Method for catalyzing hemicellulose to generate xylulose by using high-stability enzyme cascade reactor

Abstract

The invention belongs to the technical field of xylulose production, and discloses a method for catalyzing hemicellulose to generate xylulose by an immobilized enzyme cascade reactor. According to the method, hemicellulose is used as a raw material, aminated macroporous resin Namberlyst-15 is used as a carrier, xylanase and xylose isomerase are subjected to cross-linking adsorption to obtain an immobilized double-enzyme Namberlyst-15-Xyn-XI cascade reactor, and the xylulose is produced by the immobilized double-enzyme Namberlyst-15-Xyn-XI cascade reactor. According to the method, efficient immobilization and recycling of xylanase and xylose isomerase are achieved, the catalytic efficiency and selectivity are improved, the problems of high raw material cost and poor enzyme stability in the xylulose production process are solved, technical reserve is provided for the xylulose production process, and meanwhile technical support is provided for construction of a high-stability enzyme cascade reactor.

Description

Technical Field

[0001] The invention belongs to the technical field of xylulose production, and particularly relates to a method for producing xylulose by using immobilized enzyme to catalyze hemicellulose. Background Art

[0002] Xylulose, as an important monosaccharide and also a member of the rare sugars, has various biological functions and application values, such as serving as an intermediate product of bioenergy and a raw material for biopolymers, and has received widespread attention.

[0003] Xylulose can be synthesized through many pathways, such as the degradation of lignin, the metabolism of plant cell walls, etc. Among them, the preparation by enzymatic reaction with xylitol and arabinose as raw materials is currently widely used. However, xylitol and arabinose have the disadvantages of limited availability, high cost and low yield, which greatly limits the production of xylulose. Xylose and xylulose are isomers, and xylose is the basic component unit of hemicellulose, which can be obtained by hydrolyzing hemicellulose catalyzed by xylanase, which provides an important inspiration. Xylose can be obtained by xylanase with hemicellulose as raw material, and then xylose can be isomerized to xylulose under the action of xylose isomerase, so that the production cost of xylulose can be reduced and the application of xylulose can be promoted. So far, there has been no research report on the cascade reaction of xylanase and xylose isomerase to realize the conversion of hemicellulose into xylulose.

[0004] The key to achieving efficient dual-enzyme cascade reaction is to provide a good reaction space for the cascade reaction, that is, to build a cascade reaction platform so that the substrate can be transferred in the platform, promote the contact between the substrate and the enzyme, and reduce the mass transfer barrier. To achieve the above concept, the best strategy is to fix the dual enzymes on a porous carrier, build an enzyme cascade reactor, and use the large pore size of the carrier to provide space for the enzyme and the substrate to fully contact. At the same time, fixing the dual enzymes on the carrier can also achieve the recycling of the dual enzymes, further improving the efficiency of enzymatic hydrolysis.

[0005] As a macroporous resin, Amberlyst-15 can provide a relatively stable microenvironment for enzymes, reducing the impact of external factors such as temperature and pH fluctuations on enzymes, and is an ideal enzyme immobilization carrier; at the same time, Amberlyst-15 is acidic and can catalyze the hydrolysis of hemicellulose to produce xylose (Liu Minghe, Sang Yali, Tang Qiyue, et al. Research on the Hydrolysis of Corn Cobs Catalyzed by Solid Acid Amberlyst-15 to Prepare Xylose [J]. Journal of Chifeng University: Natural Science Edition, 2020, 36(3): 25-27). If Amberlyst-15 is used as a carrier to load xylanase and xylose isomerase on it, it not only provides a platform for the cascade reaction of the two enzymes, but also can utilize the acid catalytic properties of the carrier to assist the hydrolysis of hemicellulose, thus achieving the synergy of enzyme catalysis and inorganic catalysis, accelerating the conversion of hemicellulose to xylulose.

