阅读说明：本技术 固定化酶及其制备方法和应用 (Immobilized enzyme and preparation method and application thereof ) 是由 刘婧 吴成亮 张伟 高先亭 于 2021-08-13 设计创作，主要内容包括：本发明公开了一种固定化酶及其制备方法和应用,该固定化酶包括：水凝胶中空纤维管,所述水凝胶中空纤维管具有纤维网状的管壁和所述管壁围合形成的管腔；以及酶,其中,所述酶固定于所述水凝胶中空纤维管的管壁内。利用同轴打印技术,实现将酶固定于水凝胶中空纤维管中,回收简单方便,扩展了固定化酶的应用范围,且能够有效提高酶与底物的结合,提高酶的反应效率。该固定化酶可广泛用于化妆品、食品、环保和医药等领域,具有广泛应用前景。(The invention discloses an immobilized enzyme and a preparation method and application thereof, wherein the immobilized enzyme comprises the following components: the hydrogel hollow fiber tube is provided with a fiber net-shaped tube wall and a tube cavity formed by enclosing the tube wall; and an enzyme, wherein the enzyme is immobilized within the wall of the hydrogel hollow fiber tube. The enzyme is fixed in the hydrogel hollow fiber tube by utilizing the coaxial printing technology, the recovery is simple and convenient, the application range of the immobilized enzyme is expanded, the combination of the enzyme and a substrate can be effectively improved, and the reaction efficiency of the enzyme is improved. The immobilized enzyme can be widely used in the fields of cosmetics, foods, environmental protection, medicines and the like, and has wide application prospect.)

1. An immobilized enzyme, comprising:

the hydrogel hollow fiber tube is provided with a fiber net-shaped tube wall and a tube cavity formed by enclosing the tube wall;

and an enzyme, wherein the enzyme is embedded and fixed in the tube wall of the hydrogel hollow fiber tube.

2. The immobilized enzyme of claim 1, wherein the hydrogel hollow fiber tube is made of at least one material selected from sodium alginate hydrogel and chitosan hydrogel.

3. The immobilized enzyme according to claim 1, wherein the enzyme is at least one selected from the group consisting of trypsin, pectinase, glycosidase, and alkaline protease.

4. A preparation method of immobilized enzyme is characterized by comprising the following steps:

providing a first solution and a second solution, wherein the first solution is a cross-linking agent solution, and the second solution is a mixed solution of a polymer and an enzyme;

and introducing the first solution into an inner sheath flow, introducing the second solution into an outer sheath flow, and carrying out coaxial printing to obtain the immobilized enzyme with the enzyme immobilized in the wall of the hydrogel hollow fiber tube.

5. The method of claim 4, wherein the first solution, the crosslinking agent is selected from calcium chloride at a concentration of 1% to 3% (w/v);

in the second solution, the polymer is selected from sodium alginate, the concentration of the sodium alginate is 1% -3% (w/v), and the concentration of the enzyme is 0.05-10 mg/mL.

6. The method of claim 4, wherein the first solution is a mixed solution of a crosslinking agent and an inert thickener.

7. The method according to claim 6, wherein the inert thickener is present in the first solution at a concentration of 1 to 20% by mass.

8. The method of claim 4, wherein the flow rate of the first solution is 1-3mL/min and the flow rate of the second solution is 1-3 mL/min.

9. Use of the immobilized enzyme of any one of claims 1 to 3 in cosmetics.

10. The preparation method of the antioxidant active peptide is characterized by comprising the following steps:

providing crude extract of seaweed protein;

adding the immobilized enzyme of claim 1 into the crude seaweed protein extract, performing concussion and enzymolysis, and taking out the immobilized enzyme to obtain the seaweed protein polypeptide, wherein the immobilized enzyme is trypsin immobilized by a sodium alginate hydrogel hollow fiber tube.

Technical Field

The invention belongs to the technical field of enzyme immobilization, and particularly relates to an immobilized enzyme, a preparation method of the immobilized enzyme, and application of the immobilized enzyme.

Background

The enzyme is a biocatalyst with the characteristics of high efficiency, specificity and the like, and generally plays a catalytic role under the conditions of normal temperature, normal pressure and isothermy. However, enzymes leave their specific environment and are poorly stable and difficult to recycle, and these problems in these applications can be overcome by enzyme immobilization techniques.

