Construction method of cellulose enzymolysis micro-homogeneous system and recovery method of cellulase

Abstract

The invention belongs to the technical field of cellulose hydrolysis, and discloses a construction method of a cellulose enzymolysis micro-homogeneous system and a recycling method of cellulase. In a water-phase system, a micro-homogeneous system is formed between the modified cellulase and cellulose, and the cellulose is subjected to enzymolysis; the preparation method comprises the following steps: introducing a polyethylene glycol derivative polyethylene glycol dibenzaldehyde FA-PEG-FA into an amino terminal of cellulase to obtain the modified cellulase, which is marked as FA-Cell. According to the method, a polyethylene glycol derivative is introduced to the amino terminal of the cellulase, so that the cellulase and cellulose form a micro-homogeneous reaction system under an aqueous medium, the enzymatic hydrolysis is prompted to be transited from a heterogeneous phase to a homogeneous phase, and the efficient recovery of the cellulase is realized by utilizing the pH sensitive characteristic of a dynamic Schiff base bond.

Description

Technical Field

[0001] The present invention relates to the technical field of cellulose hydrolysis, and particularly relates to a method for constructing a cellulose enzymatic hydrolysis micro-homogeneous system and a method for recycling cellulase. Background Art

[0002] For a long time, abundant renewable cellulose biomass has been regarded as the most promising alternative to fossil fuel refining, and can be used to produce various platform chemicals, fuels, etc. In the refining process of cellulose biomass, enzymatic hydrolysis is an indispensable step, and this method has been studied as a key method for converting cellulose biomass into fermentable sugars.

[0003] Cellulase is a key catalyst in biomass conversion. However, cellulase still faces many challenges in catalytic applications. One of the most important problems is that most of the existing enzymatic hydrolysis reaction systems are heterogeneous reaction systems. In such heterogeneous reaction systems, the effective contact efficiency between cellulase and the substrate cellulose is relatively low, resulting in limited enzymatic hydrolysis efficiency. In addition, the difficulty in recycling cellulase is also an urgent problem to be solved. The non-reusability of enzymes limits their promotion in industrial applications. Therefore, it is particularly important to develop an innovative method that can improve the enzymatic hydrolysis reaction system, increase the contact efficiency between enzymes and substrates, and at the same time achieve efficient recycling of cellulase. Summary of the Invention

[0004] In order to solve the problems in the prior art that the effective contact efficiency between cellulase and the substrate cellulose in the heterogeneous enzymatic hydrolysis reaction system is relatively low, resulting in limited enzymatic hydrolysis efficiency, etc., the present invention provides a method for constructing a cellulose enzymatic hydrolysis micro-homogeneous system and a method for recycling cellulase.

[0005] To solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A method for constructing a cellulose enzymatic hydrolysis micro-homogeneous system, in an aqueous phase system, a micro-homogeneous system is formed between the modified cellulase and cellulose and cellulose is enzymatically hydrolyzed; a polyethylene glycol-based derivative polyethylene glycol dibenzaldehyde FA-PEG-FA is introduced at the amino terminus of the cellulase to obtain the modified cellulase, denoted as FA-Cell.

[0007] The polyethylene glycol dibenzaldehyde of the present invention can generally be prepared by an esterification reaction of polyethylene glycol with p-formylbenzoic acid. The molecular structural formula of the polyethylene glycol-based derivative polyethylene glycol dibenzaldehyde FA-PEG-FA is shown as follows:

[0008]

[0009] Further, dissolve the cellulase in a phosphate buffer with a pH of about 4.8, add the polyethylene glycol-based derivative polyethylene glycol dibenzaldehyde FA-PEG-FA and carry out the reaction. After the reaction is completed, dialysis is performed to generate the modified cellulase FA-Cell.

[0010] Further, the reaction temperature of the above modification reaction is 25-55 °C, and the reaction time is 3-48 h.

[0011] Further, it also includes a method for capturing and recovering cellulose. The capture and recovery method is as follows: after the enzymatic hydrolysis reaction is completed, filter to remove unreacted substrates, add polyethyleneimine PEI to the filtrate, adjust the pH value to alkaline to form a dynamic Schiff base gel, wash with water to remove the reaction product reducing sugar, and acidify the system to the reaction pH range to release the modified cellulase, thereby realizing the capture and recovery of the modified cellulase.

