CN112063612A

CN112063612A - Separated type multienzyme-MOF (Metal organic framework) microcapsule and preparation method thereof

Info

Publication number: CN112063612A
Application number: CN202010804688.4A
Authority: CN
Inventors: 罗志刚; 齐亮
Original assignee: South China University of Technology SCUT; Guangzhou Institute of Modern Industrial Technology
Current assignee: South China University of Technology SCUT; Guangzhou Institute of Modern Industrial Technology
Priority date: 2020-08-12
Filing date: 2020-08-12
Publication date: 2020-12-11
Anticipated expiration: 2040-08-12
Also published as: CN112063612B

Abstract

The invention discloses a separated multienzyme-MOF microcapsule and a preparation method thereof, and the preparation method comprises the following steps: (1) preparing a compound of the MOF material and glucose oxidase by an autogrowth method; (2) dissolving horseradish catalase, acrylamide, sodium bisulfite and potassium persulfate in deionized water, and uniformly mixing by ultrasonic waves to obtain a water phase; dispersing the compound prepared in the step (1) in long-chain alkane to be used as an oil phase; mixing oil and water phases by a high-speed shearing machine to obtain stable Pickering emulsion; and then, sealing the Pickering emulsion, introducing nitrogen to remove oxygen to initiate self-polymerization reaction in an aqueous phase, and obtaining the separated multienzyme-MOF microcapsule by centrifugal washing and vacuum drying. The method has mild conditions and simple and convenient operation, and greatly keeps the activity of the enzyme; the separated multienzyme structure with coexisting 'muramidase' and 'core enzyme' is prepared, and the efficiency of the cascade catalytic reaction is improved.

Description

Separated type multienzyme-MOF (Metal organic framework) microcapsule and preparation method thereof

Technical Field

The invention relates to the field of micro-nano materials, in particular to a separated multienzyme-MOF (metal organic framework) microcapsule based on surface self-growth and a Pickering emulsion system and a preparation method thereof.

Background

The importance of the multi-enzyme catalysis technology is mainly reflected in the biological synthesis process. The method has the advantages that the rational design is carried out on the catalysis process, the cascade catalysis is realized in a certain space, the reaction steps and the initial investment of reaction equipment can be effectively reduced, the loss of intermediate products in the separation process is reduced, and a green and efficient way is provided for realizing product diversification, energy conservation and emission reduction. However, due to the difference and complexity of the multi-enzyme system structure, how to realize the efficient synergy among multiple enzymes, the controllable transfer of substrates and intermediate products and the simple and convenient reuse of multiple enzymes are important subjects in the research of multi-enzyme immobilization systems.

In 2000, researchers firstly use electrostatic acting force to fix glucose oxidase and horseradish peroxidase in a layered manner, so that the preparation of a separated multi-enzyme catalytic system is realized. Subsequently, researchers have constructed different cellular-type multienzyme catalytic systems by using polymer vesicle coating and other technologies and applied the cellular-type multienzyme catalytic systems to the field of biocatalysis, and the results show that cellular immobilization of enzymes can improve the cascade catalytic efficiency to a certain extent compared with random co-immobilization of multienzymes. This is mainly due to the fact that regionalized separation of enzymes can effectively reduce the inhibition of the interaction between enzyme molecules. However, the current enzyme immobilization methods such as covalent bonding, electrostatic adsorption or cross-linking polymerization require modification treatment on enzyme or carrier in advance, inevitably increase the complexity of the preparation process, and are difficult to form controllable separated multi-enzyme configuration.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, and designs a multienzyme microcapsule which directionally distributes double enzymes in a capsule shell and a capsule cavity respectively on the basis of a Pickering emulsion and MOF surface self-growth system to achieve the aim of cascade catalysis, so as to realize the technical problems of efficient cooperation among the multienzymes and controllable transfer of substrates and intermediate products in a multistage reaction.

The invention also aims to provide a separated multienzyme MOF microcapsule based on surface self-growth and Pickering emulsion system prepared by the method.

