阅读说明：本技术 一种有序定向共固定的酶膜反应器及其制备方法和应用 (Ordered oriented co-immobilized enzyme membrane reactor and preparation method and application thereof ) 是由 叶鹏 祝黛莲 于 2019-07-10 设计创作，主要内容包括：本发明公开了一种有序定向共固定的酶膜反应器及其制备方法和应用,基于渗透汽化膜组件,所述渗透汽化膜组件包括复合有微孔滤膜的渗透汽化膜,所述微孔滤膜上按照酶催化级联反应顺序依次固定有反应所需的组合酶的固定化酶,所述组合酶为辣根过氧化物酶和葡萄糖氧化酶的组合、酪氨酸酶和碱性磷酸酶的组合、长链醇氧化酶和ω-转氨酶的组合以及乙醇脱氢酶、环己酮单加氧酶和脂肪酶的组合中的任一种,有利于提高催化反应效率,将固定化酶集合于一张微孔滤膜上,提高多步催化中间产物转移效率；通过设置微孔滤膜使得反应与分离一体,利于反应平衡向有利的方向移动；反应条件温和,酶固定化方法较为简单。(the invention discloses an orderly oriented co-immobilized enzyme membrane reactor and a preparation method and application thereof, based on a pervaporation membrane component, the pervaporation membrane component comprises a pervaporation membrane compounded with a microporous filter membrane, immobilized enzymes of combined enzymes required by reaction are sequentially fixed on the microporous filter membrane according to an enzyme catalysis cascade reaction sequence, the combined enzymes are any one of a combination of horseradish peroxidase and glucose oxidase, a combination of tyrosinase and alkaline phosphatase, a combination of long-chain alcohol oxidase and omega-transaminase and a combination of alcohol dehydrogenase, cyclohexanone monooxygenase and lipase, and are beneficial to improving the catalytic reaction efficiency, the immobilized enzymes are integrated on one microporous filter membrane, and the transfer efficiency of multi-step catalytic intermediate products is improved; reaction and separation are integrated by arranging the microporous filter membrane, so that the reaction balance is favorably moved to a favorable direction; the reaction condition is mild, and the enzyme immobilization method is simpler.)

1. the enzyme membrane reactor is characterized in that the enzyme membrane reactor is based on a pervaporation membrane assembly, the pervaporation membrane assembly comprises a pervaporation membrane compounded with a microporous filter membrane, immobilized enzymes of combined enzymes required by reactions are sequentially fixed on the microporous filter membrane according to an enzyme catalysis cascade reaction sequence, and the combined enzymes are any one of a combination of horseradish peroxidase and glucose oxidase, a combination of tyrosinase and alkaline phosphatase, a combination of long-chain alcohol oxidase and omega-transaminase and a combination of alcohol dehydrogenase, cyclohexanone monooxygenase and lipase.

2. an ordered oriented co-immobilized enzyme membrane reactor according to claim 1 wherein the organic macromolecular support of the immobilized enzyme is made of metal organic framework Material (MOF).

3. The ordered oriented co-immobilized enzyme membrane reactor of claim 2, wherein the metal organic framework Material (MOF) is ZIF-8 nanoparticles.

4. The ordered oriented co-immobilized enzyme membrane reactor of claim 1, wherein the pervaporation membrane is a poly (dimethoxysiloxane)/polyvinylidene fluoride (PDMS/PVDF) composite membrane, the microfiltration membrane is a PVDF membrane, and the immobilized enzyme is immobilized on the microfiltration membrane by dead-end filtration.

5. the ordered oriented co-immobilized enzyme membrane reactor of claim 4, wherein the pore size of the PVDF membrane is 1-3 μm, and the immobilization amount of all immobilized enzymes per unit area of the PVDF membrane is 0.1-0.5 mg/cm 2.

6. The ordered oriented co-immobilized enzyme membrane reactor of claim 1, wherein the enzyme membrane reactor has a permeate flux of 21 to 22 g-m-2-h "1.

7. The process of any of claims 1-6 wherein the process comprises the steps of:

First, an immobilized enzyme solution of a combination enzyme is prepared

Step 1: respectively dissolving the combined enzyme in deionized water to obtain a plurality of enzyme solutions;

step 2: mixing the multiple enzyme solutions obtained in the step 1 with a mixed solution respectively, reacting for 30min at room temperature, and standing for 3h to obtain multiple reaction solutions, wherein the mixed solution comprises a zinc nitrate solution and a 2-methylimidazole solution;

and step 3: centrifuging the multiple reaction solutions obtained in the step 2 at 6000r/min for 10min, and respectively collecting multiple white powders;

And 4, step 4: washing the multiple kinds of white powder obtained in the step 3 with deionized water for 3 times respectively, dispersing the white powder in the deionized water respectively, freeze-drying, and collecting multiple kinds of immobilized enzymes in the form of white powder;

And 5: putting a plurality of immobilized enzymes into the deionized water again respectively, and performing ultrasonic dispersion to obtain an immobilized enzyme solution of the combined enzyme;

second, dead-end filtration, fixation and compounding

Fixing the immobilized enzyme solution of the combined enzyme obtained in the first step on a microporous filter membrane through dead-end filtration in sequence, washing with deionized water at least twice, compounding the microporous filter membrane on a pervaporation membrane, and fixing the pervaporation membrane in a pervaporation membrane component.