[0012] Furthermore, add the captured and recovered modified cellulase to a new substrate to carry out the next round of enzymatic hydrolysis reaction, and the modified cellulase can be captured and utilized repeatedly for multiple times.

[0013] Further, during the capture and recovery of the modified cellulase, the concentration of the polyethyleneimine PEI is 0.1-0.6 mol / L.

[0014] During the enzymatic hydrolysis reaction, the concentration of the modified cellulase is 0.25-20 mg / mL, and the concentration of cellulose is 5-50 mg / mL; the enzymatic hydrolysis reaction time is 15-60 min, and the enzymatic hydrolysis reaction temperature is 25-65 °C.

[0015] The present invention provides a method for constructing a micro-homogeneous system for cellulose enzymatic hydrolysis and a method for recycling cellulase. The present invention constructs a micro-homogeneous cellulose enzymatic hydrolysis reaction system through a simple method. By introducing a polyethylene glycol-based derivative at the amino terminus of the cellulase, the modified cellulase FA-Cell is obtained. By introducing polyethylene glycol dibenzaldehyde FA-PEG-FA at the amino terminus of the cellulase, the fiber chains of cellulose undergo solvation with the polyethylene glycol groups, loosening the dense structure of cellulose, greatly improving its dispersibility in the solution, and making the reaction system present a micro-homogeneous system (as shown in Figure 1 )), thereby strengthening the effective contact between the substrate and the enzyme, improving the catalytic activity of the enzyme, and promoting the efficiency of the enzymatic hydrolysis reaction.

[0016] In addition, after the cellulase hydrolysis reaction is completed, the present invention adds a gel factor, polyethyleneimine (PEI), to solve the problem of cellulase recovery. The Schiff base bond formed between the aldehyde group with an aromatic structure and the primary amine exhibits excellent sensitivity. Under acidic conditions, the Schiff base dynamic covalent bond will not form, while under neutral or alkaline conditions, the Schiff base dynamic covalent bond between the aldehyde group of the polyethylene glycol dibenzaldehyde modified cellulase and the amino group of polyethyleneimine (PEI) can be rapidly generated, thereby changing the solubility of the bonded compound. Therefore, the present invention can simply achieve the sensitive recovery of modified cellulase by simply adjusting the pH value of the system and adding polyethyleneimine (PEI).

[0017] As can be seen from the above technical solutions, the present invention forms a micro-homogeneous reaction system between cellulase and cellulose in an aqueous medium by introducing a polyethylene glycol derivative at the amino terminus of cellulase, promotes the transition of the enzymatic hydrolysis reaction from heterogeneous to homogeneous, and realizes the efficient recovery of cellulase by utilizing the pH-sensitive characteristics of the dynamic Schiff base bond. Brief Description of the Drawings

[0018] Figure 1 It is a schematic diagram of the solvation of the fiber chain of cellulose and the polyethylene glycol derivative;

[0019] Figure 2 It is a comparison chart of the catalytic activities of natural cellulase and the modified cellulase provided by the present invention;

[0020] Figure 3 It is a chart of the recycling performance of the modified cellulase FA-Cell provided by the present invention. Detailed Embodiments

[0021] The present invention discloses a method for constructing a cellulase hydrolysis micro-homogeneous system and a method for recycling cellulase. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted 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 those skilled in the art can obviously make changes or appropriate alterations and combinations to 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.

[0022] In order to enable those skilled in the art to better understand the present invention, the following further detailed description of the present invention will be made in conjunction with specific embodiments.

[0023] Comparative Example 1

[0024] 1) Dissolve natural cellulase in a phosphate buffer solution with a pH of 4.8 to obtain a cellulase Cell solution;

[0025] 2) Mix the cellulase Cell solution obtained in step 1) with cellulose. The concentrations of the cellulase and cellulose are 0.5 mg / mL and 10 mg / mL respectively. Conduct an enzymatic hydrolysis reaction at 50 °C for 30 min. After the reaction, measure the content of the product reducing sugar by ultraviolet spectroscopy.

[0026] Example 1

[0027] 1) Dissolve natural cellulase in a phosphate buffer with a pH of about 4.8, then add polyethylene glycol dibenzaldehyde FA-PEG-FA. The reaction temperature is 35 °C and the reaction time is 24 h. After the reaction, conduct dialysis to generate a modified cellulase FA-Cell solution.

