阅读说明：本技术 一种利用杨木酶法制备高浓度单糖溶液的方法 (Method for preparing high-concentration monosaccharide solution by using poplar enzyme method ) 是由 张军华 文沛瑶 徐勇 贾丽丽 于 2020-04-30 设计创作，主要内容包括：本发明涉及一种利用杨木酶法制备高浓度单糖溶液的方法,将杨木与乙酸-双氧水溶液混合,进行预处理反应；将所述预处理反应完毕的预处理液与杨木进行固液分离,将预处理后的杨木滤渣和碱液混合,进行碱预处理；将所述碱预处理后的处理液和杨木碱处理滤渣进行固液分离,将碱处理滤渣水洗至中性,得到预处理原料；将所述预处理的原料、柠檬酸钠缓冲液和纤维素酶混合,进行酶解。该方法采用两步预处理脱除了杨木中大量的木质素和乙酰基,提高了杨木中葡聚糖的含量和酶水解效率,实现了利用杨木制备高浓度单糖溶液。该方法具有酶解液单糖浓度高、酶解效率高、试剂可回收、操作简单等特点,适于杨木生物转化制备生物能源和高值化学品。(The invention relates to a method for preparing a high-concentration monosaccharide solution by using a poplar enzyme method, which comprises the steps of mixing poplar with an acetic acid-dioxygen aqueous solution, and carrying out a pretreatment reaction; carrying out solid-liquid separation on the pretreated liquid after the pretreatment reaction and the poplar, mixing the pretreated poplar filter residue with alkali liquor, and carrying out alkali pretreatment; carrying out solid-liquid separation on the treating fluid after the alkali pretreatment and the poplar alkali treatment filter residue, and washing the alkali treatment filter residue to be neutral to obtain a pretreatment raw material; and mixing the pretreated raw materials, a sodium citrate buffer solution and cellulase for enzymolysis. The method adopts two-step pretreatment to remove a large amount of lignin and acetyl in poplar, improves the content of glucan in poplar and the enzymatic hydrolysis efficiency, and realizes the preparation of high-concentration monosaccharide solution by using poplar. The method has the characteristics of high monosaccharide concentration of the enzymolysis liquid, high enzymolysis efficiency, recoverable reagent, simple operation and the like, and is suitable for preparing biological energy and high-value chemicals by poplar biotransformation.)

1. A method for preparing a high-concentration monosaccharide solution by using a poplar enzyme method is characterized by comprising the following steps of:

(1) mixing poplar wood with acetic acid-hydrogen peroxide solution for pretreatment reaction;

(2) carrying out solid-liquid separation on the pretreated liquid after the pretreatment reaction and the poplar, mixing the pretreated poplar filter residue with alkali liquor, and carrying out alkali pretreatment;

(3) carrying out solid-liquid separation on the treating fluid after the alkali pretreatment and the poplar alkali treatment filter residue, and washing the alkali treatment filter residue to be neutral to obtain a pretreatment raw material;

(4) and mixing the pretreated raw materials, a sodium citrate buffer solution and cellulase for enzymolysis.

2. The poplar enzymolysis method according to claim 1, wherein the ratio of the weight of the pretreated raw material to the volume of the sodium citrate buffer solution in the step (4) is 150-250 mg: 1 mL; the pH value of the sodium citrate buffer solution is 5.0-6.0.

3. The poplar enzymatic hydrolysis method according to claim 1 or 7, wherein the dosage of the cellulase in the step (4) is that 20-40 FPU of cellulase is added per gram of pretreated raw material.

4. The alkali treatment method according to claim 1, wherein the alkali solution in the step (2) has a mass concentration of 0.1 to 0.5%.

5. The alkali treatment method according to claim 1 or 4, wherein the volume ratio of the mass of the pretreated filter residue to the alkali liquor in the step (2) is 1g: 8-10 mL.

