CN112626061B - Multienzyme ordered co-immobilization method for improving efficiency of cascade catalytic system - Google Patents

Abstract

The invention relates to a multienzyme ordered co-immobilization method for improving efficiency of a cascade catalytic system. The catalytic material comprises a first enzyme and a second enzyme which are orderly co-immobilized on the surface of a solid-phase substrate, and when the catalytic material catalyzes a multi-enzyme cascade reaction, the second enzyme in the catalytic material can be immediately used for catalyzing the latter reaction after the first enzyme of the catalytic material catalyzes the former reaction, so that the catalytic efficiency of the whole multi-enzyme cascade reaction can be remarkably improved.

Description

Multienzyme ordered co-immobilization method for improving efficiency of cascade catalytic system

Technical Field

The invention relates to the technical field of enzyme engineering and enzyme immobilization, in particular to a multienzyme ordered co-immobilization method for improving efficiency of a cascade catalytic system. In addition, the invention also relates to the application of the orderly co-immobilized sucrose synthase and glycosyltransferase in the efficient preparation of the saponin pharmaceutical active compounds.

Background

Biocatalyst enzymes have found wide application in production practice. The enzyme immobilization combines free enzyme with a solid-phase matrix carrier through a physical or chemical method, which is helpful for separating a biocatalyst from a reaction solution, realizes the recycling of the enzyme and reduces the production cost. Because the complex catalytic system mostly involves continuous reactions in which various enzymes participate, multi-enzyme cascade catalysis is formed. The development of co-immobilization of a multi-enzyme system not only can organically combine catalytic properties of different enzymes to promote enzyme catalytic efficiency, but also is beneficial to improving the stability of enzyme molecules and is beneficial to storage and transportation. However, in most cases, conventional multi-enzyme co-immobilization methods (including cross-linking, entrapment and direct attachment to a functional substrate surface) often result in random distribution of various enzyme molecules, resulting in random co-immobilization between the enzymes in the cascade. Because of steric hindrance or excessive dispersion among enzyme molecules in the cascade multienzyme random co-immobilization, the cooperativity of the cascade multienzyme catalytic reaction can be obviously reduced. Many functionally closely related multienzymes have evolved in nature to localize and co-localize at organelles and cell membranes, or to form multienzyme complexes. The special spatial adjacent arrangement of the multiple enzymes can generate a substrate channel effect under certain conditions, avoid the solution diffusion of reaction intermediates, promote the transmission of the intermediates among enzyme molecules, maintain the local high concentration of metabolites and facilitate the occurrence of efficient multi-step metabolic reactions in organisms. Initiated by the phenomenon of co-positioning substrate channels in the space of the cascade multienzyme in the nature, the ordered co-immobilization system of the cascade multienzyme is designed and constructed, and the efficiency of multi-step continuous reaction in the artificial cascade system is hopefully promoted by the adjacent array arrangement of related enzyme catalytic sites. Methods for ordered co-immobilization of multiple enzymes have been reported that mainly utilize scaffold-mediated co-localization, e.g., using DNA origami, RNA or peptides as scaffolds to co-immobilize multiple enzymes. However, many of the enzymes of interest (e.g., dehydrogenases, catalases and many glycosyltransferases) are mostly oligomeric enzymes composed of multiple subunits. The stoichiometry of the polyase which is beneficial to orderly co-immobilization of scaffold molecules is complex, so that the technology of constructing the orderly co-immobilization multisubunit enzyme based on the molecular scaffold is complex, and the enzyme-scaffold connection design is required to be widely optimized.

Disclosure of Invention

The invention aims to provide a multi-enzyme co-immobilized array catalytic material capable of remarkably improving the catalytic efficiency of multi-enzyme cascade reaction.

