Enzyme co-immobilization strategy based on biomolecule click reaction and application of enzyme co-immobilization strategy

Abstract

The invention relates to the technical field of biology, in particular to an enzyme co-immobilization strategy based on biomolecule click reaction and application of the enzyme co-immobilization strategy, and the enzyme co-immobilization strategy comprises the following specific steps: S1, preparing magnetic microspheres of which the surfaces are modified by haloalkane; s2, preparing a protein complex; s3, preparing a modular complex; and S4, preparing the immobilized multi-enzyme modular complex. The invention successfully establishes a plug-and-play method for assembling and immobilizing the multi-enzyme module, which is adjustable in proportion, adjustable in sequence and convenient to recycle. The immobilization system provided by the invention is simple in preparation method, can be recycled for multiple times for catalytic reaction, not only can immobilize pure enzyme, but also can realize specific immobilization of target protein in a cell lysis solution, effectively solves the problem of immobilization of protein with an unstable purification form, and has a good application prospect. The immobilization period is shortened; and the specificity of a protein immobilization method is poor.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, to an enzyme co-immobilization strategy based on biomolecule click reaction and its application, to a multi-enzyme immobilization method, and specifically studies a method for constructing an assembly module based on a protein and an isopeptide bond-connected molecular adhesive of a haloalkane dehalogenase (HaloTag)-specific dehalogenation reaction, and for directional immobilization of a target functional enzyme using magnetic beads as a carrier, belonging to the field of protein immobilization technology. Background Art

[0002] Biological enzymes have high catalytic efficiency and strong specificity, and usually play a catalytic role under mild conditions such as normal temperature and pressure. However, they are unstable when separated from cells and their specific environment, and it is difficult to reuse enzymes in industrial production. Therefore, the research on immobilized enzymes quickly emerged as an important bioengineering technology. In 1916, Nelson first reported that the invertase adsorbed on charcoal maintained its catalytic activity well, which opened the prelude to the research on enzyme immobilization.

[0003] Enzyme immobilization methods can be divided into traditional methods and new methods according to the time of their introduction. Traditional methods are further divided into physical methods and chemical methods. Physical methods include adsorption and embedding methods, which non-covalently bind to the target enzyme, while chemical methods include binding and cross-linking methods, which covalently bind to the target enzyme. New methods derived from traditional methods but different from them focus on the innovation of immobilization materials, fixation methods and directional fixation. Early enzyme immobilization methods have certain defects, such as the easy loss of adsorbed and fixed enzymes, the difficulty of diffusion of enzyme substrates and products after embedding and fixation, the stable but easy to cause enzyme inactivation of covalent binding methods, and the poor mechanical properties of cross-linked enzymes.

[0004] Attaching a short peptide tag is the most effective way to immobilize the protein of interest. It is usually connected to the C-terminus or N-terminus of the protein, which has the least disturbance on the protein function. Therefore, researchers have designed a method of irreversibly binding peptide tags, which is based on two profound discoveries. The first is that in 2007, researchers first discovered the spontaneous formation of isopeptide bonds in Gram-positive streptococcal pilus proteins. It refers to the amide bond formed by at least one non-α-position amino or carboxyl group between amino acids in the protein sequence, which is widely present in the CnaB domain of pilus proteins of Gram-positive bacteria. The second key discovery is that Howarth et al. also realized that the CnaB domain can be split into two fragments, with the split point between the two β chains closest to the C-terminus of the two fragments. The larger fragment is called Catcher and the smaller fragment is called Tag. The two split fragments are isopeptide bond adhesive pairs, which can spontaneously recombine into isopeptide bonds under certain conditions.