6. The alkali treatment method according to claim 1 or 4, wherein the temperature of the alkali pretreatment in the step (2) is 60 to 80 ℃ and the time is 1 to 2 hours.

7. The poplar acetic acid-hydrogen peroxide pretreatment method according to claim 1, wherein the acetic acid-hydrogen peroxide solution in the step (1) has a volume percentage of 60% of acetic acid hydrogen peroxide.

8. The poplar acetic acid-hydrogen peroxide pretreatment method according to claim 1, wherein the ratio of the mass of the poplar in the step (1) to the volume of the acetic acid-hydrogen peroxide solution is 1g: 8-10 mL.

9. The high-concentration monosaccharide solution obtained by the poplar enzymatic hydrolysis method according to any one of claims 1 to 8, wherein the concentration of the high-concentration monosaccharide solution is higher than 140 g/mL.

Technical Field

The invention belongs to the technical field of forestry biomass biodegradation and conversion, and particularly relates to a method for preparing a high-concentration monosaccharide solution by poplar enzymolysis.

Background

In the process of biomass biotransformation, high-concentration sugar liquor can be obtained by adopting high-substrate-concentration enzymolysis, and the fermentation for preparing biological energy and chemicals can reduce the cost of subsequent fermentation and purification, thereby having great significance for high-value utilization of biomass.

The poplar has fast growth speed, short growth period, strong environmental adaptability and wide planting area, and is an important raw material for producing biological energy and biomass chemicals.

Due to the strong degradation-resistant barrier and high lignin content of poplar, the problems of low enzymolysis yield, large enzyme dosage and the like exist in the enzymolysis process, so that the preparation of high-concentration monosaccharide enzymolysis liquid by adopting high-concentration poplar in the enzymolysis process is very difficult. For example, when the poplar was pretreated by Tian D et al in 2017, Biotechnol Biofuels.10:157 in two steps and then hydrolyzed with addition of up to 32mg of cellulase per gram of dry matter, the yield of glucose from poplar was only 70%. Zhang et al reported that when the concentration of the substrate for the enzymatic hydrolysis of pretreated poplar was 20%, the cellulose conversion rate was less than 85% and the glucose concentration was up to 158mg/mL using a special stirring device (Bioresource Technology,2009,100 (23): 5890-. Chandra et al (Bioresource Technology,2016,199: 135-141) carried out enzymatic hydrolysis on pretreated poplar with 10% substrate concentration, and the enzymatic hydrolysis yield of the poplar was only 80% with the addition of 20mg of protein/g substrate of cellulase, wherein the glucose concentration was lower than 61mg/mL, the xylose concentration was lower than 3mg/mL, and the monosaccharide concentration was lower than 64 mg/mL. Hu et al (Bioresource Technology, 2015,186: 149-. Therefore, the problems of low enzymolysis yield, low monosaccharide concentration of enzymolysis liquid and the like generally exist in the enzymatic hydrolysis process of poplar treated by the traditional pretreatment mode.

Disclosure of Invention

The invention aims to provide a method for preparing a high-concentration monosaccharide solution by using poplar. The acetic acid-hydrogen peroxide and sodium hydroxide provided by the invention are used for treating poplar in sequence, so that the enzymolysis yield of the poplar and the monosaccharide concentration in enzymolysis liquid can be greatly improved; the monosaccharide concentration of the prepared poplar enzymatic hydrolysate is more than 250 mg/mL.

In order to achieve the above object, the present invention provides the following technical solutions;

the invention provides a method for preparing a high-concentration monosaccharide solution by using a poplar enzyme method, which comprises the following steps of:

(1) mixing poplar wood with acetic acid-hydrogen peroxide solution for pretreatment reaction;

(2) carrying out solid-liquid separation on the pretreated liquid after the pretreatment reaction and the poplar, mixing the pretreated poplar filter residue with alkali liquor, and carrying out alkali pretreatment;

(3) carrying out solid-liquid separation on the treating fluid after the alkali pretreatment and the poplar alkali treatment filter residue, and washing the alkali treatment filter residue to be neutral to obtain a pretreatment raw material;

(4) and mixing the pretreated raw materials, a sodium citrate buffer solution and cellulase for enzymolysis.