In a first aspect of the present invention, there is provided a catalytic material for a multi-enzyme co-immobilized array, the catalytic material comprising:

1) a multienzyme nano self-assembly body comprises an element enzyme and a mate enzyme of more than two cascade catalytic systems and a histidine tag fused on the element enzyme and/or the mate enzyme, wherein,

the element enzyme is an oligomeric enzyme molecule, and a self-reaction peptide segment or a self-reaction chemical pair is fused on a subunit of the oligomeric enzyme molecule, and a histidine tag is optionally fused;

the pairing enzyme is a functional enzyme molecule, and a self-reaction peptide segment or a self-reaction chemical pair is fused on a subunit of the functional enzyme molecule, and a histidine tag is optionally fused;

the multienzyme nano self-assembly is obtained by spontaneous linking reaction between the element enzyme and the pairing enzyme through respective fused self-reaction peptide fragments or self-reaction chemical pairs; and

2)Ni²⁺: NTA surface-functionalized solid substrate, Ni²⁺Binding to the surface of the solid phase substrate through NTA;

the multi-enzyme nano self-assembly is connected with the Ni through the histidine tag²⁺Coordinate bonding to said Ni²⁺: immobilization of NTA surface functionalized solid phase substrate.

In another preferred embodiment, the multi-enzyme nano self-assembly comprises the elementary enzymes and the companion enzymes of a cascade catalytic system of class 2-5, preferably class 2-3, more preferably class 2 (e.g. class 1 elementary enzyme and class 1 companion enzyme).

In another preferred embodiment, the histidine tag is multivalent, such as valency 2.

In another preferred embodiment, the self-reactive peptide fragment is selected from the group consisting of: SpyTag, SpyCatcher, or a combination thereof.

In another preferred embodiment, the self-reacting chemical pair is selected from the group consisting of: a snoottag/snootcat, favidin/biotin, a disulfide bond, or a combination thereof.

In another preferred embodiment, the SpyTag or SpyCatcher is formed by a flexible polypeptide chain [ (AG)₃PEG]₅Linked to a subunit of said element enzyme and/or said partner enzyme.

In another preferred embodiment, the partzyme is selected from the group consisting of: sucrose synthase SUS, glucose dehydrogenase, or a combination thereof; and/or

The pairing enzyme is selected from the group consisting of: a glycosyltransferase UGTm, a cytochrome oxidase P450m, or a combination thereof.

In another preferred example, the partzyme (or the first enzyme) is sucrose synthase SUS.

In another preferred example, SpyTag is fused to a subunit of the sucrose synthase SUS.

In another preferred embodiment, the companion enzyme (or second enzyme) is the glycosyltransferase UGTm.

In another preferred embodiment, the glycosyltransferase is a glycosyltransferase, UGTm, having a SpyCatcher fused to a subunit.

In another preferred embodiment, the molar ratio of the element enzyme to the SpyTag peptide fragment is 1: 4.

in another preferred embodiment, the molar ratio of the pairing enzyme to the SpyCatcher peptide fragment is 1: 2.

in another preferred example, in the multi-enzyme nano self-assembly, the molar ratio of the first enzyme to the second enzyme is 1: 2.

in another preferred example, in the multi-enzyme nano self-assembly, the molar ratio of the subunit or monomer of the element enzyme to the histidine tag is 1: 3-15 (preferably 1: 4-12, more preferably 1: 5-10); and/or

The molar ratio of the subunit or monomer of the pairing enzyme to the histidine tag is 1: 3-15 (preferably 1: 4-12, more preferably 1: 5-10).

In another preferred embodiment, 10-100% mole of the catalytic material is fused with the histidine tag (preferably 30-100%, more preferably 50-100%, most preferably 80-100%); and/or

10-100% mole of the companion enzyme is fused with the histidine tag (preferably 30-100%, more preferably 50-100%, most preferably 80-100%).

In another preferred embodiment, the solid phase substrate is a shape functional profile and is selected from the group consisting of: resin microspheres, agarose microspheres, magnetic bead microspheres, or a combination thereof.

In another preferred embodiment, the solid phase substrate is selected from the group consisting of: polystyrene microspheres, agarose microspheres, or a combination thereof.

In another preferred embodiment, the particle size of the microspheres is 50-250um, preferably 50-200um, more preferably 50-150 um.

In another preferred embodiment, the multi-enzyme nano self-assembly and Ni²⁺Is 0.3 to 5, preferably 0.5 to 4, more preferably 1 to 3.

