Preparation and use method of microchannel immobilized cross-linked enzyme aggregate (CLEAs) reactor

Abstract

The invention provides a preparation method and a use method of a microchannel immobilized cross-linked enzyme aggregate (CLEAs) reactor. Comprising the following steps: S1, preparing an enzyme solution; s2, preparation of a precipitant-cross-linking agent mixed solution: mixing a precipitant acetone and a cross-linking agent glycerol diglycidyl ether in proportion to obtain the precipitant-cross-linking agent mixed solution; s3, preparation of a micro-channel reactor: drawing an AUTO CAD drawing of the micro-channel reactor, inputting the AUTO CAD drawing into a laser engraving machine, and obtaining the micro-channel reactor after engraving is completed; s4, preparation of a microchannel immobilized CLEAs reactor: firstly, carrying out surface modification on the reactor by using PEI, then simultaneously injecting an enzyme solution and a precipitant-cross-linking agent mixed solution into the microchannel through a Y-shaped tube, carrying out enzyme protein molecule precipitation and cross-linking to form CLEAs, and immobilizing the CLEAs on the surface of the microchannel wall through the cross-linking action of a cross-linking agent and the adsorption action of the surface of the microchannel to obtain the microchannel immobilized CLEAs reactor. And thus, the micro-channel immobilized CLEAs reactor is obtained. According to the invention, efficient and continuous use of the micro-channel reactor and CLEAs is realized, and the method is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of preparation and application of enzyme membrane reactors, and particularly to the preparation and use method of a microchannel immobilized cross-linked enzyme aggregate (CLEAs) reactor. Background Art

[0002] Enzyme immobilization technology is divided into two types: carrier-based immobilization technology and carrier-free immobilization technology. Carrier-based immobilization technology refers to a technology that uses carrier materials to bind or confine enzymes within a certain space, retaining their catalytic activity and allowing for recycling and repeated use. However, since it is necessary to introduce an enzyme immobilization carrier without catalytic activity, it not only reduces the catalytic activity per unit volume of the enzyme and the product yield, but also increases the use cost. Carrier-free immobilization technology refers to a technology that directly precipitates and cross-links biological enzyme protein molecules, such as cross-linked enzyme aggregates (CLEAs) and cross-linked enzyme crystals (CLECs). In particular, CLEAs technology has good application prospects due to its high catalytic efficiency per unit volume, low requirement for the purity of the original enzyme, and simple preparation method. However, CLEAs also have disadvantages such as large adhesiveness, mass transfer being easily affected by particle size, and inconvenient use, which greatly limits its practical application.

[0003] Microchannel reactors have advantages such as a large specific surface area, good mass and heat transfer, easy regulation of reaction parameters, and high reaction efficiency per unit volume. As the advantages of microchemical technology in production safety, process intensification, economy, etc. are increasingly recognized, microchannel reactors have shown good application prospects in microchemical engineering, confined spaces (such as space stations, submarines, deep submersibles, etc.). The CLEAs technology has the same invention background as microchannel reactors and microchemical technology, that is, it emphasizes the miniaturization of the reaction vessel and high output efficiency per unit volume. Combining the CLEAs technology with microchannel reactors can not only give play to the advantages of microchannel reactors, but also overcome the disadvantage of strong adhesiveness of CLEAs, promoting the application of enzyme membrane reactors in the field of biochemistry. Summary of the Invention

[0004] The present invention provides a preparation and use method of an enzyme membrane reactor that combines the characteristics of microchannel fluid and the catalytic function of CLEAs, aiming to promote the industrial application of enzyme membrane reactors.

[0005] The preparation of the microchannel immobilized CLEAs reactor includes the following steps:

[0006] S1. The biological enzyme used is urease, which is isolated from Canavalia ensiformis. An appropriate amount of urease is added to a phosphate buffer solution, and after stirring and dissolving, a urease solution is obtained;

[0007] S2. The precipitant for preparing CLEAs is acetone, and the crosslinking agent is diglycidyl ether of glycerol. Acetone and diglycidyl ether of glycerol are fully mixed to obtain a precipitant-crosslinking agent mixture;

[0008] S3. The microchannel reactor is made of polymethyl methacrylate (PMMA), with a microchannel diameter of 1.0 mm, an "S" shape, and a total length of 50 cm. Before use, the microchannel reactor is soaked in a NaOH solution at 60 °C for 30 min, taken out and washed with deionized water to remove NaOH, and then soaked in a 5% polyetherimide (PEI) solution for 30 min. After taking it out, the inner wall of the microchannel is dried with nitrogen.

