WO2018214445A1 - 利用pH响应型木质素两性表面活性剂促进木质纤维素酶解和回收纤维素酶的方法 - Google Patents

利用pH响应型木质素两性表面活性剂促进木质纤维素酶解和回收纤维素酶的方法 Download PDF

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WO2018214445A1
WO2018214445A1 PCT/CN2017/113139 CN2017113139W WO2018214445A1 WO 2018214445 A1 WO2018214445 A1 WO 2018214445A1 CN 2017113139 W CN2017113139 W CN 2017113139W WO 2018214445 A1 WO2018214445 A1 WO 2018214445A1
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lignin
cellulase
amphoteric surfactant
responsive
lignocellulose
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English (en)
French (fr)
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楼宏铭
蔡诚
邱学青
詹雪娟
杨东杰
庞煜霞
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华南理工大学
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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
    • C12P19/14—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
    • C—CHEMISTRY; METALLURGY
    • C13—SUGAR INDUSTRY
    • C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
    • C13K1/00—Glucose; Glucose-containing syrups
    • C13K1/02—Glucose; Glucose-containing syrups obtained by saccharification of cellulosic materials
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • 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
    • 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 relates to the technical field of lignocellolysis, in particular to a method for promoting lignocellulose digestion and recovery of cellulase by using pH-responsive lignin amphoteric surfactant.
  • Lignocellulosic bio-refining fuel ethanol is one of the effective and viable technologies to replace gasoline.
  • the ability of cellulase to efficiently hydrolyze lignocellulosic substrates is a key technical bottleneck.
  • cellulase directly hinders the industrialization of cellulosic ethanol because of its low vitality, high consumption and high price.
  • Efficient recycling of cellulase is an important way to reduce the cost of bioethanol.
  • cellulase recovery and utilization technologies mainly include ultrafiltration membrane recovery, cellulase immobilization recovery and fresh substrate resorption adsorption.
  • Ultrafiltration membrane recovery can simultaneously recover endonuclease, exonuclease and ⁇ -glucosidase in cellulase, and can obtain higher cellulase recovery efficiency, but ultrafiltration membrane recovery exists in expensive equipment, ultrafiltration membrane Easy to block, time-consuming, and costly issues.
  • the immobilization of cellulase can maintain the stability of cellulase and facilitate the recovery of cellulase, but there is a large mass transfer barrier between immobilized enzyme and cellulose solid particles, and the immobilization process will seriously affect the enzyme activity. Therefore, in the lignocellulose hydrolysis system, it is currently limited to the immobilized recycling of cellobiase.
  • Cellulase has high stability and strong adsorption to cellulose, which makes the adsorption of enzymes through fresh substrate adsorption a potential way to reduce the cost ratio of cellulase, but adding fresh substrate to recover cellulase is less efficient, while It also causes lignin enrichment in the enzymatic hydrolysis system, which has a negative effect on the enzymatic saccharification of cellulose, and the method cannot recover cellobiase.
  • Lignin is the second largest natural polymer material in the world in terms of quantity second only to cellulose.
  • the pulp and paper industry receives about 50 million tons of lignin by-products every year, but more than 95% of lignin is still mainly used as industrial
  • the waste of pulp and the discharge of black liquor from papermaking not only cause waste of resources, but also pollute the environment, and comprehensive development and utilization of it has practical significance for economic development and environmental protection.
  • the cellulosic ethanol industry itself also produces a large amount of enzymatic lignin, which can be of great significance if it can be used to reduce the production cost of bioethanol.
  • Lin et al. used a water-soluble lignin polyoxyethylene ether (EHL-PEG) formed by enzymatic hydrolysis of lignin and polyethylene glycol via epichlorohydrin to promote enzymatic saccharification of corn stover.
  • EHL-PEG water-soluble lignin polyoxyethylene ether
  • the 72h enzymatic hydrolysis efficiency of corn stover increased from 16.7% to 70.1%, while PEG4600 was 52.3%.
  • the object of the present invention is to provide an efficiency of not only effectively improving the enzymatic hydrolysis of lignocellulose, It recovers cellulase and expands the application of industrial lignin. It requires no additional equipment, is easy to operate, and is environmentally friendly. It uses a pH-responsive lignin amphoteric surfactant to promote the hydrolysis of lignocellulose and recover cellulase.
  • the method of the present invention first proposes a method for promoting enzymatic hydrolysis of lignocellulose by pH-responsive lignin amphoteric surfactant and recovering cellulase by simple pH adjustment.
  • the invention uses a pulping and papermaking by-product lignin or enzymatic lignin as a raw material, and is chemically reacted into a pH-responsive lignin amphoteric surfactant, which is used for strengthening the enzymatic hydrolysis of lignocellulose and cellulase. Recycling.
