CN105524959A - Method for promoting enzymatic hydrolysis of lignocellulose - Google Patents
Method for promoting enzymatic hydrolysis of lignocellulose Download PDFInfo
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- CN105524959A CN105524959A CN201610090238.7A CN201610090238A CN105524959A CN 105524959 A CN105524959 A CN 105524959A CN 201610090238 A CN201610090238 A CN 201610090238A CN 105524959 A CN105524959 A CN 105524959A
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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Abstract
The invention discloses a method for promoting enzymatic hydrolysis of lignocellulose. The method comprises the following steps: (1) mixing lignocellulose and a pH regulation solution for obtaining a first liquid-solid mixture, wherein the lignocellulose is subjected to pretreatment in advance, and the pH value of the pH regulation solution is 4.0-10.0; (2) adding a metal salt solution to the first liquid-solid mixture, and reacting for preset time to obtain a second liquid-solid mixture; (3) separating the second liquid-solid mixture to obtain a solid; and (4) enabling the solid to be subjected to enzymatic hydrolysis by utilizing cellulose. With the adoption of the method, the invalid adsorption of lignin for the cellulose can be effectively reduced, the enzymatic hydrolysis of the lignocellulose is promoted, and the saccharifying efficiency of the cellulose is improved; and the method is low in cost, and easy for popularization and use.
Description
Technical field
The present invention relates to biomass chemical field, particularly promote the method for lignocellulose enzymatic hydrolysis.
Background technology
Lignocellulose is one of the abundantest biomass of nature content, have every year in the world and produce more than 4,000 ten thousand tons of lignocellulosic materials, account for 50% of global biomass total amount, comprise agroforestry waste, waste paper, energy crop etc., wherein major part does not add utilization and abandons.In lignocellulose composition, carbohydrate content is up to 75%, can be used as the important sources of fermentable sugars for the production of liquid fuel and other chemical.In recent years along with the continuous consumption of fossil energy and becoming increasingly conspicuous of environmental problem, the exploitation of the Wood Adhesives from Biomass production energy and chemical receives the extensive concern of global all circles.
The primary chemical composition of lignocellulose comprises Mierocrystalline cellulose (35 – 55wt.%), hemicellulose (20 – 35wt.%) and xylogen (5 – 30wt.%).The relative content of three kinds of main ingredients is different with the difference of raw material.Mierocrystalline cellulose is the polysaccharide polymer with crystalline texture be connected to form by β-Isosorbide-5-Nitrae-glycosidic link by D-glucopyranose, containing 300-15000 glucosyl group.Hemicellulose is the heterogeneous polysaccharide be made up of different monose (wood sugar, Ah 's sugar, seminose and semi-lactosi etc.), and hemicellulose side chain is more, containing free hydrogen bond in molecular chain, has hydrophilicity, and there is not crystalline texture.Xylogen is the aromatic polymer polymkeric substance of the class complexity be connected to form by carbon-carbon bond, ehter bond by benzene oxide unit.Lignocellulose is mainly present in plant cell wall, is a kind of material on micro-, nanoscale with complex construction.First Mierocrystalline cellulose forms fine cellulose, has constructed the skeleton structure of cell walls, and hemicellulose and xylogen are wrapped in outside Mierocrystalline cellulose as " weighting agent ".Therefore, lignocellulose often needs pre-treatment to destroy this structure to improve cellulosic enzymatic hydrolysis characteristic.But still there is xylogen in pretreated lignocellulose matrix, and xylogen can cause cellulase hydrolysis efficiency to reduce to the ineffective adsorption of cellulase.For reducing the ineffective adsorption of Mierocrystalline cellulose on cellulase, it is found that and add the transformation efficiency that tensio-active agent can improve lignocellulose enzymolysis in enzymolysis process, reducing xylogen to the ineffective adsorption of enzyme, thus the consumption of reduction cellulase.Existing research shows, be nonionic surface active agent the most significantly, and most of ionogenic surfactant does not have promoter action to enzymolysis to enzymolysis promoter action.After conventional promotion pre-treatment, the nonionic surface active agent of lignocellulose enzymolysis is PEG and Tween series, but these nonionic surface active agent are often expensive, and inhibited for the microorganism in subsequent fermentation production process.
Therefore, be necessary to find reduction xylogen to the inexpensive method of cellulase ineffective adsorption.
Summary of the invention
The present invention is intended at least to solve one of technical problem existed in prior art.For this reason, it is with low cost that one object of the present invention is to propose one, effectively can reduce xylogen to cellulase ineffective adsorption, promotes lignocellulose enzymatic hydrolysis, improve the method for cellulosic saccharification efficiency.
