CN113046233B - Microsphere-membrane integrated enzyme reactor and preparation method and application thereof - Google Patents

Microsphere-membrane integrated enzyme reactor and preparation method and application thereof Download PDF

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CN113046233B
CN113046233B CN202110276197.1A CN202110276197A CN113046233B CN 113046233 B CN113046233 B CN 113046233B CN 202110276197 A CN202110276197 A CN 202110276197A CN 113046233 B CN113046233 B CN 113046233B
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罗建泉
刘露轲
万印华
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Institute of Process Engineering of CAS
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    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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Abstract

The invention relates to a microsphere-membrane integrated enzyme reactor and a preparation method and application thereof, wherein the microsphere-membrane integrated enzyme reactor comprises enzyme-carrying microspheres and a membrane component; the enzyme-loaded microsphere comprises a microsphere, a modified layer on the surface of the microsphere and enzyme loaded on the modified layer. The microsphere-membrane integrated enzyme reactor combines an enzyme immobilization technology with a membrane reactor, and has the characteristics of high enzyme loading and high catalytic efficiency; the membrane reactor has the advantages of hydrodynamics of the membrane reactor, and can intercept enzyme to realize high utilization rate of the enzyme; and the online separation of a macromolecular substrate and a micromolecular product can be realized, and the flow operation is simplified. In conclusion, the integrated enzyme reactor has the advantages of simple structure, high efficiency, energy conservation, high equipment integration level, small occupied area, low equipment investment and easy industrial and large-scale application.

Description

Microsphere-membrane integrated enzyme reactor and preparation method and application thereof
Technical Field
The invention belongs to the technical field of enzyme reactors, and relates to a microsphere-membrane integrated enzyme reactor and a preparation method and application thereof.
Background
With the development of modern biotechnology, enzymes are widely applied to the fields of medicine, food processing, wastewater treatment and the like, and have the advantages of high catalytic efficiency, strong specificity, greenness, no pollution and the like. The enzymatic in vitro catalysis reaction needs to be carried out in a certain container so as to regulate and control the reaction conditions and the reaction rate, and the reaction container and the accessory equipment thereof are called an enzyme reactor. The reactor may be classified into a stirred tank reactor, a bubbling reactor, a packed bed reactor, a fluidized bed reactor, an enzyme membrane reactor, etc., according to the structure of the reactor.
CN210856150U discloses an enzyme reactor with a two-layer stirring structure, wherein an upper layer stirring arm and a lower layer stirring arm rotate reversely, and the arrangement of a circulating component is matched to improve the fluidity between an enzyme reaction reagent and a related chemical reagent, increase the contact between the reaction reagent and related materials, and improve the enzyme reaction effect. However, the free enzyme has low operational stability and is easily inactivated during use. Moreover, the biological enzymes are expensive and difficult to recover, resulting in high production cost and difficulty in continuous production.
CN211255950U discloses a continuous production and enzyme recovery device of phenylglycine, which structurally comprises an enzyme reactor, an enzyme separator and a product storage tank, realizes synchronous enzyme separation in the preparation process of phenylglycine, reduces the enzyme dosage, improves the enzyme catalysis efficiency and prolongs the service life of the enzyme. However, the device has low enzyme recovery rate, complex structure and high energy consumption.
The enzyme stability can be improved through enzyme immobilization, the enzyme can be recycled, and the production cost is reduced. CN209974663U discloses a high-efficiency isooctyl palmitate production device, and the enzyme membrane reactor is a horizontal tank body. The immobilized enzyme membrane layers are arranged at intervals, and reactants are sequentially contacted with the lipase in the circulation process, so that the retention time is prolonged, and the full reaction is ensured; the thickness of the membrane layer is reduced from the inlet direction to the outlet direction of the membrane reactor in sequence so as to adjust the reaction speed of the reaction liquid and reduce the short circuit or blockage phenomenon of reactants in the immobilized enzyme membrane layer, so that the average retention time is shortened, and the reaction is facilitated. The reactor is compact in equipment, but mass transfer resistance is increased due to cascade of multiple layers of films, enzyme carrying amount is low due to limited enzyme carrying sites of the films, and production efficiency of the reactor is low. In addition, the separation function of the enzyme immobilized carrier alone in the reactor cannot be sufficiently exhibited.
At present, the production efficiency of continuous enzyme reactors is mainly limited by enzyme activity, effective enzyme loading and mass transfer. Therefore, designing a novel enzyme reactor to solve the contradiction between enzyme loading capacity and mass transfer kinetics is of great significance to the realization of industrial application of immobilized enzymes.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a microsphere-membrane integrated enzyme reactor and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a microsphere-membrane integrated enzyme reactor comprising enzyme-loaded microspheres and a membrane module;
the enzyme-loaded microsphere comprises a microsphere, a modified layer on the surface of the microsphere and enzyme loaded on the modified layer.