  • the lignin amphoteric surfactant of the present invention can not only recover cellulase, but also recycle itself after recycling.
  • a method for promoting lignocellulosic enzymatic hydrolysis and recovery of cellulase using a pH-responsive lignin amphoteric surfactant comprising the steps of: adding lignocellulose to a buffer solution, and then adding pH-responsive lignin amphoteric surfactant and fiber
  • the enzyme enzyme controls the pH of the mixed solution to be 4.0-6.2, and is heated to 40-60 ° C for 24 to 96 hours to obtain a saccharification hydrolyzate of lignocellulose, which is obtained by solid-liquid separation to obtain an enzymatic hydrolyzed liquid, and then the enzymatic hydrolyzed liquid is adjusted.
  • the pH precipitates the pH-responsive lignin amphoteric surfactant and cellulase and recycles them.
  • the pH-responsive lignin amphoteric surfactant has a pH obtained by chemically reacting a cationic group and/or an anionic group with a derivative of industrial lignin or lignin as a raw material.
  • a lignin amphoteric surfactant responsive to the anionic group being a carboxyl group, a sulfonic acid group or a phosphate group; the cationic group being a quaternary ammonium group or an amine group.
  • the industrial lignin is alkali lignin, organic solvent lignin or biomass refining lignin
  • the lignin derivative is lignosulfonate, lignin carboxylate, lignin phosphate, lignin Quaternary ammonium salt or lignin amine salt.
  • the pH-responsive lignin amphoteric surfactant is sulfonated quaternized lignin, sulfonated aminated lignin, sulfated quaternized lignin, sulfated aminated lignin, phosphorylated quaternized Lignin, phosphorylated aminated lignin, carboxylated quaternized lignin or carboxylated aminated lignin.
  • the pH-responsive lignin amphoteric surfactant has an anion or cationic group content of greater than 0.3 mmol/g lignin.
  • the content of different anionic and cationic groups in the pH-responsive lignin amphoteric surfactant is 0.3-3 mmol/g lignin to ensure the sensitivity of the pH response of the lignin amphoteric surfactant.
  • the pH of the reconditioning enzyme liquid pH causes the pH-responsive lignin amphoteric surfactant and cellulase to precipitate from 2.5 to 10.0.
  • the method for obtaining an enzymatic liquid by solid-liquid separation after completion of enzymatic hydrolysis is a natural sedimentation method, a decantation method, a filtration method, and characterized in that the centrifugation method or a combination of these methods is used.
  • the lignocellulose is pine, eucalyptus, poplar, ash, sea buckthorn, arbor, fir, birch, corn cob, corn stalk, wheat straw, bagasse, straw, rice husk, edible fungus substrate And at least one of the peanut shells.
  • the amount of the buffer is 5 to 50 times the mass of the lignocellulose, and the pH response type amphoteric table
  • the mass ratio of the surfactant to the lignocellulose is from 2 to 40:100.
  • the buffer solution of the present invention may be a buffer solution system suitable for a conventional cellulase, such as a buffer having a pH of 4.5 to 6.2 and an ionic strength of 5 to 200 mmol/L.
  • acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer or phosphate buffer is acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer or phosphate buffer.
  • the cellulase is used in an amount of from 3 to 30 FPU/g based on the glucan content in the lignocellulose.
  • the pH-responsive lignin amphoteric surfactant of the invention is completely dissolved in the buffer in the pH range of enzymatic hydrolysis (4.0-6.2), and the pH of the enzymatic hydrolyzed liquid is adjusted (pH>6.2 after the end of enzymatic hydrolysis). Or pH ⁇ 4.0 can be conveniently precipitated from the solution. Adjust the pH of the enzymatic hydrolysate to a minimum of 2.5 and a maximum of 10.0. Excessively high and too low pH will result in the inactivation of cellulase.
  • the acid which may be used in the regulation of the enzymatic hydrolysis liquid is an organic acid or a mineral acid (such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, acetic acid, formic acid, maleic acid, etc.); the base which may be used is a conventional alkali. (such as sodium hydroxide, potassium hydroxide, calcium oxide, calcium hydroxide, etc.).
  • the mechanism of the present invention is: since the solubility of the pH-responsive lignin amphoteric surfactant can be adjusted with pH, when the pH is 4.0-6.2, the pH-responsive lignin amphoteric surfactant is completely dissolved in the buffer to reduce the cellulose.
  • the inefficient adsorption of the enzyme on lignin promotes the enzymatic hydrolysis of lignocellulose; when pH>6.2 or pH ⁇ 4.0, the pH-responsive lignin amphoteric surfactant precipitates due to its interaction with cellulase ( Electrostatic action, hydrophobic action, hydrogen bonding), so the cellulase in the solution will precipitate together when the pH-responsive amphoteric surfactant is precipitated.