[0009] S4. When immobilizing CLEAs of urease in the microchannel reactor, the urease solution and the precipitant-crosslinking agent mixture are simultaneously injected into the treated microchannel reactor through a Y-shaped tube (1), and their flow rates are respectively controlled by a microfluidic injection pump for enzyme solution (2) and a microfluidic injection pump for the precipitant-crosslinking agent mixture (3). The Y-shaped tube is connected to the microchannel reactor through a flange (4). The pH value of the phosphate buffer solution for preparing the urease solution is 6.0 - 8.0, the urease concentration is 6.0 - 10.0 mg / ml, and the flow rate is 100 - 300 μl / min; the volume fraction of the precipitant in the mixture is 30% - 50%, the volume fraction of the crosslinking agent is 1.0% - 3.0%, and the flow rate of the precipitant-crosslinking agent mixture is 100 - 300 μl / min. The flow rate ratio of the enzyme solution to the precipitant-crosslinking agent mixture is 1:1. During the flow of the reaction solution, the protein molecules in the enzyme solution react with the precipitant and crosslinking agent in the mixture to form CLEAs, and CLEAs are immobilized on the inner wall of the microchannel (5) through the crosslinking action of the crosslinking agent and the adsorption action of the microchannel wall, and thus a microchannel-immobilized urease CLEAs reactor (6) can be obtained.

[0010] The application purpose of the present invention is achieved through the following technical means:

[0011] When the microchannel-immobilized urease CLEAs reactor (7) catalyzes a reaction, the filtered human urine is injected into a liquid storage bottle (8), the peristaltic pump (9) is started, so that the urine enters the reactor from one end and continuously flows, reacts with the urease CLEAs immobilized on the inner wall of the microchannel, and the hydrolyzed product ammonium ions flow out from the other end of the reactor with the wastewater and are collected in a liquid collection bottle (10). The urine flow rate is 600 - 900 μl / min, which is controlled by a flow meter (11).

[0012] The present invention has the following advantages:

[0013] 1. Compared with a microchannel-immobilized free enzyme reactor, the microchannel-immobilized CLEAs reactor has a higher enzyme activity per unit volume and a higher product yield.

[0014] 2. The large specific surface area of the microchannel reactor and the adhesion of CLEAs enable CLEAs to be uniformly and firmly fixed on the inner wall surface of the microchannel, making use of the advantages of the microchannel and overcoming the disadvantages of CLEAs.

[0015] 3. The combination of the microchannel reactor and CLEAs provides a new idea for the application of bioenzymes and enzyme membrane microreactors in the field of microchemical engineering.

[0016] 4. By parallel or series connection, increasing the number of microchannel-immobilized CLEAs reactors can easily achieve the scale-up of the reactor, which is beneficial to industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram for the preparation of the microchannel-immobilized CLEAs reactor of the present invention: wherein: (1) - Y-shaped tube; (2) - microfluidic injection pump for enzyme solution; (3) - microfluidic injection pump for precipitant-crosslinking agent mixture; (4) - flange; (5) - microchannel; (6) - microchannel reactor.

[0018] Figure 2 Schematic diagram for the application of the microchannel-immobilized CLEAs reactor of the present invention: wherein: (7) - microchannel-immobilized urease CLEAs reactor; (8) - liquid storage bottle; (9) - peristaltic pump; (10) - liquid collection bottle; (11) - flowmeter.

[0019] Figure 3 Schematic cross-sectional view of the microchannel-immobilized CLEAs reactor of the present invention: wherein: (12) - cross-section of the microchannel-immobilized CLEAs reactor; (13) - urease protein molecule; (14) - urease CLEAs; (15) - crosslinking agent molecule.

[0020] Figure 4 SEM image of CLEAs in the microchannel-immobilized CLEAs reactor of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention will be further described below in conjunction with embodiments:

[0022] Example 1: Prepare as Figure 1When preparing the microchannel-immobilized urease CLEAs reactor shown below: ① Prepare a phosphate buffer solution with pH = 6.0; ② Prepare a urease (Canavalia ensiformis) solution with a concentration of 10.0 mg / ml using the phosphate buffer solution; ③ Preparation of the microchannel-immobilized CLEAs reactor: Inject 5 ml of the urease solution with a concentration of 10.0 mg / ml into the microchannel reactor (6) through a microfluidic injection pump (2) at a flow rate of 100 μl / min; simultaneously, inject 5 ml of the precipitant-crosslinker mixture into the microchannel reactor (6) through a microfluidic injection pump (3). The volume fraction of acetone as the precipitant in the mixture is 50%, and the volume fraction of diglycidyl ether of glycerol as the crosslinker is 3.0%. The flow rate is 100 μl / min. When the solution is used up, the microchannel-immobilized urease CLEAs reactor is obtained.