Preferably, the volume ratio of the weight of the pretreatment raw material in the step (4) to the sodium citrate buffer solution is 150-250 mg: 1 mL; the pH value of the sodium citrate buffer solution is 5.0-6.0.

Preferably, the dosage of the cellulase in the step (4) is that 20-40 FPU of cellulase is added to each gram of pretreated raw material.

Preferably, the mass concentration of the alkali liquor in the step (2) is 0.1-0.5%.

Preferably, the volume ratio of the mass of the pretreated filter residue to the alkali liquor in the step (2) is 1g: 8-10 mL.

Preferably, the temperature of the alkali pretreatment in the step (2) is 60-80 ℃, and the time is 1-2 h.

Preferably, the volume percentage of the acetic acid hydrogen peroxide solution in the acetic acid-hydrogen peroxide solution in the step (1) is 60%.

Preferably, the volume ratio of the mass of the poplar in the step (1) to the acetic acid-hydrogen peroxide solution is 1g: 8-10 mL.

The high-concentration monosaccharide solution obtained by the poplar enzymolysis method is characterized in that the concentration of the high-concentration monosaccharide solution is higher than 140g/mL

According to the invention, the acetic acid-hydrogen peroxide and alkali are utilized to improve the degradability of poplar, remove a large amount of lignin and acetyl in poplar and greatly improve the glucan component of poplar, and on the basis, cellulase can further carry out enzymolysis on poplar and improve the concentration of monosaccharide in an enzymolysis solution. The embodiment shows that the method for pretreating and hydrolyzing the poplar can enable the monosaccharide concentration of the enzymolysis liquid to reach more than 250mg/mL, and can greatly improve the monosaccharide concentration in the poplar enzymolysis liquid and the enzymolysis efficiency of the poplar.

Detailed Description

The invention provides a method for preparing a high-concentration monosaccharide solution by using poplar, which comprises the following steps:

(1) mixing poplar wood with acetic acid-hydrogen peroxide solution for pretreatment reaction;

(2) carrying out solid-liquid separation on the pretreated liquid after the pretreatment reaction and the poplar, mixing the pretreated poplar filter residue with alkali liquor, and carrying out alkali pretreatment;

(3) carrying out solid-liquid separation on the treating fluid after the alkali pretreatment and the poplar alkali treatment filter residue, and washing the alkali treatment filter residue to be neutral to obtain a pretreatment raw material;

(4) and mixing the pretreated raw materials, a sodium citrate buffer solution and cellulase for enzymolysis.

The invention mixes poplar wood with acetic acid-hydrogen peroxide solution for pretreatment reaction. In the present invention, the volume ratio of the mass of poplar to the acetic acid-dioxygen aqueous solution is preferably 1g: 8-10 mL; the volume percentage of the acetic acid hydrogen peroxide in the acetic acid-hydrogen peroxide solution is preferably 60%. The acetic acid-hydrogen peroxide treatment can remove a large amount of lignin in the poplar wood, improve the degradability of the poplar wood and realize the pretreatment of the poplar wood. The source of the acetic acid-dioxygen aqueous solution is not particularly limited, and the acetic acid-dioxygen aqueous solution can be prepared by itself. When self-prepared, the preparation of the aqueous acetic acid-dioxygen solution is preferably: mixing 99% acetic acid and 30% hydrogen peroxide in equal volume, diluting the mixed solution with water, and adding sulfuric acid to obtain acetic acid-hydrogen peroxide solution. The sulfuric acid can catalyze acetic acid and hydrogen peroxide to generate peroxyacetic acid, so that the solution becomes a stronger oxidizing mixed solution. Acetic acid-hydrogen peroxide pretreatment of poplar can remove a large amount of lignin component in poplar and greatly retain its polysaccharide composition.