  • the present invention has the following advantages and beneficial effects:
  • the pH-responsive lignin amphoteric surfactant of the invention has no inhibitory effect on the enzymatic hydrolysis of pure cellulose, and the yield of enzymatic saccharification of lignocellulose can be increased by 16.9 to 78.7%.
  • the cellulase recovery in the present invention is simple, time-consuming, and requires no additional equipment, and the cellulase can be quickly recovered by simply adjusting the pH.
  • the invention uses industrial lignin and lignin derivatives as raw materials, and applies them to strengthen lignocellulose enzymatic hydrolysis and cellulase recovery, avoids environmental pollution, and is beneficial to comprehensive utilization of biomass resources.
  • the present invention can avoid the deactivation of cellulase during the immobilization reaction as compared with the currently widely studied cellulase immobilization on a temperature-responsive or pH-responsive polymer to recover cellulase.
  • Figure 1 is a graph showing the pH response of TCSL-N28 in pure water
  • FIG. 2 is a flow chart of a pH-responsive lignin amphoteric surfactant for promoting lignocellulosic hydrolysis and recovery of cellulase.
  • the reagents used in the following examples are commercially available.
  • the microcrystalline cellulose model in the examples is PH101 (purchased from Sigma Aldrich), cellulase is currently widely used Cellic CTec2, and the substrate contains dilute acid pretreated eucalyptus (Alder-DA) and Acidic sulphite treated pine (pine-SPORL).
  • the glucose concentration in the hydrolyzate of the examples was determined by a biosensor analyzer (SBA-40E, Shandong Institute of Biological Sciences).
  • the examples relate to three pH-responsive lignin amphoteric surfactants, one obtained by quaternization of sulfonated lignin (TCSL, produced by Hunan Channel Shenhua Forestry Co., Ltd.), and the isoelectric point is less than 4.0; Alkali lignin (KL, produced by Hunan Xiangjiang Paper Co., Ltd.) was obtained by quaternization, and the isoelectric point was greater than 7.0.
  • KL produced by Hunan Xiangjiang Paper Co., Ltd.
  • One is obtained by amination of sodium lignosulfonate (SL, which is derived from poplar acid sodium sulfite papermaking waste liquor, produced by Jilin Shijie Paper Co., Ltd.), and the isoelectric point is less than 4.0.
  • the specific synthesis methods of three pH-responsive lignin amphoteric surfactants are as follows:
  • the peristaltic pump will have 43.08g mass fraction of 65wt% (3-chloro-2-hydroxypropyl) trimethylammonium chloride solution
  • microcrystalline cellulose 100 parts by mass of microcrystalline cellulose, added to 5000 parts by mass of a pH 4.8, ionic strength of 50 mmol / L of acetic acid - sodium acetate buffer solution, adding 5 parts by mass of TCSL-N25, and then adding 10FPU / g to microcrystalline fiber
  • the cellulase of the mass meter is reacted at 50 ° C for 24 h.
  • the enzymatic hydrolysate is obtained by centrifugation, the pH of the enzymatic liquid is adjusted to 2.8, and a large amount of precipitate is formed after the solution, and then centrifuged, and then obtained.
  • the solid was added to the same sample as the initial enzymatic conditions (substrate and buffer) and re-digested for 24 h (without supplementation of cellulase and lignin amphoteric surfactant), and two enzymatic hydrolysiss were determined by a biosensor analyzer.
  • the glucose content, the statistical results are shown in Table 1.
  • the enzymatic hydrolyzed liquid is obtained by centrifugation, the pH of the enzymatic liquid is adjusted to 3.0, and a large amount of precipitate is formed in the solution, followed by centrifugation, and then The obtained solid was added to the same sample as the initial enzymatic conditions (substrate and buffer) for further enzymolysis for 48 h (without supplementation of cellulase and lignin amphoteric surfactant), and twice by biosensor analyzer.
  • the glucose content of the enzymatic hydrolysis is shown in Table 1.
  • the obtained solid was added to the same sample as the initial enzymatic conditions (substrate and buffer) and then hydrolyzed again for 72 h (without supplementation of cellulase and lignin amphoteric surfactant), and determined by a biosensor analyzer.
  • the glucose content of the two enzymatic hydrolysis is shown in Table 1.
  • eucalyptus-DA 100 parts by mass of eucalyptus-DA, added to 5000 parts by mass of acetic acid-sodium acetate buffer solution having a pH of 4.0 and an ionic strength of 5 mmol/L, adding 10 parts by mass of KL-N40, and then adding 10 FPU/g to the substrate.
  • the cellulase of the dextran meter is reacted at a temperature of 50 ° C for 48 h. After the reaction is completed, the enzymatic hydrolyzed liquid is obtained by centrifugation, the pH of the enzymatic liquid is adjusted to 7.0, and a large amount of precipitate is formed in the solution, followed by centrifugation, and then obtained.
