solvent, make the cellulose regenerated of dissolving, filter, be separated the pretreated cellulose materials of acquisition;
5. get a certain amount of regenerated cellulose, add buffered soln and cellulase carries out enzymolysis, obtain glucose solution.
Further, a kind of method improving cellulase hydrolysis sugar yield provided by the invention, it can also have following features: cellulosic material used is: the one of made plant cellulose or above-mentioned at least two kinds of cellulosic mixtures in Microcrystalline Cellulose, alpha-cellulose, cotton and wood pulp, bamboo slurry, agriculture and forestry organic waste material stalk, crop seeds cot.Comprise specifically: straw, wheat straw, reed straw, cotton stalk, peanut straw, Pericarppium arachidis hypogaeae, maize straw, corn page, mealie skin, bagasse are that raw material extracts the cellulosic a kind of or mixture of at least two kinds.And various fiber-rich element is starched or cellulosic obtain manner is unrestricted.Particularly, when Mierocrystalline cellulose, xylogen, hemicellulose coexist, as directly utilized maize straw for cellulosic material, be also applicable to this patent.
A kind of method improving cellulase hydrolysis sugar yield provided by the invention, wherein, the detailed process of (2) step is:
(a): will wait stoichiometricly can form CO 2the organic bases of switching mode ionic compound and alcohol or amine, Mierocrystalline cellulose, organic solvent add in dissolution kettle, cover reactor, pass into CO 2, form CO 2switching mode ionic compound-Mierocrystalline cellulose or formation CO 2switching mode ionic compound and organic solvent-cellulosic mixing solutions, the CO formed 2switching mode ionic compound has following constitutional features:
Wherein cationic structural is one or more in following structure:
A series, R 1, for carbonatoms be the alkyl of 1-6, carbonatoms is oxyethyl group polyethylene glycol groups or the methoxy poly (ethylene glycol) base of 3-10, R 2, R 3, R 4for independently methyl;
B series, n=1 or n=2; M=1-6; R is independently hydrogen, methyl or ethyl; R 1be the polyethylene glycol groups of 4-8 for independently hydrogen, the carbonatoms alkyl that is 1-6, the carbonatoms oxyethyl group polyethylene glycol groups that is 3-10, methoxy poly (ethylene glycol) base, carbonatoms are the hydroxylated alkyl groups of 1-6 or carbonatoms; R 2, R 3, R 4, R 5for independently hydrogen or methyl or ethyl;
Wherein anion structure is one or more in structure:
Wherein:
A series, the alkyl of R to be carbonatoms be 1-2, carbonatoms are oxyethyl group polyethylene glycol groups or the methoxy poly (ethylene glycol) base of 3-4, R 1for the polyethoxye ether of carbonatoms to be the alkyl of 1-3 or carbonatoms be 3-6;
B series, R 1, R 2, R 3, R 4, R 5, R 6, for independently hydrogen or for carbonatoms be the alkyl R of 1-2 7, R 8, R 9, R 10, R 11, be independently hydrogen;
C series, R is the H that on phenyl ring, optional position replaces, methyl or methoxy.
Preferably, wherein cationic structural feature is as follows:
Wherein:
N=1 or n=2; R 1for independently hydrogen or carbonatoms are the alkyl of 1-6.
Its anion structure has the one in following preferred structure feature:
Wherein: the alkane of R to be carbonatoms be 1-3; R 1for carbonatoms is the alkyl of 1-3.
In mixing solutions, Mierocrystalline cellulose mass percentage is 2%-20%.Preferred mass percentage composition is 4%-10%.
CO is filled with in mixing solutions 2the preferred CO of pressure 0.1MPa-15MPa. 2cO in pressure 0.5MPa-6MPa. mixing solutions 2ionic compound molar content is the preferred molar content of 5%-99%. is 5%-40%.