[0005] In the prior art, some strategies for enzyme co-immobilization are also disclosed. For example, CN107760666B discloses that multiple enzymes are co-immobilized on the surface of multifunctional magnetic nanoparticles modified with dopamine and its derivatives; CN101613694A discloses a magnetic / functionalized silica composite microsphere carrier used for immobilized enzymes; CN117701549A discloses that magnetic microspheres coupled with affinity ligands are mixed with crude enzyme solution, which can directly capture target proteins from crude proteins and self-assemble to form magnetic multi-enzyme complexes; CN111978404A discloses a one-step directional protein immobilization method based on Halo-tag specific dehalogenation reaction, in which the Halo-tag gene is recombined to the inactive C-terminus of the target receptor protein, and the haloalkanoic acid is modified to the surface of the separation medium through an acylation reaction. After induction of expression, the specific dehalogenation reaction between the C-terminal dehalogenase of the recombinant receptor and the haloalkane on the surface of the separation medium is used to achieve the one-step directional covalent immobilization of the Halo-tag recombinant receptor in the cell lysate.

[0006] Halo-Tag is a covalently bound ligand protein tag modified with haloalkane dehalogenase as a template, which allows ligands with different functions to be connected and fused to it, and forms a stable and irreversible covalent bond with chloroalkane. It is connected in series with the Catcher in the isopeptide bond adhesive pair through a flexible chain, and then the target enzyme connected to the Tag in the isopeptide bond adhesive pair is captured. At present, there are 5 pairs of isopeptide bond molecular adhesive pairs reported, which provide a solid foundation for the research method of the present invention. Summary of the invention

[0007] The main purpose of the present invention is to provide an enzyme co-immobilization strategy based on biomolecule click reaction: plug-and-play, adjustable order and loading capacity, specifically studying the method of constructing an assembly module based on the protein of the specific dehalogenation reaction of halogenated alkane dehalogenase (HaloTag) and the molecular adhesive connected by isopeptide bonds, and using magnetic beads as carriers to directional immobilize the target functional enzyme. This method can directionally immobilize the target protein and co-immobilize multiple target proteins, solving the problems of low specificity and long cycle in the current protein immobilization method.

[0008] In order to achieve the above object, the technical solution of the present invention is achieved as follows:

[0009] In a first aspect, the present invention provides a magnetic microsphere whose surface is modified by a haloalkane (also referred to as a haloalkane-modified fixed carrier), wherein the haloalkane is R-(CH2) n -C(=O)-, wherein n is 1 to 10, and R is selected from fluorine, chlorine, bromine, and iodine. Further, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0010] Furthermore, the preparation process of the magnetic microspheres whose surfaces are modified by chlorinated alkanes is as follows:

[0011] Step A: preparing magnetic microspheres with active amino groups on the surface;

[0012] Step B: The magnetic microspheres obtained in step A are reacted with DIEA, chloroalkanoic acid and HATU to obtain a product.

[0013] Furthermore, the chloroalkanoic acid is 6-chlorohexanoic acid.

[0014] Furthermore, the step A can be obtained by conventional techniques. The embodiment of the present application discloses the use of a modified coprecipitation method to prepare Fe3O4 magnetic nanoparticles coated with oleic acid on the outer layer, and the epoxy groups are modified on the surface of the magnetic nanoparticles to form micron-sized magnetic PMMA-DVB-GMA microspheres, and then 1,6-hexanediamine is added for further modification, and finally magnetic microspheres with active amino groups on the surface are prepared. The magnetic microspheres in step A are preferably amino-modified magnetic oleic acid-coated Fe3O4 microspheres.

[0015] In a second aspect, the present invention provides a protein complex constructed by fusion expression of SpyCatcher, DogCatcher and HaloTag.

[0016] Furthermore, the protein complex is a HaloTag-SpyCatcher-DogCatcher fusion protein (3MOD for short), which has an amino acid sequence as shown in SEQ ID No: 1.

[0017] In a third aspect, the present invention provides a modular complex, which is assembled by combining the protein complex of the present invention with one or more target enzymes.

[0018] Furthermore, the target enzyme is selected from at least one of ValDH-SpyTag, ValDH-DogTag, FDH-SpyTag, FDH-DogTag, BsGDH-SpyTag, and BsGDH-DogTag.

[0019] Furthermore, the ValDH-SpyTag has an amino acid sequence as shown in SEQ ID No: 2.

[0020] Furthermore, the ValDH-DogTag has an amino acid sequence as shown in SEQ ID No: 3.