According to an aspect of the present invention, the invention provides a kind of method promoting lignocellulose enzymatic hydrolysis.According to embodiments of the invention, the method comprises the following steps:
(1) mixed with pH regulator solution by described lignocellulose, to obtain the first liquid-solid mixture, wherein said lignocellulose is in advance through pre-treatment, and the pH value of described pH regulator solution is 4.0-10.0;
(2) in described first liquid-solid mixture, add metal salt solution, and react the scheduled time, to obtain the second liquid-solid mixture;
(3) from described second liquid-solid mixture, separation obtains solid;
(4) cellulase is utilized to make described solid carry out enzymatic hydrolysis.
Contriver is surprised to find, and utilizes the method effectively can reduce xylogen to cellulase ineffective adsorption, promotes lignocellulose enzymatic hydrolysis, improve cellulosic saccharification efficiency, and the method is with low cost, is easy to promote the use of.
According to embodiments of the invention, in step (1), described pre-treatment is dilute acid pretreatment, steam explosion pre-treatment, alkaline purification or organic solvent pre-treatment.
According to embodiments of the invention, in step (1), the pH value of described pH regulator solution is 4.5-7.0.
According to embodiments of the invention, described pH regulator solution is sulphuric acid soln or hydrochloric acid soln.
According to embodiments of the invention, the mass volume ratio of described lignocellulose and described pH regulator solution is 1:5-1:30.Thus, the enzymatic hydrolysis efficiency of follow-up lignocellulose can be improved.
According to embodiments of the invention, in step (2), the metal ion in described metal salt solution is trivalent and/or divalent-metal ion.
According to embodiments of the invention, described metal ion is at least one being selected from aluminum ion, iron ion, ferrous ion, mn ion, cobalt ion, cupric ion, calcium ion, magnesium ion, nickel ion and barium ion.
According to embodiments of the invention, described metal ion is at least one being selected from calcium ion, magnesium ion, cobalt ion and nickel ion.Thus, the enzymatic hydrolysis efficiency of follow-up lignocellulose can be improved.
According to embodiments of the invention, in step (2), in described first liquid-solid mixture, add metal salt solution, and react 0.1-3 hour at 20 DEG C-80 DEG C.Thus, the enzymatic hydrolysis efficiency of follow-up lignocellulose can be improved.
According to embodiments of the invention, in step (2), the addition of described metal salt solution for making liquid metal salt concn in described second liquid-solid mixture be 0.01-1g/L, preferred 0.1g/L.Thus, the enzymatic hydrolysis efficiency of follow-up lignocellulose can be improved.
According to embodiments of the invention, in step (3), obtain solid by filtration or centrifugal separation from described second liquid-solid mixture.
According to embodiments of the invention, in step (4), under the condition of pH4.5-5.5, carry out described enzymatic hydrolysis.Thus, the enzymatic hydrolysis efficiency of lignocellulose can be improved.
According to embodiments of the invention, utilize the pH value of the enzymatic hydrolysis system in inorganic acid solution regulating step (4) to 4.5-5.5.Thus, the metal ion generation desorption be adsorbed onto on xylogen can be avoided.
According to preferred embodiments more of the present invention, described inorganic acid solution is sulphuric acid soln or hydrochloric acid soln.
According to embodiments of the invention, in step (4), the usage quantity of described cellulase is 5-40FPU/g solid, preferred 5-15FPU/g solid.
According to embodiments of the invention, in step (4), under gradient increased temperature condition, carry out described enzymatic hydrolysis.Thus, the metal ion generation desorption be adsorbed onto on xylogen because temperature Rapid Variable Design brings can be avoided.
According to embodiments of the invention, described gradient increased temperature is that staged heats up.
According to preferred embodiments more of the present invention, it is be incubated 2 hours, until temperature keeps temperature-resistant after rising to 50 DEG C after temperature often raises 5 DEG C that described staged heats up.Thus, the phenomenon being adsorbed onto the metal ion generation desorption on xylogen because temperature Rapid Variable Design brings can be reduced.
According to embodiments of the invention, comprise further: supplement in the enzymatic hydrolysis system in step (4) and add described metal salt solution.According to some embodiments of the present invention, described metal salt solution is identical with the metal salt solution in step (2).