In mixed solvent, organic solvent is one or more in following solvent: dimethyl sulfoxide (DMSO) (DMSO), N-Methyl pyrrolidone (NMP), tetramethyl-urea, tetraethyl urea, N, N-dimethyl-imidazolinone, N, N, dimethyl formamide, N, N,-diethyl acetamide, pyrrolidone, 2-piperazine ketone, ε-caprolactam, N, N-dimethyl propylene thiazolinyl urea, tetramethylene sulfone or piperylene sulfone.Preferred dimethyl sulfoxide (DMSO) (DMSO), N, N-dimethyl-imidazolinone, N, N, one or more in dimethyl formamide, tetramethylene sulfone or piperylene sulfone.
B () heats, stirring and dissolving Mierocrystalline cellulose, and obtain cellulose solution, its dissolution conditions specific features is:
Temperature range: 30 DEG C-150 DEG C.Dissolution time scope: be 0.1 hour-24 hours.
Preferred dissolution condition is: temperature range: 50 DEG C-100 DEG C; Dissolution time scope: be 0.5 hour-10 hours.
A kind of method improving cellulase hydrolysis sugar yield provided by the invention, wherein, the detailed process of (4) step is:
A () system temperature is reduced to room temperature, release CO 2pressure, adds anti-solvent in system.This process, its type of cooling is unrestricted.The object adding anti-solvent is that the Mierocrystalline cellulose of dissolving is well regenerated, thus conveniently can realize pretreated cellulosic separation, thus obtains activated cellulose sample;
Anti-solvent described in it is water or C 1-C 3lower aliphatic alcohols, the volume ratio of solvent and reaction mixture is 0.5:1-10:1; Preferably, the volume ratio of solvent and reaction mixture is 1:1-5:1.Recycle to simply realize solvent system, the solvent particular methanol added or ethanol.
B () precipitation separation product out, sepn process can by various mode, and separate mode is unrestricted, as long as can by solid-liquid separation, preferred centrifugation, filters.
C (), in order to remove solvent, is reduced residual solvent to the impact of the efficiency of follow-up enzymolysis, is utilized water or C 1-C 3lower aliphatic alcohols to be separated obtain solid cellulose ester sample wash, purifying, drying.Wherein used aqueous solvent or C 1-C 3lower aliphatic alcohols relative to quality product ratio is: 1:1-100:1, and washing 1-10 time, obtains product after drying.Preferred solvent consumption is 20:1-50:1 relative to quality product, washing 2-3 time.Its drying means is unrestricted.
A kind of cellulase hydrolysis that improves provided by the invention produces sugared method, and main science feature is, based on the change of Mierocrystalline cellulose physics, chemical structure in cellulosic dissolving regenerative process, if cellulose crystals structure is from cellulose I type to the transformation of cellulose II type, and the accumulation form etc. between cellulose chain and chain.These changes, will improve the contact area of cellulose chain and cellulase greatly, thus improve the changing effect of cellulase, improve cellulosic enzymic hydrolysis efficiency.
The transformation of cellulose crystals structure, we are confirmed by Wide angle X-ray diffraction, specifically see Fig. 2; Its surface topography simultaneously, we are confirmed by scanning electron microscope, specifically see Fig. 3.
A kind of method improving cellulase hydrolysis sugar yield provided by the invention, wherein, the detailed process of (5) step is:
Get a certain amount of regenerated cellulose, add buffered soln and cellulase carries out enzymolysis, obtain glucose solution.It is characterized in that: the pH value of described damping fluid is consistent with the use range of selected cellulase, to ensure the activity at utmost playing enzyme.Wherein, described cellulase is that viride, Trichodermareesei, aspergillus niger, healthy and free from worry wood are mould, any one in Aspergillus phoenicis.Along with the appearance of various new enzyme or compound enzymic preparation, therefore, the use kind of enzyme is unrestricted.The consumption of enzyme is calculated as 5FPU/ gram-50FPU/ gram relative to cellulosic usage quantity, is more preferably 10FPU/ gram-30FPU/ gram.In enzymolysis process, solid-to-liquid ratio, stirring velocity, and temperature is not particularly limited, but in order to obtain better hydrolysis result, in enzymolysis process, solid-to-liquid ratio is preferred 1:5, and stirring velocity is 100 ~ 400 revs/min, carries out enzymolysis 5-72 hour in 40 ~ 60 DEG C under a certain steady temperature.