[0023] Use the above microchannel-immobilized urease CLEAs reactor to treat human urine filtered by a 0.45 μm mixed cellulose membrane. Take the result of treating urine with the microchannel-immobilized free enzyme urease reactor as a control. The preparation method of this reactor is similar to that of the microchannel-immobilized urease CLEAs reactor of this patent. The only difference is that the precipitant (acetone)-crosslinker (diglycidyl ether of glycerol) mixture in the preparation method of this patent is replaced with a crosslinker (diglycidyl ether of glycerol) solution. Take 200 ml of urine with a urea concentration of 500 mM, place it in a 250 ml storage bottle (8), and input it into the microchannel-immobilized urease CLEAs reactor (7) through a peristaltic pump (9) at a flow rate of 600 μl / min. The urea removal rate is 76%. Compared with the urea removal rate of 49% of the microchannel-immobilized free enzyme urease reactor, it is increased by 55.1%.

[0024] Example 2: When preparing the microchannel-immobilized urease CLEAs reactor as Figure 1 shown below: ① Prepare a phosphate buffer solution with pH = 7.0; ② Prepare a urease (Canavalia ensiformis) solution with a concentration of 8.0 mg / ml using the phosphate buffer solution; ③ Preparation of the microchannel-immobilized CLEAs reactor: Inject 5 ml of the urease solution with a concentration of 8.0 mg / ml into the microchannel reactor (6) through a microfluidic injection pump (2) at a flow rate of 150 μl / min; simultaneously, inject 5 ml of the precipitant-crosslinker mixture into the microchannel reactor (6) through a microfluidic injection pump (3). The volume fraction of acetone as the precipitant in the mixture is 40%, and the volume fraction of diglycidyl ether of glycerol as the crosslinker is 2.0%. The flow rate is 150 μl / min. When the solution is used up, the microchannel-immobilized urease CLEAs reactor is obtained.

[0025] The human urine filtered by a 0.45 μm mixed cellulose membrane was treated with the above enzyme membrane reactor. 200 ml of urine with a urea concentration of 500 mM was taken and placed in a 250 ml reservoir bottle (8), and then input into the microchannel-immobilized urease CLEAs reactor (7) through a peristaltic pump (9) at a flow rate of 700 μl / min. The urea removal rate was 69%. Compared with the urea removal rate of 49% of the microchannel-immobilized free enzyme urease reactor, it was increased by 40.8%.

[0026] Example 3: When preparing the microchannel-immobilized urease CLEAs reactor as Figure 1 shown: ① Prepare a phosphate buffer solution with pH = 8.0; ② Prepare a urease (Canavalia ensiformis) solution with a concentration of 6.0 mg / ml using the phosphate buffer solution; ③ Preparation of the microchannel-immobilized CLEAs reactor: Inject 5 ml of the urease solution with a concentration of 6.0 mg / ml into the microchannel reactor (6) through a microfluidic injection pump (2) at a flow rate of 200 μl / min; at the same time, inject 5 ml of the precipitant-crosslinking agent mixture into the microchannel reactor (6) through a microfluidic injection pump (3). The volume fraction of acetone as the precipitant in the mixture was 30%, and the volume fraction of glycerol diglycidyl ether as the crosslinking agent was 1.0%, and the flow rate was 200 μl / min. When the solution was exhausted, the microchannel-immobilized urease CLEAs reactor was obtained.

[0027] The human urine filtered by a 0.45 μm mixed cellulose membrane was treated with the above enzyme membrane reactor. 200 ml of urine with a urea concentration of 500 mM was taken and placed in a 250 ml reservoir bottle (8), and then input into the microchannel-immobilized urease CLEAs reactor (7) through a peristaltic pump (9) at a flow rate of 800 μl / min. The urea removal rate was 65%. Compared with the urea removal rate of 49% of the microchannel-immobilized free enzyme urease reactor, it was increased by 32.7%.

