WO2024227405A1 - 一种提高木质纤维素酶水解效率的方法 - Google Patents

一种提高木质纤维素酶水解效率的方法 Download PDF

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WO2024227405A1
WO2024227405A1 PCT/CN2024/088996 CN2024088996W WO2024227405A1 WO 2024227405 A1 WO2024227405 A1 WO 2024227405A1 CN 2024088996 W CN2024088996 W CN 2024088996W WO 2024227405 A1 WO2024227405 A1 WO 2024227405A1
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lignocellulose
enzymatic hydrolysis
residue
bamboo
extract
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French (fr)
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李勉
杨武龙
陈天影
杨铭乾
唐艳军
徐伟冬
吴强
秦淑芳
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Zhejiang Huakang Pharmaceutical Co Ltd
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Zhejiang Huakang Pharmaceutical Co Ltd
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Priority to JP2025531150A priority Critical patent/JP2025538267A/ja
Priority to EP24799863.6A priority patent/EP4621060A4/en
Publication of WO2024227405A1 publication Critical patent/WO2024227405A1/zh
Priority to US19/223,333 priority patent/US20250290107A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21B—FIBROUS RAW MATERIALS OR THEIR MECHANICAL TREATMENT
    • D21B1/00—Fibrous raw materials or their mechanical treatment
    • D21B1/04—Fibrous raw materials or their mechanical treatment by dividing raw materials into small particles, e.g. fibres
    • D21B1/06—Fibrous raw materials or their mechanical treatment by dividing raw materials into small particles, e.g. fibres by dry methods
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C1/00—Pretreatment of the finely-divided materials before digesting
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C1/00—Pretreatment of the finely-divided materials before digesting
    • D21C1/04—Pretreatment of the finely-divided materials before digesting with acid reacting compounds
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C3/00—Pulping cellulose-containing materials
    • D21C3/04—Pulping cellulose-containing materials with acids, acid salts or acid anhydrides
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C3/00—Pulping cellulose-containing materials
    • D21C3/20—Pulping cellulose-containing materials with organic solvents or in solvent environment
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the invention belongs to the technical field of preparing lignocellulose, and particularly relates to a method for improving the enzymatic hydrolysis efficiency of lignocellulose.
  • Lignocellulose is mainly composed of cellulose, hemicellulose and lignin, among which the effective saccharification of cellulose is the key to the production of chemicals and liquid fuels. To achieve efficient conversion of lignocellulose, it is necessary to start from destroying the anti-degradation barrier of lignocellulose and find an efficient and green pretreatment solvent system.
  • the pretreatment solvent system can effectively break the original compact structure of biomass and reduce the anti-degradation barrier, thus facilitating the conversion of carbohydrates into fermentable monosaccharides or fuel ethanol, etc.
  • the selection of green, mild and efficient pretreatment solvents is of great significance for the enzymatic saccharification of lignocellulose.
  • Deep eutectic solvent As a new type of ionic liquid, is formed by a simple physical mixture of hydrogen bond donors and hydrogen bond acceptors. It can effectively remove lignin or hemicellulose, has little effect on cellulose degradation, and can also recycle the solvent for reuse.
  • the technical problem to be solved by the present invention is to provide a method for improving the efficiency of enzymatic hydrolysis of lignocellulose, using a low-cost, green and environmentally friendly low eutectic solvent system to enzymatically hydrolyze lignocellulose.
  • the method has the advantages of low equipment requirements, simple operation, and can effectively improve the efficiency of enzymatic hydrolysis.
  • the present invention is achieved by providing a method for improving the efficiency of enzymatic hydrolysis of lignocellulose, comprising the following steps:
  • Step 1 crush the agricultural and forestry biomass raw materials into a particle size of 60-80 mesh, extract them with a toluene-ethanol solution, and dry them at a constant temperature to obtain the extract-free raw materials;
  • Step 2 weighing metal salt and propylene glycol respectively at a molar ratio of 1:10 to 1:124 and mixing them, and stirring the mixture at a temperature of 80 to 90° C. and a stirring speed of 180 rpm to obtain a uniform and transparent type IV low eutectic solvent; wherein the metal salt is any one of aluminum chloride, zinc chloride, and ferric chloride;
  • Step 3 weighing the extract-free raw material prepared in step 1, adding it to the type IV low eutectic solvent prepared in step 2 at a solid-liquid ratio of 1:5 to 1:15, reacting at a temperature of 80 to 120° C. and a stirring speed of 300 to 500 rpm for 2 to 4 hours to obtain a pretreated mixture;
  • Step 4 Separate the pretreated mixture into solid and liquid by centrifugation or vacuum filtration to obtain filter residue and filtrate, respectively; wash and desalt the filter residue with ethanol or deionized water until no metal salt component is detected in the washing liquid; and then dry the washed filter residue to obtain the lignocellulosic residue after pretreatment of the agricultural and forestry biomass raw materials;
  • Step 5 Add the pretreated lignocellulose residue to an acetic acid buffer solution with a pH of 4.8 at a certain substrate concentration, then add cellulose complex enzyme, and react for a period of time in an air bath shaker at 45-50°C and 140-150r/min. Finally, fermentable sugars are obtained.
