CN102702366B - Cellulose sample and application thereof - Google Patents
Cellulose sample and application thereof Download PDFInfo
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Abstract
The invention discloses a cellulose sample and application thereof, wherein the cellulose sample is prepared by a method comprising the following steps of: a, respectively dissolving microcrystalline cellulose and mixture of the microcrystalline cellulose and different quantities of lignin into ionic liquid to obtain a lot ionic liquid solution; b, respectively transferring the ionic liquid solution into reversed solvent to regenerate and reconstruct; c, respectively filtering the regenerated and reconstructed ionic liquid solution to obtain a plurality of regenerated celluloses as cellulose samples after freezing and drying the solution. The cellulose sample disclosed by the invention can adjust single influencing factor on enzyme hydrolysis, so as to improve the accuracy of a conclusion about an experiment of affecting enzyme hydrolysis.
Description
Technical field
The present invention relates to lignocellulose processing technology field, specifically a kind of Mierocrystalline cellulose sample and application thereof.
Background technology
Day by day serious along with the minimizing of petroleum resources and global environment crisis, becomes the choosing (Kunkes etc., 2008) of the certainty of countries in the world taking renewable resources as development of raw materials new forms of energy.Taking food such as corn, sugarcane and soybean as raw material,, as raise food price and increase forest harvesting because of its obvious limitation through fermentation or refining this approach of production first-generation biofuel, and being rejected gradually (Fairley, 2011).The approach of thereupon preparing s-generation biofuel taking renewable lignocelluloses of non-grain such as agriculture and forestry organic waste material, municipal wastes, draft section plant, leaf wood and needlebushs as raw material becomes various countries' research and development and business-like focus (Somma etc., 2010; Fairley, 2011).For example European Union proposes the year two thousand twenty, and its domestic more than 10% traffic fuel will be substituted by biofuel.United States Government also proposes similar target, will reach 36,000,000,000 gallons (Buyx etc., 2011) to the output of U.S.'s biofuel in 2022.Lignocellulosic material main component is Mierocrystalline cellulose (35%～50%), hemicellulose (20%～40%) and xylogen (15%～25%).In lignocellulosic material, these three kinds of main components form the support skeleton of plant materials, wherein Mierocrystalline cellulose composition microfibre, form the mesh skeleton of fiber finer cell wall, hemicellulose and xylogen are connected and are filled between fiber and microfibre with covalent linkage, form a kind of similar reinforced concrete staggered " concrete structure ".The degree of lignification of lignocellulosic material, the unhomogeneity of each component and complicacy, cell walls itself contain or the factor such as the fermentation inhibitor that generates in conversion process is considered to lignocellulosic material and chemical reagent and enzyme is shown to anti-degradation property immanent cause (Iiyama etc., 1994; Cosgrove etc., 2005; Wyman etc., 2005; Himmel etc., 2007).For efficient economy taking lignocellulose as development of raw materials biofuel, its anti-degradation property must be overcome.For reaching this target, (Sticklen etc., 2006 except studying aspect plant engineering and enzyme engineering two; Abramson etc., 2010), more strategy is that biomass are carried out to pre-treatment, to improve its degradability.Report that pretreated mode may be summarized to be: biological process, Physical, chemical method and physico-chemical processes.For the difference of various pretreatment processs and report (Himmel etc., 2007 in a large number of summary comment of relative merits separately; Hendriks etc., 2009; Alvira etc., 2010).
The content of cellulosic degree of crystallinity and xylogen is considered to all most important two factors in lignocellulosic material enzymic hydrolysis factor that affect, and their kinetics and final yields to enzymolysis process play keying action (Nishiyama etc., 2002; Zhu etc., 2008).Natural cellulose is a kind of semi-crystalline polymer, be called as cellulose Ⅰ, it is a kind of polymorphism mixture (being made up of the mixing of anorthic crystal I α and monoclinic crystal I β), and its existence is the major obstacle place (Samayam etc., 2011) of cellulose hydrolysis.In addition, research also shows, (Lee etc., 2009 except content of lignin; Studer etc., 2011), ratio (Ximenes etc., 2010 of distribution, Syringa oblata Lindl. base (S) and the lignum-vitae base (G) of xylogen; Studer etc., 2011) and the functional group of xylogen etc. cellulase hydrolysis is also had to impact (Nakagame etc., 2011; Kumar etc., 2011).Xylogen mainly contains two kinds of mechanism to the restraining effect of cellulase hydrolysis: (1) forms physical barrier, and prevention Mierocrystalline cellulose contacts with enzyme; (2) adsorbing fiber element enzyme, the activity (Pan etc., 2005) of reduction cellulase.For disclosing the relation between these two influence factors and cellulase hydrolysis, understand them separately on the impact of enzymic hydrolysis speed and effect seem particularly important (Zhu etc., 2008).