[0028] Example 4: When preparing the same as Figure 1When preparing the microchannel-immobilized urease CLEAs reactor shown below, ① prepare a phosphate buffer solution with pH = 6.5; ② prepare a urease (Canavalia ensiformis) solution with a concentration of 9.5 mg / ml using the phosphate buffer solution; ③ preparation of the microchannel-immobilized CLEAs reactor: Inject 5 ml of the urease solution with a concentration of 9.5 mg / ml into the microchannel reactor (6) through a microfluidic injection pump (2) at a flow rate of 250 μl / min; simultaneously, inject 5 ml of the precipitant-crosslinker mixture into the microchannel reactor (6) through a microfluidic injection pump (3). The volume fraction of the precipitant acetone in the mixture is 50%, and the volume fraction of the crosslinker glycerol diglycidyl ether is 2.0%. The flow rate is 250 μl / min. When the solution is used up, the microchannel-immobilized urease CLEAs reactor is obtained.

[0029] Use the above enzyme membrane reactor to treat human urine filtered by a 0.45 μm mixed cellulose membrane. Take 200 ml of urine with a urea concentration of 500 mM, place it in a 250 ml reservoir bottle (8), and input it into the microchannel-immobilized urease CLEAs reactor (7) through a peristaltic pump (9) at a flow rate of 650 μl / min. The urea removal rate is 71%. Compared with the urea removal rate of 49% of the microchannel-immobilized free enzyme urease reactor, it is increased by 44.9%.

[0030] Example 5: When preparing the Figure 1 microchannel-immobilized urease CLEAs reactor shown below, ① prepare a phosphate buffer solution with pH = 7.5; ② prepare a urease (Canavalia ensiformis) solution with a concentration of 7.5 mg / ml using the phosphate buffer solution; ③ preparation of the microchannel-immobilized CLEAs reactor: Inject 5 ml of the urease solution with a concentration of 7.5 mg / ml into the microchannel reactor (6) through a microfluidic injection pump (2) at a flow rate of 300 μl / min; simultaneously, inject 5 ml of the precipitant-crosslinker mixture into the microchannel reactor (6) through a microfluidic injection pump (3). The volume fraction of the precipitant acetone in the mixture is 40%, and the volume fraction of the crosslinker glycerol diglycidyl ether is 1.5%. The flow rate is 300 μl / min. When the solution is used up, the microchannel-immobilized urease CLEAs reactor is obtained.

[0031] Use the above enzyme membrane reactor to treat human urine filtered by a 0.45 μm mixed cellulose membrane. Take 200 ml of urine with a urea concentration of 500 mM, place it in a 250 ml reservoir bottle (8), and input it into the microchannel-immobilized urease CLEAs reactor (7) through a peristaltic pump (9) at a flow rate of 750 μl / min. The urea removal rate is 73%. Compared with the urea removal rate of 49% of the microchannel-immobilized free enzyme urease reactor, it is increased by 49.0%.

[0033] Example 6: When preparing the microchannel-immobilized urease CLEAs reactor as Figure 1 shown: ① Prepare a phosphate buffer solution with pH = 6.5; ② Prepare a urease (Canavalia ensiformis) solution with a concentration of 7.0 mg / ml using the phosphate buffer solution; ③ Preparation of the microchannel-immobilized CLEAs reactor: Inject 5 ml of the urease solution with a concentration of 7.0 mg / ml into the microchannel reactor (6) through a microfluidic injection pump (2) at a flow rate of 200 μl / min; simultaneously, inject 5 ml of the precipitant-crosslinker mixture into the microchannel reactor (6) through a microfluidic injection pump (3). The volume fraction of the precipitant acetone in the mixture is 50%, and the volume fraction of the crosslinker glycerol diglycidyl ether is 2.5%. The flow rate is 200 μl / min. When the solution is exhausted, the microchannel-immobilized urease CLEAs reactor is obtained.

[0035] Use the above enzyme membrane reactor to treat human urine filtered by a 0.45 μm mixed cellulose membrane. Take 200 ml of urine with a urea concentration of 500 mM, place it in a 250 ml storage bottle (8), and input it into the microchannel-immobilized urease CLEAs reactor (7) through a peristaltic pump (9) at a flow rate of 850 μl / min. The urea removal rate is 70%. Compared with the urea removal rate of 49% of the microchannel-immobilized free enzyme urease reactor, it is increased by 42.9%.