[0021] Furthermore, the FDH-SpyTag has an amino acid sequence as shown in SEQ ID No:4.

[0022] Furthermore, the FDH-DogTag has an amino acid sequence as shown in SEQ ID No:5.

[0023] Furthermore, the BsGDH-SpyTag has an amino acid sequence as shown in SEQ ID No:6.

[0024] Furthermore, the BsGDH-DogTag has an amino acid sequence as shown in SEQ ID No:7.

[0025] Furthermore, the molar ratio of the target enzyme to the protein complex is 1:1 to 5. Preferably, it is 1:1, 1:1.5, 1:2, 1:3, 1:4 or 1:5. Furthermore, when there are two target enzymes, the molar ratio of the two is 1:1, 1:2, 1:3, etc.

[0026] Further, the target enzyme is expressed by fusion of the preceding target enzyme and the molecular adhesive. Further, the preceding target enzyme is selected from at least one of formate dehydrogenase (FDH), glucose dehydrogenase (GDH), valine dehydrogenase (ValDH), threonine deaminase (L-TD), fatty acid photodecarboxylase (CvFAP) and lipase (TLL), preferably at least one of ValDH, FDH and BsGDH; or it can be in the form of purified enzyme or crude enzyme solution.

[0027] Furthermore, the molecular adhesive is selected from at least one of DogTag and SpyTag. The N- or C-terminus of the preceding target enzyme is fused with the N- or C-terminus of the molecular adhesive.

[0028] Furthermore, the modular complex is self-assembled by incubating the protein complex of the present invention with one or more target enzymes under the conditions of 25-30° C. and PBS (100 mM) buffer at pH 7.5.

[0029] In a third aspect, the present invention provides an immobilized multi-enzyme modular complex, which is completed by incubating the modular complex with magnetic microspheres whose surfaces are modified with chlorinated alkanes.

[0030] Furthermore, the molar ratio of the magnetic microspheres modified with chlorinated alkanes on the surface to the modular complex is 1:1 to 5. Preferably, it is 1:1, 1:1.5, 1:2, 1:3, 1:4 or 1:5. The modular complex is fixed by covalently binding the HaloTag at the front end of 3MOD to the chlorinated alkanes on the microspheres. There is no effective method to measure the number of chlorinated alkanes modified on the microspheres. Therefore, the connection amount of the microspheres and the modular complex is calculated by calculating the amount of the modular complex loaded on the microspheres, that is, Table 2. By determining the loading capacity of 3MOD on 1g of microspheres, ValDH and FDH with a molar ratio of 1:1:1.5 to 3MOD are added and incubated with 3MOD to obtain a modular complex, and then incubated with the microspheres for immobilization.

[0031] Furthermore, the immobilized multi-enzyme modular complex is completed by incubating the magnetic microspheres whose surfaces are modified with chlorinated alkanes with the modular complex in a PBS (100 mM) buffer at 25-30° C. and pH 7.5.

[0032] In a fourth aspect, the present invention provides an enzyme co-immobilization method based on a biomolecule click reaction, the specific steps of which are as follows:

[0033] S1: Preparation of magnetic microspheres modified with halogenated alkanes;

[0034] S2: Preparation of protein complex;

[0035] S3: Preparation of modular complexes;

[0036] S4: Preparation of immobilized multi-enzyme modular complexes.

[0037] In another aspect, the present invention provides a use of an immobilized multi-enzyme modular complex in a catalytic reaction.

[0038] Furthermore, the catalytic reaction is a dual enzyme cascade catalysis of α-ketobutyric acid to generate L-2-aminobutyric acid. Furthermore, the catalytic reaction dehydrogenates ammonium formate under the catalysis of formate dehydrogenase or glucose dehydrogenase, and simultaneously utilizes the coenzyme NAD + Regenerate NADH and drive valine dehydrogenase to catalyze the conversion of α-ketobutyric acid to produce L-2-aminobutyric acid product.