  • the solid was added to the same sample as the initial enzymatic conditions (substrate and buffer) for another 48 h (without cellulase and lignin amphoteric surfactant), and two enzymatic assays were determined by biosensor analyzer.
  • the glucose content, the statistical results are shown in Table 1.
  • microcrystalline cellulose 100 parts by mass of microcrystalline cellulose was added to 5000 parts by mass of a citric acid-sodium citrate buffer solution having a pH of 4.8 and an ionic strength of 5 mmol/L, and 25 parts by mass of TCSL-N28 was added, and then 20 FPU/g was added thereto.
  • the cellulase of the crystalline cellulose mass meter is reacted at 50 ° C for 24 h.
  • the enzymatic hydrolyzed liquid is obtained by centrifugation, the pH of the enzymatic hydrolyzed liquid is adjusted to 3.0, and a large amount of precipitate is formed in the solution, followed by centrifugation, and then The obtained solid was added to the same sample as the initial enzymatic conditions (substrate and buffer) and re-digested for 48 h (without supplementation of cellulase and lignin amphoteric surfactant), and the enzyme was measured by a biosensor analyzer.
  • the glucose content of the solution is shown in Table 1.
  • eucalyptus-DA 250 parts by mass of eucalyptus-DA, added to 5000 parts by mass of a phosphate buffer solution having a pH of 6.0 and an ionic strength of 5 mmol/L, adding 15 parts by mass of ASL, and then adding 20 FPU/g to the dextran in the substrate.
  • the cellulase is reacted at 50 ° C for 48 h.
  • the enzymatic hydrolysis liquid is obtained by centrifugation, the pH of the enzymatic liquid is adjusted to 3.2, a large amount of precipitate is formed in the solution, and then centrifugation is carried out, and the obtained solid is added thereto.
  • the lignin amphoteric surfactant can effectively promote the enzymatic hydrolysis of lignocellulose, and can recover a certain cellulase, and the lignin amphoteric surfactant can also be recycled in the process.