Nearly allly report that pertinent literature all confirms that the content that reduces the cellulosic degree of crystallinity of substrate and xylogen will significantly improve effect (Sticklen etc., 2006 of enzymic hydrolysis; Abramson etc., 2010; Chundawat etc., 2011).But research was all to analyze for the raw material after different pretreatments mostly in the past, thereby obtain related conclusions.And in fact these methods all have some limitations, when changing an influence factor, other influences factor also changes thereupon, is difficult to obtain the impact effect (Zhu etc., 2008) of single influence factor to enzymic hydrolysis.For one of overcoming defect, investigator's custom selects Microcrystalline Cellulose (Avicel) as Mierocrystalline cellulose model thing, in enzymic hydrolysis system, add the various xylogen of different content as the sample of experiment, to investigate the impact of xylogen on cellulase hydrolysis.Even if but like this, still have some limitations.Because Mierocrystalline cellulose is a complex construction being cross-linked with each other with hemicellulose and xylogen in cell walls, is not what be separated from each other.Defective effect in aforesaid method the accuracy of conclusion.
In view of above-mentioned existing investigation xylogen and Mierocrystalline cellulose are to the problem existing in the method for Enzymatic Hydrolysis, the practical experience of the inventor based on for many years and abundant expertise are actively studied and are innovated, Mierocrystalline cellulose sample and the application thereof for enzymic hydrolysis of a kind of novelty of final invention, to solve defect of the prior art.
Summary of the invention
In order to solve the above-mentioned problems in the prior art, the invention provides a kind of Mierocrystalline cellulose sample, Mierocrystalline cellulose sample of the present invention can be adjusted the single influence factor to enzymic hydrolysis, thereby has improved about the accuracy of conclusion that affects enzymic hydrolysis experiment.
In order to solve the problems of the technologies described above, the present invention has adopted following technical scheme:
Mierocrystalline cellulose sample, it is made by following steps:
A. Microcrystalline Cellulose and Microcrystalline Cellulose are dissolved in respectively in ionic liquid from the mixture of the xylogen of different amounts, obtain multiple ionic liquid solutions;
B. above-mentioned multiple ionic liquid solutions are transferred to respectively to the reconstruct of regenerating in anti-phase solvent;
C. the ionic liquid solution after regeneration reconstruct is filtered respectively, and after lyophilize, obtain the multiple regenerated celluloses as fiber sample.
Further, described ionic liquid is 1-ethyl-3-methylimidazole acetate ([C
2mim] OAc) ionic liquid.
Further, the quality of described Microcrystalline Cellulose and xylogen and with the mass ratio of ionic liquid be 1:11～25.Be preferably 1:11.5-19, more preferably 1:19.
Further, the temperature that described Microcrystalline Cellulose and xylogen dissolve in ionic liquid is 80-130 DEG C.Be preferably 110-130 DEG C.
Further, described Microcrystalline Cellulose and xylogen adopt oil bath heating while dissolving in ionic liquid, and logical nitrogen protection.
Further, described ionic liquid solution after cellulose regenerated reconstruct filters by nitrocellulose filter.
Further, described anti-phase solvent is deionized water, acetone adds water mixture or ketone adds alcohol mixture.
Another object of the present invention is for a kind of application of above-mentioned Mierocrystalline cellulose sample is provided, to realize accurate analysis xylogen and the impact of Mierocrystalline cellulose on enzymic hydrolysis.Its technical scheme is as follows:
Above-mentioned arbitrary described Mierocrystalline cellulose sample is analyzing xylogen and Mierocrystalline cellulose to the application in the impact of enzymic hydrolysis.Particularly analyzing xylogen and fiber to the application in the impact of enzymic hydrolysis speed and transformation efficiency.