[0039] The present invention has the following advantages:

[0040] The present invention provides an enzyme co-immobilization strategy based on biomolecule click reaction: plug-and-play, adjustable order and loading capacity. This method is based on the study of molecular adhesives connected by isopeptide bonds, and proposes to use SpyCatcher, DogCatcher and HaloTag to construct a modular protein called 3MOD (3module), capture the target protein connected to SpyTag and DogTag through spontaneous specific reaction, and covalently bind HaloTag to magnetic beads with surface modified chlorinated alkanes through specific dehalogenation reaction, successfully establishing a plug-and-play, adjustable ratio, adjustable order and conveniently recyclable multi-enzyme module assembly and immobilization method.

[0041] The immobilization system provided by the present invention has a simple preparation method and can be recycled multiple times for catalytic reactions. It can not only immobilize pure enzymes, but also achieve specific immobilization of target proteins in cell lysates, effectively solving the problem of immobilization of proteins in unstable purified form, shortening the immobilization cycle, and poor specificity of protein immobilization methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0043] Figure 1 This is a diagram of the preparation process of haloalkanoic acid-modified magnetic beads (separation medium);

[0044] Figure 2 is the enzyme immobilization flow chart (single enzyme);

[0045] Figure 3 It is the enzyme immobilization flow chart (double enzyme, loading capacity adjustment);

[0046] Figure 4 It is the flow chart of enzyme immobilization (double enzyme, sequential regulation);

[0047] Figure 5 It is the flow chart of enzyme immobilization (direct immobilization of cell lysate);

[0048] Figures 6 to 9 The scanning electron microscope images, energy spectra and particle size statistical characterization images of the magnetic beads surface after modification of the magnetic beads at different stages and enzyme immobilization;

[0049] Fig.10 The elemental surface scan shows the successful modification of chloroalkane (Cl) on the surface of magnetic beads and the successful immobilization of the target protein (N);

[0050] Fig.11 A reaction process diagram of an actual catalytic reaction after enzyme immobilization using the method of the present invention, including catalytic reaction process diagrams of single enzyme immobilized magnetic beads, multi-load single enzyme immobilized magnetic beads, and dual enzyme immobilized magnetic beads in different orders;

[0051] Fig.12 Changes in molecular weight of tunable fusion proteins are shown;

[0052] Figures 13 to 21 A plug-in and display diagram for implementing plug-and-play for the enzyme-immobilized magnetic beads of the present invention;

[0053] Fig. 22 The molecular weights of the fusion proteins are shown in order;

[0054] Fig.23 The SDS-PAGE electrophoresis verification diagram and catalytic reaction process diagram of the target protein immobilized from cell lysate using this method; Fig.23 A is the capture of target protein in cell-free extract by 3MOD module protein; Fig.23 B is the catalytic reaction of immobilized magnetic beads in cell-free extract;

[0055] Fig.24 To investigate the reusability of dual-enzyme immobilized microspheres. DETAILED DESCRIPTION

[0056] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many detailed descriptions are intended to enable the present application to be better understood. However, those skilled in the art can effortlessly recognize that some of the features can be omitted in different situations, or can be replaced by other materials and methods. In some cases, some operations related to the present application are not shown or described in the specification, in order to avoid the core part of the present application being overwhelmed by too much description, and for those skilled in the art, it is not necessary to describe these related operations in detail, and they can fully understand the related operations according to the description in the specification and the general technical knowledge in the art.

[0057] In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various implementations. At the same time, the steps or actions in the method description can also be interchanged or adjusted in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the specification and the drawings are only for the purpose of clearly describing a certain embodiment and are not meant to be a required sequence, unless otherwise specified that a certain sequence must be followed.

[0058] The serial numbers assigned to the components in this article, such as "first", "second", etc., are only used to distinguish the objects described and do not have any order or technical meaning.