It should be noted that, method of the present invention has adsorbing principle to realize to metal ion according to xylogen.Pretreated lignocellulose solid is toward contact residual lignin, and this part xylogen has irreversible ineffective adsorption effect to cellulase.And adopt relatively inexpensive metal ion can reduce cellulase loss to shield xylogen to the adsorption site of cellulase.Therefore, method disclosed by the invention is mainly in order to promote the enzymatic hydrolysis of the solid still containing xylogen after pre-treatment, particularly conventional dilute acid pretreatment, steam explosion pre-treatment, because most of lignin still remains in solids, thus the promoter action of metal ion is more remarkable.PH is the important factor affecting xylogen adsorbing metal ions.Metal ion treatment and cellulase solution preocess are separated by method provided by the invention, namely in enzymolysis advance row metal ion processing, and thus can adjust ph as required.Preferably, the pH of metal ion treatment is 4.0-10.0, more preferably 4.5-7.0.Process under subacidity or neutrallty condition, can ensure that xylogen surface is combined to carry out coordination with metal ion with enough negative charges.The shielding of the metal ion adopted to xylogen adsorption site has material impact.Although xylogen heavy metal ion, such as Pb
2+, Gd
2+etc. having the strongest adsorptive power, but method provided by the invention preferably uses non-heavy metal ion, because heavy metal ion often causes the inactivation of albumen in follow-up enzymolysis process.Optional metal ion comprises, one or more mixing in aluminum ion, iron ion, ferrous ion, mn ion, cobalt ion, cupric ion, calcium ion, magnesium ion, nickel ion, barium ion; Preferably, described preferred metal ion is divalent-metal ion; Further preferably, described metal ion is calcium ion, magnesium ion, cobalt ion and nickel ion.This is because these metal ions can not cause cellulase inactivation on the one hand, on the other hand, be discharged in enzymolysis solution as desorb occurs these metal ions in enzymolysis process, it also can be used as the trace quantity mineral substance element in follow-up fermentation process needed for microorganism growth.Because different metal ion is different for the screening ability of xylogen adsorption site, therefore, the concentration of its optimum is also different.In method provided by the invention, preferable alloy ionic concn is 0.01-1g/L, and too high concentration of metal ions can suppress cellulosic enzymolysis on the contrary.
And then, according to other embodiments of the present invention, in the step (4) of method of the present invention, should avoid using organic acid buffer liquid when carrying out cellulase catalytic hydrolysis.This is because, usual cellulosic enzymolysis carries out in the acetic acid or citrate buffer solution of pH4.8, but organic acid often has certain sequestering power for metal ion, enzymolysis is carried out in damping fluid, the metal ion generating portion desorption be adsorbed on xylogen can be caused, thus reduce or eliminate the shielding effect of metal ion to xylogen avtive spot.Further, as previously mentioned, under the condition of pH4.5-5.5, carry out described enzymatic hydrolysis, and described enzymatic hydrolysis carries out in non-organic acid buffered soln; Preferably, described pH4.5-5.5 is regulated by sulfuric acid or hydrochloric acid, thus, can avoid the negative impact using organic acid buffer solution to bring.
It should be noted that, term " first ", " second " only for describing object, and can not be interpreted as instruction or hint relative importance or imply the quantity indicating indicated technical characteristic.Thus, be limited with " first ", the feature of " second " can express or impliedly comprise one or more these features.Further, in describing the invention, except as otherwise noted, the implication of " multiple " is two or more.
Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Embodiment
Embodiments of the invention are described below in detail.Embodiment described below is exemplary, only for explaining the present invention, and can not be interpreted as limitation of the present invention.
Below in conjunction with embodiment, the solution of the present invention is made an explanation.It will be understood to those of skill in the art that the following examples only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition in embodiment, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
Embodiment 1
Metal ion screens
Straw is extremely neutral through 0.5% dilute sulphuric acid pre-treatment 1h after washing at 140 DEG C, to obtain through pretreated straw (i.e. pretreated lignocellulose).The sulphuric acid soln of above-mentioned pretreated straw and pH4.8 is mixed to get liquid-solid mixture, and solids content is 5%.Adding in different metal salts solution to liquid-solid mixture respectively makes metal salt concentrations in liquid phase be 0.05-1g/L, at room temperature subsequently, rotating speed is cross after processing 1 hour in the shaking table of 150rpm to filter liquid, use the cellulase consumption of 15FPU/g solid again, initial pH4.8,50 DEG C, under the condition of 150rpm, carry out enzymolysis.Wherein, enzymatic hydrolysis system temperature rises to 50 DEG C from room temperature and adopts gradient increased temperature mode, and insulation 2 hours after namely often raising 5 DEG C, until temperature keeps temperature-resistant after rising to 50 DEG C.Control group does not add metal salt solution, result, and different metal ion processing group is compared with control group, and increase (+) or the reduction (-) of enzymolysis transformation efficiency are as shown in table 1.As known from Table 1, suitable metal salt concentrations is 0.1g/L, and corresponding metal ion volumetric molar concentration is 0.25-1.1mmol/L.After can obviously promoting 120h, the metal ion of cellulase transformation efficiency has Ca
2+, Ni
2+, Fe
3+, Ba
2+, Cu
2+, Zn
2+, Mg
2+, enzymolysis transformation efficiency improves 5-13%.