Further, described analysis xylogen and Mierocrystalline cellulose comprise Mierocrystalline cellulose sample are carried out to enzymic hydrolysis the impact of enzymic hydrolysis.
Compared with prior art, beneficial effect of the present invention is:
1. the inventive method utilizes the full solution of ionic liquid system that Mierocrystalline cellulose and xylogen are reconstructed, thereby has studied cellulose crystallity and the impact of content of lignin on cellulase hydrolysis.Eliminated in prior art and changed an influence factor, the defect that other influences factor also changes thereupon, has accurately reflected the impact on cellulase hydrolysis of cellulose crystallity and content of lignin.
2. the inventive method and tradition are to adding in enzymic hydrolysis system compared with the investigation method of different content xylogen, and in reconstruct sample, xylogen shows different inhibition mechanism to the impact of enzymic hydrolysis.
3. the xylogen in the reconstruct sample that the inventive method adopts will form physical barrier to cellulase and stop itself and cellulosic absorption, and the xylogen particle on surface will be by adsorbing fiber element enzyme the enzymatic hydrolyzation with reduction sample.
4. show when the ionic liquid pretreatment lignocellulosic material by the inventive method research, after processing, the content of xylogen is not clearly on the impact of enzymic hydrolysis in sample, and that Mierocrystalline cellulose crystal formation changes cellulose Ⅱ into by cellulose Ⅰ is very large on the impact of enzymic hydrolysis.
Brief description of the drawings
Fig. 1 is the histogram that sample of the present invention carries out the cellulose conversion rate of enzymic hydrolysis;
Fig. 2 is the linear graph of the dependency of content of lignin and 36 hours enzymatic hydrolyzations;
Fig. 3 is the X-ray diffractogram of each sample of the embodiment of the present invention;
The scintigram of the electron microscope that Fig. 4 A-Fig. 4 F is associated sample of the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail, but not as a limitation of the invention.
In the present embodiment, ionic liquid, [C
2mim] OAc), purchased from Lanzhou chemical physics institute, purity>=98.5%.Microcrystalline Cellulose, the polymerization degree is 210～240, purchased from Chemical Reagent Co., Ltd., Sinopharm Group.Xylogen adopts milled wood lignin quality (MWL), and from ball milling Triploid of Populus Tomentosa sample, extraction and purifying obtain.Triploid of Populus Tomentosa used is life in 6 years, picks up from Shandong Province, and its Klason content of lignin is 19.9% according to TAPPI standard test.Cellulase (Celluclast1.5L) and beta-glucosidase are bought and are obtained from Sigma Aldrich.Other agents useful for same are all that analytical pure or reagent are pure, directly buy and use, and do not carry out further purifying.
Mierocrystalline cellulose sample, it is made by following steps:
A. Microcrystalline Cellulose and Microcrystalline Cellulose are dissolved in respectively in ionic liquid from the mixture of the xylogen of different amounts, obtain multiple ionic liquid solutions;
B. above-mentioned multiple ionic liquid solutions are transferred to respectively to the reconstruct of regenerating in anti-phase solvent;
C. the ionic liquid solution after regeneration reconstruct is filtered respectively, and after lyophilize, obtain the multiple regenerated celluloses as fiber sample.