The enzyme immobilization technique is a biotechnology in which free enzymes are bound to a certain space or an insoluble carrier, and the free flow of the free enzymes is restricted, so that the free enzymes can exert a catalytic effect for a long time and can be recovered and utilized. After the enzyme is immobilized, the interaction with the immobilized substrate may change the spatial structure except the enzyme catalytic active center, thereby improving the stability of the enzyme in strong pH, high temperature or organic solvent, and simultaneously separating the target product from the enzyme reaction system. In this way, the immobilized enzyme can be removed by simple mechanical separation or centrifugation, thereby eliminating the need to use complicated product separation techniques such as dialysis. In summary, enzyme immobilization has the greatest advantage of creating a new enzyme system that can be reused in successive catalytic reaction cycles, reducing production costs. Because of a series of advantages of immobilized enzymes, the immobilized enzymes are widely applied to the industrial fields of medicines, foods, water treatment and the like.

The current immobilized enzyme technology mainly comprises: adsorption, entrapment, cross-linking and covalent bonding. Wherein, the adsorption method is mainly based on physical adsorption, and the enzyme is easy to fall off in the reaction process of the enzyme and a substrate. The cross-linking method and the covalent binding method are mainly based on chemical bond fixation, and the spatial conformation of the enzyme is changed after the fixation, so that an active center cannot be exposed, and the binding of the enzyme and a substrate is influenced. The enzyme immobilized based on the gel microsphere embedding technology at present has high stability and high activity. However, if the size of the gel microsphere is relatively small, it is troublesome to recover the enzyme, and if the size of the gel microsphere is relatively large, the enzyme at the center cannot be bound to the substrate, and in addition, a substrate inhibition effect occurs, thereby affecting the enzyme reaction efficiency. In addition, the traditional embedding method has the defects that only small molecular substrates and products can diffuse through a high polymer net frame, and the traditional embedding method is not suitable for enzymes of which the substrates and the products are large molecules, so that the application range of the immobilized enzyme is limited.

Disclosure of Invention

In view of the above, the present invention provides an immobilized enzyme, a preparation method and an application thereof, wherein the immobilized enzyme fixes the enzyme in the tube wall of the hydrogel hollow fiber tube, is easy and convenient to recover, can be used for diffusion of macromolecular substrates and products, and can effectively improve the combination of the enzyme and the substrates, thereby improving the reaction efficiency of the enzyme.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention firstly provides an immobilized enzyme, which comprises:

the hydrogel hollow fiber tube is provided with a fiber net-shaped tube wall and a tube cavity formed by enclosing the tube wall;

and an enzyme, wherein the enzyme is embedded and fixed in the tube wall of the hydrogel hollow fiber tube.

Furthermore, the hollow fiber tube is made of at least one material selected from sodium alginate hydrogel and chitosan hydrogel.

Further, the enzyme is selected from at least one of trypsin, pectinase, glycosidase and alkaline protease.

The invention further provides a preparation method of the immobilized enzyme, which comprises the following steps:

providing a first solution and a second solution, wherein the first solution is a cross-linking agent solution, and the second solution is a mixed solution of a polymer and an enzyme;

and introducing the first solution into an inner sheath flow, introducing the second solution into an outer sheath flow, and carrying out coaxial printing to obtain the immobilized enzyme with the enzyme immobilized in the wall of the hydrogel hollow fiber tube.

Further, in the first solution, the cross-linking agent is selected from calcium chloride, and the concentration of the calcium chloride is 1-3% (w/v);

in the second solution, the polymer is selected from sodium alginate, the concentration of the sodium alginate is 1% -3% (w/v), and the concentration of the enzyme is 0.05-10 mg/mL.

Further, the first solution is a mixed solution of a cross-linking agent and an inert thickening agent.

Further, the concentration of the inert thickener in the first solution is 1-20%.

Further, the flow rate of the first solution is 1-3mL/min, and the flow rate of the second solution is 1-3 mL/min.

The invention also provides the application of the immobilized enzyme in cosmetics.

The invention further provides a preparation method of the antioxidant active peptide, which comprises the following steps:

providing crude extract of seaweed protein;

adding the immobilized enzyme into the crude seaweed protein extract, performing concussion and enzymolysis, and taking out the immobilized enzyme to prepare the seaweed protein polypeptide, wherein the immobilized enzyme is trypsin immobilized by a sodium alginate hydrogel hollow fiber tube.