  • Figure 1 is a plot of pH responsiveness of TCSL-N28 in pure water, illustrating that TCSL-N28 has a sensitive pH response, dissolves at pH > 4.0, and precipitates at pH ⁇ 4.0.
  • Figure 2 is a process flow diagram of lignin amphoteric surfactant to promote lignocellulose hydrolysis and recovery of cellulase by pH adjustment. The process does not involve complicated processes, no additional equipment, low energy consumption, and environmental protection. .

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Abstract

利用pH响应型木质素两性表面活性剂促进木质纤维素酶解和回收纤维素酶的方法。该方法将木质纤维素加入缓冲溶液中，再加入pH响应型木质素两性表面活性剂和纤维素酶，控制混合溶液的pH为4．0-6．2，加热至40-60℃温度下反应24-96h，得到木质纤维素的糖化水解液，通过固液分离后得到酶解液体，再调节酶解液体pH使pH响应型木质素两性表面活性剂和纤维素酶沉淀出来，循环利用。该方法不仅可以有效地提高木质纤维素的酶解效率，回收一定的纤维素酶，而且扩大了工业木质素的应用，不需要额外的设备，操作简单，绿色环保。

Description

利用pH响应型木质素两性表面活性剂促进木质纤维素酶解和回收纤维素酶的方法 技术领域
本发明涉及木质纤维素酶解技术领域，特别是涉及利用pH响应型木质素两性表面活性剂促进木质纤维素酶解和回收纤维素酶的方法。
背景技术
木质纤维素生物炼制燃料乙醇是替代汽油有效可行的技术之一。在这一炼制过程中，纤维素酶能否高效水解木质纤维素底物是关键的技术瓶颈。同时，纤维素酶因其具有活力低、耗量大和价格贵等特点，直接阻碍了纤维素乙醇的工业化。实现纤维素酶的高效回收利用是降低生物乙醇成本的重要途径。目前，纤维素酶回收利用技术主要有超滤膜回收、纤维素酶的固定化回收和新鲜底物重吸附法回收。
超滤膜回收可以同时回收纤维素酶中的内切酶，外切酶和β-葡萄糖苷酶，且可以得到较高纤维素酶回收效率，但是超滤膜回收存在着设备贵、超滤膜易堵塞、操作费时、成本高等问题。纤维素酶的固定化可以保持纤维素酶的稳定且利于纤维素酶的回收利用，但是固定化酶与纤维素固体颗粒之间存在很大的传质障碍，且固定化过程会严重影响酶活，因此在木质纤维素酶解体系中目前仅限于纤维二糖酶的固定化回收再用。纤维素酶具有高稳定性和对纤维素的强吸附的特性，使得酶通过新鲜底物吸附回收成为降低纤维素酶成本比例的潜在途径，但是添加新鲜底物回收纤维素酶效率较低，同时也会造成酶解体系中木质素富集，对纤维素的酶解糖化起到消极作用，且该方法无法回收纤维二糖酶。
木质素是自然界中在数量上仅次于纤维素的第二大天然高分子材料，制浆造纸工业每年都会得到5000万吨左右的木质素副产品，但超过95％的木质素仍然主要作为工业制浆的废弃物，造纸黑液的排放不仅造成资源的浪费，同时又污染环境，对其进行综合开发利用对经济的发展和环境保护都具有现实意义。纤维素乙醇工业本身也会产生大量的酶解木质素，若能将其应用于降低生物乙醇的生产成本方面将会有巨大的意义。
Lou等发现低分子量高磺化度的木质素可以有效地促进纯纤维素的酶解，高分子量低磺化度的木质素会抑制纤维素的酶解。提出了木质素磺酸盐可以与纤维素酶形成复合物来稳定纤维素酶，并通过改变纤维素的酶解条件得到了验证(cellulose,2014.21:1351-1359.)。
Wang等发现纤维素酶与磺化木质素结合后，纤维素酶与木质纤维素底物中的木质素之间的静电排斥作用增加了，纤维素酶在木质素上的无效吸附减小了，更多的纤维素酶可以参与到纤维素的水解中，实验观察到添加磺化木质素可使预处理后的白杨树和美国黑松的葡萄糖产率分别提高了25.9％和31.8％(biotechnology for biofuels,2013.6:1-10.)。
Lin等将一种由酶解木质素和聚乙二醇通过环氧氯丙烷交联合成的水溶性的木质素聚氧乙烯醚(EHL-PEG)用于促进玉米秸秆的酶解糖化。在EHL-PEG作用下，玉米秸秆的72h酶解效率从16.7％提高到70.1％，而PEG4600为52.3％。当木质纤维素中木质素含量越高，EHL-PEG强化酶解的效果比PEG4600越显著(Bioresource Technology,2015,185:165-170.)。
但是这些研究只是将木质素改性后应用于促进木质纤维素的酶解，并没有在酶解结束后将仍然具有活性的纤维素酶加以回收利用。