In a second aspect of the present invention, there is provided a method for preparing the catalytic material of the first aspect of the present invention, comprising the steps of:

1) providing a first reaction solution, a second reaction solution and a third reaction solution, wherein the first reaction solution contains an element enzyme, the second reaction solution contains a pairing enzyme, and the third reaction solution contains Ni²⁺: NTA surface functionalized solid phase substrate, wherein,

the element enzyme is a tetrameric molecule, and the element enzyme is fused with a SpyTag and an optional histidine tag;

the companion enzyme is a dimer molecule, and the companion enzyme is fused with a SpyCatcher and an optional histidine tag;

2) mixing the first reaction solution and the second reaction solution to obtain a fourth reaction solution, and carrying out self-assembly reaction to obtain a multienzyme nano self-assembly;

3) and (3) mixing the product obtained in the step 2) with the third reaction solution to obtain a fifth reaction solution, and reacting to obtain the multienzyme co-immobilized array catalytic material.

In another preferred embodiment, the pH of the fourth reaction solution is 4.5 to 8.5.

In another preferred embodiment, the pH of the fourth reaction solution is 4.5 to 7.5, preferably 4.5 to 7.2.

In a third aspect of the present invention, there is provided a use of the catalytic material of the multi-enzyme co-immobilized array according to the first aspect of the present invention, for a use selected from the group consisting of:

1) used for catalyzing the synthesis of saponin compounds;

2) for self-cycling to produce UDP-glucose (UDPG) as a sugar donor.

The fourth aspect of the invention provides a method for synthesizing saponin compounds, which comprises the following steps:

1) reacting sucrose with UDP by using the multi-enzyme co-immobilized array catalytic material as an enzyme to obtain UDPG;

2) directly taking the product obtained in the step 1) as a raw material, and reacting the product with protopanaxadiol PPD to obtain Uridine Diphosphate (UDP) and saponin Rh 2.

In another preferred embodiment, the reaction efficiency of the process is 50 to 100% (preferably 50 to 95%, more preferably 50 to 90%).

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1: designing glycosyltransferase UGTm of ordered co-immobilized dimer and tetramer sucrose synthase SUS to form a cascade enzyme catalytic array; A) firstly, SpyCatcher/SpyTag is fused to the subunit ends of UGTm and SUS, and a stable covalent isopeptide bond is spontaneously formed between SpyCatcherUGTm and SpyTagSUS and the multi-enzyme nano self-assembly is formed by oligomerization of multi-subunit enzyme. Further utilizing a plurality of polyhistidine tags carried on the constitutive enzymes in the multienzyme nano self-assembly to be further fixed on Ni2 +: on the NTA surface functionalized microsphere, a co-immobilized biological catalytic material with ordered multi-enzyme array arrangement is established. B) An artificial cascade catalytic system consisting of glycosyltransferase UGTm and sucrose synthase SUS; in this ordered co-immobilized multi-enzyme array of constructed UGTm-SUS, the conversion of ginsenoside protopanaxadiol PPD to rare ginsenoside Rh2 and the regeneration of the expensive sugar donor UDPG can be facilitated.

FIG. 2: quaternary structure of the polyprotein enzyme used for construction. (A) The structure of UGT51 (PDB: 5GL5) from Saccharomyces cerevisiae. (B) The structure of SUS, a sucrose synthase derived from Arabidopsis thaliana (PDB: 3S 28). The N and C termini are marked as red and blue circles, respectively. .

FIG. 3: constructs expressing the SpyTag/SpyCatcher fusion enzyme and spontaneous covalent binding reactions between SpyCatcher and SpyTag. (A) pET28a-SpyCatcherUGT plasmid. SpyC: a SpyCatcher domain; LL: (GGGGS)2 junction element. (B) pET28a-SpyTagSUS plasmid. SpyT: a SpyTag domain; LL: [ (AG)3PEG ]5 junction element. (C) Structure and molecular mechanism of SpyCatcher/SpyTag chemical pairing. .

FIG. 4: gel filtration chromatography of fused or unfused enzymes (a) elution profiles of spycatcheergtm and unfused UGTm. (B) Elution profiles of SpyTagSUS and unfused SUS counterparts. .

FIG. 5: the relative enzyme activities of SpyCatcherUGTm, SpyTagSUS and non-fusion enzymes were compared. All measurements were performed at least 3 times and the error bars represent the standard error. .