Embodiment 2
Each metal ion species is on the impact of cellulase hydrolysis during different enzyme dosage
Pre-treatment straw raw material used, metal ion treatment and enzymolysis process temperature, pH parameter are with embodiment 1.Adopt 0.1g/L metal salt concentrations, and the cellulase consumption of 5-40FPU/g solid.Control group does not add metal ion, and after 120h, cellulosic enzymolysis transformation efficiency is as shown in table 2.
Table 2 different metal ion under different cellulase consumption on 120h after the impact of cellulose conversion rate
As seen from the above table, under different cellulase consumption, metal ion all has certain promoter action to cellulose conversion rate, and when lower cellulase consumption metal ion, particularly Mg
2+show the most significant promoter action.
Embodiment 3
The impact of pH
Pre-treatment straw raw material used and enzymolysis process are with embodiment 1.Adopt 0.1g/LMgCl
2concentration, changes MgCl
2the pH value of system during straw after solution-treated dilute acid pretreatment, the solid after process adopts the cellulase consumption of 5FPU/g solid to carry out enzymolysis under corresponding pH.Not add the pre-treatment straw of metal ion for contrast, after 120h, cellulosic enzymolysis transformation efficiency is as shown in table 3.
Table 3pH is to MgCl
2the impact of process dilute acid pretreatment straw enzymolysis
As seen from the above table, pH is to Mg
2+promoter action there is remarkably influenced, when processing under lower or higher pH, the promoter action of metal ion becomes remarkable, and optimum pH is near 5.0.
Embodiment 4
The impact of enzymolysis process gradient increased temperature
Pre-treatment straw raw material used is with embodiment 1.Adopt 0.1g/LMgCl
2solution processes through pretreated straw under pH4.8 condition, adopts the cellulase consumption of 10FPU/g solid to carry out enzymolysis afterwards.Enzymatic hydrolysis system temperature rises to 50 DEG C from room temperature and adopts two schemes.Scheme one: for non-gradient heats up, namely temperature is increased to 50 DEG C from room temperature rapid (in 5min).Scheme two: be gradient increased temperature, 2 hours are incubated after temperature often raises 5 DEG C, until temperature keeps temperature-resistant after rising to 50 DEG C, namely temperature 30 DEG C, 35 DEG C, 40 DEG C, be incubated 2h respectively at 45 DEG C of temperature, total heating-up time (containing every section of 2h soaking time) that temperature rises to 50 DEG C is 8-8.5h.As a result, the 120h enzymolysis cellulose conversion rate of scheme one is 75.1 ± 1.5%, and the enzymolysis transformation efficiency of scheme two is 78.2 ± 0.7%.Thus, scheme two better effects if is shown.
Embodiment 5
Different pretreatments raw material ratio comparatively
Described metal ion treatment and enzymolysis process are as the scheme two of embodiment 4.After metal ion treatment different pretreatments, the enzymolysis cellulose conversion rate of raw material is as shown in table 4.
Wherein, dilute acid pretreatment is 0.5% dilution heat of sulfuric acid in the liquid-solid ratio (L/kg) of 10:1, processes 30min at 160 DEG C; Alkaline purification is employing 1% sodium hydroxide solution in the liquid-solid ratio (L/kg) of 10:1, processes 30min at 160 DEG C; Steam explosion pre-treatment is that the raw material of water content 50% is incubated 5min after being steam heated to 200 DEG C, lets out to normal pressure by pressure then suddenly; Organic solvent be employing 50% ethanol 10:1 liquid-solid ratio (L/kg), add 0.1% sulfuric acid, process 30min at 160 DEG C.
