阅读说明：本技术 超临界二氧化碳耦合NaOH预处理玉米秸秆提高其酶解转化木糖效率的方法 (Method for improving enzymolysis and xylose conversion efficiency of corn straws by supercritical carbon dioxide coupled NaOH pretreatment ) 是由 徐建 顾帅令 吴海军 左宗涛 魏无忌 胡世杰 张楷 徐霞 于 2020-12-24 设计创作，主要内容包括：本发明公开了生物质预处理和农业废弃物综合利用技术领域的超临界二氧化碳耦合NaOH预处理玉米秸秆提高其酶解转化木糖效率的方法,包括以下步骤：(1)将玉米秸秆粉加入至碱溶液中,搅拌均匀后得到混合料；(2)将混合料在超临界CO-2条件下预处理；(3)将预处理后的混合料经无纺布抽滤,得到滤液和滤渣；(4)取干燥后的滤渣放入柠檬酸三钠缓冲液中,并向柠檬酸三钠缓冲液中加入酸性木聚糖酶,酶水解18-24h,得到木糖；本发明创新性使用超临界二氧化碳耦合NaOH预处理玉米秸秆,加强酶水解制备木糖效率,同时,预处理方式简单方便,强化试剂(NaOH)廉价易得,处理温度相对较低,节约能耗,并且最大程度上保留半纤维素,保证酶水解效率。(The invention discloses a method for improving the efficiency of enzymolysis and xylose conversion of corn straws by pretreating the corn straws with supercritical carbon dioxide coupled with NaOH, which belongs to the technical field of biomass pretreatment and comprehensive utilization of agricultural wastes, and comprises the following steps: (1) adding the corn straw powder into an alkali solution, and uniformly stirring to obtain a mixture; (2) subjecting the mixture to supercritical CO 2 Pretreating under the condition; (3) carrying out suction filtration on the pretreated mixture through non-woven fabrics to obtain filtrate and filter residues; (4) putting the dried filter residue into a trisodium citrate buffer solution, adding acidic xylanase into the trisodium citrate buffer solution, and performing enzymatic hydrolysis for 18-24h to obtain xylose; the invention innovatively uses supercritical carbon dioxide coupled NaOH to pretreat the corn straws, enhances the efficiency of preparing xylose by enzymatic hydrolysis, simultaneously has simple and convenient pretreatment mode, cheap and easily obtained strengthening reagent (NaOH), relatively low treatment temperature, energy consumption saving and furthestThe hemicellulose is reserved, and the enzymatic hydrolysis efficiency is ensured.)

1. The method for improving the efficiency of enzymolysis and xylose conversion of corn straws by pretreating the corn straws with supercritical carbon dioxide coupled with NaOH is characterized by comprising the following steps of:

(1) crushing, washing and drying corn straws to obtain corn straw powder, adding the corn straw powder into an alkaline solution, and uniformly stirring to obtain a mixture;

(2) subjecting the mixture to supercritical CO₂Pretreating for 50-70min at 35-70 deg.C;

(3) carrying out suction filtration on the pretreated mixture through non-woven fabrics to obtain filtrate and filter residues, washing the filter residues with deionized water, and drying the washed filter residues to remove water;

(4) and (3) putting the dried filter residue into a trisodium citrate buffer solution, wherein the pH of the trisodium citrate buffer solution is 5, adding acidic xylanase into the trisodium citrate buffer solution, and performing enzymatic hydrolysis for 18-24h to obtain xylose.

2. The method for improving the efficiency of enzymolysis conversion of xylose by pretreating corn stalks with supercritical carbon dioxide coupled with NaOH according to claim 1, wherein in the step (1), the particle size of the corn stalk powder is 30-50 meshes.

3. The method for improving the efficiency of enzymatic conversion of xylose by corn stalks pretreated by supercritical carbon dioxide coupled with NaOH according to claim 1, wherein in the step (1), the aqueous alkali is 1 wt% NaOH solution.

4. The method for improving the efficiency of enzymatic hydrolysis conversion of xylose by pretreating corn straws by coupling supercritical carbon dioxide and NaOH according to claim 1, wherein in the step (2), the time for pretreating the supercritical carbon dioxide is 60min, and the treatment temperature is 35 ℃.

5. The method for improving the efficiency of enzymatic hydrolysis and xylose conversion of corn stalks by pretreatment of the corn stalks with supercritical carbon dioxide coupled with NaOH according to claim 1, wherein in the step (3), the washed filter residues are dried in an oven at 45 ℃ for 12 hours.