Use Mierocrystalline cellulose sample application of the present invention in analyzing xylogen and the impact of Mierocrystalline cellulose on enzymic hydrolysis, especially for analyzing xylogen and the impact of fiber on enzymic hydrolysis speed and transformation efficiency.Emphasis is to adopt Mierocrystalline cellulose sample of the present invention to carry out enzymic hydrolysis, then carries out correlation analysis.Enzymic hydrolysis and correlation analysis all can adopt prior art.Describe with preferred embodiment below.Wherein the regeneration reconstruct of Mierocrystalline cellulose sample is as follows: for ensureing that Mierocrystalline cellulose and xylogen can dissolve completely, take four parts of 600mg Microcrystalline Celluloses, then after mixing with the milled wood lignin quality (MWL) of 0mg, 100mg, 200mg and 300mg respectively, be placed in respectively the there-necked flask of 50mL, then in there-necked flask, add 20g[C respectively
2mim] OAc.There-necked flask is placed in to IKA well heater (IKA basic model suit, Germany) thereupon and at 90 DEG C, carries out oil bath heating 3 hours, stir speed (S.S.) is 600 revs/min, logical nitrogen protection.After treating that Mierocrystalline cellulose and xylogen dissolve completely, hot ionic liquid solution is transferred to the reconstruct of regenerating in the deionized water of approximately 70 DEG C of 400mL, and stirred continuously and healthily 1 hour.Sample after reconstruct filter by nitrocellulose filter and obtain (aperture is 0.45 μ m,
), and repeatedly rinse to remove residual ionic liquid with deionized water, last lyophilize obtains four kinds of regeneration reconstruct Mierocrystalline celluloses.
With Microcrystalline Cellulose raw material (sample 1), regenerate the separately Mierocrystalline cellulose (sample 2) of reconstruct of Microcrystalline Cellulose, the Mierocrystalline cellulose (sample 3) of Microcrystalline Cellulose and 100mg milled wood lignin quality regeneration reconstruct, the Mierocrystalline cellulose (sample 4) of Microcrystalline Cellulose and 200mg milled wood lignin quality regeneration reconstruct, regenerate separately xylogen that the Mierocrystalline cellulose (sample 2) of reconstruct adds different amounts of the Mierocrystalline cellulose (sample 5) of Microcrystalline Cellulose and 300mg milled wood lignin quality regeneration reconstruct and Microcrystalline Cellulose carries out relevant experiment exam xylogen and Mierocrystalline cellulose to Enzymatic Hydrolysis as sample according to existing method.Wherein using in sample 2 respectively additional 14.3%, 25.0% and 33.3% MWL sample as sample 6, sample 7 and sample 8.Wherein additional MWL sample is the mass percent of sample 2.
Related experiment comprises enzymic hydrolysis.Enzymolysis carries out in the erlenmeyer flask of 50mL, concrete enzymatic hydrolysis condition is: take 0.1g sample as in flask, add the sodium acetate buffer solution of the 50mM that 10mL pH value is 4.8, then add 40 μ L tetracycline antibioticses and 20 μ L cycloheximide, finally add the cellulase of 35FPU/g and the beta-glucosidase of 37.5IU/g, flask is placed in to air table and starts enzymolysis, shaking table is set to 50 DEG C, 150 revs/min, enzymolysis 36 hours.After enzymolysis finishes, get 100 μ L enzymolysis solutions on the whizzer of 10000 revs/min centrifugal 10 minutes, supernatant liquor is used for detecting the content of glucose in enzymolysis solution.Detect and adopt high-efficiency anion exchange ion chromatogram to measure (ICS3000, Dai An, the U.S.), chromatographic column is changed to PA100 analytical column.All enzymolysis and detect all repetitive operations, to reduce error.
Fig. 1 is the histogram that above-mentioned sample carries out the cellulose conversion rate of enzymic hydrolysis in 36 hours.As shown in Figure 1, sample 2 significantly increases than the enzymatic hydrolyzation of sample 1, is increased to 98.6% by original 51.6%.Reason mainly contains two aspects, i.e. the increase of sample specific surface area (Dadi etc., 2007; Silva etc., 2011) and Mierocrystalline cellulose crystal formation change cellulose Ⅱ (Samayam etc., 2011) into by cellulose Ⅰ.Two kinds of reasons are also proven in XRD and sem analysis.For sample 3, sample 4 and sample 5, its 36 hours enzymatic hydrolyzations are respectively 97.8%, 91.7% and 85.5%.And three samples all contain the lignin component of high level, are respectively 12.8%, 22.8% and 32.6%.These results show, even if contain the xylogen of high level after reconstruct in sample, its 36 hours enzymatic hydrolyzations are all more than 85%., 36 hours enzymatic hydrolyzations that are mixed to get sample 6, sample 7 and sample 8 by regeneration Microcrystalline Cellulose interpolation different content xylogen but significantly drop to 67.4% from 85.0%.Should point out, in sample 6-8, the content of xylogen is the content a little more than xylogen in sample 3-5.In two kinds of different investigation modes, cellulosic enzymatic hydrolyzation differs greatly, and concrete reason is that xylogen shows different inhibition mechanism.