  • the solvent system prepared by metal salts and glycerol in the present invention is a type IV deep eutectic solvent system.
  • Glycerol is an organic solvent commonly used in pretreatment. When used alone for pretreatment, it requires a higher temperature and a longer time to achieve the effect of removing lignin. The removal effect can be improved in an acidic environment.
  • the Lewis acidity provided by the metal salt can increase the removal rate of lignin, effectively degrade hemicellulose, and swell rather than dissolve cellulose.
  • the type IV deep eutectic solvent system of the present invention has high stability and is easy to prepare, and is a green solvent with more application prospects.
  • the agricultural and forestry biomass is bamboo.
  • step 4 the drying condition of the washed filter residue is drying at 80° C. to constant weight.
  • step five the substrate concentration of the lignocellulosic residue is 2-10wt%
  • the cellulose complex enzyme is Novozymes Cellic @ CTec2
  • the addition amount of the cellulose complex enzyme is 15-50FPU/g substrate
  • the enzyme hydrolysis reaction time is 3h-48h.
  • the method of improving the enzymatic hydrolysis efficiency of lignocellulose of the present invention uses a type IV low eutectic solvent prepared by metal salt-propylene glycol to pretreat the lignocellulose in the agricultural and forestry biomass raw materials, and then enzymatically hydrolyzes the pretreated lignocellulose residue, which has the characteristics of low cost, easy preparation, mild treatment conditions, short treatment time, etc., and has low requirements for equipment, easy to control the reaction, and the whole process is green and clean.
  • the cellulose looseness of the pretreated lignocellulose residue is improved, and a large amount of hemicellulose and lignin are removed, which is conducive to promoting the contact between cellulase and substrate, thereby improving the efficiency of cellulase hydrolysis.
  • FIG1 is a schematic flow diagram of a method for improving the efficiency of enzymatic hydrolysis of lignocellulose according to the present invention
  • Figure 2 is a scanning electron microscope image of bamboo powder before (a1, a2, a3) and after (b1, b2, b3) pretreatment with a type IV deep eutectic solvent synthesized by ferric chloride-propylene glycol;
  • FIG3 is a schematic diagram comparing the enzymatic hydrolysis efficiency (i.e., glucose conversion rate) of various embodiments of the present invention and comparative examples;
  • FIG. 4 is a schematic diagram showing a comparison of the hemicellulose removal rate and the lignin removal rate of the bamboo residue after pretreatment according to various embodiments of the present invention.
  • a preferred embodiment of the method for improving the enzymatic hydrolysis efficiency of lignocellulose of the present invention comprises the following steps:
  • Step 1 Crush the agricultural and forestry biomass raw materials to a particle size of 60-80 mesh, and extract them with a toluene-ethanol solution with a volume ratio of 2:1. The extract is extracted and dried at a constant temperature of 80°C to obtain the extract raw material.
  • the agricultural and forestry biomass is bamboo.
  • Step 2 Weigh the metal salt and glycerol at a molar ratio of 1:10 to 1:124, respectively, and mix the mixture at a temperature of 80 to 90°C and a stirring speed of 180 rpm to obtain a uniform and transparent type IV low eutectic solvent.
  • the metal salt is any one of aluminum chloride, zinc chloride, and ferric chloride.
  • the molar ratio of the metal salt to glycerol is preferably 1:124. According to the final pretreatment effect, the metal salt is preferably ferric chloride.
  • Step 3 Weigh the extract-free raw material prepared in step 1, add it to the type IV deep eutectic solvent prepared in step 2 at a solid-liquid ratio of 1:5 to 1:15, react at a temperature of 80 to 120° C. and a stirring speed of 300 to 500 rpm for 2 to 4 hours to obtain a pretreated mixture.
  • the solid-liquid ratio is preferably 1:10.
  • Step 4 Use centrifugal separation or vacuum filtration to separate the pretreated mixture into solid and liquid to obtain filter residue and filtrate respectively, wash and desalt the filter residue with ethanol or deionized water until no metal salt component is detected in the washing liquid, and then dry the washed filter residue to obtain the lignocellulosic residue after pretreatment of the agricultural and forestry biomass raw materials; the drying condition of the washed filter residue is to dry it at 80°C to constant weight.
  • Step 5 Add the pretreated lignocellulose residue to an acetate buffer solution with a pH of 4.8 at a certain substrate concentration, then add cellulose complex enzyme, and react for a period of time in an air bath shaker at 45-50° C. and a rotation speed of 140-150 r/min to obtain fermentable sugar.
  • the substrate concentration of the lignocellulose residue is 2-10wt%
  • the cellulose complex enzyme is Novozymes Cellic @ CTec2
  • the addition amount of cellulose complex enzyme is 15-50FPU/g substrate
  • the enzyme hydrolysis reaction time is 3h-48h.
