阅读说明：本技术 一种木糖渣预处理改善酶解糖化效果的方法 (Method for improving enzymolysis saccharification effect by pretreating xylose residues ) 是由 王朝宇 迟雪文 毕艳红 卢艺惠 赵祥杰 李明依 于 2019-10-24 设计创作，主要内容包括：本发明公开了一种木糖渣预处理改善酶解糖化效果的方法,包括如下步骤：称取木糖渣,加入低共熔溶剂(Deep Eutectic Solvent,DES)进行预处理,反应结束后对其进行固液分离；将分离的固体中加入KOH溶液进行预处理,反应结束后对其进行固液分离,分别收集固体和液体部分；将固体部分洗涤至中性即得到预处理后的木糖渣,进行酶解糖化,测定酶解液中的葡萄糖浓度。本发明木糖渣清洁高效预处理的方法可以改善纤维素酶水解效率低的问题,从而减少纤维素酶的用量,提高还原糖的转化效率,为木糖渣的综合利用提供一定的理论依据。(The invention discloses a method for improving enzymolysis saccharification effect by pretreating types of xylose residues, which comprises the following steps of weighing the xylose residues, adding a Deep Eutectic Solvent (DES) for pretreatment, carrying out solid-liquid separation after the reaction is finished, adding a KOH solution into the separated solid for pretreatment, carrying out solid-liquid separation after the reaction is finished, respectively collecting solid and liquid parts, washing the solid part to be neutral to obtain the pretreated xylose residues, carrying out enzymolysis saccharification, and determining the glucose concentration in an enzymolysis solution.)

1, kinds of xylose slag pretreatment improves the enzymolysis saccharification effect method, its characteristic lies in, including the following step:

(1) weighing the xylose residues, adding DES (data encryption standard) for pretreatment, performing solid-liquid separation after the reaction is finished, and respectively collecting solid and liquid parts;

(2) adding KOH solution into the solid separated in the step (1) for pretreatment, carrying out solid-liquid separation on the solid after the reaction is finished, and respectively collecting the solid part and the liquid part;

(3) and (3) washing the solid part separated in the step (2) to be neutral to obtain pretreated xylose residue, carrying out enzymolysis saccharification, and determining the concentration of glucose in the enzymolysis liquid.

2. The method for improving enzymatic saccharification effect of xylose residue pretreatment of claim 1, wherein the DES is prepared by mixing choline chloride: lactic acid was added in a molar ratio of 1: 1-5.

3. The method for improving enzymolysis saccharification effect of xylose residue pretreatment of claim 1, wherein the conditions of DES pretreatment of xylose residue are as follows: preferably, the pretreatment time is 0.5-6h, the pretreatment solid-to-liquid ratio is 1:8-1:30g/mL, and the pretreatment temperature is 60-130 ℃.

4. The method for improving enzymatic saccharification effect of xylose residue pretreatment of claim 1, wherein the mass fraction of KOH solution is 5-30 wt.%.

5. The method for improving enzymatic saccharification effect of xylose residue pretreatment of claim 1, wherein the pretreatment conditions of the KOH solution are as follows: the treatment time is 30-180min, the treatment solid-liquid ratio is 1:6-1:18g/mL, and the treatment temperature is 50-100 ℃.

6. The method for improving enzymatic saccharification effect of xylose residue pretreatment of claim 1, wherein the enzymatic saccharification is that the pretreated xylose residue is added with citric acid buffer solution to adjust pH, sodium azide solution is added, then cellulase is added, and after reaction, HPLC is adopted to measure glucose concentration in the enzymatic hydrolysate.

7. The method for improving enzymatic saccharification effect of xylose residue pretreatment of claim 6, wherein said cellulase is added in an amount of 3-40 FPU/g-substrate, solid loading is 2-20 wt.%, and enzymatic hydrolysis is performed at pH 4.8 and temperature 50 ℃ for 2-96 h.

8, use of the liquid produced by the method for improving enzymolysis saccharification effect of xylose residue pretreatment as claimed in claim 1 in the preparation of potassium fertilizer, lignin-based fertilizer and compound fertilizer.

Technical Field

The invention belongs to the technical field of biochemical engineering, and particularly relates to a method for improving enzymolysis saccharification effect by pretreating kinds of xylose residues.

Background

Meanwhile, the problems of ecological damage, environmental pollution and the like caused in the development and utilization process of fossil energy are gradually highlighted, the global climate is greatly influenced by the large emission of greenhouse gases, and the ecological safety and the sustainable development of the human society are seriously challenged due to the frequent occurrence of various natural disasters.

The annual output of Chinese wood fiber biomass resources is over 7.5 hundred million tons, and the Chinese wood fiber biomass resources are main raw materials in the industrial fields of chemical industry, papermaking, textile and the like, but the utilization rate of the Chinese wood fiber biomass resources is not high, so that if the wood fiber biomass resources can be subjected to processing pretreatment to produce biomass fuel or bulk chemicals with high added values, the global energy crisis can be relieved to the extent of , and the pressure of fossil energy is relieved.

The xylose residue is a product obtained by extracting xylose from corncobs through dilute acid treatment, and more than 500 million tons of xylose residue is generated in China every year, wherein most of the xylose residue is combusted to generate heat, so that environmental problems and waste of cellulose materials are caused.

Disclosure of Invention

The invention aims to solve the problems in the prior art, and provides a method for improving enzymolysis saccharification effect by types of xylose residues through pretreatment.