[0006] Using the large pore size of the resin to adsorb and immobilize enzymes is a common method for preparing enzyme cascade reactors, but it has problems such as poor stability and low reuse rate. The silane coupling agent reacts with the active functional groups of Amberlyst-15, introduces active groups that covalently react with the enzyme on the resin, and prepares the enzyme cascade reactor by covalent fixation, which can effectively solve the problems existing in the preparation of enzyme cascade reactors and promote the development of the process of converting hemicellulose to xylulose. Summary of the invention

[0007] In order to solve the technical problems in the prior art such as high raw material cost and poor stability of constructed cascade reactors in the production process of xylulose, the present invention provides a method for constructing a high-stable enzyme cascade reactor to catalyze the production of xylulose from hemicellulose.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] Using hemicellulose as raw material, the macroporous resin Amberlyst-15 was aminated, and xylanase and xylose isomerase were immobilized on Amberlyst-15 using the adsorption-crosslinking method. A highly stable enzyme cascade reactor was constructed to produce xylulose.

[0010] The amination method of the macroporous resin Amberlyst-15 of the present invention is:

[0011] (1) The macroporous resin Amberlyst-15 carrier was added to a 95:5% (v / v) ethanol / water solution containing 5% silane coupling agent KH550 (3-aminopropyltriethoxysilane), stirred at 50° C. for 2 h, and then filtered to separate the precipitate.

[0012] (2) Take a certain amount of precipitate, add 200 ml of anhydrous ethanol, use an ultrasonic cleaning machine, continue ultrasonic cleaning for 10 minutes, then filter and separate the precipitate, and repeat three times.

[0013] (3) The obtained precipitate was dried in an oven at 50° C. for 12 h to obtain the carrier NAmberlyst-15.

[0014] The preparation method for constructing a high-stable enzyme cascade reactor of the present invention is:

[0015] Weigh 0.5 g of NAmberlyst-15 carrier and suspend it in the enzyme solution, and carry out the covalent binding reaction for 4 h at 150 r / min and 50° C. After the reaction, filter and wash the precipitate with phosphate buffer, and dry to obtain the immobilized enzyme.

[0016] The carrier NAmberlyst-15 obtained after amination is used to immobilize the xylanase and xylose isomerase in a mixed manner to obtain a cascade reactor of immobilized dual enzymes NAmberlyst-15-Xyn-XI.

[0017] The method for constructing a high-stable enzyme cascade reactor provided by the present invention to catalyze hemicellulose to generate xylulose, hydrolyzes hemicellulose to generate xylose, and then isomerizes to xylulose, thereby increasing the yield of xylulose. At the same time, the cascade reactor can be reused, thereby greatly reducing production costs. The technology for constructing a high-stable enzyme cascade reactor provided by the present invention can improve the efficiency of the enzyme catalysis method, thereby solving a series of problems existing therein. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a comparison chart of the accumulation of xylulose produced by hemicellulose catalyzed by free enzyme and by immobilized enzyme;

[0019] Figure 2 This is a diagram showing the reusability of the immobilized enzyme provided by the present invention. DETAILED DESCRIPTION

[0020] The present invention discloses a method for constructing a high-stable enzyme cascade reactor to catalyze hemicellulose to produce xylulose. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0021] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in detail below in conjunction with specific implementation methods.

[0022] In the embodiment of the present invention:

[0023] The silane coupling agent KH550 was hydrolyzed in a 95:5 (v / v) ethanol / water solution overnight before use, and the mass concentration of the silane coupling agent KH550 was 5%.

[0024] The xylanase and xylose isomerase were purchased through conventional channels and stored in the laboratory for use.

[0025] Comparative Example 1

[0026] Under the conditions of pH 6.0 and reaction temperature of 50°C, 50 mg / mL of hemicellulose was catalyzed to produce xylulose using xylanase and xylose isomerase. 0.5 g of hemicellulose was catalyzed in 10 mL of reaction system, and the reaction time was 16 h.

[0027] Finally, the concentration of xylulose in the reaction solution was determined by high performance liquid chromatography. The yield of xylulose after free enzyme catalyzed hemicellulose was 7.69 mg / mL. The hemicellulose conversion rate was 22%.

[0028] Example 1

[0029] Under the conditions of pH 6.0 and reaction temperature 50°C, NAmberlyst-15-Xyn-XI enzyme cascade reactor was used to catalyze 50 mg / mL of hemicellulose to produce xylulose. 0.5 g of hemicellulose was catalyzed in 10 mL of reaction system, and the reaction time was 16 h.