[0059] The expression and purification methods of the fusion protein of the present invention are as follows:

[0060] The above recombinant gene plasmid verified by sequencing was introduced into E. coli.BL21 (DE3) competent E. coli by chemical transformation, and the revived bacterial solution was spread on LB solid medium containing 50μg / mL kanamycin, and a single colony was picked and inoculated into 5mL LB liquid medium containing 50μg / mL kanamycin, and cultured at 37°C, 200rpm for 6-8h. According to the inoculum volume of 2%, it was transferred to 250mL fresh TB liquid medium containing 50μg / mL kanamycin, and cultured at 37°C, 200rpm until OD600 reached about 1.1-1.5 (about 3.5h), and IPTG with a final concentration of 0.5mM was added, and the overnight induction culture was carried out at 18°C, 200rpm for about 20h.

[0061] After the culture was completed, the culture solution was centrifuged at 12000g and 4°C for 10 min, the supernatant was discarded, and the bacteria were washed and centrifuged twice with PBS (pH 7.5, 100 mM) buffer and stored at -80°C for future use.

[0062] The collected bacteria were resuspended in PBS (pH 7.5, 100mM) buffer, ultrasonically disrupted in an ice bath, centrifuged at 4°C, 12000rpm to remove cell debris, and the supernatant was filtered with a 0.22μm filter membrane. If the target protein cell lysate is used for immobilization, it can be directly used for enzyme immobilization; if the pure enzyme is used for immobilization, the target protein is purified according to the Ni-NTA gravity column purification method; 3MOD protein needs to be used in pure enzyme form in this method, and the specific purification method is as follows:

[0063] Sample pretreatment: resuspend the cells in loading buffer, disrupt them by ultrasonic wave in an ice bath, centrifuge at 12000 rpm at 4°C to remove cell debris, and filter the supernatant with a 0.22 μm filter membrane;

[0064] Loading: Rinse the nickel column with 50 mL (10 column volumes) of loading buffer, slowly load the pretreated sample onto the column, and place on ice for 0.5 h to allow the protein to fully bind to the nickel column;

[0065] Washing: Wash the nickel column with 50 mL (10 column volumes) of washing buffer I to elute the impurities;

[0066] Elution of target protein: Elute the target protein with elution buffer, collect the eluate into multiple tubes and number them sequentially;

[0067] Electrophoresis verification: Perform SDS-PAGE electrophoresis analysis on the collected eluate to determine the purity of the target protein and the number of the tube in which it is located.

[0068] Ultrafiltration and storage: After electrophoresis verification, the samples in the selected tubes are combined and transferred to ultrafiltration tubes. The protein samples are desalted and concentrated by low-temperature (4°C) centrifugal ultrafiltration. The ultrafiltered protein samples are added with glycerol at a final concentration of 20%, quickly frozen in liquid nitrogen, and stored at -80°C.

[0069] Example 1 Plug and Play

[0070] like Figures 1 to 5 The present invention provides an enzyme co-immobilization strategy based on biomolecule click reaction: plug-and-play, adjustable sequence and loading capacity, specifically comprising the following steps:

[0071] Step 1: Synthesis of fixed carrier

[0072] like Figure 1 As shown, Fe3O4 magnetic nanoparticles with oleic acid as the outer layer were prepared by modified coprecipitation method, and the surface of magnetic nanoparticles was modified with epoxy groups to form micrometer-sized magnetic PMMA-DVB-GMA microspheres. 1,6-hexanediamine was then added for further modification, and finally a magnetic microsphere fixed carrier with active amino groups on the surface was prepared.

[0073] Step 2: Chlorinated alkanes to modify the fixed carrier

[0074] like Figure 1 As shown, the magnetic microspheres with active amino groups were added with DIEA, 6-chlorohexanoic acid and HATU, and reacted at room temperature for 4 hours. After the reaction, the fixed carrier modified with chlorinated alkane was obtained by washing.