The microsphere-membrane integrated enzyme reactor combines an enzyme immobilization technology with a membrane reactor, and has the characteristics of high enzyme loading and high catalytic efficiency; the reactor also has the hydrodynamic advantages of a membrane reactor, such as short mass transfer path, small resistance and high separation efficiency, in the reactor, part of products transferred along the radial direction can directly permeate the membrane to enter the subsequent flow without flowing through the whole microsphere packed column, and the purposes of shortening the mass transfer path and reducing the mass transfer resistance are achieved; meanwhile, the enzyme can be trapped, so that the high utilization rate of the enzyme is realized; and the online separation of a macromolecular substrate and a micromolecular product can be realized, and the flow operation is simplified. In conclusion, the integrated enzyme reactor has the advantages of simple structure, high efficiency, energy conservation, high equipment integration level, small occupied area, low equipment investment and easy industrial and large-scale application.
Preferably, the microsphere-membrane integrated enzyme reactor further comprises back-flushing and pulsed counter-flow operating elements.
In the actual operation process, the microsphere-membrane integrated enzyme reactor can be connected with a back washing and pulse reverse flow operation element, and the back washing and pulse reverse flow operation are utilized, so that the utilization rate of immobilized enzyme is further improved, and the reactor can be ensured to stably operate for a long time.
Preferably, the filling amount of the enzyme-loaded microspheres is 50-95% of the flow channel space of the membrane module, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc., and other specific values within the numerical range can be selected, which is not described herein again. Preferably 50 to 70%.
The filling amount of the enzyme-loaded microspheres is specially selected to be 50-95%, particularly 50-70% of the flow channel space of the membrane module, because the enzyme loading amount and the mass transfer kinetic property can be balanced to the maximum extent in the numerical range, and the comprehensive working efficiency of the reactor is maximized.
Preferably, the membrane module comprises any one of or a combination of at least two of a tubular membrane module, a flat-plate membrane module and a hollow fiber membrane module, for example, a combination of a tubular membrane module and a flat-plate membrane module, a combination of a tubular membrane module and a hollow fiber membrane module, and the like, and any other combination mode can be selected, which is not described herein again. Tubular membrane modules are preferred.
Preferably, the membrane material in the membrane module comprises any one or a combination of at least two of polysulfone, polyethersulfone, sulfonated polyethersulfone, polyacrylonitrile, polyvinylidene fluoride, polyvinyl alcohol or regenerated cellulose; for example, the combination of polysulfone and polyethersulfone, and the combination of sulfonated polyethersulfone and polyacrylonitrile, any other combination modes can be selected, and the details are not repeated herein.
Preferably, the membrane in the membrane module comprises a microfiltration membrane or an ultrafiltration membrane.
Preferably, the material of the modification layer includes tannic acid and polyelectrolyte.
Tannic acid in the modified layer is plant polyphenol, is rich in catechol groups, is adhered to the surface of the microsphere through various interactions, can provide a large number of reaction sites for enzyme immobilization after secondary polyelectrolyte grafting, remarkably improves the enzyme-carrying quantity of the microsphere and the stability of the immobilized enzyme, and is beneficial to the reuse of the immobilized enzyme.
Preferably, the polyelectrolyte comprises any one or a combination of at least two of polyethyleneimine, polyallylamine and salts thereof, polyarginine or polyglutamic acid; the combination of at least two of the compounds, such as the combination of polyethyleneimine and polyallylamine and salts thereof, the combination of polyarginine and polyglutamic acid, and the like, can be selected in any combination manner, and is not repeated herein.
Preferably, the mass ratio of the tannic acid to the microspheres is 1:5-1:20, such as 1:5, 1:6, 1:7, 1:8, 1:10, 1:12, 1:14, 1:15, 1:16, 1:17, 1:18, 1:20, and the like, and other specific values in the numerical range can be selected, which is not described herein again.
Preferably, the mass ratio of the tannic acid to the polyelectrolyte is 1:2-2:1, for example, 1:2, 2:3, 1:1, 3:2, 2:1, etc., and other specific values in the value range can be selected, which is not described herein again.
The mass ratio of the tannic acid to the microspheres, the mass ratio of the tannic acid to the polyelectrolyte, the particle size of the microspheres, the type of the polyelectrolyte and the type of microsphere materials comprehensively influence the size of the modified microspheres, further influence the enzyme-carrying amount and the behavior of the enzyme-carrying microspheres in the reactor, and further influence the working efficiency of the reactor.
Preferably, the enzyme is supported on the modified layer by physical adsorption or chemical covalent bond.
Preferably, the enzyme comprises a hydrolase, an oxidoreductase or an isomerase.
Preferably, the enzyme comprises psicose isomerase, rhamnose isomerase, galactosidase, L-arabinose isomerase, xylitol dehydrogenase, sucrase, amylase, protease, cellulase or dextranase.
Preferably, the particle size of the microspheres is 0.02-3mm, such as 0.02mm, 0.1mm, 0.5mm, 1mm, 2mm, 3mm, etc., and other specific values within the numerical range can be selected, which is not described herein again.