The purpose of the invention is realized by the following technical scheme:

a preparation method of a separated type multienzyme-MOF microcapsule comprises the following steps:

(1) preparing a complex of MOF material (ZIF-8) and Glucose Oxidase (GOD) by an autogrowth method;

(2) preparing a separated multienzyme-MOF microcapsule by using a Pickering emulsion system: dissolving horseradish catalase (HRP), acrylamide, sodium bisulfite and potassium persulfate in deionized water, and uniformly mixing by ultrasonic waves to obtain a water phase; dispersing the compound prepared in the step (1) in long-chain alkane to be used as an oil phase; mixing oil and water phases by a high-speed shearing machine to obtain stable Pickering emulsion; and then, sealing the Pickering emulsion, introducing nitrogen to remove oxygen to initiate self-polymerization reaction in an aqueous phase, and obtaining the separated multienzyme-MOF microcapsule by centrifugal washing and vacuum drying.

Preferably, the preparation of the complex of MOF material and glucose oxidase of step (1): preparing aqueous solution of glucose oxidase, zinc salt and 2-methylimidazole, stirring, mixing, reacting, centrifuging, washing and collecting the product.

Preferably, the mass fraction of the glucose oxidase aqueous solution in the step (1) is as follows: 3-5 mg/mL.

Preferably, the mass fraction of the zinc salt aqueous solution is: 40-65 mg/mL.

Preferably, the mass fraction of the 2-methylimidazole water solution is as follows: 80-120 mg/mL.

Preferably, the volume ratio of the aqueous glucose oxidase solution to the aqueous zinc salt solution to the aqueous 2-methylimidazole solution is: (1-2): (1-3): (5-10).

Preferably, the reaction temperature in the step (1) is 25-40 ℃, the reaction time is 0.5-2h, and the stirring speed is 100-120 rpm.

Preferably, the centrifugation speed in the centrifugation washing is 5000-8000rpm, the centrifugation time is 3-5min, and the washing times are 3-5.

Preferably, the mass fractions of horseradish catalase, acrylamide, sodium bisulfite and potassium persulfate in the aqueous phase in the step (2) are respectively 5-8mg/mL, 0.3-0.5mg/mL, 0.02-0.04mg/mL and 0.05-0.1 mg/mL.

Preferably, the mass fraction of the compound prepared in the step (1) in the long-chain alkane is 10-30 mg/mL.

Preferably, the volume ratio of the addition of the aqueous phase to the oil phase is (0.5-1): (2-5).

Preferably, the self-polymerization reaction in the step (2) is carried out under the conditions of nitrogen introduction and oxygen removal for 10-30min, the reaction temperature is 30-45 ℃ and the reaction time is 3-6 h.

Preferably, the time of the ultrasound in the step (2) is 5-10 min; the ultrasonic frequency is 20KHZ-60 KHZ; the high-speed shearing condition is that the rotating speed is 8000-10000rpm, and the time is 1-3 min.

Preferably, the centrifugal washing conditions in step (2) are a centrifugal speed of 3000-.

Preferably, the Zn salt in the step (2) is one of zinc nitrate, zinc chloride and zinc sulfate.

Preferably, the long-chain alkane is C6-C20 alkane, and more preferably one of heptane, decane, dodecane and tetradecane.

A separated multienzyme-MOF microcapsule based on surface self-growth and Pickering emulsion system, wherein glucose oxidase is distributed on the capsule wall and horseradish peroxidase is distributed in the capsule cavity, and the microcapsule is prepared by any one of the methods.

The invention is based on the fact that the precursor of the MOF material can realize self-growth on the surface, namely under mild conditions, the metal ions and organic ligands of the MOF material can spontaneously combine on the surface of the enzyme to form a finished body. Therefore, the surface self-growth method realizes effective immobilization of an enzyme while preserving the structure and properties of the enzyme. On the basis, the MOF material carried by the enzyme is used as a basic particle to wrap the second enzyme again by using a particle copolymerization effect under a Pickering emulsion system, so that a separated multi-enzyme structure with coexisting 'wall enzyme' and 'core enzyme' is formed.