Fig. 2 is the linear graph of the dependency of content of lignin and 36 hours enzymatic hydrolyzations.Wherein dotted line is obtained by sample 6-8, and solid line is obtained by sample 3-5.As can be seen from the figure, between the content of xylogen and cellulase hydrolysis rate, exist obvious negative linear correlation,, along with the content of xylogen in sample increases, cellulosic enzymatic hydrolyzation is corresponding reduction also.But, two kinds of not only relation conefficient differences of relation line that different investigation modes obtain, and also there is obvious difference in slope.The relation conefficient of dotted line has reached 0.9998, and the relation conefficient of solid line only has 0.8812.In addition, the slope of dotted line is wanted obviously than the height of solid line.This result shows that in two kinds of different investigation modes, xylogen shows different inhibition mechanism to the impact of cellulase hydrolysis rate.For sample 6-8, cellulosic enzymatic hydrolyzation is almost only subject to the impact of content of lignin.This restraining effect main manifestations is that xylogen passes through adsorbing fiber element enzyme, thereby reduces the activity of cellulase.Pan etc. (2005) study discovery, and except xylogen self is to the physical barrier of cellulase, the adsorption between cellulase and xylogen is a conclusive influence factor that causes cellulase hydrolysis rate to decline.But, except content of lignin affects enzymic hydrolysis, should also there is other influence factor in the sample 3-5 obtaining for reconstruct.
Cellulose crystallity is analyzed: the X-ray diffractogram of each sample that Fig. 3 is the embodiment of the present invention.As shown in Figure 3, the crystallinity index of sample 1-5 is respectively 60.3%, 45.0%, 42.9%, 42.5% and 40.7%.Ownership and the mark of the characteristic diffraction peak of the characteristic diffraction peak to cellulose Ⅰ (1-10), (110) and (200) crystal face and cellulose Ⅱ (1-10), (110) and (020) crystal face are shown in Fig. 3 (Samayam etc., 2011).From Fig. 3, can find, change cellulose Ⅱ through the crystal formation of Microcrystalline Cellulose in ionic liquid sample 2-5 after treatment into by cellulose Ⅰ.In reconstruct sample, along with the increase of amorphous content of lignin, corresponding crystallinity index reduces slightly, but the enzymatic hydrolyzation of counter sample also decreases.For the sample after reconstruct, the slight variation of cellulose crystallity can't have a huge impact final enzymic hydrolysis.The research of Park etc. (2010) also shows the slight variation of cellulose crystallity index to be associated with enzymic hydrolysis efficiency.
Fig. 4 A-Fig. 4 F is respectively the scintigram (SEM picture) of sample 1 in the embodiment of the present invention, sample 2, xylogen (MWL), sample 3, sample 4 and the sample 5 surperficial electron microscopes that amplify 10000 times.As can be observed from Figure, the surface of untreated Microcrystalline Cellulose sample presents fibrous and very fine and close.After ionic liquid is processed, the fibrous morphology on surface disappears and generates with a large amount of holes.This surface changes in (2010) such as Li also to be observed during with same ion liquid treatment switchgrass.Research shows, this modification of surface morphology will significantly increase specific surface area (Dadi etc., 2007 of reconstruct sample; Silva etc., 2011).For reconstruct sample, the increase of specific surface area and the reduction of degree of crystallinity are the major causes that causes its enzymic hydrolysis efficiency to increase.In addition can observe MWL by SEM figure and be rendered as multiple dimensioned nano particle.