8. the method of claim 7, wherein in step 1, the concentration of any enzyme solution is (3.0-5.2) g/L; in the step 2, the volume ratio of the zinc nitrate solution to the 2-methylimidazole solution to any enzyme solution is 2: 20: (1-2); the concentration of the zinc nitrate solution is 0.25-0.36mol/L, and the concentration of the 2-methylimidazole solution is 1.11-1.42 mol/L.

9. The use of the ordered oriented co-immobilized enzyme membrane reactor of any one of claims 1 to 6, wherein based on the ordered oriented co-immobilized enzyme membrane reactor, a liquid storage tank is further provided on one side of the ordered oriented co-immobilized enzyme membrane reactor, the enzyme membrane reactor is connected with the liquid storage tank through a feed pipe and a feed liquid pump, and the enzyme membrane reactor is also connected with the liquid storage tank through a first discharge pipe on the same side; and a cold trap, a drying tower, a buffer bottle and a vacuum pump are sequentially arranged on the other side of the enzyme membrane reactor, the enzyme membrane reactor is connected with the cold trap through a second discharge pipe, a pump is arranged between the cold trap and the drying tower, and the initial substrate passes through the enzyme membrane reactor which is orderly and directionally co-fixed to collect the permeate.

Technical Field

The invention belongs to the technical field of reactors, and particularly relates to an orderly oriented co-immobilized enzyme membrane reactor, and a preparation method and application thereof.

Background

enzymes act as biocatalysts, increasing the rate of a wide range of diverse chemical reactions occurring in the body. It is an indispensable component for a living body to participate in important chemical reaction processes essential to life, such as DNA replication and transcription, protein synthesis, primary and secondary metabolism, signal transduction, and cellular defense mechanisms. Enzymes are widely used in the fields of biocatalysis, biosensing, biomedical devices, and the like. However, the enzyme has problems of low thermal stability, poor stability of organic solvents, high cost, poor reusability, etc., so that its wide application is greatly hindered.

The enzyme immobilization technique is one of effective methods for overcoming the disadvantages of free enzymes, and shows a very good effect in improving the stability of enzymes under storage and reaction conditions. People usually fix the immobilized enzyme on different macromolecular carriers by using an enzyme immobilization technology and then prepare the immobilized enzyme reactor, so that the time of enzyme catalytic reaction can be shortened, and the reusability of enzyme is improved.

disclosure of Invention

The invention aims to overcome the problem of low conversion efficiency of the conventional immobilized enzyme reactor and provide an orderly oriented co-immobilized enzyme membrane reactor.

The second purpose of the invention is to overcome the problem of complex preparation method of the existing immobilized enzyme reactor and provide the preparation method of the ordered oriented co-immobilized enzyme membrane reactor.

the invention also aims to provide application of the orderly oriented co-immobilized multienzyme reactor.

the technical scheme for solving the technical problems is as follows:

An orderly oriented co-immobilized enzyme membrane reactor is based on a pervaporation membrane assembly, wherein the pervaporation membrane assembly comprises a pervaporation membrane compounded with a microporous filter membrane, immobilized enzymes of combined enzymes required by reaction are sequentially immobilized on the microporous filter membrane according to an enzyme catalysis cascade reaction sequence, and the combined enzymes are any one of a combination of horseradish peroxidase and glucose oxidase, a combination of tyrosinase and alkaline phosphatase, a combination of long-chain alcohol oxidase and omega-transaminase and a combination of alcohol dehydrogenase, cyclohexanone monooxygenase and lipase.

preferably, the organic macromolecular carrier of the immobilized enzyme is made of a metal organic framework Material (MOF).

preferably, the metal organic framework Material (MOF) is ZIF-8 nanoparticles.

preferably, the pervaporation membrane is a polydimethoxysiloxane/polyvinylidene fluoride (PDMS/PVDF) composite membrane, the microfiltration membrane is a PVDF membrane, and the immobilized enzyme is fixed on the microfiltration membrane by a dead-end filtration method.

Preferably, the aperture of the PVDF membrane is 1-3 μm, and the fixation amount of all immobilized enzymes on the PVDF membrane per unit area is 0.1-0.5 mg/cm 2.

Preferably, the permeation flux of the orderly oriented co-immobilized enzyme membrane reactor is 21-22 g.m < -2 > h < -1 >.