Preferably, the material of the microsphere comprises any one or a combination of at least two of polystyrene, polyglycidyl methacrylate, glucomannan, chitosan or sodium alginate; the combination of at least two of the above-mentioned compounds, such as the combination of polystyrene and poly glycidyl methacrylate, the combination of glucomannan and chitosan, the combination of chitosan and sodium alginate, etc., can be selected from any other combination modes, and are not described herein again.
In a second aspect, the present invention provides a method for preparing a microsphere-membrane integrated enzyme reactor according to the first aspect, the method comprising the steps of:
(1) modifying the microspheres to obtain surface-modified microspheres;
(2) carrying out enzyme immobilization on the surface-modified microspheres obtained in the step (1) to obtain enzyme-loaded microspheres;
(3) loading the enzyme-loaded microspheres obtained in the step (2) into a membrane module to obtain the microsphere-membrane integrated enzyme reactor.
The preparation method of the microsphere-membrane integrated enzyme reactor is simple and easy to operate, and is very suitable for industrial and large-scale production.
Preferably, the method for modifying microspheres in step (1) comprises: mixing the microspheres with a mixed solution of tannic acid and polyelectrolyte, and reacting to obtain the product;
or mixing the microspheres with a tannic acid solution, reacting, and mixing with a polyelectrolyte solution, wherein the mixing ratio of the tannic acid solution to the polyelectrolyte solution is 1:2-2:1 (such as 1:2, 2:3, 1:1, 3:2, 2:1, etc.), and reacting to obtain the microsphere.
Preferably, the solvent of the solution is a Tris-HCl buffer solution.
Preferably, the concentration of the tannic acid in the solution is 1 to 10g/L, e.g., 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, etc.; the concentration of the polyelectrolyte in the solution is 1-10g/L, such as 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L and the like, and other specific values in the above numerical range can be selected, and are not repeated herein.
Preferably, the temperature of the reaction is 15-35 ℃, such as 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃ and the like; the time is 2-24h, for example, 2h, 3h, 5h, 6h, 7h, 10h, 11h, 12h, 18h, 20h, 22h, 24h, etc., and other specific values within the above numerical range can be selected, which is not described herein again.
The temperature and time of the reaction determine whether tannic acid and polyelectrolyte can be supported on the surface of the microsphere to the maximum extent and the degree of excellence in stability.
Preferably, the method for immobilizing an enzyme in step (2) comprises: mixing the surface modified microspheres with an enzyme solution, and reacting to obtain the product.
Preferably, the concentration of the enzyme solution is 0.1-5g/L, such as 0.1g/L, 0.5g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, and the like, and other specific values in the numerical range can be selected, which are not described in detail herein.
Preferably, the temperature of the reaction is 15-35 ℃, such as 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃ and the like; the time is 1-8h, for example, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, etc., and other specific values within the above numerical range can be selected, which is not described herein again.
The temperature and time of the reaction determine whether the enzyme can be maximally supported on the surface of the polyelectrolyte and the degree of excellence in stability.
Preferably, step (3) further comprises: loading the free enzyme which is not fixed in the step (2) into a membrane module.
Preferably, step (3) further comprises: connecting the back washing and pulse reverse flow operation elements.
Preferably, the interval period of the back washing and the pulse reverse flow operation is 30min-12h, such as 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 10h, 12h and the like; a duration of 30-240s, e.g., 30s, 50s, 80s, 100s, 120s, 150s, 180s, 200s, 240s, etc.; the flux is 1.5-3 times of the filtration flux, such as 1.5 times, 2 times, 2.5 times, 3 times, and other specific values within the above numerical range can be selected, and are not described herein again.
In a third aspect, the present invention provides the use of a microsphere-membrane integrated enzyme reactor according to the first aspect for the preparation of an enzyme-catalysed product.
Such as glucose-fructose syrup, rare sugars, polypeptides, and the like.
Compared with the prior art, the invention has the following beneficial effects:
the microsphere-membrane integrated enzyme reactor combines an enzyme immobilization technology with a membrane reactor, and has the characteristics of high enzyme loading and high catalytic efficiency; the reactor also has the fluid mechanics advantages of a membrane reactor, such as short mass transfer path, small resistance and high separation efficiency, in the reactor, part of products transferred along the radial direction can directly permeate the membrane to enter the subsequent flow without flowing through the whole microsphere packed column, and the purposes of shortening the mass transfer path and reducing the mass transfer resistance are achieved; meanwhile, the enzyme can be trapped, so that the high utilization rate of the enzyme is realized; and the online separation of a macromolecular substrate and a micromolecular product can be realized, and the flow operation is simplified. In conclusion, the integrated enzyme reactor has the advantages of simple structure, high efficiency, energy conservation, high equipment integration level, small occupied area, low equipment investment and easy industrial and large-scale application.