  • the substrate concentration of the lignocellulose reaction residue is preferably 2wt%, and the greater the substrate concentration, the longer it takes for the cellulose enzyme hydrolysis to reach the plateau phase.
  • the cellulose complex enzyme is Novozymes Cellic @ CTec2, and the different addition amounts of cellulose complex enzymes have different effects on the time and effect required for enzyme hydrolysis, and affect the experiment and future production costs.
  • the addition amount of cellulose enzyme is 15FPU/g substrate, and the enzyme hydrolysis reaction time is 3h-48h.
  • the enzyme hydrolysis reaction time can be further optimized to 48h according to the optimization of lignocellulose pretreatment residue, substrate concentration, cellulose complex enzyme usage, substrate enzyme hydrolysis effect, and saving enzyme hydrolysis time.
  • the present invention uses metal salt-glycerol to synthesize type IV low eutectic solvent, which has the characteristics of being cheap, easy to obtain, recyclable during utilization, and green and environmentally friendly. Moreover, metal salt-glycerol has a good removal effect on hemicellulose and lignin, and has little dissolution effect on cellulose, thereby achieving efficient conversion of cellulose.
  • the method for improving the enzymatic hydrolysis efficiency of lignocellulose of the present invention is further illustrated by the following examples and comparative examples.
  • the raw bamboo was crushed and sieved to 60-80 mesh, extracted with toluene-ethanol in a volume ratio of 2:1, and then dried at a constant temperature of 80°C. Dry to constant weight to obtain the extract raw material.
  • bamboo residue pretreated with type IV low eutectic solvent synthesized by ferric chloride-glycerol is obtained.
  • the bamboo residue is added to an acetic acid buffer solution with a pH of 4.8 at a substrate concentration of 5%, and a cellulose complex enzyme (Cellic @ CTec2) with a substrate of 50FPU/g bamboo residue is added.
  • the reaction is carried out in an air shaker at 50°C and 140r/min for 48h to obtain fermentable sugar-glucose.
  • the enzymatic hydrolysis efficiency of the pretreated bamboo residue was measured to be 46.63%.
  • the raw material of bamboo was used as a control, and the bamboo was directly hydrolyzed by enzymes without pretreatment with the metal salt-propylene glycol synthesis type IV low eutectic solvent, and the efficiency of direct enzymatic hydrolysis of bamboo was analyzed.
  • the steps include:
  • the raw bamboo was crushed into 60-80 meshes, extracted with toluene-ethanol and then dried at a constant temperature of 80°C.
  • a certain amount of raw bamboo without extract was added to an acetate buffer with a pH of 4.8 at a substrate concentration of 2%, and a cellulose complex enzyme (Cellic @ CTec2) with a content of 15 FPU/g of bamboo substrate was added.
  • the enzymatic hydrolysis efficiency was 17.78%.
  • Example 1 The enzymatic hydrolysis efficiency of Example 1 is 2.6 times that of the comparative example, indicating that the deep eutectic solvent pretreatment method of the present invention can significantly improve the enzymatic hydrolysis efficiency of bamboo.
  • the raw material of bamboo was crushed and sieved to 60-80 mesh, extracted with toluene-ethanol in a volume ratio of 2:1, and then dried at a constant temperature of 80°C to constant weight to obtain the raw material of extract.
  • Ferric chloride and glycerol were weighed in a ratio of 1:124 and stirred at a speed of 180rpm until a uniform transparent liquid.
  • a certain amount of raw material of extract was weighed and added to the ferric chloride-glycerol solvent in a solid-liquid ratio of 1:10, stirred at 300rpm at 100°C and reacted for 3h.
  • bamboo residues were added to an acetic acid buffer solution with a pH of 4.8 at a substrate concentration of 2%, and a cellulose complex enzyme (Cellic @ CTec2) with a substrate of 15 FPU/g was added. The mixture was reacted in an air shaker at 50°C and 140 r/min for 48 hours to obtain fermentable sugar, glucose. Finally, the enzymatic hydrolysis rate of the pretreated bamboo residues was measured to be 67.87%, which is 3.8 times that of the unpretreated bamboo in the control group.
  • the raw bamboo was crushed and sieved to 60-80 mesh, extracted with toluene-ethanol in a volume ratio of 2:1, and then dried at a constant temperature of 80°C. Dry to constant weight to obtain the extract raw material.
  • bamboo residue pretreated with type IV low eutectic solvent synthesized by ferric chloride-glycerol is obtained.
  • the bamboo residue is added to an acetic acid buffer solution with a pH of 4.8 at a substrate concentration of 2%, and a cellulose complex enzyme (Cellic @ CTec2) with a substrate of 15FPU/g bamboo residue is added.
  • the fermentable sugar-glucose is obtained by reacting in an air shaker at 50°C and 140r/min for 48h.