FIG. 6: (A) SDS-PAGE analysis of SpyCatcher/SpyTag mediated UGTm-SUS self-assembly. The purified enzyme and the assembled UGTm-SUS multienzyme nanoclusters were subjected to 10% SDS-PAGE operation and stained with coomassie brilliant blue. Lane M, protein molecular weight; lane 1, purified recombinant SpyTagSUS protein; lane 2, purified recombinant spycatchereugtm protein; lane 3, assembled SpyCatcherUGTm-SpyTagSUS protein. (B) The purity of the assembled SpyCatcherUGTm-SpyTagSUS protein was determined by densitometry and was 91%.

FIG. 7: Spycatcher/SpyTag mediated irreversible covalent binding at various pHs. Lane M: protein markers (kDa), lane 1: SpyTagSUS, lane 2: spycatcheergtm, lane 3: self-assembly in Tris-HCl buffer pH 7.0, lane 4: self-assembly in Tris-HCl buffer pH 7.5, lane 5: self-assembly in Tris-HCl buffer pH 8.0, lane 6: self-assembly in Tris-HCl buffer pH 8.5.

FIG. 8: (A) construct (B) of pACYDuet-6XHis-SpyCatcherUGTm-SpyTagSUS for in vivo protein co-expression and self-assembly 6XHis-SpyCatcherUGTm-SpyTagSUS self-assembled in crude E.coli lysate by Ni + -NTA affinity chromatography was analyzed by 10% SDS-PAGE. Lane M: protein markers (kDa), lane 1: cell lysate, lane 2: supernatant, lane 3: washed samples, lane 4: eluted sample. .

FIG. 9: characterization of the immobilized UGTm-SUS Components on functionalized microspheres. A) Analyzing the immobilized material by a Broadford method; after thorough washing of the incubated microspheres in the enzyme solution, the support was examined for attached proteins using Bradford solution; blank control: a Broadford solution; non-immobilized material: freshly prepared microspheres in a Broadford solution; immobilization material: the enzyme-immobilized microspheres were in Broadford solution. (B) optical microscope images of enzyme-immobilized or non-immobilized functionalized microspheres. Examples of convex-concave surface morphologies are indicated by blue arrows. The non-immobilized functionalized microspheres showed a smooth surface (inset).

FIG. 10: optical microscopy images of functionalized microspheres of immobilized UGTm-SUS self-assemblies. Examples of the concavo-convex surface morphology are indicated by blue arrows. .

FIG. 11: UGTm-SUS array ordered co-immobilized representative electron microscopy images. A) Scanning electron microscope images of immobilized UGTm-SUS self-assemblies on the surface of the support. After fixation, the samples were lyophilized and sputter coated with nanogold before analysis. The appearance of a granular structure in the immobilized material was observed under a scanning electron microscope. B) Transmission electron microscopy images of immobilized UGTm-SUS self-assemblies. The fine particles arranged orderly on the surface can be seen under a transmission electron microscope. C) Atomic force microscopy images of immobilized UGTm-SUS self-assemblies. The height of the microstructure of the immobilized multienzyme complex is about 20 nm.

FIG. 12: multienzyme immobilization rate, UDPG regeneration efficiency, and ordered co-immobilized UGTm-SUS array storage stability and repeated utility analysis. A) UGTm-SUS self-assembly and multi-enzyme mixture immobilization efficiency analysis on the support at 20 ℃ and pH 7.0; the amount of immobilized protein was determined from the difference between the total protein used for immobilization and the residual amount in the solution after immobilization. B) And (5) analyzing conversion efficiency. The conversion is shown as the percentage of PPD converted to Rh2, the final product. C) Orderly co-immobilization, random co-immobilization system and initial conversion rate of free multi-enzyme nano self-assembly under different UDP concentrations. D) Storage stability at 4 ℃. All measurements were performed at a protein concentration of 40 μ M; E) the ordered co-immobilized UGTm-SUS array is reusable after multiple recovery cycles; data represent the mean ± standard deviation of three measurements.

FIG. 13: HPLC analysis of the final product Rh2 formed from PPD by concerted catalysis of an ordered fixed UGTm-SUS cascade.

FIG. 14: SEM pictures of microspheres before loading.

FIG. 15: scanning electron micrographs of the catalytic material obtained after random immobilization of the multienzyme.

FIG. 16: scanning electron microscope photographs of the microspheres obtained by orderly immobilizing the multienzyme.