It dissolves pretreatment time to the impact of follow-up enzymolysis efficiency, and we are by illustrating the cellulosic hydrolysis result obtained under different pretreatments condition.Specifically see Fig. 4.
The present invention uses CO 2switching mode ionic compound or CO 2the mixed solvent that switching mode ionic compound and organic solvent form is solvent, carries out dissolving pre-treatment to Mierocrystalline cellulose, and have technique simple, solvent cost is low, easy to operate, and solvent can be recycled, and cellulase hydrolysis speed is fast, sugar yield advantages of higher.
Accompanying drawing explanation
Fig. 1 schema of the present invention;
The different cellulosic material of Fig. 2 and the comparative analysis of pre-treating cellulosic material Wide angle X-ray diffraction;
Fig. 3 cellulosic material and pretreated cellulose materials scanning electron microscope study;
Glucose yield after cellulase hydrolysis under Fig. 4 different pretreatments condition;
Fig. 5 cellulosic material and pretreated cellulose materials enzymolysis kinetics are studied.
Embodiment
Further illustrate the method for the present invention's description below by way of specific embodiment, but be not limited to content of the present invention.
Embodiment 1
Fig. 1 is a kind of schematic flow sheet improving the method for cellulase hydrolysis sugar yield provided by the invention.As shown in Figure 1, it carries out in accordance with the following steps:
1. be raw material with Mierocrystalline cellulose;
2. can with CO 2one or more raw materials of compound in reaction formation switching mode ionic compound (Switchable ionic compounds) and Mierocrystalline cellulose are mixed to form predissolve system;
Or can with CO 2reaction forms one or more raw materials of compound in switching mode ionic compound and Mierocrystalline cellulose and is mixed to form predissolve system to the organic solvent that Mierocrystalline cellulose has a good swelling action;
Or by CO 2one or more and Mierocrystalline cellulose in switching mode ionic compound (Switchable ionic compounds) are mixed to form predissolve system;
Or by CO 2one or more and Mierocrystalline cellulose in switching mode ionic compound and predissolve system is mixed to form to the organic solvent that Mierocrystalline cellulose has a good swelling action;
3. pass into CO toward system 2, by the heating of this mixed system, stirring and dissolving, release CO 2after pressure, obtain cellulose solution;
4. in cellulose solution, add anti-solvent, make the cellulose regenerated of dissolving, filter, be separated the pretreated cellulose materials of acquisition;
5. get a certain amount of regenerated cellulose, add buffered soln and cellulase carries out enzymolysis, obtain glucose solution.
Concrete techniqueflow is as Fig. 1.
Embodiment 2
By methyl alcohol (0.039 mole, 0.8 gram), tetramethyl guanidine (0.039 mole, 4.5 grams), DMSO (12.2 grams), the 2.0 grams of mixing of corn stalk fiber element, add in autoclave, cover tightly kettle cover, pass into CO 2, maintain pressure at 0.2MPa, be heated to 50 DEG C, dissolve 60 minutes.Cooling, release CO 2, open reactor, under agitation, in system, add 100ml anhydrous methanol, make the cellulose regenerated of dissolving, filter, collect filtrate.Again by filter residue 2*100ml anhydrous methanol washing, wash 1h at every turn, to remove solvent completely, then dry, obtain and dissolve pretreated Mierocrystalline cellulose.Filter, collect filter residue, lyophilize, obtains regenerated cellulose.Be raw material with regenerated cellulose, add NaAc_HAc buffer solution, solid-to-liquid ratio is 1:10, and viride addition is 15FPU/g, shaking table speed: 100 revs/min, and enzymatic hydrolysis condition is pH4.5, temperature 50 C, and enzymolysis time is 24 hours.Glucose yield: 95%.