After poplar is pretreated by acetic acid-hydrogen peroxide, the invention carries out solid-liquid separation on pretreatment liquid after the pretreatment reaction and poplar, mixes pretreated poplar filter residue with alkali liquor and carries out alkali pretreatment. In the invention, the mass of the pretreated filter residue and the volume ratio of the alkali liquor are preferably 1g: 8-10 mL; the mass concentration of the alkali liquor is preferably 0.1-0.5%, and more preferably 0.2-0.4%; the alkali liquor with the concentration can avoid the condition that a large amount of acetyl is not enough to be removed in the enzymolysis process due to too low concentration, can also avoid the condition that the removal of a large amount of lignin and polysaccharide components is not beneficial to hydrolysis caused by excessive alkali liquor, and can also effectively reduce the loss of the polysaccharide components. The alkali liquor can remove acetyl in the poplar wood, and can improve the enzymolysis efficiency of the poplar wood. The solute in the alkali liquor is not particularly limited, and preferably comprises one or more of sodium hydroxide, potassium hydroxide and calcium hydroxide; the alkali pretreatment is preferably carried out at a temperature of 60-80 ℃, more preferably 60-75 ℃, and in the embodiment of the invention, the temperature of the alkali pretreatment is specifically 60 ℃; the time is preferably 1-2 h. The alkali pretreatment is preferably carried out in a sealed reaction kettle, the material of the liner of the reaction kettle is preferably polytetrafluoroethylene, the reaction kettle with the liner made of polytetrafluoroethylene does not contain reducing materials such as metals, and the reaction kettle is corrosion-resistant and not easy to cause explosion. The proper alkali pretreatment temperature and time enable acetyl groups of the poplar to be removed and simultaneously reserve a large amount of polysaccharide components and a small amount of necessary lignin components in the alkali treatment process, residual acetyl groups in the poplar can be greatly removed after the poplar is subjected to alkali pretreatment after acetic acid-hydrogen peroxide pretreatment, and the accessibility of cellulase to the poplar is improved, so that the hydrolysis of the poplar is promoted.

After the alkali treatment is finished, carrying out solid-liquid separation on the treatment liquid after the alkali pretreatment and the poplar alkali treatment filter residue, and washing the alkali treatment filter residue to be neutral to obtain the pretreatment raw material. According to the invention, the filter residue is washed to be neutral by water, so that residual alkali liquor in the raw material can be removed, and the interference of the residual alkali liquor on subsequent hydrolysis is reduced.

After the pretreated raw material is obtained, the pretreated raw material, a sodium citrate buffer solution and cellulase are mixed for enzymolysis. In the invention, the volume ratio of the mass of the pretreatment raw material to the sodium citrate buffer solution is 150-250 mg: 1mL, more preferably 200-250 mg: 1 mL; the pH value of the sodium citrate buffer solution is 5-6. The dosage of the cellulase is preferably 20-40 FPU (fermented glutinous rice) cellulase added to each gram of filter residue. In the present invention, the temperature of the enzymatic hydrolysis is preferably 50 ℃; the enzymolysis time is preferably 168 h. The source of the cellulase is not particularly limited in the invention, and the cellulase conventional in the field can be adopted. After the raw materials are washed to be neutral, cellulase is added, and the sodium citrate buffer solution can buffer the release of organic acid in the hydrolysis process, so that the hydrolysis pH is ensured to be stabilized at the optimal activity of the enzyme.

The concentration of the high-concentration monosaccharide solution is preferably higher than 140mg/mL, and further preferably 150-250 mg/mL, so that subsequent high-concentration product conversion is facilitated, and the distillation and purification cost in the later-stage conversion process is greatly reduced.

The method for preparing a monosaccharide solution with high concentration by using the poplar enzymatic method provided by the invention is described in detail below with reference to examples, but the method is not to be construed as limiting the scope of the invention.