Detailed Description

The inventor designs and prepares a catalytic material for directionally and orderly fixing a multienzyme array on a solid substrate through long-term and deep research, when the multienzyme ordered co-immobilized catalytic material is used for catalytic reaction, a first enzyme of the catalytic material catalyzes a first reaction product and is a substrate of a subsequent enzyme in a cascade reaction, because multienzyme molecules in a system are assembled into array arrangement, the substrate is prevented from randomly diffusing, the conveying distance of the substrate among the multienzymes is shortened, the catalytic reaction of the subsequent enzyme can be quickly started, the conversion of a former reaction product can remove the product inhibition effect common in enzyme catalysis, the reaction balance moves towards the direction of a final product, and the multienzyme coordinated catalytic efficiency is improved. The construction of the multi-enzyme co-immobilized array catalytic material provided by the invention is that a self-assembly body is provided with a plurality of His labels, and the His labels can be directly combined with the surface of a substrate material with Ni-NTA (nitrilotriacetic acid) functionalization without introducing other additional modification. The fixing method is simple, the operation steps are few, the operation can be completed quickly, and the high activity of the biocatalyst can be maintained. Compared with other immobilization utilizing single fusion protein His label mediated immobilization, the method has the advantages of high obvious immobilization efficiency, strong stability and difficult separation from a substrate material by utilizing the population effect that the assembly body has a plurality of His labels. In the multi-enzyme co-immobilized array catalytic material, the His labels fused on each subunit of the oligomerase are utilized for carrying out substrate combination to form multi-point combination of the oligomerase and the substrate, so that the inactivation of the oligomerase caused by the dissociation of common subunits in the application of the oligomerase can be effectively avoided, the overall stability of the oligomerase catalytic material is improved, and the cyclic reuse of the oligomerase catalytic material is facilitated. On this basis, the inventors have completed the present invention.

Term(s) for

As used herein, the term"UDP" refers to uridine diphosphate having the structural formula

As used herein, the term "UDPG" refers to uridine diphosphate glucose, having the structural formula

As used herein, the term "PPD" refers to protopanaxadiol, of the formula

As used herein, the term "Rh 2" refers to ginsenoside having the structural formula

The term "MENCs" refers to polymerase nanoclusters, and particularly refers to a supermolecular nanocluster-like catalytic material formed by self-assembly among multiple enzymes.

The invention relates to a novel method for orderly co-immobilizing cascade multienzyme, which can improve the overall efficiency of multienzyme cascade catalysis. In particular to a novel technology for realizing orderly co-immobilization of multienzyme on a solid phase substrate by utilizing self-assembly of the oligoenzyme and mediation of rich polyhistidine affinity labels on a self-assembly body. The multi-enzyme cascade array arrangement is obtained by the multi-enzyme co-immobilization technology, so that a substrate channel effect is easily formed among different enzyme molecules in a cascade system, the transfer of reaction intermediates among different enzyme molecules is promoted, and the cooperative catalysis efficiency is improved; the co-immobilization method can effectively enhance the stability of the multi-subunit enzyme and strengthen the overall performance of the biological cascade catalytic system. The method comprises the following steps: multiple oligomeriases in a cascade system, such as: dimeric glycosyltransferase UGT51 and tetrameric sucrose synthase are respectively fused with SpyCatcher and SpyTag self-reaction chemical pairs for recombinant expression, oligomerization of the enzymes and SpyCather/SpyTag spontaneous covalent reaction are utilized to assemble a multienzyme nano cluster module, and the multi-enzyme nano cluster module is further assembled by virtue of abundant self-assembly modulesThe poly-histidine tag of (1) directly immobilizing the multi-enzyme assembly on the functionalized Ni²⁺NTA polystyrene microsphere, so as to prepare multienzyme ordered co-immobilized material simply and quickly. The invention can be used for enhancing the catalytic performances such as efficiency, stability and the like of a cascade multienzyme system, and the orderly co-immobilized cascade multienzyme technology has important application prospect in the preparation of nano bioreactors and biological detectors.