6. The method for improving the efficiency of enzymolysis and xylose conversion of corn straws pretreated by supercritical carbon dioxide coupled with NaOH according to claim 1, wherein 50g of corn straw powder and 500ml of aqueous alkali are taken in the step (1), and the mixture is obtained after uniform stirring.

7. The method for improving the efficiency of enzymatic conversion of xylose by corn stalks pretreated by supercritical carbon dioxide coupled with NaOH according to claim 6, wherein in the step (4), 2g of dried filter residue is taken and put into 100ml of trisodium citrate buffer solution.

Technical Field

The invention relates to the technical field of biomass pretreatment and comprehensive utilization of agricultural wastes, in particular to a method for improving the efficiency of enzymolysis and xylose conversion of corn straws by pretreating the corn straws with supercritical carbon dioxide coupled with NaOH.

Background

Biomass energy is used as a new renewable energy source to replace petrochemical resources, and is a necessary means for solving the problems of human environment, resources and energy sources in the future. The biological refining, taking the production of biological energy as an example, comprises the following basic processes: biomass collection, pretreatment, component separation, fermentation or chemical conversion, product recovery and byproduct comprehensive utilization. Among them, biomass pretreatment occupies an important position, and the search for an efficient and convenient pretreatment method requires continuous research and test.

China is a big agricultural country, the corn planting areas are widely distributed, the corn straws are mainly used as compost and as simple fuel for combustion, and the corn straws are rarely used for higher value. Corn stover, as a type of lignocellulose, is composed primarily of cellulose, hemicellulose, and lignin, and although lignocellulosic biomass has long been considered as a sustainable resource for the production of biofuels and other chemicals by saccharification and fermentation, the conversion of hemicellulose before it is not pretreated is very low, primarily due to the complexity of its structure.

In the prior art, research finds that supercritical carbon dioxide is a potential pretreatment means, but the pretreatment effect on lignocellulose is very limited due to the fact that the structure of lignocellulose is extremely complex, most typically, hydrogen bond linkage between cellulose and hemicellulose, and lignin-carbohydrate complex (LCC) formed by connecting lignin and hemicellulose through chemical bonds such as ether bond, acetal bond, ester bond, carbon-carbon bond and the like, and the ubiquitous structure causes the common obstacles for re-separation and utilization of lignocellulose.

Therefore, the method for improving the efficiency of enzymatic conversion of xylose by pretreating the corn straws by coupling supercritical carbon dioxide and NaOH is provided to solve the problems.

Disclosure of Invention

The invention aims to provide a method for improving the efficiency of enzymolysis and xylose conversion of corn straws by pretreating the corn straws with supercritical carbon dioxide coupled with NaOH, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following scheme to realize the following steps: the method for improving the efficiency of enzymatic conversion of xylose by pretreating corn straws by coupling supercritical carbon dioxide and NaOH specifically comprises the following steps:

(1) crushing, washing and drying corn straws to obtain corn straw powder, adding the corn straw powder into an alkaline solution, and uniformly stirring to obtain a mixture;

(2) subjecting the mixture to supercritical CO₂Pretreating for 50-70min at 35-70 deg.C;

(3) carrying out suction filtration on the pretreated mixture through non-woven fabrics to obtain filtrate and filter residues, washing the filter residues with deionized water, and drying the washed filter residues to remove water;

(4) and (3) putting the dried filter residue into a trisodium citrate buffer solution, wherein the pH of the trisodium citrate buffer solution is 5, adding acidic xylanase into the trisodium citrate buffer solution, and performing enzymatic hydrolysis for 18-24h to obtain xylose.

Preferably, in the step (1), the grain size of the corn straw powder is 30-50 meshes.

Preferably, in step (1), the alkali solution is a 1 wt% NaOH solution.

Preferably, in the step (2), the time of the supercritical carbon dioxide pretreatment is 60min, and the treatment temperature is 35 ℃.

Preferably, in the step (3), the washed filter residue is dried in an oven at 45 ℃ for 12 hours.

Preferably, in the step (1), 50g of corn straw powder and 500ml of aqueous alkali are taken and uniformly stirred to obtain a mixture.

Preferably, in step (4), 2g of the dried filter residue is taken and put into 100ml of trisodium citrate buffer.