Drawings
FIG. 1 is a schematic representation of surface modified microspheres of the present invention (wherein 1-tannic acid, 2-polyelectrolyte);
FIG. 2 is a schematic representation of enzyme-loaded microspheres of the present invention (wherein 1-tannic acid, 2-polyelectrolyte, 3-enzyme);
FIG. 3 is a schematic of a microsphere-membrane integrated enzyme reactor in accordance with the present invention (wherein 1-tannic acid, 2-polyelectrolyte, 3-enzyme, 4-membrane modules);
FIG. 4 is a graph of the results of the long-term operation stability of high fructose corn syrup produced by a microsphere-membrane integrated enzyme reactor.
Detailed Description
The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitation of the present invention.
The styrene microspheres referred to in the following examples and comparative examples were obtained from cangzhou baoyen adsorbent materials science and technology ltd, model number D301R, and particle size was 0.32-1.25 mm; the tubular membrane component is purchased from Xiamen Shidao membrane science and technology Limited, and has the model of Ceramem-0100, the membrane material of polyethersulfone and the membrane of ultrafiltration membrane (10 kDa); the flat-plate membrane component is prepared from polyethersulfone and microfiltration membrane, which are membrane materials of Beijing Bishui Water resources science and technology GmbH.
Example 1
This example provides a microsphere-membrane integrated enzyme reactor, which is prepared as follows:
(1) tannic acid and polyethyleneimine were dissolved in Tris-HCl buffer (pH 8.5, 10mM), respectively, to prepare 2g/L of tannic acid solution and polyethyleneimine solution, respectively, and then mixed at a volume ratio of 1: 1. Adding 5mL of a tannin and polyethyleneimine mixed solution into styrene microspheres, shaking at 20 ℃ for 18h, standing, removing an upper layer solution, adding deionized water, and washing until the solution is colorless to obtain surface-modified microspheres; the schematic diagram is shown in FIG. 1 (wherein 1-tannic acid, 2-polyelectrolyte).
(2) Preparing 2% glutaraldehyde solution, adding 0.1g of the surface-modified microspheres obtained in the step (1) into 10mL of glutaraldehyde solution, activating the mixture for 2 hours at 20 ℃ in a shaking table, and cleaning to remove unreacted solvent. Then adding 5mL of sucrose invertase solution with the concentration of 0.1g/L, and continuing to oscillate for 4 hours to obtain enzyme-loaded microspheres; the schematic diagram is shown in FIG. 2 (wherein 1-tannic acid, 2-polyelectrolyte, 3-enzyme).
(3) Filling the enzyme-loaded microspheres obtained in the step (2) into a tubular membrane module, wherein the filling amount of the microspheres accounts for 60% of the flow channel space of the membrane module, so as to obtain the microsphere-membrane integrated enzyme reactor; the schematic diagram is shown in FIG. 3 (wherein 1-tannic acid, 2-polyelectrolyte, 3-enzyme, 4-membrane module).
Example 2
This example provides a microsphere-membrane integrated enzyme reactor, which is prepared by the following steps:
(1) tannic acid and polyethyleneimine were dissolved in Tris-HCl buffer (pH 8.5, 10mM), respectively, to prepare 2g/L of tannic acid solution and polyethyleneimine solution, respectively. Adding 5mL of tannic acid solution into styrene microspheres, oscillating for 9 hours at 20 ℃, adding the polyethyleneimine solution, continuously oscillating for 9 hours, standing, removing the upper-layer solution, adding deionized water, and washing until the solution is colorless to obtain surface-modified microspheres;
(2) adding 5mL of sucrose invertase solution with the concentration of 1g/L into 0.1g of the surface-modified microspheres, and oscillating for 4 hours at 20 ℃ in a shaking table to obtain enzyme-loaded microspheres;
(3) filling the enzyme-loaded microspheres obtained in the step (2) into a flat-plate membrane module, wherein the filling amount of the microspheres accounts for 60% of the flow channel space of the membrane module, and thus obtaining the microsphere-membrane integrated enzyme reactor.
Example 3
This example provides a microsphere-membrane integrated enzyme reactor, which is prepared as follows:
(1) tannic acid and polyacrylamide were dissolved in Tris-HCl buffer (pH 8.5, 10mM) to prepare 2g/L of tannic acid solution and polyacrylamide solution, respectively. Firstly adding 5mL of tannic acid solution into styrene microspheres, oscillating for 9h at 20 ℃, then adding the polyacrylamide solution, continuously oscillating for 9h, standing, removing the upper layer solution, adding deionized water, and washing until the solution is colorless to obtain surface-modified microspheres;
(2) adding 5mL of sucrose invertase solution with the concentration of 1g/L into 0.1g of the surface-modified microspheres, and oscillating for 4 hours at 20 ℃ in a shaking table to obtain enzyme-loaded microspheres;
(3) and (3) filling the enzyme-loaded microspheres obtained in the step (2) into a tubular membrane module, wherein the filling amount of the microspheres accounts for 60% of the flow channel space of the membrane module, so as to obtain the microsphere-membrane integrated enzyme reactor.