  • the enzymatic hydrolysis efficiency of the pretreated bamboo residue was measured to be 91.13%, which is 5.1 times that of the unpretreated bamboo residue in the control example.
  • the raw material of bamboo was crushed and sieved to 60-80 mesh, extracted with toluene-ethanol in a volume ratio of 2:1, and then dried at a constant temperature of 80°C to constant weight to obtain the raw material of extract.
  • Ferric chloride and glycerol were weighed in a ratio of 1:15 and stirred at a speed of 180rpm until a uniform transparent liquid.
  • a certain amount of raw material of extract was weighed and added to the ferric chloride-glycerol solvent in a solid-liquid ratio of 1:5, stirred at 300rpm at 100°C and reacted for 3h.
  • bamboo residues were added to an acetic acid buffer solution with a pH of 4.8 at a substrate concentration of 10%, and a cellulose complex enzyme (Cellic @ CTec2) with a substrate of 50 FPU/g of bamboo residues was added, and the mixture was reacted in an air shaker at 50°C and 140 r/min for 48 hours to obtain fermentable sugar, glucose. Finally, the enzymatic hydrolysis rate of the pretreated bamboo residues was measured to be 69.41%, which is 3.9 times the enzymatic hydrolysis efficiency of the unpretreated bamboo in the control group.
  • the raw material of bamboo was crushed and sieved to 60-80 mesh, extracted with toluene-ethanol in a volume ratio of 2:1, and then dried at a constant temperature of 80°C to constant weight to obtain the raw material of extract.
  • Zinc chloride and glycerol were weighed in a ratio of 1:124 and stirred at a speed of 180rpm until a uniform transparent liquid.
  • a certain amount of raw material of extract was weighed and added to the zinc chloride-glycerol solvent in a solid-liquid ratio of 1:10, stirred at 300rpm at 100°C and reacted for 3h.
  • bamboo residues were added to an acetic acid buffer solution with a pH of 4.8 at a substrate concentration of 2%, and a cellulose complex enzyme (Cellic @ CTec2) with a substrate of 15 FPU/g of bamboo residues was added, and the mixture was reacted in an air shaker at 50°C and 140 r/min for 48 hours to obtain fermentable sugar, glucose. Finally, the enzymatic hydrolysis efficiency of the pretreated bamboo residues was measured to be 24.75%, which is 1.4 times that of the unpretreated bamboo residues in the control group.
  • the raw material of bamboo was crushed and sieved to 60-80 mesh, extracted with toluene-ethanol in a volume ratio of 2:1, and then dried at a constant temperature of 80°C to constant weight to obtain the raw material of extract.
  • Aluminum chloride and glycerol were weighed in a ratio of 1:124 and stirred at a speed of 180rpm until a uniform transparent liquid.
  • a certain amount of raw material of extract was weighed and added to the aluminum chloride-glycerol solvent in a solid-liquid ratio of 1:10, stirred at 300rpm at 100°C and reacted for 3h.
  • bamboo residues were added to an acetic acid buffer solution with a pH of 4.8 at a substrate concentration of 2%, and a cellulose complex enzyme (Cellic @ CTec2) with a substrate of 15 FPU/g of bamboo residues was added, and the mixture was reacted in an air shaker at 50°C and 140 r/min for 48 hours to obtain fermentable sugar, glucose. Finally, the enzymatic hydrolysis efficiency of the pretreated bamboo residues was measured to be 59.78%, which is 3.4 times that of the unpretreated bamboo residues in the control group.
  • the surface of the bamboo fiber of the unpretreated bamboo powder is relatively smooth and intact, as shown in Figures 2-a1, 2-a1, and 2-a1, and is closely arranged; while the surface of the bamboo fiber of the bamboo powder pretreated with the type IV low eutectic solvent synthesized by ferric chloride-propylene glycol of the present invention is rough, with deeper grooves, as shown in Figures 2-b1, 2-b2, and 2-b3.
  • the fibers are broken in many places, and the degree of damage on the fracture surface is high, which greatly increases the contact area between cellulase and cellulose and the adsorption capacity of cellulose for cellulase, thereby improving the enzymatic hydrolysis efficiency of cellulose.
  • the pretreatment process conditions and pretreatment solvent system have different degrees of influence on the removal of lignin and hemicellulose components.
  • the removal rate can reflect the destruction of the structural density of lignocellulosic biomass to a certain extent. Therefore, the removal of lignin and hemicellulose can promote the enzymatic hydrolysis of lignocellulosic biomass and improve the efficiency of enzymatic hydrolysis.