Embodiment 3
By ethylene glycol (0.018 mole, 1.14 grams), tetramethyl guanidine (0.036 mole, 4.14 grams), DMSO (12.7 grams), Microcrystalline Cellulose 1.0 grams mixing, adds in autoclave, covers tightly kettle cover, pass into CO 2, maintain pressure at 0.2MPa, be heated to 50 DEG C, dissolve 60 minutes.Cooling, release CO 2, open reactor, under agitation, in system, add 100ml anhydrous methanol, make the cellulose regenerated of dissolving, filter, collect filtrate.Again by filter residue 2*100ml anhydrous methanol washing, wash 1h at every turn, to remove solvent completely, then dry, obtain and dissolve pretreated Mierocrystalline cellulose.Filter, collect filter residue, lyophilize, obtains regenerated cellulose.Be raw material with regenerated cellulose, add citric acid-sodium citrate buffer, solid-to-liquid ratio is 1:20, and viride addition is 20FPU/g, shaking table speed: 400 revs/min, and enzymatic hydrolysis condition is pH4.8, temperature 50 C, and enzymolysis time is 24 hours.Different time, glucose analysis is carried out in sampling, the results are shown in Figure 5.Unpretreated cellulase hydrolysis very slow (B curve), by patent pretreatment process of the present invention, cellulase hydrolysis efficiency significantly improves, and can find from Fig. 5 A curve, by 24 hours enzymolysis, glucose yield nearly 100%.
Embodiment 4
By glycerine (0.03 mole, 2.76 grams), tetramethyl guanidine (0.01 mole, 1.15 grams), DMSO (14.0 grams), wheatgrass cellulose 3.0 grams mixing, adds in autoclave, covers tightly kettle cover, pass into CO 2, maintain pressure at 1MPa, be heated to 60 DEG C, dissolve 120 minutes.Cooling, release CO 2, open reactor, under agitation, in system, add 200ml dehydrated alcohol, make the cellulose regenerated of dissolving, filter, collect filtrate.Again filter residue is used 2*200ml absolute ethanol washing, wash 1h at every turn, to remove solvent completely, then dry, obtain and dissolve pretreated Mierocrystalline cellulose.Filter, collect filter residue, lyophilize, obtains regenerated cellulose.Be raw material with regenerated cellulose, add citric acid-sodium citrate buffer, solid-to-liquid ratio is 1:5, and Trichodermareesei addition is 20FPU/g, shaking table speed: 200 revs/min, and enzymatic hydrolysis condition is pH5.5, temperature 50 C, and enzymolysis time is 48 hours.Glucose yield 95%.
Embodiment 5 by methyl alcohol (0.04 mole, 1.28 grams), 1,8-diazacyclo [5,4,0] hendecene-7(0.04 mole, 6.09 grams), DMSO (11.0 grams), cotton wood cellulose 2.0 grams mixing, adds in autoclave, covers tightly kettle cover, pass into CO 2, maintain pressure at 0.1MPa, be heated to 60 DEG C, dissolve 240 minutes.Cooling, release CO 2, open reactor, under agitation, in system, add 200ml dehydrated alcohol, make the cellulose regenerated of dissolving, filter, collect filtrate.Again filter residue is used 2*200ml absolute ethanol washing, wash 1h at every turn, to remove solvent completely, then dry, obtain and dissolve pretreated Mierocrystalline cellulose.Filter, collect filter residue, lyophilize, obtains regenerated cellulose.Be raw material with regenerated cellulose, add citric acid-sodium citrate buffer, solid-to-liquid ratio is 1:5, and the mould addition of healthy and free from worry wood is 10FPU/g, shaking table speed: 200 revs/min, and enzymatic hydrolysis condition is pH4.8, temperature 50 C, and enzymolysis time is 48 hours.Glucose yield 96%.