Example 4
This example provides a microsphere-membrane integrated enzyme reactor, which is prepared by the following steps:
(1) tannic acid and polyallylamine were dissolved in Tris-HCl buffer (pH 8.5, 10mM) to prepare 5g/L tannic acid solution and polyallylamine solution, respectively. Firstly adding 5mL of tannic acid solution into styrene microspheres, oscillating for 7h at 20 ℃, then adding the polyacrylamide solution, continuously oscillating for 7h, standing, removing the upper layer solution, adding deionized water, and washing until the solution is colorless to obtain surface-modified microspheres;
(2) adding 5mL of dextran enzyme solution with the concentration of 2g/L into 0.1g of the surface-modified microspheres, and oscillating for 3 hours at 20 ℃ in a shaking table to obtain enzyme-loaded microspheres;
(3) and (3) filling the enzyme-loaded microspheres obtained in the step (2) into a tubular membrane module, wherein the filling amount of the microspheres accounts for 60% of the flow channel space of the membrane module, so as to obtain the microsphere-membrane integrated enzyme reactor.
Example 5
This example provides a microsphere-membrane integrated enzyme reactor, which is prepared by the following steps:
(1) tannic acid and polyacrylamide were dissolved in Tris-HCl buffer (pH 8.5, 10mM) to prepare a tannic acid solution and a polyacrylamide solution at a concentration of 7g/L, respectively. Adding 5mL of tannic acid solution into styrene microspheres, oscillating for 5 hours at 20 ℃, adding the polyacrylamide solution, continuously oscillating for 5 hours, standing, removing the upper layer solution, adding deionized water, and washing until the solution is colorless to obtain surface-modified microspheres;
(2) adding 5mL of a psicose 3-epimerase solution with the concentration of 3g/L into 0.1g of the surface-modified microspheres, and oscillating for 2 hours at 20 ℃ in a shaking table to obtain enzyme-loaded microspheres;
(3) and (3) filling the enzyme-loaded microspheres obtained in the step (2) into a tubular membrane module, wherein the filling amount of the microspheres accounts for 60% of the flow channel space of the membrane module, so as to obtain the microsphere-membrane integrated enzyme reactor.
Example 6
This example provides a microsphere-membrane integrated enzyme reactor, which is prepared by a method different from that of example 1 only in the step (3): and (3) filling all the enzyme-loaded microspheres obtained in the step (2) and the residual unloaded free enzymes into a tubular membrane module, wherein the filling amount of the microspheres accounts for 60% of the flow channel space of the membrane module, so as to obtain the microsphere-membrane integrated enzyme reactor.
Example 7
This example provides a microsphere-membrane integrated enzyme reactor, which is prepared by a method different from that of example 1 only in the step (3): and (3) filling the enzyme-loaded microspheres obtained in the step (2) into a tubular membrane module, wherein the filling amount of the microspheres accounts for 45% of the flow channel space of the membrane module, so as to obtain the microsphere-membrane integrated enzyme reactor.
Example 8
This example provides a microsphere-membrane integrated enzyme reactor, which is prepared by a method different from that of example 1 only in the step (3): filling the enzyme-loaded microspheres obtained in the step (2) into a tubular membrane component, wherein the filling amount of the microspheres accounts for 75% of the flow channel space of the membrane component, so as to obtain the microsphere-membrane integrated enzyme reactor.
Comparative example 1
This comparative example provides a microsphere-membrane integrated enzyme reactor, the preparation method of which differs from example 1 only in that the microspheres used are not surface modified, as follows:
(1) adding 5mL of sucrose invertase solution with the concentration of 0.1g/L into 0.1g of styrene microspheres, and oscillating for 4h at 20 ℃ to obtain enzyme-loaded microspheres;
(3) and (3) filling the enzyme-loaded microspheres obtained in the step (2) into a tubular membrane module, wherein the filling amount of the microspheres accounts for 60% of the flow channel space of the membrane module, so as to obtain the microsphere-membrane integrated enzyme reactor.
Comparative example 2
This comparative example provides a microsphere-membrane integrated enzyme reactor, the preparation method of which differs from example 1 only in that the enzyme used is a free enzyme, as follows:
and filling 5mL of sucrose invertase solution with the concentration of 0.1g/L into the tubular membrane module to obtain the microsphere-membrane integrated enzyme reactor.