A preparation method of an ordered oriented co-immobilized enzyme membrane reactor is characterized by comprising the following steps:

First, an immobilized enzyme solution of a combination enzyme is prepared

step 1: respectively dissolving the combined enzyme in deionized water to obtain a plurality of enzyme solutions;

step 2: mixing the multiple enzyme solutions obtained in the step 1 with a mixed solution respectively, reacting for 30min at room temperature, and standing for 3h to obtain multiple reaction solutions, wherein the mixed solution comprises a zinc nitrate solution and a 2-methylimidazole solution;

And step 3: centrifuging the multiple reaction solutions obtained in the step 2 at 6000rpm for 10min, and respectively collecting multiple white powders;

And 4, step 4: respectively washing the multiple kinds of white powder obtained in the step 3 with deionized water for 3 times, respectively dispersing the white powder in the deionized water, freeze-drying, and collecting multiple kinds of immobilized enzymes in the form of white powder;

and 5: putting the various white powders into the deionized water again respectively, and performing ultrasonic dispersion to obtain an immobilized enzyme solution of the combined enzyme;

Second, dead-end filtration, fixation and compounding

Fixing the immobilized enzyme solution of the combined enzyme obtained in the first step on a microporous filter membrane through dead-end filtration in sequence, washing with deionized water at least twice, compounding the microporous filter membrane on a pervaporation membrane, and fixing the pervaporation membrane in a pervaporation membrane component.

Preferably, in the step 1, the concentration of any enzyme solution is (3.0-5.2) g/L; in the step 2, the volume ratio of the zinc nitrate solution to the 2-methylimidazole solution to any enzyme solution is 2: 20: (1-2); the concentration of the zinc nitrate solution is 0.25-0.36mol/L, and the concentration of the 2-methylimidazole solution is 1.11-1.42 mol/L.

the application of the ordered oriented co-immobilized enzyme membrane reactor is based on the ordered oriented co-immobilized enzyme membrane reactor, wherein a liquid storage tank is also arranged on one side of the ordered oriented co-immobilized enzyme membrane reactor, the enzyme membrane reactor is connected with the liquid storage tank through a feed pipe and a feed liquid pump, and the same side of the enzyme membrane reactor is also connected with the liquid storage tank through a first discharge pipe; and a cold trap, a drying tower, a buffer bottle and a vacuum pump are sequentially arranged on the other side of the enzyme membrane reactor, the enzyme membrane reactor is connected with the cold trap through a second discharge pipe, a pump is arranged between the cold trap and the drying tower, and the initial substrate passes through the enzyme membrane reactor which is orderly and directionally co-fixed to collect the permeate.

Specifically, the air pressure at the downstream of the pervaporation membrane module is adjusted by a pump between the cold trap and the drying tower, so that the air pressure difference is generated between the upstream and the downstream of the pervaporation membrane, and after the air pressure is adjusted to a certain pressure, the pervaporation is started: the raw materials in the liquid storage tank are conveyed to one side of the enzyme membrane reactor through the feed pipe by the feed liquid pump, the raw materials move to the other side of the enzyme membrane reactor under the action of air pressure difference, so that the raw materials are subjected to cascade reaction under the action of various immobilized enzymes on the microfiltration membrane, reaction substrates are converted to form products, the products are enriched on the other side of the pervaporation membrane to form permeate, and if the reaction substrates conveyed to the pervaporation membrane module by the feed liquid pump are excessive, then the permeate is returned to the liquid storage tank through the first discharge pipe, and is conveyed to the cold trap through the discharge pipe under the action of the pump, dry ice is arranged in the cold trap and is used for liquefying the permeate to form permeate, and for the gas which cannot be liquefied in time, the drying tower absorbs water vapor, and a buffer bottle is arranged in front of the vacuum pump in order to prevent the gas of the drying tower from entering the vacuum pump under the action of the vacuum pump.

Compared with the prior art, the invention has the beneficial effects that:

The arrangement sequence of the immobilized enzymes is in accordance with the catalytic sequence of the enzyme cascade reaction, which is beneficial to improving the catalytic reaction efficiency, and the immobilized enzymes are integrated on a microporous filter membrane, so that the transfer efficiency of the multi-step catalytic intermediate product is improved; reaction and separation are integrated by arranging the microporous filter membrane, so that the reaction balance is favorably moved to a favorable direction; the reaction condition is mild, and the enzyme immobilization method is simpler.

drawings

FIG. 1 is a schematic structural diagram of the present invention.

FIG. 2 is a schematic diagram of an apparatus for converting glucose.

the labels in the figure are: 1-pervaporation membrane component, 2-microporous filter membrane, 3-pervaporation membrane, 4-enzyme membrane reactor, 5-cold trap, 6-pump, 7-drying tower, 8-buffer bottle, 9-vacuum pump, 10-liquid storage tank and 11-liquid pump.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in 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.