[0075] Step 3: Multi-enzyme self-assembly to form a modular complex

[0076] 1) SpyTag is fused with the target enzyme ValDH for expression, and the target enzyme ValDH-SpyTag is obtained after purification;

[0077] 2) SpyCatcher, DogCatcher and HaloTag were fused and expressed to construct a 3MOD protein complex; the primers were designed as shown in Table 1 below, and the HaloTag gene, (GGGS)4-SpyCatcher gene and (GGGS)4-DogCatcher gene synthesized and subcloned into pET28a(+) by a biotechnology company were recombined, and primers were constructed based on the HaloTag of pET28a(+) to insert the (GGGS)4-SpyCatcher gene and the (GGGS)4-DogCatcher gene in sequence to construct a 3MOD protein complex;

[0078] Table 1. Primer pairs used to construct 3MOD protein complex (HaloTag-SpyCatcher-DogCatcher)

[0079]

[0080]

[0081] 3) The target enzyme ValDH-SpyTag and the 3MOD protein complex were incubated at a molar ratio of 1:1 at 25-30°C, pH 7.5 PBS (100 mM) buffer, so that the multi-enzyme self-assembled to form a modular complex 3MOD-ValDH-SpyTag. The SDS-PAGE electrophoresis verification diagram is shown in Fig.12 and Fig. 22 .

[0082] Step 4: The multi-enzyme modular complex is fixed to the modified carrier

[0083] The chlorinated alkane-modified fixed carrier was washed four times with binding buffer [0.1M Tris-HCl, 0.15M NaCl, 0.01% IGEPAL CA-630 (Sigma-Aldrich), pH 7.6] and finally resuspended in 100 mL binding buffer. The multi-enzyme self-assembly to form the modular complex 3MOD-ValDH-SpyTag was added and incubated in PBS (100mM) buffer at 25-30°C, pH 7.5 for 3 hours to complete the fixation of the multi-enzyme modular complex. The catalytic process of the actual reaction is detailed in the attached figure. Fig.11 , the magnetic bead protein loading capacity is shown in Table 2 below.

[0084]

[0085]

[0086] Example 2 Multiple Loading

[0087] like Figure 1 As shown, for the rest of the unclear parts, please refer to the common knowledge or the contents described in Example 1. The present invention provides an enzyme co-immobilization strategy based on biomolecule click reaction: plug-and-play, with adjustable sequence and loading capacity, specifically comprising the following steps:

[0088] Step 1 and Step 2 are the same as those in Example 1.

[0089] Step 3: Multi-enzyme self-assembly to form a modular complex

[0090] 1) SpyTag and DogTag were fused with the target enzyme ValDH for expression, and the purified target enzymes ValDH-SpyTag and ValDH-DogTag were obtained;

[0091] 2) SpyCatcher, DogCatcher and HaloTag were fused and expressed to construct the 3MOD protein complex;

[0092] 3) The fusion expressed and purified target enzymes ValDH-SpyTag, ValDH-DogTag and 3MOD protein complex were incubated at a molar ratio of 1:1:1 at 25-30°C, pH 7.5 PBS (100 mM) buffer, so that multiple enzymes self-assembled to form a modular complex 3MOD-ValDH-SpyTag-ValDH-DogTag. The SDS-PAGE electrophoresis verification diagram is shown in Figures 12 and Fig. 22 .

[0093] Step 4: The multi-enzyme modular complex is fixed to the modified carrier

[0094] The chlorinated alkane-modified fixed carrier and the multi-enzyme self-assembled modular complex were incubated in PBS (100 mM) buffer at 25-30°C, pH 7.5 for 2-4 hours to complete the fixation of the multi-enzyme modular complex. The catalytic process of the actual reaction is shown in the attached figure. Fig.11 , the magnetic bead protein loading capacity is shown in Table 2.

[0095] Example 3 Multiple Loading

[0096] like Figure 1 As shown, for the rest of the unclear parts, please refer to the common knowledge or the contents described in Example 1. The present invention provides an enzyme co-immobilization strategy based on biomolecule click reaction: plug-and-play, with adjustable sequence and loading capacity, specifically comprising the following steps:

[0097] Step 1 and Step 2 are the same as those in Example 1.