Comparative example 3
The comparative example provides a microsphere packed bed reactor, the preparation method of which is as follows:
(1) tannic acid and polyethyleneimine were dissolved in Tris-HCl buffer (pH 8.5, 10mM), respectively, to prepare 2g/L of tannic acid solution and polyethyleneimine solution, respectively, and then mixed at a volume ratio of 1: 1. Adding 5mL of a tannin and polyethyleneimine mixed solution into styrene microspheres, shaking at 20 ℃ for 18h, standing, removing an upper layer solution, adding deionized water, and washing until the solution is colorless to obtain surface-modified microspheres;
(2) preparing 2% glutaraldehyde solution, adding 0.1g of the surface-modified microspheres obtained in the step (1) into 10mL of glutaraldehyde solution, activating the mixture for 2 hours at 20 ℃ in a shaking table, and cleaning to remove unreacted solvent. Then adding 5mL of sucrose invertase solution with the concentration of 0.1g/L, and continuing to oscillate for 4 hours to obtain enzyme-loaded microspheres;
(3) and (3) filling the enzyme-loaded microspheres obtained in the step (2) into a glass column, wherein the filling amount of the microspheres accounts for 60% of the flow channel space of the glass column, so as to obtain the microsphere packed bed reactor.
Application examples 1-3 and application examples 6-8
Six methods for producing high fructose corn syrup by utilizing a microsphere-membrane integrated enzyme reactor are provided, which specifically comprise the following steps:
the feed liquid tank was filled with an aqueous sucrose solution at a concentration of 150mM, a feed flow rate of 0.2mL/min and a reaction temperature of 35 ℃ to the microsphere-membrane integrated enzyme reactor prepared in examples 1 to 3 and 6 to 8, and the aqueous sucrose solution was continuously pumped into the reactor, hydrolyzed by sucrose invertase, and the membrane permeate was introduced into the product tank to collect the product. And after the reactor continuously runs for 10 hours, backwashing is carried out for 1min, and the backwashing flux is 1.5 times of the material liquid flux.
Application example 4
The method for producing the micromolecule dextran by utilizing the microsphere-membrane integrated enzyme reactor comprises the following steps:
dextran solution (average molecular weight 4 ten thousand) is filled in a feed liquid tank, the concentration is 50g/L, the feeding flow rate is controlled to be 0.2mL/min, the reaction temperature is 35 ℃, the dextran solution is continuously pumped into the microsphere-membrane integrated enzyme reactor prepared in the embodiment 4, after the hydrolysis by the dextranase, membrane permeate enters a product tank, and a product is collected. And after the reactor continuously runs for 3 hours, backwashing is carried out for 3min, and the backwashing flux is 1.5 times of the feed liquid flux.
Application example 5
The method for producing the psicose by using the microsphere-membrane integrated enzyme reactor comprises the following steps:
the feed liquid tank was filled with a fructose aqueous solution at a concentration of 20g/L, the feed flow rate was controlled at 0.2mL/min, the reaction temperature was 35 ℃ and fructose solution was continuously pumped into the microsphere-membrane integrated enzyme reactor prepared in example 5, and after hydrolysis by psicose 3-epimerase, the membrane permeate entered the product tank to collect the product. And after the reactor continuously runs for 3 hours, backwashing is carried out for 3min, and the backwashing flux is 1.5 times of the feed liquid flux.
Comparative application examples 1 to 3
Three methods for producing high fructose corn syrup by utilizing a microsphere-membrane integrated enzyme reactor are provided, which specifically comprise the following steps:
the feed liquid tank was filled with an aqueous sucrose solution at a concentration of 150mM, the feed flow rate was controlled at 0.2mL/min, the reaction temperature was 35 ℃ and an aqueous sucrose solution was continuously pumped into the microsphere-membrane integrated enzyme reactor prepared in comparative examples 1 to 3, and after hydrolysis by sucrose invertase, the membrane permeate was fed into the product tank to collect the product. And after the reactor continuously runs for 10 hours, backwashing is carried out for 1min, and the backwashing flux is 1.5 times of the material liquid flux.
Comparative application example 4
A method for producing high fructose corn syrup by using a microsphere-membrane integrated enzyme reactor is provided, which is different from the application example 1 only in that the reactor is continuously operated but not subjected to back washing operation.
Evaluation test:
(1) enzyme loading per unit of microsphere
And respectively measuring the protein contents of the original enzyme solution, the fixed supernatant and the washing solution by using a Coomassie brilliant blue method. The enzyme loading of the unit microsphere can be obtained according to the mass conservation.
Figure BDA0002976732730000131
Wherein, C0、CAIAnd CWRespectively representing the concentrations of protein in the original enzyme solution, the fixed supernatant and the washing solution, mg/ml; v.v. of0、VAIAnd VWRespectively representing the volumes of original enzyme solution, fixed supernatant and washing solution, ml; m isMicRepresents the mass of the microspheres, mg. The results are shown in Table 1.
(2) Monitoring of operating pressure
In the application example and the comparative application example, a certain pressure needs to be applied to the reactor by using a vacuum pump during the enzyme catalysis reaction so as to ensure that the flow rate of the material is constant, and the monitoring result of the operation pressure is shown in table 1.
(3) Conversion of product
The reactor maintains constant feed liquid flux, and the feed liquid inlet volume is the same as the product outflow volume, so the product conversion rate can be calculated according to the following formula.