Embodiment 6
By ethylene glycol (0.018 mole, 1.14 grams), tetramethyl guanidine (0.036 mole, 4.14 grams), DMF (12.0 grams), Pine cellulose 2.0 grams mixing, adds in autoclave, covers tightly kettle cover, pass into CO 2, maintain pressure at 2.0MPa, be heated to 50 DEG C, dissolve 300 minutes.Cooling, release CO 2, open reactor, under agitation, in system, add 100ml anhydrous methanol, make the cellulose regenerated of dissolving, filter, collect filtrate.Again by filter residue 2*100ml anhydrous methanol washing, wash 1h at every turn, to remove solvent completely, then dry, obtain and dissolve pretreated Mierocrystalline cellulose.Filter, collect filter residue, lyophilize, obtains regenerated cellulose.Be raw material with regenerated cellulose, add dilution heat of sulfuric acid, solid-to-liquid ratio is 1:10, and Aspergillus phoenicis addition is 10FPU/g, shaking table speed: 200 revs/min, and enzymatic hydrolysis condition is pH4.8, temperature 50 C, and enzymolysis time is 24 hours.Glucose yield nearly 95%.
Embodiment 7
By methyl alcohol (0.4 mole, 12.8 grams), tetramethyl guanidine (0.4 mole, 46.0 grams), Microcrystalline Cellulose 6.0 grams mixing, adds in autoclave, covers tightly kettle cover, pass into CO 2, maintain pressure at 0.2MPa, be heated to 80 DEG C, dissolve 240 minutes.Cooling, release CO 2, open reactor, under agitation, in system, add 200ml anhydrous methanol, make the cellulose regenerated of dissolving, filter, collect filtrate.Again by filter residue 2*200ml anhydrous methanol washing, wash 1h at every turn, to remove solvent completely, then dry, obtain and dissolve pretreated Mierocrystalline cellulose.Filter, collect filter residue, lyophilize, obtains regenerated cellulose.Be raw material with regenerated cellulose, add citric acid-sodium citrate buffer, solid-to-liquid ratio is 1:5, and Trichodermareesei addition is 30FPU/g, shaking table speed: 200 revs/min, and enzymatic hydrolysis condition is pH6.0, temperature 50 C, and enzymolysis time is 5 hours.Glucose yield: 85%.This embodiment illustrates, is not having under organic solvent, CO 2switching mode ionic compound as solvent, can dissolve pretreatment of fiber element, thus improves its enzymolysis efficiency.
Patent of the present invention also comprises and first synthesizes CO 2switching mode ionic compound, and then carry out dissolving pre-treatment with Mierocrystalline cellulose mixing, can obtain same usefulness, we are explained by following embodiment:
Embodiment 8
By ethylene glycol (0.18 mole, 11.4 grams), tetramethyl guanidine (0.36 mole, 41.4 grams), adds in autoclave, covers tightly kettle cover, pass into CO 2, maintain pressure at 0.5MPa, react 10 minutes, release CO 2, open reactor, then add 10 grams of Mierocrystalline celluloses, cover reactor, fill the CO as 0.5MPa 2, increase the temperature to 50 DEG C, dissolve 60 minutes.Cooling, release CO 2, open reactor, under agitation, in system, add 100ml anhydrous methanol, make the cellulose regenerated of dissolving, filter, collect filtrate.Again by filter residue 2*100ml anhydrous methanol washing, wash 1h at every turn, to remove solvent completely, then dry, obtain and dissolve pretreated Mierocrystalline cellulose.Filter, collect filter residue, lyophilize, obtains regenerated cellulose.Be raw material with regenerated cellulose, add citric acid-sodium citrate buffer, solid-to-liquid ratio is 1:10, and Aspergillus phoenicis addition is 5FPU/g, shaking table speed: 200 revs/min, and enzymatic hydrolysis condition is pH5, temperature 50 C, and enzymolysis time is 24 hours.Glucose yield: 95%.