For sample 3-5, there is obvious variation with the amount of adding MWL in its configuration of surface.Along with the increase of MWL in sample, the holes that form on regeneration Microcrystalline Cellulose sample 2 surfaces in reconstruct sample not only aperture diminish, and number also obviously reduces.This result shows in ionic liquid treating processes, and MWL nanoparticle can carry out good reconstruct with Microcrystalline Cellulose, thereby has reduced the hole showing.For reconstruct sample, MWL will form physical barrier and stop cellulase to contact with cellulosic.In addition, reconstruct sample is along with its specific surface area of increase of MWL also will reduce, and then reduces its enzymic hydrolysis efficiency.Compared with regeneration Microcrystalline Cellulose sample 2, another considerable change of reconstruct sample surfaces is that surface has spherical particle to generate.Similarly spherical particle is all found on the surface of dilute acid pretreatment and ammonia fiber blasting procedure (AFEX) processing biomass samples, and be proved the xylogen and other degraded products (Selig etc., 2007 that mainly come from preprocessing process new deposition; Chundawat etc., 2011; Sannigrahi etc., 2011).Clearly the treatment condition in this research are compared with AFEX with dilute acid pretreatment and are all wanted gentle, and only 90 DEG C of service temperatures, lower than the second-order transition temperature (100 160 DEG C of –) (Samayam etc., 2011) of xylogen.In addition, Singh etc. (2009) finds that in the time of the switchgrass of water regeneration of ionic liquid processing the regeneration polysaccharide fraction that reclaims have certain repulsive interaction to wooden.Based on above analysis, this research thinks that the spherical particle of reconstruct sample surfaces is mainly derived from the MWL of regeneration.And along with the increase of MWL, the diameter of these spherical particles also increases to some extent.The MWL particle of Surface Creation is by adsorbing fiber element enzyme, thus reduction enzymolysis efficiency
Above embodiment is only exemplary embodiment of the present invention, is not used in restriction the present invention, and protection scope of the present invention is defined by the claims.Those skilled in the art can, in essence of the present invention and protection domain, make various amendments or be equal to replacement the present invention, this amendment or be equal to replacement and also should be considered as dropping in protection scope of the present invention.
Claims (7)
1. Mierocrystalline cellulose sample, is characterized in that, it is made by following steps:
A. Microcrystalline Cellulose and Microcrystalline Cellulose are dissolved in respectively in ionic liquid from the mixture of the xylogen of different amounts, obtain multiple ionic liquid solutions;
B. above-mentioned multiple ionic liquid solutions are transferred to respectively to the reconstruct of regenerating in anti-phase solvent;
C. the ionic liquid solution after regeneration reconstruct is filtered respectively, and after lyophilize, obtain the multiple regenerated celluloses as Mierocrystalline cellulose sample;
Wherein, described ionic liquid is 1-ethyl-3-methylimidazole acetate ionic liquid.
2. Mierocrystalline cellulose sample according to claim 1, is characterized in that, the quality of described Microcrystalline Cellulose and xylogen and with the mass ratio of ionic liquid be 1:11～25.
3. Mierocrystalline cellulose sample according to claim 1, is characterized in that, the temperature that described Microcrystalline Cellulose and xylogen dissolve in ionic liquid is 80-130 DEG C.
4. Mierocrystalline cellulose sample according to claim 1, is characterized in that, described Microcrystalline Cellulose and xylogen adopt oil bath heating while dissolving in ionic liquid, and logical nitrogen protection.
5. Mierocrystalline cellulose sample according to claim 1, is characterized in that, described ionic liquid solution after cellulose regenerated reconstruct filters by nitrocellulose filter.
6. Mierocrystalline cellulose sample according to claim 1, is characterized in that, described anti-phase solvent is deionized water, acetone adds water mixture or ketone adds alcohol mixture.
7. the Mierocrystalline cellulose sample described in claim 1-6 any one, analyzing xylogen and Mierocrystalline cellulose to the application in the impact of enzymic hydrolysis, carries out enzymic hydrolysis to Mierocrystalline cellulose sample and analyzes xylogen and the impact of fiber on enzymic hydrolysis speed and transformation efficiency.
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| 殷友利.离子液体再生纤维素的酶解表征与固载酶应用.《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》》.2010, |
| 离子液体再生纤维素的酶解表征与固载酶应用;殷友利;《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》》;20100802;正文第22页3.2.1和3.2.3，25页图3-2，26页图3－3 * |
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