Compared with the prior art, the invention has the following advantages and effects:

1) the preparation of the enzyme-carrying particle stabilizer does not need a complex process, has mild conditions and simple and convenient operation, and greatly keeps the activity of the enzyme.

2) The surface self-growth and Pickering emulsion particle copolymerization effect are utilized to form a separated multienzyme structure with coexisting 'wall enzyme' and 'core enzyme', and the efficiency of the cascade catalytic reaction is improved.

Drawings

FIG. 1 is a bar graph of the absorbance of the final product of a tandem catalysis experiment using partitioned multienzyme-MOF microcapsules in various examples.

Figure 2 bar graph of enzyme activity stability test of partitioned multienzyme-MOF microcapsules in example 1.

FIG. 3 bar graph of viability stability test of free enzyme (not immobilized).

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. The enzymes used in the present invention were purchased from sigma.

Example 1

(1) 12mg GOD was dissolved in 4mL of deionized water, 160mg zinc nitrate was dissolved in 4mL of deionized water, 1.6g 2-methylimidazole was dissolved in 20mL of deionized water, and the three solutions were mixed and stirred at 100rpm for 0.5h at 20 ℃ at a constant speed, at which time the solution gradually turned milky white. Subsequently, the product was washed 3 times by centrifugation at 5000rpm for 3min using deionized water.

(2) 25mg of HRP, 1.5mg of acrylamide, 0.1mg of sodium bisulfite and 0.25mg of potassium persulfate were dissolved in 5mL of deionized water, and uniformly mixed by sonication at 20 ℃ and 20KHZ frequency for 5min to obtain an aqueous phase. 100mg of ZIF-8@ GOD prepared in step (1) was dispersed in 10mL heptane as the oil phase. Mixing the water phase and the oil phase according to the volume ratio of 0.5:2, and homogenizing for 1min at the rotating speed of 8000rpm by a high-speed shearing machine to obtain the stable Pickering emulsion. Pouring the emulsion into a 50mL three-neck round-bottom flask with a condenser tube, introducing nitrogen to remove oxygen for 10min, and then initiating self-polymerization at 30 ℃ for reaction for 3 h. Subsequently, the product was centrifuged and washed 3 times with ethanol at 3000rpm for 3min and dried under vacuum to obtain cellular-MOF microcapsules with glucose oxidase as the wall and horseradish peroxidase in the cavity.

Example 2

(1) 20mg GOD was dissolved in 4mL of deionized water, 390mg zinc nitrate was dissolved in 6mL of deionized water, 4.8g 2-methylimidazole was dissolved in 40mL of deionized water, and the three solutions were mixed and stirred at 120rpm for 2 hours at 40 ℃ at a constant speed, at which time the solution gradually turned milky white. Subsequently, the product was washed 5 times by centrifugation at 8000rpm for 5min using deionized water.

(2) 40mg of HRP, 2.5mg of acrylamide, 0.2mg of sodium bisulfite and 0.5mg of potassium persulfate were dissolved in 5mL of deionized water, and uniformly mixed by sonication at 30 ℃ and 60KHZ frequency for 10min to obtain an aqueous phase. 300mg of ZIF-8@ GOD prepared in step (1) was dispersed in 10mL of decane as an oil phase. Mixing the water phase and the oil phase according to the volume ratio of 1:5, and homogenizing for 3min at the rotating speed of 10000rpm by a high-speed shearing machine to obtain the stable Pickering emulsion. The emulsion is poured into a 50mL three-mouth round-bottom flask with a condenser tube, nitrogen is introduced to remove oxygen for 30min, and then self-polymerization is initiated at 45 ℃ to react for 6 h. Subsequently, the product was centrifuged and washed 5 times with ethanol at 5000rpm for 5min and dried under vacuum to obtain cellular-MOF microcapsules with glucose oxidase as the wall and horseradish peroxidase in the cavity.