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Abstract

一种提高木质纤维素酶水解效率的方法，包括步骤：将农林生物质原料破碎至60～80目粒径，用甲苯-乙醇溶液进行抽提，恒温烘干得到去抽提物原料；按摩尔比为1:10～1:124分别称取金属盐和丙三醇进行混合，混合物在80～90℃的温度条件下，以180rpm的搅拌速度得到均一透明的Ⅳ型低共熔溶剂；称取去抽提物原料加入到Ⅳ型低共熔溶剂中，在80～120℃的温度条件下，以300～500rpm的搅拌速度反应2h～4h，得到预处理混合物；对预处理混合物进行固液分离，分别得到滤渣和滤液，然后将清洗后的滤渣进行烘干，得到农林生物质原料预处理后的木质纤维素残渣；对木质纤维素残渣进行酶解得到可发酵性糖。

Description

一种提高木质纤维素酶水解效率的方法 技术领域
本发明属于制备木质纤维素技术领域，特别涉及一种提高木质纤维素酶水解效率的方法。
背景技术
木质纤维素主要是由纤维素、半纤维素和木质素组成，其中纤维素的有效糖化是生产化学品和液体燃料的关键。要实现木质纤维素高效转化，必须从破坏木质纤维素抗降解屏障出发，寻找高效且绿色环保的预处理溶剂体系。
预处理溶剂体系能够有效打破生物质原有的致密性结构，降低抗降解屏障，从而利于碳水化合物向可发酵单糖的转化或燃料乙醇的转化等。而绿色环保、温和、高效预处理溶剂的选择对木质纤维素的酶解糖化具有重要意义。
低共熔溶剂(DES)作为一类新型离子液体，由氢键供体和氢键受体经简单的物理混合而成，可有效去除木质素或半纤维素，且对纤维素降解作用小，还可对溶剂进行回收再利用。
发明内容
本发明所要解决的技术问题在于，提供一种提高木质纤维素酶水解效率的方法，采用一种成本低廉、绿色环保的低共熔溶剂体系对木质纤维素进行酶水解，该方法具有对设备要求低、操作简单、可有效提高酶水解效率等优点。
本发明是这样实现的，提供一种提高木质纤维素酶水解效率的方法，包括如下步骤：
步骤一、将农林生物质原料破碎至60～80目粒径，用甲苯-乙醇溶液进行抽提，恒温烘干得到去抽提物原料；
步骤二、按摩尔比为1:10～1:124分别称取金属盐和丙三醇进行混合，混合物在80～90℃的温度条件下，以180rpm的搅拌速度得到均一透明的Ⅳ型低共熔溶剂；其中，金属盐为氯化铝、氯化锌、氯化铁中任意一种；
步骤三、称取步骤一制备的去抽提物原料，按固液比1:5～1:15加入到步骤二制备的Ⅳ型低共熔溶剂中，在80～120℃的温度条件下，以300～500rpm的搅拌速度反应2h～4h，得到预处理混合物；
步骤四、采用离心分离或减压抽滤方式对预处理混合物进行固液分离，分别得到滤渣和滤液，将滤渣用乙醇或去离子水清洗脱盐至洗液检测不到金属盐组分，然后将清洗后的滤渣进行烘干，得到农林生物质原料预处理后的木质纤维素残渣；
步骤五、将预处理后的木质纤维素残渣按一定的底物浓度加入到pH为4.8的醋酸缓冲液中，再加入纤维素复合酶，在45～50℃、140～150r/min转速的空气浴摇床中，反应一段时间 后，得到可发酵性糖。
目前，将低共熔溶剂(DES)分为四类，本发明采用金属盐和丙三醇制备的溶剂体系为Ⅳ型低共熔溶剂体系。丙三醇是预处理中常用的有机溶剂，单独用于预处理时需要较高温度和较长时间才能达到脱除木质素的效果，在酸性环境中可以改善脱除效果，金属盐提供的路易斯酸性可提高木质素的脱除率，有效降解半纤维素，并对纤维素有润胀而非溶解。本发明的Ⅳ型低共熔溶剂体系具有稳定性高、易制备，是更具应用前景的绿色溶剂。
进一步地，在步骤一中，所述农林生物质为竹材。
进一步地，在步骤四中，所述清洗后的滤渣的烘干条件是在80℃下烘干至恒重。
进一步地，在步骤五中，所述木质纤维素残渣的底物浓度为2～10wt％，纤维素复合酶为诺维信Cellic@CTec2，纤维素复合酶的添加量为15～50FPU/g底物，酶水解反应时间为3h～48h。
与现有技术相比，本发明的提高木质纤维素酶水解效率的方法，采用金属盐-丙三醇制备的Ⅳ型低共熔溶剂对农林生物质原料中的木质纤维素进行预处理，然后再对预处理后的木质纤维素残渣进行酶水解，具有成本低廉、容易制备，处理条件温和、处理时间短等特点，而且对设备的要求较低，反应易于控制，整个过程绿色清洁。另一方面，经过预处理后的木质纤维素残渣的纤维素疏松度得到提高，脱除了大量半纤维素及木质素，有利于促进纤维素酶与底物的接触，从而提高纤维素酶水解效率。