[0028] 2) Mix the modified cellulase FA-Cell solution obtained in step 1) with cellulose and conduct an enzymatic hydrolysis reaction. The reaction system presents a micro-homogeneous system. The concentrations of the modified cellulase and cellulose are 0.5 mg / mL and 10 mg / mL respectively. Conduct an enzymatic hydrolysis reaction at 50 °C for 30 min. After the reaction, measure the content of the product reducing sugar by ultraviolet spectroscopy.

[0029] The catalytic activity comparison chart of natural cellulase and the new cellulase is as Figure 2 shown. It can be seen from the figure that the catalytic activity of the modified cellulase obtained in Example 1 is significantly higher than that of the unmodified cellulase in Comparative Example 1. Specifically, the catalytic activity of the modified cellulase provided by the present invention is 116.12% higher than that of natural cellulase. This result indicates that the formation of the micro-homogeneous system provided by the present invention can effectively improve the catalytic activity of cellulase.

[0030] Example 2

[0031] 1) Dissolve natural cellulase in a phosphate buffer with a pH of about 4.8, then add a polyethylene glycol-based derivative FA-PEG-FA. The reaction temperature is 35 °C and the reaction time is 24 h to obtain a modified cellulase FA-Cell solution.

[0032] 2) Add the modified cellulase solution obtained in step 1) to cellulose for an enzymatic hydrolysis reaction. After the reaction, filter to remove the unreacted substrate. Add 0.2 mol / L of polyethyleneimine PEI to the filtrate, adjust the pH to alkaline to form a gel. After washing to remove the product reducing sugar, adjust the pH value to alkaline (adjust the pH value to about 9) to liquefy the gel, and continue to add fresh substrate cellulose for the next round of reaction. Repeat this cycle 5 times. During the enzymatic hydrolysis reaction, the concentration of the modified cellulase is 20 mg / mL and the concentration of cellulose is 30 mg / mL.

[0033] The modified cellulase provided by the present invention was recycled 5 times. Taking the initial enzyme activity of each modified cellulase as 100% in the first cycle, it was further tested, and finally the results of its three tests were compared to evaluate its reusability in the actual process. As Figure 3 shown, after 5 consecutive operations, the remaining enzyme activity of the modified cellulase FA-Cell could still be maintained at ≥80.00%.

[0034] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for constructing a cellulase hydrolysis micro-homogeneous system, characterized in that: In an aqueous phase system, a micro-homogeneous system is formed between the modified cellulase and cellulose, and the cellulose is enzymatically hydrolyzed; a polyethylene glycol-based derivative, polyethylene glycol dibenzaldehyde FA-PEG-FA, is introduced at the amino terminus of the cellulase to obtain the modified cellulase, denoted as FA-Cell.

2. The construction method according to claim 1, characterized in that: The cellulase is dissolved in a phosphate buffer with a pH of about 4.8, and the polyethylene glycol-based derivative polyethylene glycol dibenzaldehyde FA-PEG-FA is added for a modification reaction. After the reaction is completed, dialysis is performed to generate the modified cellulase FA-Cell.

3. The construction method according to claim 3, characterized in that: The reaction temperature of the modification reaction is 25 - 55 °C, and the reaction time is 3 - 48 h.

4. The construction method according to claim 1, characterized in that: It also includes a method for capturing and recovering cellulose. The capturing and recovering method is that after the enzymatic hydrolysis reaction, the unreacted substrate is removed by filtration. Polyethyleneimine PEI is added to the filtrate, and the pH value is adjusted to alkaline to form a dynamic Schiff base gel. The reaction product reducing sugar is removed by washing with water, and the system is acidified to the reaction pH range to release the modified cellulase, successfully achieving the capture and recovery of the modified cellulase.

5. The construction method according to claim 4, characterized in that: The modified cellulase after capture and recovery is continuously added to a new substrate for the next round of enzymatic hydrolysis reaction, and the modified cellulase can be captured and utilized repeatedly for multiple times.

6. The construction method according to claim 4 or 5, characterized in that: The concentration of the polyethyleneimine PEI is 0.1 - 0.6 mol / L.

7. The construction method according to claim 1, characterized in that: In the enzymatic reaction, the concentration of the modified cellulase is 0.25 - 20 mg / mL, and the concentration of cellulose is 5 - 50 mg / mL.

8. The construction method according to claim 1 or 7, characterized in that: The reaction time of the enzymatic hydrolysis reaction is 15 - 60 min, and the reaction temperature is 25 - 65 °C.

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