Embodiment 9
By 2-(3-hydroxypropyl)-1,1,3,3-tetramethyl guanidine (0.05 mole, 8.7g), DMSO (14.0 grams), wheatgrass cellulose 3.0 grams mixing, adds in autoclave, covers tightly kettle cover, pass into CO 2, maintain pressure at 0.2MPa, be heated to 80 DEG C, dissolve 120 minutes.Cooling, release CO 2, open reactor, under agitation, in system, add 200ml anhydrous methanol, make the cellulose regenerated of dissolving, filter, collect filtrate.Again by filter residue 2*200ml anhydrous methanol washing, wash 1h at every turn, to remove solvent completely, then dry, obtain and dissolve pretreated Mierocrystalline cellulose.Filter, collect filter residue, lyophilize, obtains regenerated cellulose.Be raw material with regenerated cellulose, add citric acid-sodium citrate buffer, solid-to-liquid ratio is 1:10, and Trichodermareesei addition is 20FPU/g, shaking table speed: 500 revs/min, and enzymatic hydrolysis condition is pH5, temperature 50 C, and enzymolysis time is 24 hours.Glucose yield: 99%.
A kind of method improving cellulase hydrolysis efficiency of patent protection of the present invention, one of its essential characteristic is that it well recycles character, and we are explained by embodiment below
Embodiment 10
By ethylene glycol (0.018 mole, 1.14 grams), tetramethyl guanidine (0.036 mole, 4.14 grams), DMSO (12.0 grams), straw cellulose 2.0 grams mixing, adds in autoclave, covers tightly kettle cover, pass into CO 2, maintain pressure at 2.0MPa, be heated to 80 DEG C, dissolve 300 minutes.Cooling, release CO 2, open reactor, under agitation, in system, add 100ml anhydrous methanol, make the cellulose regenerated of dissolving, filter, collect filtrate.Again by filter residue 2*100ml anhydrous methanol washing, wash 1h at every turn, to remove solvent completely, then dry, obtain and dissolve pretreated Mierocrystalline cellulose.Filter, collect filter residue, lyophilize, obtains regenerated cellulose.Be raw material with regenerated cellulose, add citric acid-sodium citrate buffer, solid-to-liquid ratio is 1:10, and Trichodermareesei addition is 10FPU/g, and enzymatic hydrolysis condition is pH5, temperature 50 C, and enzymolysis time is 24 hours.Glucose yield nearly 95%.Collection, merging filtrate, remove methyl alcohol by the method for distillation, collects methyl alcohol and be used for next step cellulosic regenerative process, residual solution 17.25g, solvent reclamation rate >99%.The solvent of regeneration is directly used in next step cellulose dissolution, adds Mierocrystalline cellulose 2.0 grams mixing, add in autoclave, cover tightly kettle cover, pass into CO in system 2, maintain pressure at 2.0MPa, be heated to 60 DEG C, dissolve 300 minutes.Cooling, release CO 2, open reactor, under agitation, in system, add 100ml anhydrous methanol, make the cellulose regenerated of dissolving, filter, collect filtrate.Again by filter residue 2*100ml anhydrous methanol washing, wash 1h at every turn, to remove solvent completely, then dry, obtain and dissolve pretreated Mierocrystalline cellulose.Filter, collect filter residue, lyophilize, obtains regenerated cellulose.Be raw material with regenerated cellulose, add citric acid-sodium citrate buffer, solid-to-liquid ratio is 1:10, and cellulase addition is 10FPU/g, shaking table speed: 200 revs/min, and enzymatic hydrolysis condition is pH5, temperature 50 C, and enzymolysis time is 24 hours.Glucose yield nearly 95%.Explanation system has and well recycles character.
The Mierocrystalline cellulose of occurring in nature generally all coexists with hemicellulose, xylogen, in order to better economy, directly utilizes the Mierocrystalline cellulose in lignocellulose to be also applicable to patent of the present invention for raw material, is specially explained with following examples:
Embodiment 11
By ethylene glycol (0.018 mole, 1.14 grams), tetramethyl guanidine (0.036 mole, 4.14 grams), DMSO (12.7 grams), the mixing of 1.0 grams, maize straw powder, adds in autoclave, covers tightly kettle cover, pass into CO 2, maintain pressure at 6.0MPa, be heated to 100 DEG C, dissolve 10 hours.Cooling, release CO 2, open reactor, under agitation, in system, add 100ml anhydrous methanol, make the cellulose regenerated of dissolving, filter, collect filtrate.Again by filter residue 2*100ml anhydrous methanol washing, wash 1h at every turn, to remove solvent completely, then dry, obtain and dissolve pretreated Mierocrystalline cellulose.Filter, collect filter residue, lyophilize, obtains regenerated cellulose.Be raw material with regenerated cellulose, add NaAc_HAc buffer solution, solid-to-liquid ratio is 1:10, and Trichodermareesei addition is 50FPU/g, shaking table speed: 200 revs/min, and enzymatic hydrolysis condition is pH5, temperature enzymolysis time is 72 hours.Glucose yield: 90%.