Table 40.1g/LMgCl
2the enzymolysis cellulose conversion rate of process different pretreatments raw material compares
As seen from the above table, MgCl
2all promoter action is had to the sample after different pretreatments, but more obvious for the facilitation effect of sample after dilute acid pretreatment and steam explosion pre-treatment.
In the description of this specification sheets, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referred to the schematic representation of above-mentioned term.And the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiment or example.
Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.
Claims (10)
1. promote a method for lignocellulose enzymatic hydrolysis, it is characterized in that, comprise the following steps:
(1) mixed with pH regulator solution by described lignocellulose, to obtain the first liquid-solid mixture, wherein said lignocellulose is in advance through pre-treatment, and the pH value of described pH regulator solution is 4.0-10.0;
(2) in described first liquid-solid mixture, add metal salt solution, and react the scheduled time, to obtain the second liquid-solid mixture;
(3) from described second liquid-solid mixture, separation obtains solid;
(4) cellulase is utilized to make described solid carry out enzymatic hydrolysis.
2. method according to claim 1, is characterized in that, in step (1), described pre-treatment is dilute acid pretreatment, steam explosion pre-treatment, alkaline purification or organic solvent pre-treatment.
3. method according to claim 1, is characterized in that, in step (1), the pH value of described pH regulator solution is 4.5-7.0,
Optionally, described pH regulator solution is sulphuric acid soln or hydrochloric acid soln,
Optionally, the mass volume ratio of described lignocellulose and described pH regulator solution is 1:5-1:30.
4. method according to claim 1, is characterized in that, in step (2), the metal ion in described metal salt solution is trivalent and/or divalent-metal ion,
Optionally, described metal ion is at least one being selected from aluminum ion, iron ion, ferrous ion, mn ion, cobalt ion, cupric ion, calcium ion, magnesium ion, nickel ion and barium ion,
Preferably, described metal ion is at least one being selected from calcium ion, magnesium ion, cobalt ion and nickel ion.
5. method according to claim 1, is characterized in that, in step (2), adds metal salt solution, and react 0.1-3 hour in described first liquid-solid mixture at 20 DEG C-80 DEG C,
Optionally, in step (2), the addition of described metal salt solution for making liquid metal salt concn in described second liquid-solid mixture be 0.01-1g/L, preferred 0.1g/L.
6. method according to claim 1, is characterized in that, in step (3), obtains solid by filtration or centrifugal separation from described second liquid-solid mixture.
7. method according to claim 1, is characterized in that, in step (4), under the condition of pH4.5-5.5, carries out described enzymatic hydrolysis,
Optionally, utilize the pH value of the enzymatic hydrolysis system in inorganic acid solution regulating step (4) to 4.5-5.5,
Preferably, described inorganic acid solution is sulphuric acid soln or hydrochloric acid soln.
8. method according to claim 1, is characterized in that, in step (4), the usage quantity of described cellulase is 5-40FPU/g solid, preferred 5-15FPU/g solid.
9. method according to claim 1, is characterized in that, in step (4), under gradient increased temperature condition, carries out described enzymatic hydrolysis,
Optionally, described gradient increased temperature is that staged heats up,
Preferably, described staged heats up is be incubated 2 hours, until temperature keeps temperature-resistant after rising to 50 DEG C after temperature often raises 5 DEG C.
10. method according to claim 1, is characterized in that, comprises further:
Supplement in the enzymatic hydrolysis system in step (4) and add described metal salt solution.
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| CN106671236A (en) * | 2016-12-17 | 2017-05-17 | 福建神采新材料科技有限公司 | Bamboo joint breaking charging machine |
| CN109266704A (en) * | 2018-09-19 | 2019-01-25 | 昆明理工大学 | A method of promoting lignocellulosic enzyme hydrolysis |
| CN115821616A (en) * | 2022-11-08 | 2023-03-21 | 湖北中烟工业有限责任公司 | Preparation method of cigarette carton material |
| CN115821616B (en) * | 2022-11-08 | 2024-05-31 | 湖北中烟工业有限责任公司 | Preparation method of carton material for cigarettes |
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Cited By (4)
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| CN106671236A (en) * | 2016-12-17 | 2017-05-17 | 福建神采新材料科技有限公司 | Bamboo joint breaking charging machine |
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| CN115821616A (en) * | 2022-11-08 | 2023-03-21 | 湖北中烟工业有限责任公司 | Preparation method of cigarette carton material |
| CN115821616B (en) * | 2022-11-08 | 2024-05-31 | 湖北中烟工业有限责任公司 | Preparation method of carton material for cigarettes |
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Application publication date: 20160427 |