Multimeric enzymes first undergo hierarchical self-assembly driven by multimeric oligomerization, where a SpyTag/SpyCatcher covalent pairing reaction produces highly ordered supramolecular structure multienzyme nanocluster assemblies MENCs. Subsequently, the multienzyme nanocluster assembly module can be further immobilized on appropriate Ni through poly-histidine tags constituting enzymes²⁺: NTA functionalized microspheres or microplates. The established polymerase co-immobilization forms an ordered biological catalysis array of a plurality of enzymes in spatial adjacent arrangement, and provides substrate channels for different biocatalysts so as to realize better cascade performance.

The design couples the genetic fused SpyTag/SpyCatcher covalent chemical reaction pair with the spontaneous oligomerization of the multi-subunit enzyme, and the modular self-assembly of the multi-enzyme can be rapidly obtained. The SpyTag/SpyCatcher covalent chemical reaction pair derived from the CnaB2 domain has the advantage of spontaneously forming stable isopeptide bonds under various conditions, and is widely applied to the field of protein chemistry, such as biological coupling, vaccine synthesis, preparation of thermostable enzymes and the like. CnaB2 was split into two parts, SpyTag (13 amino acid peptide stretch) and Spycatcher (116 amino acid peptide stretch); asp in SpyTag and Lys in Spycatcher can spontaneously react to form an isopeptide covalent bond.

The His-tag (His-tag), also known as polyhistidine tag, consists of 6 to 10 consecutive histidine residues. The His tag may be conjugated to Ni under normal or denaturing conditions²⁺、Co²⁺The transition metal ions form coordinate bonds and are selectively bonded to the metal ions. The commonly used His tag is a 6XHis tag, which has a small structure and a molecular weight of 0.8kDa, and has a low possibility of affecting the function of the fusion protein. Chelation of metal ions by mediator ligands can be used to purify His-tagged proteins. Four coordinate nitrilotrisAfter acetic acid (NTA) is combined with Ni, a very stable structure can be formed, and the characteristics of high protein loading capacity and low metal ion shedding are achieved. The Ni-NTA medium becomes the best selective functional material for purifying the 6 × His label recombinant protein. Ni-NTA agarose gel resin filler and Ni-NTA magnetic bead form products have been provided on the market. The His tag for protein recombinant expression and purification is convenient to use and low in cost, and is the most widely applied affinity tag.

At present, the field of biocatalysis needs a multienzyme ordered co-immobilization technology which has simple immobilization process, can effectively avoid random distribution of enzyme molecules, quickly and efficiently forms site-specific binding of multienzyme in a cascade catalytic system on a solid phase substrate, and thus forms a high-density cascade enzyme ordered catalytic functional unit on a solid phase carrier. Furthermore, ordered immobilization of the multimeric enzyme on a support may improve the stability of the multimeric enzyme under harsh reaction conditions by mitigating inactivation associated with subunit dissociation. The development is convenient, the high-efficiency multienzyme ordered co-immobilization technology has potential application in manufacturing advanced nano bioreactors or other enzyme catalytic sensing devices.

The invention provides a multienzyme ordered co-immobilization method, which is simple to operate and good in repeatability, and can greatly improve the stability and reusability of immobilized enzymes, thereby reducing the production cost of biological manufacturing.

In the construction of an ordered co-immobilized multi-enzyme array, dimeric glycosyltransferase UGT51 mutant (UGTm, S81A/L82A/V84A/K92A/E96K/S129A/N172D) from Saccharomyces cerevisiae and tetrameric sucrose synthase (SUS) from Arabidopsis thaliana were selected as model enzymes for the artificial cascade catalytic system. The orderly co-immobilized glycosyltransferase and sucrose synthase enhance the multi-enzyme synergistic effect in the artificial cascade catalysis approach, and have important application value in the fields of preparing natural glucoside drug molecules by biocatalysis and sugar derivatization.

The ordered co-immobilization method of the multienzyme comprises the following steps:

(1) analysis of molecular Structure of enzyme, sucrose synthase SUS based on which is a tetrameric molecule composed of monomers having a molecular weight of 92kDa, andthe N-terminus is outside the fold, and relatively small SpyTag (. about.1.5 kDa) and 6XHis peptides were designed to be fused to the N-terminus of each SUS monomer. The glycosyltransferase UGTm is a dimeric molecule composed of 45kDa monomers, also with an extended N-terminus of folding, and the SpyCatcher (. about.9.5 kDa) peptide was designed to be fused to the N-terminus of each UGTm monomer, and fused at the C-terminus to a 6XHis tag. To retain sufficient rotational flexibility for enzyme-substrate interactions, flexibility ([ (AG) was introduced between the corresponding Spycatcher/SpyTag and fusion enzyme₃PEG]₅And connecting the peptide fragments.