Claims (10)

1. improve a method for cellulase hydrolysis sugar yield, it is characterized in that: it carries out in accordance with the following steps:
(1) take Mierocrystalline cellulose as raw material;
(2) Mierocrystalline cellulose is dissolved in CO 2in switching mode ionic compound or CO 2in the mixed solvent that switching mode ionic compound and organic solvent form, form cellulose solution;
(3) in cellulose solution, add anti-solvent, make the cellulose regenerated of dissolving, filter, be separated the pretreated cellulose materials of acquisition;
(4) get regenerated cellulose, add buffered soln and cellulase carries out enzymolysis, obtain glucose solution.
2. method according to claim 1, is characterized in that: cellulosic material used is: Microcrystalline Cellulose, alpha-cellulose, cotton, wood pulps, bamboo pulp and from agricultural thing stalk, crop seeds cot, be separated the one of made plant cellulose or two or more cellulosic mixture.
3. method according to claim 1, is characterized in that: described Mierocrystalline cellulose is dissolved in CO 2in switching mode ionic compound or CO 2switching mode ionic compound and organic solvent, the process forming cellulose solution is:
(1) can with CO 2one or more raw materials of compound in reaction formation switching mode ionic compound (Switchable ionic compounds) and Mierocrystalline cellulose are mixed to form predissolve system;
Or can with CO 2reaction forms one or more raw materials of compound in switching mode ionic compound and Mierocrystalline cellulose and is mixed to form predissolve system to the organic solvent that Mierocrystalline cellulose has a good swelling action;
Or by CO 2one or more and Mierocrystalline cellulose in switching mode ionic compound (Switchable ionic compounds) are mixed to form predissolve system;
Or by CO 2one or more and Mierocrystalline cellulose in switching mode ionic compound and predissolve system is mixed to form to the organic solvent that Mierocrystalline cellulose has a good swelling action;
(2) CO is passed into toward system 2, by the heating of this mixed system, stirring and dissolving, release CO 2after pressure, obtain cellulose solution;
The CO used 2the positively charged ion of switching mode ionic compound has one or more of following constitutional features:
Wherein:
A series, R 1, for carbonatoms be the alkyl of 1-6, carbonatoms is oxyethyl group polyethylene glycol groups, the methoxy poly (ethylene glycol) base of 3-10, R 2, R 3, R 4for independently methyl;
B series, n=1 or n=2; M=1-6; R is independently hydrogen, methyl, ethyl; R 1for independently hydrogen or carbonatoms are the alkyl of 1-6, carbonatoms is the oxyethyl group polyethylene glycol groups of 3-10, methoxy poly (ethylene glycol) base, carbonatoms are 1-6 hydroxylated alkyl groups or carbonatoms are the polyethylene glycol groups of 4-8; R 2, R 3, R 4, R 5for independently hydrogen, methyl or ethyl;
The CO used 2the anion structure of switching mode ionic compound have in following constitutional features one or more:
Wherein:
A series, the alkyl of R to be carbonatoms be 1-2, carbonatoms are oxyethyl group polyethylene glycol groups or the methoxy poly (ethylene glycol) base of 3-4, R 1for carbonatoms be the alkyl of 1-3, carbonatoms is the polyethoxye ether of 3-6;
B series, R 1, R 2, R 3, R 4, R 5, R 6, for independently hydrogen or for carbonatoms be the alkyl of 1-2;
C series, R is H, the methyl or methoxy that on phenyl ring, optional position replaces.