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

the immobilized enzyme is fixed in the tube wall of the hydrogel hollow fiber tube, has a higher specific surface, and can effectively improve the combination of the enzyme and a substrate, thereby improving the reaction efficiency of the enzyme and the substrate; the stability of the enzyme is obviously improved compared with that of the enzyme in a free state under the wrapping of the enzyme in the hollow fiber. And the hollow fiber tubular structure can be prepared into a large-size hydrogel structure under the condition of not influencing enzyme activity, so that the recovery after enzyme catalysis is facilitated, and a complicated means is not needed.

The invention uses the coaxial printing technology to prepare the immobilized enzyme, thereby successfully embedding and fixing the enzyme in the tube wall of the hydrogel hollow fiber tube, and the preparation method is simple, has high automation degree, can realize mass production, and can effectively realize large-scale industrialization.

Drawings

FIG. 1 is a schematic diagram of a coaxial printing system for preparing immobilized enzyme according to a preferred embodiment of the present invention;

FIG. 2 is a graph showing a comparison of the results of the absolute enzyme activities in example 1 of the present invention and comparative example 1.

In the figure: 10-a first injection device;

20-a second injection device;

30-coaxial needle cylinder, 31-inner needle cylinder and 32-outer needle cylinder;

40-coaxial needle, 41-inner needle, 42-outer needle.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the specific embodiments illustrated. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

In a first aspect, the present invention provides an immobilized enzyme comprising:

the hydrogel hollow fiber tube is provided with a fiber net-shaped tube wall and a tube cavity formed by enclosing the tube wall;

and an enzyme, wherein the enzyme is embedded and fixed in the tube wall of the hydrogel hollow fiber tube.

The immobilized enzyme is embedded and fixed in the tube wall of the hydrogel hollow fiber tube, the size of the hydrogel hollow fiber tube can reach 100-5000 mu m, the recovery method is simple, the hydrogel structure with the embedded enzyme is directly taken out during recovery, and the immobilized enzyme is suitable for diffusion of macromolecular substrates and products, and the application range of the immobilized enzyme is expanded. The hydrogel has a hollow structure on the inner side and a higher specific surface, so that the contact between the enzyme and the substrate can be effectively improved, the reaction efficiency of the enzyme and the substrate is improved, and the stability of the enzyme is obviously improved compared with that in a free state under the wrapping of the hydrogel.

In a further aspect, the material of the hydrogel hollow fiber tube in the present invention is not particularly limited, any hydrogel structural material formed by crosslinking may be a sodium alginate hydrogel or a composite material containing the sodium alginate hydrogel, or a chitosan hydrogel or a composite material containing the chitosan hydrogel, preferably, the material of the hydrogel hollow fiber tube in the present invention is at least one selected from the group consisting of a sodium alginate hydrogel and a chitosan hydrogel, and more preferably, in one or more embodiments of the present invention, the material of the hydrogel hollow fiber tube is sodium alginate.

Further, the enzyme used in the present invention is not particularly limited, and may be any acid enzyme, neutral enzyme or alkaline enzyme in the art, and specific examples include, but are not limited to, trypsin, pectinase, glycosidase, alkaline protease, and in one or more embodiments of the present invention, the enzyme is selected from trypsin.

In a second aspect, the present invention provides a method for preparing an immobilized enzyme, comprising the steps of:

providing a first solution and a second solution, wherein the first solution is a cross-linking agent solution, and the second solution is a mixed solution of a polymer and an enzyme;

and introducing the first solution into an inner sheath flow, introducing the second solution into an outer sheath flow, and carrying out coaxial printing to obtain the immobilized enzyme with the enzyme immobilized in the wall of the hydrogel hollow fiber tube.