The invention has the beneficial effects that:

the invention aims to strengthen the effect of the supercritical carbon dioxide pretreatment of corn straws by using a method of adding NaOH solution, thereby better breaking the complex cross-linking structure of lignocellulose, destroying chemical bonds among lignin, hemicellulose and hemicellulose, improving the accessibility of enzyme to the hemicellulose and further improving the enzyme catalysis efficiency; moreover, because the reaction is carried out at low temperature, the loss of hemicellulose is very little, and the guarantee of the yield is provided for the subsequent enzyme hydrolysis;

according to the invention, the corn straw is pretreated by innovatively coupling supercritical carbon dioxide with NaOH, so that the xylose preparation efficiency by enzymatic hydrolysis is enhanced, meanwhile, the pretreatment mode is simple and convenient, the strengthening reagent (NaOH) is cheap and easy to obtain, the treatment temperature is relatively low, the energy consumption is saved, the hemicellulose is retained to the greatest extent, and the enzymatic hydrolysis efficiency is ensured.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a process flow diagram of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Acid xylanase (Shandong Su Ke Han Biotechnology limited, enzyme activity is 200000U/g).

Example 1

The method for improving the efficiency of enzymatic conversion of xylose by pretreating corn straws by coupling supercritical carbon dioxide and NaOH specifically comprises the following steps:

(1) crushing, washing and drying corn straws to obtain corn straw powder, adding 50g of the corn straw powder into 500ml of a 1 wt% NaOH solution, and uniformly stirring to obtain a mixture;

(2) subjecting the mixture to supercritical CO₂Pretreating for 60min at 35 deg.C;

(3) carrying out suction filtration on the pretreated mixture through non-woven fabrics to obtain filtrate and filter residues, washing the filter residues with 1000ml of deionized water, and drying the washed filter residues in a drying oven at 45 ℃ for 12 hours;

(4) and (3) putting 2g of dried filter residue into 100ml of trisodium citrate buffer solution, wherein the pH value of the trisodium citrate buffer solution is 5, adding 0.625g of acidic xylanase into the trisodium citrate buffer solution, and performing enzymatic hydrolysis for 24h to obtain xylose.

In the step (1), the grain diameter of the crushed corn straws is 30-50 meshes.

Example 2

The method for improving the efficiency of enzymatic conversion of xylose by pretreating corn straws by coupling supercritical carbon dioxide and NaOH specifically comprises the following steps:

(1) crushing, washing and drying corn straws to obtain corn straw powder, adding 50g of the corn straw powder into 500ml of a 1 wt% NaOH solution, and uniformly stirring to obtain a mixture;

(2) subjecting the mixture to supercritical CO₂Pretreating for 60min at 50 deg.C;

(3) carrying out suction filtration on the pretreated mixture through non-woven fabrics to obtain filtrate and filter residues, washing the filter residues with 1000ml of deionized water, and drying the washed filter residues in a drying oven at 45 ℃ for 12 hours;

(4) and (3) putting 2g of dried filter residue into 100ml of trisodium citrate buffer solution, wherein the pH value of the trisodium citrate buffer solution is 5, adding 0.625g of acidic xylanase into the trisodium citrate buffer solution, and performing enzymatic hydrolysis for 24h to obtain xylose.

In the step (1), the grain diameter of the crushed corn straws is 30-50 meshes.

Example 3

The method for improving the efficiency of enzymatic conversion of xylose by pretreating corn straws by coupling supercritical carbon dioxide and NaOH specifically comprises the following steps:

(1) crushing, washing and drying corn straws to obtain corn straw powder, adding 50g of the corn straw powder into 500ml of a 1 wt% NaOH solution, and uniformly stirring to obtain a mixture;

(2) subjecting the mixture to supercritical CO₂Pretreating for 60min at 70 deg.C;

(3) carrying out suction filtration on the pretreated mixture through non-woven fabrics to obtain filtrate and filter residues, washing the filter residues with 1000ml of deionized water, and drying the washed filter residues in a drying oven at 45 ℃ for 12 hours;

(4) and (3) putting 2g of dried filter residue into 100ml of trisodium citrate buffer solution, wherein the pH value of the trisodium citrate buffer solution is 5, adding 0.625g of acidic xylanase into the trisodium citrate buffer solution, and performing enzymatic hydrolysis for 24h to obtain xylose.

In the step (1), the grain diameter of the crushed corn straws is 30-50 meshes.

Result detection

1. The test method comprises the following steps: according to the embodiment 1-3, 0.5ml of the enzyme hydrolysate is extracted at 0, 1, 4, 8, 12 and 24 hours, the enzyme hydrolysate is immediately placed into boiling water for 10min for inactivation after extraction, and a high-speed centrifuge is used for centrifugation after inactivation, wherein the rotation speed is 10000rpm, and the centrifugation time is 10 min. After centrifugation, taking the supernatant for HPLC analysis, and quantifying the xylose content in the enzyme water solution. The xylose yield obtained by the traditional method was 9.4%. Specific detection results are shown in table 1.

TABLE 1 xylose yields

Group of	Xylose yield/%)
		Example 1	33.76
Example 2	31.88
		Example 3	19.37

In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not exhaustive. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.