附图说明
图1为本发明提高木质纤维素酶水解效率的方法的流程示意图；
图2为竹粉经氯化铁-丙三醇合成的Ⅳ型低共熔溶剂预处理前(a1、a2、a3)、预处理后(b1、b2、b3)的扫描电镜图；
图3为本发明的各实施例和对比例的酶水解效率(即葡萄糖转化率)对比示意图；
图4为本发明的各实施例的毛竹残渣预处理后的半纤维素脱除率和木质素的脱除率对比示意图。
具体实施方式
为了使本发明所要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
请参照图1所示，本发明提高木质纤维素酶水解效率的方法的较佳实施例，包括如下步骤：
步骤一、将农林生物质原料破碎至60～80目粒径，用体积比2:1的甲苯-乙醇溶液进行抽 提，80℃恒温烘干得到去抽提物原料。所述农林生物质为毛竹。
步骤二、按摩尔比为1:10～1:124分别称取金属盐和丙三醇进行混合，混合物在80～90℃的温度条件下，以180rpm的搅拌速度得到均一透明的Ⅳ型低共熔溶剂。其中，金属盐为氯化铝、氯化锌、氯化铁中任意一种。金属盐与丙三醇的摩尔比优选为1:124。根据最终的预处理效果，金属盐优选为氯化铁。
步骤三、称取步骤一制备的去抽提物原料，按固液比1:5～1:15加入到步骤二制备的Ⅳ型低共熔溶剂中，在80～120℃的温度条件下，以300～500rpm的搅拌速度反应2h～4h，得到预处理混合物。固液比优选为1:10。
步骤四、采用离心分离或减压抽滤方式对预处理混合物进行固液分离，分别得到滤渣和滤液，将滤渣用乙醇或去离子水清洗脱盐至洗液检测不到金属盐组分，然后将清洗后的滤渣进行烘干，得到农林生物质原料预处理后的木质纤维素残渣；所述清洗后的滤渣的烘干条件是在80℃下烘干至恒重。
步骤五、将预处理后的木质纤维素残渣按一定的底物浓度加入到pH为4.8的醋酸缓冲液中，再加入纤维素复合酶，在45～50℃、140～150r/min转速的空气浴摇床中，反应一段时间后，得到可发酵性糖。
所述木质纤维素残渣的底物浓度为2～10wt％，纤维素复合酶为诺维信Cellic@CTec2，纤维素复合酶的添加量为15～50FPU/g底物，酶水解反应时间为3h～48h。木质纤维素反应残渣的底物浓度优选为2wt％，底物浓度越大，纤维素酶水解达到平台期所需时间越长。纤维素复合酶为诺维信Cellic@CTec2，纤维素复合酶添加量不同对酶水解所需时间及效果有所差异，且影响实验及今后生产成本。在实现高酶水解效率的同时降低生产成本，综合考虑优选纤维素酶添加量为15FPU/g底物，酶水解反应时间为3h～48h，可根据木质纤维素预处理残渣、底物浓度、纤维素复合酶使用量、底物酶水解效果的最优化及节约酶水解时间角度进一步优化酶水解反应时间为48h。
本发明使用金属盐-丙三醇合成Ⅳ型低共熔溶剂具有廉价易得、在利用过程中可循环使用、绿色环保等特点。而且，金属盐-丙三醇对半纤维素和木质素有很好的脱除作用，并对纤维素的溶解作用很小，从而实现纤维素的高效转化。
下面通过实施例和对比例进一步说明本发明的提高木质纤维素酶水解效率的方法。
实施例1
本发明第一种提高木质纤维素酶水解效率的方法的实施例，包括如下步骤：
将毛竹原料粉碎过筛取60～80目，经体积比2:1比例的甲苯-乙醇抽提后在80℃下恒温烘 干至恒重，得到去抽提物原料。按1:10的比例称取氯化铁和丙三醇，以180rpm的速度搅拌至均一透明液体。称取一定量去抽提物原料按照1:15固液比加入到氯化铁-丙三醇溶剂中，在80℃下，以300rpm的速度搅拌并反应4h。反应结束后，采用真空抽滤法进行固液分离，将分离得到的毛竹滤渣用去离子水清洗脱盐至检测不到金属盐成分，得到氯化铁-丙三醇合成的Ⅳ型低共熔溶剂预处理后的毛竹残渣。毛竹残渣按5％的底物浓度加入到pH为4.8的醋酸缓冲溶液中，加入50FPU/g毛竹残渣底物的纤维素复合酶(Cellic@CTec2)，在50℃，140r/min的空气摇床中反应48h，得到可发酵性糖——葡萄糖。最后测量得到预处理后的毛竹残渣的酶水解效率为46.63％。
对比例
毛竹原料为对照，将毛竹不经过金属盐-丙三醇合成Ⅳ型低共熔溶剂的预处理而直接进行酶水解，分析毛竹直接进行酶水解的效率。包括如下步骤：