In order to achieve the purpose, the method for improving the enzymolysis saccharification effect by pretreating kinds of xylose residues comprises the following steps:

(1) weighing the xylose residues, adding DES (Deep Eutectic Solvent, DES) for pretreatment, carrying out solid-liquid separation after the reaction is finished, and respectively collecting solid and liquid parts;

(2) adding KOH solution into the solid separated in the step (1) for pretreatment, carrying out solid-liquid separation on the solid after the reaction is finished, and respectively collecting solid and liquid parts;

(3) and (3) washing the solid part separated in the step (2) to be neutral to obtain pretreated xylose residue, carrying out enzymolysis saccharification, and determining the concentration of glucose in the enzymolysis liquid.

The xylose residue used in the invention is purchased from Futaste Co.Ltd or other commercial companies, or according to a literature diluted acid treatment method, the resource utilization and research of Zhao Zheng Chi slag [ D ] Zheng university, 2016 ] and the effect is .

Wherein the DES consists of choline chloride: lactic acid was added in a molar ratio of 1: 1-5. Preferably, the DES consists of choline chloride: lactic acid was added in a molar ratio of 1: 2, and preparing the product.

the DES is prepared by adding choline chloride and lactic acid into a reaction flask, magnetically stirring at 60-80 deg.C until the mixture becomes colorless clear liquid, transferring to a vacuum drying oven, and adding P₂O₅Fully drying the mixture for later use.

Wherein, the DES pretreatment conditions of the xylose residues are as follows: the pretreatment time is 0.5-6h, the pretreatment solid-to-liquid ratio is 1:8-1:30g/mL, and the pretreatment temperature is 60-130 ℃.

Wherein the mass fraction of the KOH solution is 5-30%.

Wherein, the pretreatment conditions of the KOH solution are as follows: the treatment time is 30-180min, the treatment solid-liquid ratio is 1:6-1:18g/mL, and the treatment temperature is 50-100 ℃.

And (3) carrying out solid-liquid separation on the xylose residue after KOH pretreatment, respectively collecting solid and liquid parts, washing the solid residue to be neutral by using tap water, and then carrying out enzymolysis saccharification.

, adding citric acid buffer solution to adjust pH, adding sodium azide solution, adding cellulase, and measuring glucose concentration in the enzymatic hydrolysate by HPLC after reaction.

Preferably, the cellulase is added in an amount of 3-40 FPU/g-substrate (i.e. pretreated xylose residue), the solid loading is 2-20 wt.%, and the enzymatic hydrolysis is carried out at pH 4.8 and temperature 50 ℃ for 2-96 h. If the enzymatic hydrolysis reaction volume is 20mL, 3g of pretreated xylose residue (dry basis) is added at a solids loading of 15 wt.%, and the total reaction volume is 20mL × 15% solids loading (solids content) is 3g dry weight by weight.

According to IUPAC standard, the definition of Filter paper enzyme activity (FPA) is 1g of solid enzyme (or 1mL of liquid enzyme), and the enzyme dosage required for generating 1 mu mol of glucose from a Filter paper substrate per hour at 50 ℃ and pH 4.8 is defined as enzyme activity units expressed by U/g (or U/mL).

After the enzymatic saccharification process is finished, quantitative enzymatic hydrolysate is taken to be subjected to High Performance Liquid Chromatography (HPLC) detection, and the content of reducing sugar in the enzymatic hydrolysate is measured, wherein the detection conditions of HPLC are shown in Table 1.

TABLE 1 conditions of liquid chromatography detection

The liquid (the black liquid collected in the steps (1) and (2)) generated in the method for improving the enzymolysis saccharification effect by pretreating the xylose residue is applied to the preparation of potassium fertilizer, lignin-based fertilizer and compound fertilizer.

According to the method, the DES and KOH pretreatment is adopted to remove lignin and hemicellulose wrapped on the surface of the cellulose, so that the highly ordered and tightly arranged regions of the cellulose are damaged, pores are generated on the surface of the cellulose, more cellulose is exposed, the porosity and porosity are increased, the accessibility of the cellulose by the cellulase is improved, and the enzymolysis saccharification efficiency is promoted.

Has the advantages that: compared with the prior art, the invention has the following advantages:

according to the invention, the DES and KOH are adopted to pretreat the xylose residues, so that the wrapping of lignin and hemicellulose on cellulose is damaged, the lignin is removed, the hemicellulose is degraded, the crystal structure of the cellulose is changed, the accessibility and the porosity of the cellulose are improved, the contact of cellulase and a substrate is promoted, and further the cellulose hydrolysis yield and the reducing sugar yield are improved.

The method adopts eutectic solvents DES and KOH solution for pretreatment, has mild reaction conditions, reduces the damage to instruments and equipment, and is beneficial to industrial production and application.

Drawings

FIG. 1 is a Scanning Electron Microscope (SEM) image of xylose residue before and after pretreatment;

FIG. 2 is a Fourier Infrared Spectroscopy (FTIR) of xylose residue before and after pretreatment;

FIG. 3 is an X-ray diffraction pattern (XRD) before and after pretreatment of xylose residue;

wherein a is a xylose residue raw material; b is the xylose residue after 10 wt.% KOH pretreatment in example 2; c is the xylose residue of example 4 pretreated with 16 wt.% KOH;

observation of SEM, FTIR and XRD patterns shows that compared with the untreated xylose residue raw material, the crystallinity of the pretreated xylose residue is increased, the KOH dosage is increased, the degradation of the xylose residue is more severe, the porosity and the porosity are increased, so that the accessibility of the cellulase to the cellulose is increased, the enzymolysis saccharification efficiency is improved, but the cost is consumed due to the excessive KOH dosage, the over-hydrolysis is caused due to the excessive KOH dosage, and the 16 wt.% KOH pretreatment effect is the best.

Detailed Description

The invention is further illustrated in connection with the following examples and the accompanying drawings.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.