Figure BDA0002976732730000141
Wherein, α represents the product conversion; c0、C1Representing the reactor inlet substrate concentration and the reactor outlet product concentration, respectively.
The substrate and product concentration measuring instrument is HPLC, and the type of chromatographic column used is Bio-Rad
Figure BDA0002976732730000142
HPX-87N, the mobile phase is ultrapure water (application examples 1-3, 6-8, comparative application example 1-4); the chromatographic column is of Sugar-Pak column type, and the mobile phase is ultrapure water (A)Application example 5); the results of measuring the sugar concentration by the phenol-sulfuric acid method (application example 4) are shown in table 1.
TABLE 1
Figure BDA0002976732730000143
Figure BDA0002976732730000151
As can be seen from the data in Table 1: compared with the comparative application example 1, the enzyme loading amount of the microspheres can be obviously improved after the microspheres are modified, so that the performance of the microsphere-membrane integrated enzyme reactor is improved.
Compared with the free enzyme membrane reactor, the application example 1 and the comparative application example 2 show that the enzyme utilization rate and the enzyme reusability can be obviously improved by constructing the microsphere-membrane integrated enzyme reactor after immobilizing the enzyme, so that not only is the substrate conversion rate improved, but also the enzyme consumption in the production process is reduced, and the production cost is reduced. Compared with free enzyme, the immobilized enzyme enlarges the distribution range of the enzyme in the reactor, enlarges the effective reaction area in the reactor, lightens the membrane pollution caused by the deposition of the free enzyme on the membrane surface, reduces the operation pressure of the reactor and reduces the energy consumption of the reactor.
Compared with the application example 1 and the comparative application example 3, the microsphere-membrane integrated enzyme reactor has the advantages that the conversion rate is slightly reduced but the operation pressure is obviously reduced and the energy consumption is obviously reduced due to the shortened mass transfer path compared with the microsphere packed bed enzyme reactor. This is because in the microsphere-membrane integrated enzyme reactor, substances can flow into the subsequent flow path from the bottom outlet of the reactor through axial transfer; the product can be transferred along the radial direction, and the product after enzymolysis directly permeates the membrane to enter the subsequent flow without flowing through the whole microsphere packed column, thereby shortening the mass transfer path and reducing the mass transfer resistance.
Compared with the comparative application example 4, the application example 1 shows that the continuous operation of the reactor but no back washing and pulse reverse flow operation can cause the continuous rise of the operating pressure of the reactor, the increase of the stacking density of the microspheres and the reduction of the utilization rate of the immobilized enzyme; on the other hand, the membrane pollution is increased continuously, the membrane flux is reduced rapidly, and finally the production efficiency of the reactor is reduced. The application of the back washing and pulse reverse flow technology not only ensures that the immobilized enzyme maintains a relatively stable state, but also can remove pollutants adsorbed on the membrane surface/membrane holes in time, slow down the reduction rate of the membrane flux and effectively improve the long-term operation stability of the reactor.
According to the results of application example 6, the purpose of simultaneously utilizing immobilized enzyme and free enzyme can be achieved by adjusting the type of the membrane module, the effective enzyme content and the enzyme utilization rate in the reactor are improved, and the production efficiency of the reactor is further improved.
In addition, as can be seen from the application example 4, the online separation of the macromolecular substrate and the micromolecular product can be realized by optimizing the membrane module, the molecular weight of the product can be regulated and controlled, the process flow is greatly simplified, the energy consumption is reduced, and the equipment investment cost is reduced.
Application example 1 compared with application examples 7-8, it is known that the filling amount of the microspheres has an important influence on the pressure and the conversion rate of the reactor, and that the filling amount is too low and too high, which has a negative influence on the conversion rate of the product.
(4) Stability evaluation of microsphere-Membrane Integrated enzyme reactor
The evaluation method comprises the following steps: the reactor was constructed according to the method described in application example 6, operated continuously and the product concentration was detected, and the conversion was calculated to evaluate the long-term operation stability of the reactor.
The long-term operation stability result of the high fructose corn syrup produced by the microsphere-membrane integrated enzyme reactor is shown in figure 4, the conversion rate of the reactor can be kept stable within 36h of continuous operation, the reactor does not have a descending trend, and the reactor is expected to operate stably for a longer time.
The applicant states that the present invention is illustrated by the above examples, but the present invention is not limited to the above examples, i.e., it is not meant to be dependent on the above examples to practice the present invention. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are all within the protection scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims (23)

1. A microsphere-membrane integrated enzyme reactor is characterized by comprising enzyme-carrying microspheres and a membrane component;
the enzyme-loaded microsphere comprises a microsphere, a modification layer on the surface of the microsphere and enzyme loaded on the modification layer;
the microsphere-membrane integrated enzyme reactor also comprises a back flushing and pulse reverse flow operation element;
the filling amount of the enzyme-carrying microspheres is 50-70% of the flow channel space of the membrane module;
the material of the modified layer comprises tannic acid and polyelectrolyte, and the polyelectrolyte comprises any one or the combination of at least two of polyethyleneimine, polyallylamine and salt thereof, polyarginine or polyglutamic acid.