将毛竹原料破碎至60～80目，经甲苯-乙醇抽提后在80℃下恒温烘干。将一定量去抽提物毛竹原料按2％的底物浓度加入到pH为4.8的醋酸缓冲液中，加入15FPU/g毛竹底物的纤维素复合酶(Cellic@CTec2)，在50℃，140r/min的空气浴摇床中反应48h后酶水解效率为17.78％。
实施例1的酶水解效率是对比例的酶水解效率的2.6倍，说明本发明的低共熔溶剂预处理方法可以显著地提高毛竹酶水解效率。
实施例2
本发明第二种提高木质纤维素酶水解效率的方法的实施例，包括如下步骤：
将毛竹原料粉碎过筛取60～80目，经体积比2:1比例的甲苯-乙醇抽提后在80℃下恒温烘干至恒重，得到去抽提物原料。按1:124的比例称取氯化铁和丙三醇，以180rpm的速度搅拌至均一透明液体。称取一定量去抽提物原料按照1:10固液比加入到氯化铁-丙三醇溶剂中，在100℃下，以300rpm的速度搅拌并反应3h。反应结束后，采用真空抽滤法进行固液分离，将分离得到的毛竹滤渣用去离子水清洗脱盐至检测不到金属盐成分，得到氯化铁-丙三醇合成的Ⅳ型低共熔溶剂预处理后的毛竹残渣。毛竹残渣按2％的底物浓度加入到pH为4.8的醋酸缓冲溶液中，加入15FPU/g毛竹残渣底物的纤维素复合酶(Cellic@CTec2)，在50℃，140r/min的空气摇床中反应48h得到可发酵性糖——葡萄糖。最后测量得到预处理后的毛竹残渣的酶水解小率为67.87％，是对比例的未经预处理毛竹酶水解效率的3.8倍。
实施例3
本发明第三种提高木质纤维素酶水解效率的方法的实施例，包括如下步骤：
将毛竹原料粉碎过筛取60～80目，经体积比2:1比例的甲苯-乙醇抽提后在80℃下恒温烘 干至恒重，得到去抽提物原料。按1:124的比例称取氯化铁和丙三醇，以180rpm的速度搅拌至均一透明液体。称取一定量去抽提物原料按照1:10固液比加入到氯化铁-丙三醇溶剂中，在120℃下，以300rpm的速度搅拌并反应2h。反应结束后，采用真空抽滤法进行固液分离，将分离得到的毛竹滤渣用去离子水清洗脱盐至检测不到金属盐成分，得到氯化铁-丙三醇合成的Ⅳ型低共熔溶剂预处理后的毛竹残渣。毛竹残渣按2％的底物浓度加入到pH为4.8的醋酸缓冲溶液中，加入15FPU/g毛竹残渣底物的纤维素复合酶(Cellic@CTec2)，在50℃，140r/min的空气摇床中反应48h得到可发酵性糖——葡萄糖。最后测量得到预处理后的毛竹残渣的酶水解效率为91.13％，是对比例的未经预处理毛竹酶水解效率的5.1倍。
实施例4
本发明第四种提高木质纤维素酶水解效率的方法的实施例，包括如下步骤：
将毛竹原料粉碎过筛取60～80目，经体积比2:1比例的甲苯-乙醇抽提后在80℃下恒温烘干至恒重，得到去抽提物原料。按1:15的比例称取氯化铁和丙三醇，以180rpm的速度搅拌至均一透明液体。称取一定量去抽提物原料按照1:5固液比加入到氯化铁-丙三醇溶剂中，在100℃下，以300rpm的速度搅拌并反应3h。反应结束后，采用真空抽滤法进行固液分离，将分离得到的毛竹滤渣用去离子水清洗脱盐至检测不到金属盐成分，得到氯化铁-丙三醇合成的Ⅳ型低共熔溶剂预处理后的毛竹残渣。毛竹残渣按10％的底物浓度加入到pH为4.8的醋酸缓冲溶液中，加入50FPU/g毛竹残渣底物的纤维素复合酶(Cellic@CTec2)，在50℃，140r/min的空气摇床中反应48h得到可发酵性糖——葡萄糖。最后测量得到预处理后的毛竹残渣的酶水解小率为69.41％，是对比例的未经预处理毛竹酶水解效率的3.9倍。
实施例5
本发明第五种提高木质纤维素酶水解效率的方法的实施例，包括如下步骤：
将毛竹原料粉碎过筛取60～80目，经体积比2:1比例的甲苯-乙醇抽提后在80℃下恒温烘干至恒重，得到去抽提物原料。按1:124的比例称取氯化锌和丙三醇，以180rpm的速度搅拌至均一透明液体。称取一定量去抽提物原料按照1:10固液比加入到氯化锌-丙三醇溶剂中，在100℃下，以300rpm的速度搅拌并反应3h。反应结束后，采用真空抽滤法进行固液分离，将分离得到的毛竹滤渣用去离子水清洗脱盐至检测不到金属盐成分，得到氯化锌-丙三醇合成的Ⅳ型低共熔溶剂预处理后的毛竹残渣。毛竹残渣按2％的底物浓度加入到pH为4.8的醋酸缓冲溶液中，加入15FPU/g毛竹残渣底物的纤维素复合酶(Cellic@CTec2)，在50℃，140r/min的空气摇床中反应48h得到可发酵性糖——葡萄糖。最后测量得到预处理后的毛竹残渣的酶水解效率为24.75％，是对比例的未经预处理毛竹酶水解效率的1.4倍。