[0098] Step 3: Multi-enzyme self-assembly to form a modular complex

[0099] 1) SpyTag and DogTag were fused and expressed with the target enzymes ValDH and FDH, respectively, and the target enzymes ValDH-SpyTag, FDH-DogTag, ValDH-DogTag, and FDH-SpyTag were obtained after purification;

[0100] 2) SpyCatcher, DogCatcher and HaloTag were fused and expressed to construct the 3MOD protein complex;

[0101] 3) The target enzyme ValDH-SpyTag, FDH-DogTag and 3MOD were incubated at a molar ratio of 1:1:1.5 at 25-30°C, pH 7.5 PBS (100 mM) buffer; multiple enzymes were self-assembled to form a modular complex 3MOD-ValDH-SpyTag-FDH-DogTag, as shown in Figure 12 and SDS-PAGE electrophoresis verification. Fig. 22 ;

[0102] 4) The target enzyme ValDH-DogTag, FDH-SpyTag and 3MOD were incubated at a molar ratio of 1:1:1.5 at 25-30°C, pH 7.5 PBS (100mM) buffer to form a modular complex 3MOD-FDH-SpyTag-ValDH-DogTag. The SDS-PAGE electrophoresis verification diagram is shown in the attached figure. Fig.12 and Fig. 22 .

[0103] Step 4: The multi-enzyme modular complex is fixed to the modified carrier

[0104] The chlorinated alkane-modified fixed carrier and the multi-enzyme self-assembled modular complex were incubated in PBS (100 mM) buffer at 25-30°C, pH 7.5 for 2-4 hours to complete the fixation of the multi-enzyme modular complex. The catalytic process of the actual reaction is shown in the attached figure. Fig.11 , the magnetic bead protein loading capacity is shown in Table 1.

[0105] Example 4 Direct fixation of cell lysate

[0106] like Figure 1 As shown, for the rest of the unclear parts, please refer to the common knowledge or the contents described in Example 1. The present invention provides an enzyme co-immobilization strategy based on biomolecule click reaction: plug-and-play, with adjustable sequence and loading capacity, specifically comprising the following steps:

[0107] Step 1 and Step 2 are the same as those in Example 1.

[0108] Step 3: Multi-enzyme self-assembly to form a modular complex

[0109] 1) SpyTag and DogTag are fused with target enzymes ValDH and FDH, respectively, and the fusion-expressed target enzymes ValDH-SpyTag and FDH-DogTag are ultrasonically crushed and centrifuged to obtain a certain amount of supernatant (cell lysate);

[0110] 2) SpyCatcher, DogCatcher and HaloTag were fused and expressed to construct the 3MOD protein complex;

[0111] 3) The supernatant (cell lysate) was incubated with the purified 3MOD at 25-30°C, pH 7.5 PBS (100 mM) buffer to form a modular complex 3MOD-ValDH-SpyTag-FDH-DogTag. The SDS-PAGE electrophoresis verification diagram is shown in the attached figure. Fig.23 .

[0112] Step 4: The multi-enzyme modular complex is fixed to the modified carrier

[0113] The chlorinated alkane-modified fixed carrier and the multi-enzyme self-assembled modular complex were incubated in PBS (100 mM) buffer at 25-30°C, pH 7.5 for 2-4 hours to complete the fixation of the multi-enzyme modular complex. The catalytic process of the actual reaction is shown in the attached figure. Fig.23 .

[0114] like Figures 6 to 10 It can be seen that the magnetic bead solid carrier prepared in the embodiment of the present invention has been verified in each step of modification, the particle size reaches the micron level, the carrier surface area is large, and a large molecular weight fusion protein complex can be immobilized. Through element surface scanning, it can be seen that the surface of the magnetic beads is successfully modified with chloroalkane (Cl) and the target protein (N) is successfully immobilized.

[0115] like Fig.23 As shown, the present invention provides an enzyme co-immobilization strategy based on biomolecule click reaction: plug-and-play, adjustable sequence and loading capacity, rapid directional immobilization of the target protein can be achieved without purifying the target protein, and the operation cycle of the protein immobilization process is significantly shortened.

[0116] Example 5 Investigation on the reusability of dual enzyme immobilized microspheres

[0117] like Fig.24 As shown, the time course of each catalytic cycle was 9 h, and 100 mg of immobilized microspheres were used for the experiment, ensuring that the enzyme-immobilized microspheres could be reused 7 times with a conversion rate of >50%.