[0033] Example 7: When preparing the microchannel-immobilized urease CLEAs reactor as Figure 1 shown: ① Prepare a phosphate buffer solution with pH = 7.5; ② Prepare a urease (Canavalia ensiformis) solution with a concentration of 6.5 mg / ml using the phosphate buffer solution; ③ Preparation of the microchannel-immobilized CLEAs reactor: Inject 5 ml of the urease solution with a concentration of 6.5 mg / ml into the microchannel reactor (6) through a microfluidic injection pump (2) at a flow rate of 150 μl / min; simultaneously, inject 5 ml of the precipitant-crosslinker mixture into the microchannel reactor (6) through a microfluidic injection pump (3). The volume fraction of the precipitant acetone in the mixture is 35%, and the volume fraction of the crosslinker glycerol diglycidyl ether is 2.0%. The flow rate is 150 μl / min. When the solution is exhausted, the microchannel-immobilized urease CLEAs reactor is obtained.

[0035] The human urine filtered by a 0.45 μm mixed cellulose membrane was treated with the above enzyme membrane reactor. 200 ml of urine with a urea concentration of 500 mM was placed in a 250 ml liquid storage bottle (8) and input into the microchannel-immobilized urease CLEAs reactor (7) through a peristaltic pump (9) at a flow rate of 900 μl / min. The urea removal rate was 65%. Compared with the urea removal rate of 49% of the microchannel-immobilized free enzyme urease reactor, it increased by 32.6%.

[0033] Example 8: When preparing the microchannel-immobilized urease CLEAs reactor as Figure 1 shown: ① Prepare a phosphate buffer solution with pH = 7.0; ② Prepare a urease (Canavalia ensiformis) solution with a concentration of 8.5 mg / ml using the phosphate buffer solution; ③ Preparation of the microchannel-immobilized CLEAs reactor: Inject 5 ml of the urease solution with a concentration of 8.5 mg / ml into the microchannel reactor (6) through a microfluidic injection pump (2) at a flow rate of 250 μl / min; simultaneously, inject 5 ml of the precipitant-crosslinking agent mixture into the microchannel reactor (6) through a microfluidic injection pump (3). The volume fraction of the precipitant acetone in the mixture was 45%, and the volume fraction of the crosslinking agent glycerol diglycidyl ether was 2.0%. The flow rate was 250 μl / min. When the solution was exhausted, the microchannel-immobilized urease CLEAs reactor was obtained.

[0035] The human urine filtered by a 0.45 μm mixed cellulose membrane was treated with the above enzyme membrane reactor. 200 ml of urine with a urea concentration of 500 mM was placed in a 250 ml liquid storage bottle (8) and input into the microchannel-immobilized urease CLEAs reactor (7) through a peristaltic pump (9) at a flow rate of 900 μl / min. The urea removal rate was 69%. Compared with the urea removal rate of 49% of the microchannel-immobilized free enzyme urease reactor, it increased by 40.8%.

Claims

1. Preparation method of a microchannel-immobilized cross-linked enzyme aggregate (CLEA) reactor, characterized in that: An enzyme solution and a precipitant (acetone)-crosslinking agent (glycerol diglycidyl ether) mixed solution are simultaneously injected into a microchannel reactor made of polymethyl methacrylate (PMMA) through a Y-shaped tube. The concentration of the urease solution is 6-10 mg / ml, the pH is 6.0-8.0, and the flow rate is 100-300 μl / min; the volume fraction of the precipitant in the mixed solution is 30%-50%, the volume fraction of the crosslinking agent is 1.0%-3.0%, and the flow rate of the mixed solution is 100-300 μl / min. The precipitant-crosslinking agent mixed solution reacts with the protein molecules in the urease solution to form CLEAs, and the CLEAs are immobilized on the surface of the microchannel wall through the crosslinking action of the crosslinking agent and the surface adsorption action of the microchannel, and thus the microchannel-immobilized CLEA reactor can be obtained.

2. The method for preparing a microchannel-immobilized CLEA reactor according to claim 1, characterized in that: The urease selected is the urease extracted from Canavalia ensiformis.

3. Method for treating urine by applying the microchannel-immobilized CLEA reactor according to claim 1, characterized in that: The urine is injected into the liquid storage bottle (8), the peristaltic pump (9) is started, so that the urine enters from one end of the microchannel-immobilized CLEA reactor (7), and the flow rate is 600-900 μl / min, and the flow rate is controlled by the flow meter (11). Urea reacts with the CLEAs immobilized on the surface of the microchannel wall, and urea is decomposed into ammonium ions, and flows out from the other end of the reactor with the wastewater and flows into the liquid collection bottle (10).

4. The method according to claim 3, characterized in that: The urine described is filtered human urine.

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