Example 3

(1) 18mg GOD was dissolved in 4mL of deionized water, 300mg zinc nitrate was dissolved in 5mL of deionized water, 3.6g 2-methylimidazole was dissolved in 30mL of deionized water, and the three solutions were mixed and stirred at 25 ℃ at 120rpm for 1 hour, at which time the solution gradually turned milky white. Subsequently, the product was washed 3 times by centrifugation at 5000rpm for 3min using deionized water.

(2) 30mg of HRP, 2mg of acrylamide, 0.15mg of sodium bisulfite and 0.3mg of potassium persulfate were dissolved in 5mL of deionized water and uniformly mixed by ultrasonic waves at 25 ℃ and a frequency of 40KHZ for 10min to serve as an aqueous phase. 200mg of ZIF-8@ GOD prepared in step (1) was dispersed in 10mL of dodecane as the oil phase. Mixing the water phase and the oil phase according to the volume ratio of 1:2, and homogenizing for 2min at the rotating speed of 9000rpm by a high-speed shearing machine to obtain the stable Pickering emulsion. The emulsion is poured into a 50mL three-neck round-bottom flask with a condenser tube, nitrogen is introduced to remove oxygen for 20min, and then self-polymerization is initiated at 35 ℃ to react for 4 h. Subsequently, the product was centrifuged and washed 3 times with ethanol at 4000rpm for 5min and dried under vacuum to obtain cellular-MOF microcapsules with glucose oxidase as the wall and horseradish peroxidase in the cavity.

Example 4

In this example, no enzyme was added in steps (1) and (2), and the other steps and conditions were the same as in example 2.

Example 5

In this example, 20mg GOD enzyme and 40mg HRP enzyme were added in step (1), no enzyme was added in step (2), and other steps and conditions were the same as in example 2.

Example 6

In this example, no enzyme was added in step (1), and the enzymes added in step (2) were 20mg of GOD enzyme and 40mg of HRP enzyme, and other steps and conditions were the same as in example 2.

Determination of Material Properties

(1) Measurement of catalytic efficiency

Glucose Oxidase (GOD) catalyzes the binding of glucose to oxygen to produce gluconic acid and hydrogen peroxide. ABTS catalyzed by hydrogen peroxide with horseradish catalase (HRP)^2-Oxidation to ABTS^-And can be detected by an ultraviolet-visible spectrophotometer at the wavelength of 403 nm.

The operation method comprises the following steps: 2mL (1mM) of glucose was added to 1mL of phosphate buffered saline (10mM, pH 7.4), 0.75mg of the preparation material of example 1 and 532mM of ABTS were added to the solution, and the mixture was stirred at room temperature and mixed well to initiate a reaction. The reacted solution was then centrifuged at 10000rpm for 3min to remove residual material. And using UV-2450 type ultraviolet-visible spectrophotometer at 403nm to ABTS^-And (6) detecting. Same trueThe experiment was performed 5 more times, except that the materials prepared in the different examples were replaced, and the other steps were unchanged.

As can be seen from fig. 1, the properties of the materials of examples 1,2,3 differ due to the change of the preparation process. But overall, the product ABTS^-The absorbance of (A) is 0.7 or more, indicating that the catalysis is preferable. Examples 5 and 6 show that the catalytic efficiency of the enzyme is reduced in the non-partitioned material because no space is available between the multiple enzymes to form a proper channel for the transfer of the intermediate product. In example 4, no formation of the final product was detected, since no enzyme was present and the structure itself did not produce catalytic efficacy.

(2) Stability test

Resistance to enzymatic hydrolysis

5mg of the material prepared in example 1 was dissolved in a phosphate buffer (10mM, pH 7.4), 2mg of trypsin was added, the mixture was stirred at 37 ℃ and kept warm for 30min, and the residue was washed 3 times to remove the residual trypsin and tested for enzymatic activity. The untreated sample of example 1 served as a blank.

Ability to resist metal complexation

5mg of the material prepared in example 1 was dissolved in a phosphate buffer (10mM, pH 7.4, containing 1 wt% EDTA), stirred at 37 ℃ and kept at that temperature for 30min, the residue was washed 3 times to remove the residual EDTA and tested for enzymatic activity. The untreated sample of example 1 served as a blank.