The preparation method of the immobilized enzyme is realized based on the coaxial printing technology, the enzyme can be smoothly embedded in the tube wall of the hydrogel hollow fiber tube, the preparation method is simple, the automation degree is high, and the mass production can be realized, so that the industrialized scale is effectively realized. It is understood that the coaxial printing technology adopted by the present invention is not particularly limited, and a coaxial printing system conventional in the art can be used in the preparation method of the present invention, and fig. 1 shows the coaxial printing system adopted in one or more embodiments of the present invention, and as shown in fig. 1, the coaxial printing system comprises a first injection device 10, a second injection device 20, a coaxial syringe 30 and a coaxial needle 40, wherein the first injection device 10 and the second injection device 20 are injection pumps for respectively injecting and draining a first solution and a second solution to a designated position. Further, the coaxial cylinder 30 is composed of an inner cylinder 31 and an outer cylinder 32, the outer cylinder 32 is disposed outside the inner cylinder 31, and the outer cylinder 32 is coaxial with the inner cylinder 31, an inner sheath flow is formed inside the inner cylinder 31, and an outer sheath flow is formed between the outer cylinder 32 and the inner cylinder 31; the coaxial needle 40 is composed of an inner needle 41 and an outer needle 42, the outer needle 42 is disposed outside the inner needle 41, the outer needle 42 is coaxial with the inner needle 41, wherein the outer needle 42 is communicated with the outer barrel 32, and the inner needle 41 is communicated with the inner barrel 31. In one or more embodiments of the present invention, as shown in fig. 1, the first injection device 10 is connected to the input end of the inner syringe 31 through a pipeline, the first solution is introduced into the inner sheath flow, the second injection device 20 is connected to the input end of the outer syringe 32 through a pipeline, the second solution is introduced into the outer sheath flow, the output end of the coaxial syringe 30 is connected to the coaxial needle 40, the first solution and the second solution do not contact each other in the coaxial syringe 30 and the coaxial needle 40, when the first solution and the second solution flow out of the coaxial needle 40, the two solutions react to form the hollow fiber tube-shaped hydrogel at the moment of contact, and the enzyme is embedded and fixed in the tube wall of the hollow fiber tube, so as to achieve the immobilization of the enzyme. It is understood that the coaxial printing system in fig. 1 is only an example for making the technical solution of the present invention clearer, and other coaxial printing systems capable of achieving the same effect can be used in the preparation of the immobilized enzyme of the present invention, and are not specifically set forth herein. The preparation method of the invention can not only efficiently and rapidly carry out enzyme immobilization, but also embed bacteria and fungi capable of expressing the enzyme, and after the embedding and immobilization, the bacteria and the fungi can continuously express the enzyme, thereby being convenient for subsequent application and further improving the enzyme activity and the stability thereof.

Further, the "cross-linking agent" as used herein refers to an auxiliary agent capable of initiating cross-linking of the polymer to form the hydrogel material, and specifically, there may be mentioned, but not limited to, calcium chloride, barium chloride, etc., and preferably, the cross-linking agent in the present invention is calcium chloride which is used more commonly in the art; the "polymer" as used herein refers to any monomer capable of being crosslinked by the crosslinking agent to form a hydrogel material, and is selected according to the final hydrogel material, and specific examples include, but are not limited to, sodium alginate or a complex containing the sodium alginate, chitosan or a complex containing the chitosan, and preferably, the polymer in the present invention is sodium alginate or chitosan. The concentration of the cross-linking agent in the first solution, and the concentration of the polymer and the enzyme in the second solution can be adjusted according to the needs, so that the concentration is not particularly limited, and the adjustment of the size of the hydrogel hollow fiber tube and the embedding amount of the immobilized enzyme can be realized by adjusting the concentrations, and in one or more embodiments of the invention, the concentration of the cross-linking agent in the first solution is 1-3% (w/v);

in the second solution, the concentration of the polymer is 1-3% (w/v), and the concentration of the enzyme is 0.05-10 mg/mL.

Further, in order to improve the operability, in one or more embodiments of the present invention, the first solution is a mixed solution of calcium chloride and an inert thickener, where the inert thickener is an inert component that can increase the viscosity of the solution and does not react with the crosslinking agent and the polymer, such as glycerol, PEG, pluronic, and the like, and the inert component does not participate in the reaction and only plays a supporting role during the printing process, adjusts the viscosity of the liquid, improves the operability of the preparation process, and automatically flows out after being placed in a refrigerator for a period of time at a later stage. In one or more embodiments of the present invention, pluronic is added to the first solution as an inert material.

In a further scheme, the mass concentration of the inert thickening agent in the first solution is 1-20%.

In a further scheme, the flow rate in the method is not particularly limited, can be adjusted according to needs, and the hollow fiber tubular hydrogel is prepared by adjusting the flow rate, preferably, the flow rate of the first solution is 1-3mL/min, and the flow rate of the second solution is 1-3 mL/min; in one or more embodiments of the invention, the flow rate of the first solution is 2mL/min and the flow rate of the second solution is 1 mL/min.

The third aspect of the present invention provides the use of the immobilized enzyme according to any one of the first aspect of the present invention in the preparation of cosmetics, and it is understood that the immobilized enzyme according to the present invention can be applied to the fields of food, light industry, medicine, chemical industry, analysis, environmental protection, energy and scientific research, after being immobilized with different enzymes according to actual needs.