发明内容
为了克服木质纤维素酶解工艺现有技术所存在的酶解效率低和纤维素酶用量大等缺点和不足，本发明的目的在于提供一种不仅可以有效地提高木质纤维素的酶解效率，回收纤维素酶，而且扩大了工业木质素的应用，不需要额外的设备，操作简单，绿色环保的利用pH响应型木质素两性表面活性剂促进木质纤维素酶解并回收纤维素酶的方法。
本发明方法首次提出了利用pH响应型木质素两性表面活性剂促进木质纤维素的酶解并通过简单的调节pH回收纤维素酶的方法。本发明以制浆造纸副产物木质素或酶解木质素为原料，经过化学反应改性成pH响应型木质素两性表面活性剂，将其用于强化木质纤维素的酶解和纤维素酶的回收。本发明木质素两性表面活性剂不仅能回收纤维素酶，而且其自身也可以回收后循环利用。
本发明的目的通过下述方案实现：
利用pH响应型木质素两性表面活性剂促进木质纤维素酶解和回收纤维素酶的方法，包括以下步骤：将木质纤维素加入缓冲溶液中，再加入pH响应型木质素两性表面活性剂和纤维素酶，控制混合溶液的pH为4.0～6.2，加热至40～60℃温度下反应24～96h，得到木质纤维素的糖化水解液，通过固液分离后得到酶解液体，再调节酶解液体pH使pH响应型木质素两性表面活性剂和纤维素酶沉淀出来，循环利用。
为进一步实现本发明目的，优选地，所述pH响应型木质素两性表面活性剂为工业木质素或木质素的衍生物为原料经过化学反应引入阳离子基团和/或阴离子基团得到的具有pH响应的木质素两性表面活性剂，其中阴离子基团为羧基、磺酸基或磷酸基；所述阳离子基团为季铵基或胺基。
优选地，所述工业木质素为碱木质素、有机溶剂木质素或生物质炼制木质素，木质素的衍生物为木质素磺酸盐、木质素羧酸盐、木质素磷酸盐、木质素季铵盐或木质素胺盐。
优选地，所述pH响应型木质素两性表面活性剂为磺化季铵化木质素、磺化胺化木质素、硫酸化季铵化木质素、硫酸化胺化木质素、磷酸化季铵化木质素、磷酸化胺化木质素、羧化季铵化木质素或羧化胺化木质素。
优选地，所述pH响应型木质素两性表面活性剂中阴离子或阳离子基团的含量均大于0.3mmol/g木质素。pH响应型木质素两性表面活性剂中不同阴阳离子基团的含量为0.3～3mmol/g木质素可保证木质素两性表面活性剂pH响应的灵敏性。
优选地，所述再调节酶解液体pH使pH响应型木质素两性表面活性剂和纤维素酶沉淀出来的pH值取值为2.5-10.0。
优选地，酶解结束后通过固液分离得到酶解液体的方法为自然沉降法、倾析法、过滤法、其特征在于，所述离心法或这些方法的联合使用。
优选地，所述木质纤维素为松木、桉木、杨木、水曲柳、沙棘、伯树、杉木、桦木、玉米芯、玉米秸秆、麦秆、甘蔗渣、稻草、稻壳、食用菌基质和花生壳中的至少一种。
优选地，所述缓冲液的量为木质纤维素质量的5～50倍，pH响应型两性表 面活性剂与木质纤维素的质量比为2～40：100。本发明缓冲溶液为常规纤维素酶适用缓冲溶液系统即可，如pH＝4.5～6.2，离子强度为5～200mmol/L的缓冲液。优选为醋酸-醋酸钠缓冲液、柠檬酸-柠檬酸钠缓冲液或磷酸盐缓冲液。
优选地，所述纤维素酶的用量以木质纤维素中的葡聚糖含量计为3～30FPU/g。
本发明pH响应型木质素两性表面活性剂在酶解的pH范围内(4.0～6.2)完全溶解在缓冲液中，酶解结束后，通过调高或调低酶解液体的pH(pH>6.2或pH<4.0)可以方便地从溶液中沉淀出来。调节酶解液体的pH值最低不超过2.5，最高不超过10.0。过高和过低的pH值都会导致纤维素酶的失活。所述的调节酶解液体中可能用到的酸为有机酸或无机酸(如盐酸、硫酸、硝酸、磷酸、醋酸、乙酸、甲酸、马来酸等)；可能用到的碱为常规的碱(如氢氧化钠，氢氧化钾，氧化钙，氢氧化钙等)。
本发明的机理为：由于pH响应型木质素两性表面活性剂的溶解度可以随pH调节，当pH在4.0～6.2时，pH响应型木质素两性表面活性剂完全溶解于缓冲液中，减少纤维素酶在木质素上的无效吸附，促进木质纤维素的酶解；当pH>6.2或pH<4.0时，pH响应型木质素两性表面活性剂析出，由于其与纤维素酶存在一定的相互作用(静电作用、疏水作用、氢键作用)，所以在pH响应型两性表面活性剂析出时会将溶液中的纤维素酶一起沉淀下来。
本发明相对于现有技术，具有如下的优点及有益效果：
(1)本发明以pH响应型木质素两性表面活性剂作为酶解助剂，对纯纤维素的酶解没有抑制作用，可使木质纤维素的酶解糖化得率提高16.9～78.7％。
(2)本发明中回收纤维素酶的操作简单、耗时短、不需要额外的设备，只需要简单的调节pH就可以快速地回收纤维素酶。
(3)本发明以工业木质素和木质素衍生物为原料，将其应用于强化木质纤维素酶解和纤维素酶的回收，避免了环境污染，有利于生物质资源的综合利用。
(4)本发明与目前普遍研究的将纤维素酶固定在温度响应或pH响应聚合物上来回收纤维素酶相比可以避免纤维素酶在固定反应过程中的失活。
附图说明
图1是TCSL-N28在纯水中的pH响应性情况图；