Free enzyme stability test. 20mg of GOD enzyme and 40mg of HRP combined enzyme are used to replace the immobilized enzyme in example 1, and other steps and conditions are not changed.

As can be seen from FIGS. 2 and 3, the catalytic efficiency of the immobilized enzyme is still maintained at a higher level when the enzyme is treated with the protease and the metal complex than the free enzyme, which indicates that the MOF material can serve as an outer shell to protect the enzyme from the protease and the metal complex.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A preparation method of a separated type multienzyme-MOF microcapsule is characterized by comprising the following steps:

(1) preparing a compound of the MOF material and glucose oxidase by an autogrowth method;

(2) preparing a separated multienzyme-MOF microcapsule by using a Pickering emulsion system: dissolving horseradish catalase, acrylamide, sodium bisulfite and potassium persulfate in deionized water, and uniformly mixing by ultrasonic waves to obtain a water phase; dispersing the compound prepared in the step (1) in long-chain alkane to be used as an oil phase; mixing oil and water phases by a high-speed shearing machine to obtain stable Pickering emulsion; and then, sealing the Pickering emulsion, introducing nitrogen to remove oxygen to initiate self-polymerization reaction in an aqueous phase, and obtaining the separated multienzyme-MOF microcapsule by centrifugal washing and vacuum drying.

2. The method of claim 1, wherein the step (1) of preparing the complex of MOF material and glucose oxidase comprises: preparing aqueous solution of glucose oxidase, zinc salt and 2-methylimidazole, stirring, mixing, reacting, centrifuging, washing and collecting the product.

3. The method according to claim 2, wherein the mass fraction of the aqueous glucose oxidase solution in step (1) is as follows: 3-5 mg/mL;

the zinc salt aqueous solution comprises the following components in percentage by mass: 40-65 mg/mL;

the 2-methylimidazole water solution comprises the following components in percentage by mass: 80-120 mg/mL;

the volume ratio of the glucose oxidase aqueous solution to the zinc salt aqueous solution to the 2-methylimidazole aqueous solution is as follows: (1-2): (1-3): (5-10).

4. The method as claimed in claim 3, wherein the reaction temperature in step (1) is 25-40 ℃, the reaction time is 0.5-2h, and the stirring speed is 100-120 rpm;

the centrifugal speed in the centrifugal washing is 5000-.

5. The method according to any one of claims 1 to 4, wherein the mass fractions of horseradish catalase, acrylamide, sodium bisulfite and potassium persulfate in the aqueous phase in the step (2) are respectively 5-8mg/mL, 0.3-0.5mg/mL, 0.02-0.04mg/mL and 0.05-0.1 mg/mL;

the mass fraction of the compound prepared in the step (1) in the long-chain alkane is 10-30 mg/mL;

the volume ratio of the water phase to the oil phase is (0.5-1): (2-5).

6. The method according to claim 5, wherein the self-polymerization reaction in the step (2) is carried out under the conditions of nitrogen introduction and oxygen removal for 10-30min, the reaction temperature is 30-45 ℃ and the reaction time is 3-6 h.

7. The method according to claim 6, wherein the time of the ultrasound of step (2) is 5-10 min; the ultrasonic frequency is 20KHZ-60 KHZ; the high-speed shearing condition is that the rotating speed is 8000-10000rpm, and the time is 1-3 min.

8. The method as claimed in claim 7, wherein the centrifugal washing conditions in step (2) are a centrifugal speed of 3000-5000rpm, a centrifugal time of 3-5min, and a washing frequency of 3-5 times.

9. The method according to any one of claims 1 to 4, wherein the Zn salt in the step (2) is one of zinc nitrate, zinc chloride and zinc sulfate; the long-chain alkane is C6-C20 alkane.

10. A divided multi-enzyme MOF microcapsule prepared by the method of any one of claims 1 to 9, wherein the glucose oxidase is distributed in the wall of the capsule and the horseradish peroxidase is distributed in the cavity of the capsule.