实施例6
本发明第六种提高木质纤维素酶水解效率的方法的实施例，包括如下步骤：
将毛竹原料粉碎过筛取60～80目，经体积比2:1比例的甲苯-乙醇抽提后在80℃下恒温烘干至恒重，得到去抽提物原料。按1:124的比例称取氯化铝和丙三醇，以180rpm的速度搅拌至均一透明液体。称取一定量去抽提物原料按照1:10固液比加入到氯化铝-丙三醇溶剂中，在100℃下，以300rpm的速度搅拌并反应3h。反应结束后，采用真空抽滤法进行固液分离，将分离得到的毛竹滤渣用去离子水清洗脱盐至检测不到金属盐成分，得到氯化铝-丙三醇合成的Ⅳ型低共熔溶剂预处理后的毛竹残渣。毛竹残渣按2％的底物浓度加入到pH为4.8的醋酸缓冲溶液中，加入15FPU/g毛竹残渣底物的纤维素复合酶(Cellic@CTec2)，在50℃，140r/min的空气摇床中反应48h得到可发酵性糖——葡萄糖。最后测量得到预处理后的毛竹残渣的酶水解效率为59.78％，是对比例的未经预处理毛竹酶水解效率的3.4倍。
请参照图2所示，未预处理的竹粉的竹纤维表面相对光滑、完整，如图2-a1、图2-a1、图2-a1所示，排列紧密；而经本发明的氯化铁-丙三醇合成的Ⅳ型低共熔溶剂预处理后的竹粉的竹纤维表面粗糙，沟壑加深，如图2-b1、图2-b2、图2-b3所示，纤维多处被打断，断裂面破损程度高，大大增加纤维素酶与纤维素的接触面积以及纤维素对纤维素酶的吸附能力，进而提高了纤维素的酶水解效率。
请参照图3所示，与对比例相比，经本发明的氯化铁-丙三醇合成的Ⅳ型低共熔溶剂预处理后，木质纤维生物质的酶水解效果均有所提高，尤其是氯化铁基DES和氯化铝基DES，这与金属盐的性质密切相关，且预处理工艺条件对最终酶水解效率有不同程度的影响。
请参照图4所示，预处理工艺条件以及预处理溶剂体系对木质素和半纤维素组分的脱除有不同程度的影响。脱除率可在一定程度上反映木质纤维生物质结构致密性的破坏。因此，木质素脱除和半纤维素脱除后可促进木质纤维生物质的酶水解，提高酶水解效率。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (4)

  1. 一种提高木质纤维素酶水解效率的方法，其特征在于，包括如下步骤：
    步骤一、将农林生物质原料破碎至60～80目粒径，用甲苯-乙醇溶液进行抽提，恒温烘干得到去抽提物原料；
    步骤二、按摩尔比为1:10～1:124分别称取金属盐和丙三醇进行混合，混合物在80～90℃的温度条件下，以180rpm的搅拌速度得到均一透明的Ⅳ型低共熔溶剂；其中，金属盐为氯化铝、氯化锌、氯化铁中任意一种；
    步骤三、称取步骤一制备的去抽提物原料，按固液比1:5～1:15加入到步骤二制备的Ⅳ型低共熔溶剂中，在80～120℃的温度条件下，以300～500rpm的搅拌速度反应2h～4h，得到预处理混合物；
    步骤四、采用离心分离或减压抽滤方式对预处理混合物进行固液分离，分别得到滤渣和滤液，将滤渣用乙醇或去离子水清洗脱盐至洗液检测不到金属盐组分，然后将清洗后的滤渣进行烘干，得到农林生物质原料预处理后的木质纤维素残渣；
    步骤五、将预处理后的木质纤维素残渣按一定的底物浓度加入到pH为4.8的醋酸缓冲液中，再加入纤维素复合酶，在45～50℃、140～150r/min转速的空气浴摇床中，反应一段时间后，得到可发酵性糖。
  2. 如权利要求1所述的提高木质纤维素酶水解效率的方法，其特征在于，在步骤一中，所述农林生物质为竹材。
  3. 如权利要求1所述的提高木质纤维素酶水解效率的方法，其特征在于，在步骤四中，所述清洗后的滤渣的烘干条件是在80℃下烘干至恒重。
  4. 如权利要求1所述的提高木质纤维素酶水解效率的方法，其特征在于，在步骤五中，所述木质纤维素残渣的底物浓度为2～10wt％，纤维素复合酶为诺维信Cellic@CTec2，纤维素复合酶的添加量为15～50FPU/g底物，酶水解反应时间为3h～48h。
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