2. The microsphere-membrane integrated enzyme reactor of claim 1, wherein the membrane module comprises any one of a tubular membrane module, a flat plate membrane module or a hollow fiber membrane module or a combination of at least two thereof.
3. The microsphere-membrane integrated enzyme reactor of claim 2, wherein the membrane module comprises a tubular membrane module.
4. The microsphere-membrane integrated enzyme reactor of claim 1, wherein the membrane material in the membrane module comprises any one of polysulfone, polyethersulfone, sulfonated polyethersulfone, polyacrylonitrile, polyvinylidene fluoride, polyvinyl alcohol, or regenerated cellulose or a combination of at least two thereof.
5. The microsphere-membrane integrated enzyme reactor of claim 1, wherein the membrane in the membrane module comprises a microfiltration membrane or an ultrafiltration membrane.
6. The microsphere-membrane integrated enzyme reactor of claim 1, wherein the mass ratio of tannic acid to microspheres is from 1:5 to 1: 20.
7. The microsphere-membrane integrated enzyme reactor of claim 1, wherein the mass ratio of the tannic acid to the polyelectrolyte is from 1:2 to 2: 1.
8. The microsphere-membrane integrated enzyme reactor of claim 1, wherein the enzyme is supported on the modified layer by physical adsorption or chemical covalent bond.
9. The microsphere-membrane integrated enzyme reactor of claim 1, wherein the enzyme comprises a hydrolase, an oxidoreductase, or an isomerase.
10. The microsphere-membrane integrated enzyme reactor of claim 1, wherein the enzyme comprises psicose isomerase, rhamnose isomerase, galactosidase, L-arabinose isomerase, xylitol dehydrogenase, sucrase, amylase, protease, cellulase or dextranase.
11. The microsphere-membrane integrated enzyme reactor of claim 1, wherein the particle size of the microspheres is from 0.02mm to 3 mm.
12. The microsphere-membrane integrated enzyme reactor of claim 1, wherein the material of the microspheres comprises any one of polystyrene, polyglycidyl methacrylate, glucomannan, chitosan, or sodium alginate, or a combination of at least two thereof.
13. The method of making a microsphere-membrane integrated enzyme reactor according to any one of claims 1 to 12, wherein the method comprises the steps of:
(1) modifying the microspheres to obtain surface-modified microspheres;
(2) carrying out enzyme immobilization on the surface-modified microspheres obtained in the step (1) to obtain enzyme-loaded microspheres;
(3) loading the enzyme-loaded microspheres obtained in the step (2) into a membrane module to obtain the microsphere-membrane integrated enzyme reactor;
the step (3) further comprises: connecting the back flushing and pulse reverse flow operation elements.
14. The method for preparing a microsphere-membrane integrated enzyme reactor according to claim 13, wherein the method for modifying microspheres in step (1) comprises: mixing the microspheres with a mixed solution of tannic acid and polyelectrolyte, and reacting to obtain the product;
or mixing the microspheres with a tannic acid solution, reacting, and mixing with a polyelectrolyte solution, wherein the mixing ratio of the tannic acid solution to the polyelectrolyte solution is 1:2-2:1, and reacting to obtain the microsphere.
15. The method of making a microsphere-membrane integrated enzyme reactor of claim 14, wherein the solvent of the solution is Tris-HCl buffer solution.
16. The method of making a microsphere-membrane integrated enzyme reactor of claim 14, wherein the concentration of tannic acid in the solution is from 1 to 10 g/L; the concentration of the polyelectrolyte in the solution is 1-10 g/L.
17. The method of claim 14, wherein the reaction is carried out at a temperature of 15-35 ℃ for a period of 2-24 hours.
18. The method for preparing a microsphere-membrane integrated enzyme reactor according to claim 13, wherein the enzyme immobilization method of the step (2) comprises: mixing the surface modified microspheres with an enzyme solution, and reacting to obtain the product.
19. The method of making a microsphere-membrane integrated enzyme reactor of claim 18, wherein the concentration of the enzyme solution is 0.1 to 5 g/L.
20. The method of claim 18, wherein the reaction is at a temperature of 15-35 ℃ for 1-8 hours.
21. The method of making a microsphere-membrane integrated enzyme reactor of claim 13, wherein step (3) further comprises: loading the free enzyme which is not fixed in the step (2) into a membrane module.
22. The method of claim 13, wherein the back-flushing and pulsed counter-current operation are separated by a period of 30min to 12h, a duration of 30 to 240s, and a flux of 1.5 to 3 times the filtration flux.
23. Use of a microsphere-membrane integrated enzyme reactor according to any one of claims 1 to 12 for the preparation of an enzyme catalyzed product.
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