The fourth aspect of the invention provides a preparation method of antioxidant active peptide, which comprises the following steps:

providing crude extract of seaweed protein;

adding the immobilized enzyme of the first aspect of the invention into the crude seaweed protein extract, oscillating for enzymolysis, and taking out the immobilized enzyme to obtain the seaweed protein polypeptide, wherein the immobilized enzyme is trypsin immobilized by a sodium alginate hydrogel hollow fiber tube.

In the above steps, the crude extract of algal protein is not particularly limited, and may be obtained by a conventional crude extraction method in the art, for example, in one or more embodiments of the present invention, a proper amount of dried algal powder is weighed, soaked in distilled water at 4 ℃ for 3 days, repeatedly frozen and thawed, centrifuged, and the supernatant is taken to obtain the crude extract of algal protein. In addition, in the step of preparing the algal protein peptide, the parameters of the shaking enzymolysis are not particularly limited, and can be adjusted according to the amount of the crude extract of algal protein, and in one or more embodiments of the present invention, the shaking enzymolysis is performed at 37 ℃ for 24 hours.

The marine algae is a natural resource with rich content and wide distribution, and is rich in various trace elements, saccharides and proteins. The marine algae extract is helpful for repairing damaged skin cells, has the effects of moisturizing, repairing and regenerating skin, calming, soothing, tendering, smoothing and the like, and enables the skin to return to a healthy and balanced state; in addition, an isolating film can be formed on the surface of the skin to strengthen the defense system of the skin and resist ultraviolet injury. The hollow fiber hydrogel immobilized enzyme is applied to the preparation of seaweed protein polypeptide, so that the deep treatment of algae is realized, and the utilization and absorption of the seaweed protein are obviously improved.

The technical scheme of the invention is further clearly and completely explained by combining specific embodiments.

Example 1

Providing a first solution and a second solution

Preparing a first solution with calcium chloride concentration of 3% (w/v), and preparing a second solution with sodium alginate concentration of 3% (w/v) and trypsin concentration of 1 mg/mL.

Preparation of immobilized enzymes

By using the coaxial printing system shown in fig. 1, the first solution was introduced as an inner sheath flow at a flow rate of 2mL/min, and the second solution was introduced as an outer sheath flow at a flow rate of 1mL/min, and coaxial printing was performed to obtain an immobilized enzyme in which the enzyme was immobilized in the wall of the hydrogel hollow fiber tube.

Application example 1

Obtaining crude extract of seaweed protein

Weighing a proper amount of seaweed dry powder, soaking the seaweed dry powder in distilled water at the temperature of 4 ℃ for 3 days, repeatedly freezing and thawing, centrifuging, and taking supernatant to obtain a crude seaweed protein extracting solution.

Preparation of seaweed protein polypeptide

And (3) putting the crude seaweed protein extract into a triangular flask, adding the immobilized trypsin prepared in the example 1, performing shake enzymolysis for 24 hours (37 ℃), and taking out the immobilized trypsin to obtain the seaweed protein polypeptide.

Comparative example

In the comparative example, immobilized enzyme was obtained by a conventional gel bead embedding method in the art, and the preparation method thereof can be referred to "research on sodium alginate-immobilized pectinase", and the raw material composition was the same as in example 1.

The absolute enzyme activity in example 1 and comparative example 1 was tested, and the results are shown in fig. 2, and it can be seen that the absolute enzyme activity of the immobilized enzyme in example 1 is significantly higher than that of the comparative example.

The specific test method of enzyme activity is as follows:

(1) preparing a reagent:

(pH8.0) weighing 121.14g of Tris-methyl carbamate (Tris) solution, dissolving in 900mL of water, adjusting the pH value to 8.0 with concentrated hydrochloric acid, adding water, diluting to 1L with volumetric flask, preserving at 4 ℃, diluting 20 times when using, and preparing into 50mmol/L Tris-HCl buffer solution with pH of 8.0.

② BApNA substrate solution, 0.0435g is weighed and dissolved in 10mL dimethyl sulfoxide, and the solution is preserved at 4 ℃.

③ 10 percent of trichloroacetic acid, namely 10g of trichloroacetic acid, is dissolved in 100mL of deionized water and is stored at 4 DEG C

(2) The determination step comprises:

50 microliters of enzyme solution was dissolved in 1850 microliters of buffer, mixed and mixed uniformly, and preheated at 37 ℃ for 2 min.