[0030] Finally, the concentration of xylulose in the reaction solution was determined by high performance liquid chromatography. The yield of xylulose after hemicellulose catalyzed by the enzyme cascade reactor NAmberlyst-15-Xyn-XI was 16.09 mg / mL. Compared with the free enzyme, the catalytic efficiency of the immobilized dual enzyme increased by 109.23%, and the yield was significantly improved.

[0031] Example 2

[0032] The prepared NAmberlyst-15-Xyn-XI was used to catalyze 50 mg / mL of hemicellulose to produce xylulose at a pH of 5.5 and a reaction temperature of 50°C. 0.5 g of hemicellulose was catalyzed in a 10 mL reaction system and the reaction time was 16 h.

[0033] The enzyme cascade reactor was recycled for the above five times, with the first enzyme activity as 100%, and further tested. Finally, the five test results were compared to evaluate its reusability in the actual process. Figure 2 As shown, after 5 consecutive operations, the remaining enzyme activity of the immobilized enzyme was still 82.24%.

[0034] Example 3

[0035] 1) Amberlyst-15 particles were reacted with a 95:5 (v / v) ethanol / water solution containing 5% 3-aminopropyltriethoxysilane (APTES) at room temperature under stirring for 20 minutes, and a precipitate was obtained by filtration; ultrasonic cleaning was performed in 200 mL of ethanol for 10 minutes, and a precipitate was obtained by filtration, and the process was repeated 3 times. The precipitate was dried in an oven at 50° C. overnight to obtain the carrier NAmberlyst-15;

[0036] 2) Mixing xylanase and xylose isomerase to obtain a mixed enzyme solution, in which the concentrations of xylanase and xylose isomerase are both 25 mg / mL; suspending the carrier NAmberlyst-15 obtained in step 1) in the above-mentioned mixed enzyme solution, adding polyethylene glycol diglycidyl ether for cross-linking, and performing post-treatment after the cross-linking to obtain the enzyme cascade reactor Namberlyst-15-Xyn-XI.

[0037] The enzyme cascade reactors used in Examples 1-2 of the present invention are all prepared by Example 3.

[0038] The above description is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for catalyzing hemicellulose to produce xylulose using a high-stable enzyme cascade reactor, characterized in that: The xylanase and xylose isomerase in the process of hemicellulose hydrolysis and isomerization were dual-enzyme immobilized to obtain the NAmberlyst-15-Xyn-XI high-stable enzyme cascade reactor, and hemicellulose was used as the raw material and enzymatically hydrolyzed to produce xylulose.

2. The method according to claim 1, characterized in that The preparation method of the enzyme cascade reactor comprises the following steps: reacting Amberlyst-15 particles with an ethanol / water solution containing 3-aminopropyltriethoxysilane and treating the mixture to obtain a carrier NAmberlyst-15; and immobilizing xylanase and xylose isomerase on the obtained carrier NAmberlyst-15 to obtain an enzyme cascade reactor Namberlyst-15-Xyn-XI.

3. The method according to claim 2, characterized in that Amberlyst-15 particles were reacted with an ethanol / water solution containing 5% 3-aminopropyltriethoxysilane by mass at room temperature with stirring for 20 minutes, the volume ratio of ethanol / water solution was 95:5, and a precipitate was obtained by filtration; ultrasonic cleaning was performed in 200 mL of ethanol for 10 minutes, and a precipitate was obtained by filtration, and the process was repeated 3 times. The precipitate was dried in an oven at 50°C for 12 hours to obtain the carrier NAmberlyst-15.

4. The method according to claim 2 or 3, characterized in that Xylanase and xylose isomerase were mixed to obtain a mixed enzyme solution, wherein the concentration ratio of xylanase to xylose isomerase was 1:1, and the concentrations of both were 25 mg / mL. The obtained carrier NAmberlyst-15 was suspended in the mixed enzyme solution, and then post-processed to obtain the enzyme cascade reactor Namberlyst-15-Xyn-XI.

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