(2) The spycatcherUGTm and spytagSUS fusion enzymes, as well as the corresponding unfused enzymes, were expressed and purified in E.coli. Gel filtration chromatography analysis of the purified proteins showed that spycatchergtm and SpyTagSUS showed similar retention curves to the unfused corresponding enzymes, indicating that the introduction of the fusion tag did not have a visible effect on the expression and folding of the enzyme protein. Furthermore, analysis of UDPG conversion showed that the fused spycatchergtm and spycatchsus exhibited similar enzymatic activities to the non-fused counterparts, indicating that the spycag or SpyCatcher fusions did not significantly affect the structural and functional integrity of these enzymes.

(3) Mixing these SpyCatcher/SpyTag fusion enzymes results in the formation of covalently cross-linked enzyme self-assemblies. SDS-PAGE shows protein bands corresponding to molecular weights above 150kDa, which is substantially consistent with the theoretically calculated molecular weight of the covalently coupled SpyCatcherUGTm-SpyTagSUS subunit at 166kDa, indicating that hetero-peptide covalent bonds have been formed between different enzyme molecules to achieve self-assembly.

(4) This SpyCatcher/SpyTag self-assembly crosslinking reaction is sensitive to pH. By adjusting the pH, the fusion enzyme was incubated at a pH of 1: 1 subunit molar ratio, after 1 hour incubation at room temperature, SDS-PAGE analysis found that the cross-linking assembly rate of SpyCatcherUGTm with SpyTagSUS was at least > 90% (densitometry).

(5) The inventors also analyzed whether co-expression in E.coli BL21(DE3) cells could directly obtain self-assembled UGTm-SUS functional units for immobilization. In fact, the co-expression vector of pRSFDuet-1 vector is used to realize the self-assembly of the enzyme in vivo, namely, the covalently bound 6XHis-SpyCatcherUGTm-SpyTagSUS can be directly obtained by purification. The results of these multienzyme in vivo co-expression indicate that self-assembled products can be formed in vivo. However, in view of the high efficiency and controllability of self-assembly in the in vitro mixing process, the in vitro self-assembly enzyme material is used for immobilization of multiple enzymes.

(6) Direct immobilization of multienzyme assemblies to functionalized Ni using abundant multivalent histidine tags on self-assembled modules²⁺Because of ordered linkage among enzyme molecules formed by self-assembly on NTA solid phase materials, direct immobilization of the multienzyme nanoclusters can obtain multienzyme arrays in ordered arrangement on a solid phase substrate. In order to fix UGTm-SUS components rapidly and efficiently, the inventors used commercially available Ni-based materials²⁺: the NTA complex is surface functionalized with microspheres (about 100 to 150 μm in diameter) of polystyrene. The functionalized microspheres and UGTm-SUS assembly solution are incubated for 1 hour at 20 ℃ with gentle shaking, so that multi-enzyme immobilization is realized.

(6) In the cascade unit, the rare pharmacologically active ginsenoside Rh2 can be efficiently synthesized by glycosylating protopanaxagenin diol (PPD) with UDP to UDP-glucose (UDPG) by the glycosyltransferase UGTm. The UDP intermediate produced in this artificial cascade catalytic system can be transported directly to the adjacent sucrose synthase SUS, which catalyzes the regeneration of the expensive sugar donor UDPG in the presence of high sucrose concentrations. The design and implementation of co-immobilized UGTm-SUS arrays has the potential to produce valuable rare active glycoside molecules in an efficient and economical manner.

(7) After thorough washing with 30mM imidazole, 50mM Tris-HCl buffer (pH 7.6) and 1% Tween to remove non-specific protein adsorption, microspheres with immobilization requirements were collected for analysis by Broadford assay. A visible blue change of Broadford was observed only in the assembly of UGTm-SUS immobilized microspheres, indicating that the protein can be rapidly loaded onto the solid support.

(8) Unlike the smooth surface of the unfixed microspheres, the UGTm-SUS component-immobilized microspheres showed a distinct heterogeneous dimple surface morphology under an optical microscope.