② adding 100 microliter substrate (20mmol/L) into the mixed solution, and reacting for 20min at 37 ℃;

③ when the reaction time is over, 200 microlitre of trichloroacetic acid (10 percent) is added into the reaction system to stop the reaction, and the contrast tube is the reaction system in which the trichloroacetic acid (10 percent) is added in advance to destroy the enzyme activity.

Cooling the reaction liquid to room temperature, and measuring the light absorption value of the reaction liquid under the condition that the wavelength is 410 nm.

One control, three replicates, was set for each sample. The amount of enzyme required to convert 1 micromole of substrate BApNA per second was defined as 1 enzyme activity unit.

Example 2

Providing a first solution and a second solution

Preparing a first solution with calcium chloride concentration of 3% (w/v) and Pluronic content of 5%, and preparing a second solution with sodium alginate concentration of 3% (w/v) and trypsin concentration of 10 mg/mL.

Preparation of immobilized enzymes

By using the coaxial printing system shown in fig. 1, the first solution was introduced as an inner sheath flow at a flow rate of 3mL/min, and the second solution was introduced as an outer sheath flow at a flow rate of 3mL/min, and coaxial printing was performed to obtain an immobilized enzyme in which the enzyme was immobilized in the wall of the hydrogel hollow fiber tube.

Example 3

Providing a first solution and a second solution

Preparing a first solution with the calcium chloride concentration of 2% (w/v) and the Pluronic content of 10%, and preparing a second solution with the sodium alginate concentration of 2% (w/v) and the trypsin concentration of 5 mg/mL.

Preparation of immobilized enzymes

By using the coaxial printing system shown in fig. 1, the first solution was introduced as an inner sheath flow at a flow rate of 1mL/min, and the second solution was introduced as an outer sheath flow at a flow rate of 1mL/min, and coaxial printing was performed to obtain an immobilized enzyme in which the enzyme was immobilized in the wall of the hydrogel hollow fiber tube.

Example 4

Providing a first solution and a second solution

Preparing a first solution with the calcium chloride concentration of 1% (w/v) and the Pluronic content of 10%, and preparing a second solution with the sodium alginate concentration of 3% (w/v) and the trypsin concentration of 0.05 mg/mL.

Preparation of immobilized enzymes

By using the coaxial printing system shown in fig. 1, the first solution was introduced as an inner sheath flow at a flow rate of 2mL/min, and the second solution was introduced as an outer sheath flow at a flow rate of 2mL/min, and coaxial printing was performed to obtain an immobilized enzyme in which the enzyme was immobilized in the wall of the hydrogel hollow fiber tube.

Example 5

Providing a first solution and a second solution

Preparing a first solution with barium chloride concentration of 2% (w/v) and Pluronic content of 5%, and preparing a second solution with sodium alginate concentration of 3% (w/v) and trypsin concentration of 4 mg/mL.

Preparation of immobilized enzymes

By using the coaxial printing system shown in fig. 1, the first solution was introduced as an inner sheath flow at a flow rate of 3mL/min, and the second solution was introduced as an outer sheath flow at a flow rate of 3mL/min, and coaxial printing was performed to obtain an immobilized enzyme in which the enzyme was immobilized in the wall of the hydrogel hollow fiber tube.

Example 6

Providing a first solution and a second solution

Preparing a first solution with barium chloride concentration of 2% (w/v) and glycerol content of 20%, and preparing a second solution with sodium alginate concentration of 3% (w/v) and trypsin concentration of 3 mg/mL.

Preparation of immobilized enzymes

By using the coaxial printing system shown in fig. 1, the first solution was introduced as an inner sheath flow at a flow rate of 3mL/min, and the second solution was introduced as an outer sheath flow at a flow rate of 2mL/min, and coaxial printing was performed to obtain an immobilized enzyme in which the enzyme was immobilized in the wall of the hydrogel hollow fiber tube.

Example 7

Providing a first solution and a second solution

Preparing a first solution with the calcium chloride concentration of 2% (w/v) and the glycerol content of 20%, and preparing a second solution with the sodium alginate concentration of 3% (w/v) and the glycosidase concentration of 1 mg/mL.

Preparation of immobilized enzymes

By using the coaxial printing system shown in fig. 1, the first solution was introduced as an inner sheath flow at a flow rate of 3mL/min, and the second solution was introduced as an outer sheath flow at a flow rate of 2mL/min, and coaxial printing was performed to obtain an immobilized enzyme in which the enzyme was immobilized in the wall of the hydrogel hollow fiber tube.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.