图2是pH响应型木质素两性表面活性剂促进木质纤维素酶解和回收纤维素酶工艺流程图。
具体实施方式
为更好地理解本发明，下面结合附图和实施例对本发明作进一步的描述，但本发明的实施方式不限于此。下列实施例中所用试剂均可从市场购买得到。实施例中的微晶纤维素型号为PH101(购自西格玛奥德里奇)，纤维素酶是目前被广泛使用的Cellic CTec2，底物包含有稀酸预处理的桉木(桉木-DA)和酸性亚硫酸法处理过的松木(松木-SPORL)。实施例水解液中葡萄糖浓度是通过生物传感分析仪(SBA-40E，山东省生物科学研究院)测定的。
实施例涉及到三种pH响应型木质素两性表面活性剂，一种是由磺化木质素(TCSL，湖南通道神华林木有限公司生产)季铵化得到，等电点小于4.0；一种是由碱木质素(KL，湖南湘江纸业有限公司生产)季铵化得到，等电点大于7.0。一种是由木质素磺酸钠(SL，来源于杨木酸性亚硫酸钠造纸废液，由吉林石岘纸业有限责任公司生产)胺化得到，等电点小于4.0。三种pH响应型木质素两性表面活性剂的具体合成方法如下：
季铵化磺化木质素(TCSL-N x)：配制pH＝12的350g TCSL水溶液(TCSL占溶液质量分数为20％)，倒入500mL的三口烧瓶，用水浴锅将温度升到80℃后，用蠕动泵将1.0769x g质量分数为65wt％(3-氯-2-羟丙基)三甲基氯化铵溶液往烧瓶中缓慢滴加，控制滴加速度为1mL/min，滴加5min时加入0.3721xg质量分数为20％的NaOH水溶液，再继续滴加完(3-氯-2-羟丙基)三甲基氯化铵溶液后，在80℃下反应3个小时，将得到的反应液用纯水稀释50倍后调节到pH＝3，使产品析出；x为(3-氯-2-羟丙基)三甲基氯化铵占TCSL的质量分数，x的取值范围为10～80。
季铵化碱木质素(KL-N40)：配制pH＝12的350g KL水溶液(KL占溶液质量分数为20％)，倒入500mL的三口烧瓶，用水浴锅将温度升到80℃后，用蠕动泵将43.08g质量分数为65wt％(3-氯-2-羟丙基)三甲基氯化铵溶液往 烧瓶中缓慢滴加，控制滴加速度为1mL/min，滴加5min时加入22.91g质量分数为20％的NaOH水溶液，再继续滴加完(3-氯-2-羟丙基)三甲基氯化铵溶液后，在80℃下反应3个小时，将得到的反应液用纯水稀释50倍后调节到pH＝7，使产品析出。
胺化木质素磺酸钠(ASL)：配制pH＝12的50g SL水溶液(SL占溶液质量分数为20％)，倒入100mL的三口烧瓶，控制搅拌速度为350rpm，设定水浴锅的温度为80℃，当温度升到50℃-55℃，加入1.5g甲醛溶液(甲醛沸点低，温度低加入)，待温度上升到80℃后，加入对应的3.657g二乙胺，在80℃下反应4h，将得到的反应液用纯水稀释50倍后调节到pH＝3，使产品析出。
实施例1
取100质量份微晶纤维素，加入到5000质量份pH为4.8，离子强度为50mmol/L的醋酸-醋酸钠缓冲溶液中，加入5质量份TCSL-N25，再加入10FPU/g以微晶纤维素质量计的纤维素酶，在50℃温度下反应24h，反应结束后，离心分离获得酶解液，将酶解液体的pH调至2.8，溶液出现大量沉淀后进行离心分离，再将得到的固体加入到与初始酶解条件(底物和缓冲液)相同的样品中再次酶解24h(不补充纤维素酶和木质素两性表面活性剂)，通过生物传感分析仪测定两次酶解的葡萄糖含量，统计结果如表1所示。
实施例2
取100质量份桉木-DA，加入到5000质量份pH为4.8，离子强度为25mmol/L的柠檬酸-柠檬酸钠缓冲溶液中，加入25质量份TCSL-N28，再加入20FPU/g以底物中葡聚糖计的纤维素酶，在50℃温度下反应48h，反应结束后，离心分离获得酶解液体，将酶解液体的pH调至3.0，溶液出现大量沉淀后进行离心分离，再将得到的固体加入到与初始酶解条件(底物和缓冲液)相同的样品中再次酶解48h(不补充纤维素酶和木质素两性表面活性剂)，通过生物传感分析仪测定两次酶解的葡萄糖含量，统计结果如表1所示。
实施例3
取500质量份松木-SPORL，加入到5000质量份pH为5.5，离子强度为5mmol/L的磷酸盐缓冲溶液中，加入10质量份TCSL-N30，再加入10FPU/g 以底物中葡聚糖计的纤维素酶，在50℃温度下反应72h，反应结束后，离心分离获得酶解液体，将酶解液体的pH调至3.0，溶液出现大量沉淀后进行离心分离,再将得到的固体加入到与初始酶解条件(底物和缓冲液)相同的样品中再次酶解72h(不补充纤维素酶和木质素两性表面活性剂)，通过生物传感分析仪测定两次酶解的葡萄糖含量，统计结果如表1所示。
实施例4