4. method as claimed in claim 3, is characterized in that: the CO used 2the positively charged ion of switching mode ionic compound has the one of following preferred structure feature:
Wherein:
N=1 or n=2; R 1for independently hydrogen or carbonatoms are the alkyl of 1-6;
Its anion structure has the one in following preferred structure feature:
Wherein: the alkyl of R to be carbonatoms be 1-3; R 1for carbonatoms is the alkyl of 1-3.
5. the method according to claim 1 or 3, is characterized in that:
Described organic solvent has following feature:
Organic solvent is one or more in following solvent: dimethyl sulfoxide (DMSO) (DMSO), N-Methyl pyrrolidone (NMP), tetramethyl-urea, tetraethyl urea, N, N-dimethyl-imidazolinone, N, N, dimethyl formamide, N, N,-diethyl acetamide, pyrrolidone, 2-piperazine ketone, ε-caprolactam, N, N-dimethyl propylene thiazolinyl urea, tetramethylene sulfone, piperylene sulfone;
In mixed solvent, CO 2switching mode ionic compound volumetric molar concentration is 5%-99%; Preferred CO 2switching mode ionic compound volumetric molar concentration is: 5%-40%.
6. the method for dissolving cellulos as claimed in claim 3, is characterized in that: the CO passing into certain pressure toward system 2, the mixed solution system forming switching mode ionic compound or form switching mode ionic compound and the organic solvent that Mierocrystalline cellulose has good swelling action is formed, the selectable CO of the method 2pressure range is 0.1Mpa-15Mpa; For obtaining good solute effect, more preferably CO 2pressure range is 0.5Mpa-1.0Mpa;
Dissolving cellulos forms the process of cellulose solution: react in encloses container and carry out, the type of heating of employing is unrestricted, and its temperature range can select 30 DEG C-150 DEG C; Its temperature range more preferably 50 DEG C-80 DEG C.
7. the method according to claim 1 or 3, is characterized in that: in cellulose solution, cellulosic mass concentration is 2%-20%, and preferred concentration is 4%-10%.
8. method according to claim 1, is characterized in that:
Step (3) is at cellulosic CO 2switching mode ionic compound or CO 2add anti-solvent in the solution of the mixed solvent that switching mode ionic compound and organic solvent form, make the Cellulose precipitates of dissolving, separation; Anti-solvent described in it has following feature: it is water or C 1-C 3lower aliphatic alcohols, after itself and homogeneous reaction, the volume ratio of solution is 0.5:1-10:1; Preferably, described C 1-C 3lower aliphatic alcohols is one or more in methyl alcohol, ethanol or Virahol.
9. the method according to claim 1 or 8, is characterized in that: after the cellulose prods purification that separation obtains by step (5), drying, obtain pretreated cellulose sample;
Its purge process has following feature: use aqueous solvent or C 1-C 3(solvent phase for product quality ratio is lower aliphatic alcohols: 1:1-100:1) wash 1-5 time, obtain product after drying;
Preferred solvent is 20:1-50:1 relative to product quality, washing 2-3 time.
10. by method according to claim 1, it is characterized in that: step (4) gets a certain amount of regenerated cellulose, add buffered soln with cellulase carries out enzymolysis, acquisition glucose solution;
The pH value of described damping fluid is consistent with the use range of selected cellulase, to ensure the activity at utmost playing enzyme;
Described cellulase is that viride, Trichodermareesei, aspergillus niger, healthy and free from worry wood are mould, any one or two or more in Aspergillus phoenicis; Relative to cellulosic amount used, cellulase consumption is 5FPU/ gram-50FPU/ gram;
In enzymolysis process, stirring velocity is 50 ~ 400 revs/min, carries out enzymolysis 5-72 hour in 40 ~ 60 DEG C under a certain steady temperature.
CN201410120195.3A 2014-03-27 2014-03-27 Method for improving cellulase hydrolysis sugar field Pending CN104946701A (en)

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