[0118] In summary, the method provided by the present invention can recover the immobilized target protein multiple times for catalytic reactions, which can not only immobilize pure enzymes, but also achieve specific immobilization of target proteins in cell lysates, effectively solving the problem of immobilization of unstable proteins in purified form, shortening the immobilization cycle and the poor specificity of protein immobilization methods.

[0119] The above content is only an explanation of the preferred embodiments of the present invention, which is intended to demonstrate its core concepts and application methods, but is not intended to limit the wide applicability of the present invention. Under the premise of following the basic spirit and core principles of the present invention, any form of modification, equivalent alternative, technical improvement or innovation shall be deemed to fall within the scope of protection claimed by the present invention.

Claims

1. A magnetic microsphere whose surface is modified by a haloalkane, characterized in that: The halogenated alkane is R-(CH2) n -C(=O)-, wherein n is 1 to 10, and R is selected from fluorine, chlorine, bromine or iodine.

2. The magnetic microsphere according to claim 1, characterized in that: The preparation process of the magnetic microspheres whose surfaces are modified by chlorinated alkanes is as follows: Step A: preparing magnetic microspheres with active amino groups on the surface; Step B: The magnetic microspheres obtained in step A are reacted with DIEA, chloroalkanoic acid and HATU to obtain a product.

3. A protein complex, characterized in that It is constructed by fusion expression of SpyCatcher, DogCatcher and HaloTag.

4. The protein complex according to claim 3, characterized in that The protein complex is a HaloTag-SpyCatcher-DogCatcher fusion protein, which has an amino acid sequence as shown in SEQ ID No:

1.

5. A modular complex, characterized in that: The protein complex is assembled by combining the protein complex described in any one of claims 3 to 4 with one or more target enzymes.

6. The modular complex according to claim 5, characterized in that: The target enzyme is selected from at least one of ValDH-SpyTag, ValDH-DogTag, FDH-SpyTag, FDH-DogTag, BsGDH-SpyTag, and BsGDH-DogTag; Further, the ValDH-SpyTag has an amino acid sequence as shown in SEQ ID No: 2; Further, the ValDH-DogTag has an amino acid sequence as shown in SEQ ID No: 3; Further, the FDH-SpyTag has an amino acid sequence as shown in SEQ ID No: 4; Further, the FDH-DogTag has an amino acid sequence as shown in SEQ ID No: 5; Further, the BsGDH-SpyTag has an amino acid sequence as shown in SEQ ID No: 6; Furthermore, the BsGDH-DogTag has an amino acid sequence as shown in SEQ ID No:

7.

7. The modular complex according to claim 5, characterized in that: The molar ratio of the target enzyme to the protein complex is 1:1-5; Furthermore, the target enzyme is expressed by fusion of the preceding target enzyme and a molecular adhesive; Further, the preceding target enzyme is selected from at least one of formate dehydrogenase, glucose dehydrogenase, valine dehydrogenase, threonine deaminase, fatty acid photodecarboxylase and lipase, or is in the form of a purified enzyme or a crude enzyme solution; Furthermore, the molecular adhesive is selected from at least one of DogTag and SpyTag.

8. An immobilized multi-enzyme modular complex, characterized in that The method is completed by incubating the magnetic microspheres whose surfaces are modified with chlorinated alkanes as described in claim 1 with the modular complex as described in any one of claims 5 to 7.

9. An enzyme co-immobilization method based on biomolecule click reaction, characterized in that: The specific steps are as follows: S1: preparing the magnetic microspheres whose surfaces are modified with halogenated alkanes as claimed in claim 1; S2: preparing the protein complex according to claim 3; S3: preparing the modular composite body according to claim 5; S4: preparing the immobilized multi-enzyme modular complex according to claim 8.

10. Use of the immobilized multi-enzyme modular complex according to claim 8 in a catalytic reaction; Furthermore, the catalytic reaction is a double enzyme cascade catalysis of α-ketobutyric acid to produce L-2-aminobutyric acid.

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