取100质量份桉木-DA，加入到5000质量份pH为4.0，离子强度为5mmol/L的醋酸-醋酸钠缓冲溶液中，加入10质量份KL-N40，再加入10FPU/g以底物中葡聚糖计的纤维素酶，在50℃温度下反应48h，反应结束后，离心分离获得酶解液体，将酶解液体的pH调至7.0，溶液出现大量沉淀后进行离心分离，再将得到的固体加入到与初始酶解条件(底物和缓冲液)相同的样品中再次酶解48h(不补充纤维素酶和木质素两性表面活性剂)，通过生物传感分析仪测定两次酶解的葡萄糖含量，统计结果如表1所示。
实施例5
取100质量份微晶纤维素，加入到5000质量份pH为4.8，离子强度为5mmol/L的柠檬酸-柠檬酸钠缓冲溶液中，加入25质量份TCSL-N28，再加入20FPU/g以微晶纤维素质量计的纤维素酶，在50℃温度下反应24h，反应结束后，离心分离获得酶解液体，将酶解液体的pH调至3.0，溶液出现大量沉淀后进行离心分离,再将得到的固体加入到与初始酶解条件(底物和缓冲液)相同的样品中再次酶解48h(不补充纤维素酶和木质素两性表面活性剂)，通过生物传感分析仪测定两次酶解的葡萄糖含量，统计结果如表1所示。
实施例6
取250质量份桉木-DA，加入到5000质量份pH为6.0，离子强度为5mmol/L的磷酸盐缓冲溶液中，加入15质量份ASL，再加入20FPU/g以底物中葡聚糖计的纤维素酶，在50℃温度下反应48h，反应结束后，离心分离获得酶解液体，将酶解液体的pH调至3.2，溶液出现大量沉淀后进行离心分离，再将得到的固体加入到与初始酶解条件(底物和缓冲液)相同的样品中再次酶解48h(不补充纤维素酶和木质素两性表面活性剂)，通过生物传感分析仪测 定两次酶解的葡萄糖含量，统计结果如表1所示。
表1 木质素两性表面活性剂促进木质纤维素酶解和回收纤维素酶的情况
Figure PCTCN2017113139-appb-000001
根据表1中可以看出，木质素两性表面活性剂可以有效地促进木质纤维素的酶解，并且可以回收一定的纤维素酶，同时木质素两性表面活性剂也可以在过程中得到循环利用。
图1是TCSL-N28在纯水中的pH响应性情况图，说明了TCSL-N28具有灵敏的pH响应性，在pH>4.0时溶解，在pH<4.0时沉淀。
图2是木质素两性表面活性剂促进木质纤维素酶解和通过调节pH来回收纤维素酶的工艺流程图，过程中没有涉及到复杂的工艺，不需要额外的设备，能耗低，绿色环保。
需要说明的是，本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 利用pH响应型木质素两性表面活性剂促进木质纤维素酶解和回收纤维素酶的方法，其特征在于包括以下步骤：将木质纤维素加入缓冲溶液中，再加入pH响应型木质素两性表面活性剂和纤维素酶，控制混合溶液的pH为4.0～6.2，加热至40～60℃温度下反应24～96h，得到木质纤维素的糖化水解液，通过固液分离后得到酶解液体，再调节酶解液体pH使pH响应型木质素两性表面活性剂和纤维素酶沉淀出来，循环利用。
  2. 根据权利要求1所述的方法，其特征在于，所述pH响应型木质素两性表面活性剂为工业木质素或木质素的衍生物为原料经过化学反应引入阳离子基团和/或阴离子基团得到的具有pH响应的木质素两性表面活性剂，其中阴离子基团为羧基、磺酸基或磷酸基；所述阳离子基团为季铵基或胺基。
  3. 根据权利要求2所述的方法，其特征在于，所述工业木质素为碱木质素、有机溶剂木质素或生物质炼制木质素，木质素的衍生物为木质素磺酸盐、木质素羧酸盐、木质素磷酸盐、木质素季铵盐或木质素胺盐。
  4. 根据权利要求1或2所述的方法，其特征在于，所述pH响应型木质素两性表面活性剂为磺化季铵化木质素、磺化胺化木质素、硫酸化季铵化木质素、硫酸化胺化木质素、磷酸化季铵化木质素、磷酸化胺化木质素、羧化季铵化木质素或羧化胺化木质素。
  5. 根据权利要求1所述的方法，其特征在于，所述pH响应型木质素两性表面活性剂中阴离子或阳离子基团的含量均大于0.3mmol/g木质素。
  6. 根据权利要求1所述的方法，其特征在于，所述再调节酶解液体pH使pH响应型木质素两性表面活性剂和纤维素酶沉淀出来的pH值取值为2.5-10.0。
  7. 根据权利要求1所述的方法，其特征在于，酶解结束后通过固液分离得到酶解液体的方法为自然沉降法、倾析法、过滤法、其特征在于，所述离心法或这些方法的联合使用。
  8. 根据权利要求1所述的方法，其特征在于，所述木质纤维素为松木、 桉木、杨木、水曲柳、沙棘、伯树、杉木、桦木、玉米芯、玉米秸秆、麦秆、甘蔗渣、稻草、稻壳、食用菌基质和花生壳中的至少一种。
  9. 根据权利要求1所述的方法，其特征在于，所述醋酸-醋酸钠缓冲液、柠檬酸-柠檬酸钠缓冲液或磷酸盐缓冲液；所述缓冲液的量为木质纤维素质量的5～50倍，pH响应型两性表面活性剂与木质纤维素的质量比为2～40：100.
  10. 根据权利要求1所述的方法，其特征在于，所述纤维素酶的用量以木质纤维素中的葡聚糖含量计为3～30FPU/g。
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