(9) Field emission scanning electron microscope (FE-SEM) imaging showed the presence of a particulate nanolayer covering the support. Transmission Electron Microscopy (TEM) showed a well-defined dense distribution of uniform nanoparticles, indicating that the tiny biocatalyst assembly particles are present on the surface in a highly ordered array. The four-stage structure of the enzyme and the potential multi-point linkage of multimeric complexes may be responsible for this observed morphology. Atomic Force Microscopy (AFM) showed that the average height of the nanolayers of the highly uniform protein layer was about 20 nm. This height is consistent with the estimated geometric dimensions of the UGTm/SUS mounting structure. The assembly can be effectively fixed on the carrier, so that the ordered multi-enzyme co-immobilized array catalytic material is created.

More specifically, the invention provides an ordered co-immobilization method of cascade multienzyme, which comprises the steps of obtaining multienzyme nanoclusters by using cascade multienzyme molecules through a self-assembly technology, forming polyhistidine tags with extremely rich quantity on an enzyme assembly body by using polyhistidine tags carried by component enzymes of the multienzyme nanoclusters, and directly fixing the ordered multienzyme nanoclusters to Ni through affinity²⁺NTA functionalized microsphere or microplate surface to form multienzyme ordered co-immobilized array.

Based on the strategy, an ordered co-immobilized array of glycosyltransferase UGTm and SUS sucrose synthase SUS is constructed to realize effective in-situ regeneration of expensive glycosyl donor UDP-glucose, and the method is applied to biosynthesis of rare ginsenoside Rh2 with biological and pharmaceutical activities.

Glycosyltransferase UGTm and SUS sucrose synthase SUS are respectively fused with Spycatcher and SpyTag peptide segments and flexible linking peptide segments, and recombinant expression is carried out on heterologous expression host strains through constructing expression plasmids or co-expression plasmids, and protein purification and assembly are carried out.

The glycosyltransferase UGTm has an amino acid sequence shown as SEQ NO. 1.

The glycosyltransferase SUS has an amino acid sequence as set forth in SEQ NO: 2.

The recombinant plasmid is fused with a SpyCatcher and a 6xHis tag, and the vector of the recombinant plasmid is pET-28a (+) plasmid.

The recombinant plasmid is fused with a SpyTag and a 6xHis tag, and the vector of the recombinant plasmid is a pET-28a (+) plasmid.

The host cell is Escherichia coli BL21(DE 3).

The step of realizing multi-site glycosylation comprises the steps of taking UDP-glucose as a glycosyl donor, taking protopanaxadiol PPD as a glycosyl acceptor, adding the glycosyl transferase and sucrose synthase co-immobilized material into a reaction system containing UDP-glucose and protopanaxadiol PPD for reaction to obtain a reaction solution, and carrying out separation and detection by High Performance Liquid Chromatography (HPLC), mass spectrometry (LC-MS) and the like.

The immobilization rate was checked by determining the initial enzyme amount minus the incubation time of the protein pairs remaining in the solution for immobilization. Ordered co-immobilized UGTm-SUS arrays reach load plateau only within 30 minutes, while random immobilization, i.e., direct attachment of an equimolar mixture of 6XHis-UGTm and 6XHis-SUS to the same Ni²⁺: NTA microspheres, did not reach the loading plateau up to 60 minutes. This is probably due to the presence of abundant multivalent histidine tags in the covalent self-assembly complexes, which ensure relatively more efficient and stoichiometric immobilization, resulting in ordered immobilization showing higher immobilization rates than random immobilization. The loading capacity of UGTm-SUS self-assembly calculated by determining the amount of protein recovered after 20mM EDTA treatment was 376. mu.g protein/mg microsphere.

To analyze the biocatalytic efficiency, the inventors examined the total enzymatic activity of ordered co-immobilized UGTm-SUS arrays, randomly co-immobilized enzymes and their unbound free self-assembly counterparts. The reactions were carried out at different times and the corresponding PPD substrate conversions were summarized. Under the same reaction conditions, at the same total enzyme concentration, the ordered co-immobilized array showed about 2.9-fold and 1.7-fold higher conversions over the random co-immobilized and free self-assembled enzyme systems, respectively, in the first hour of reaction time. At 4 hours, approximately 98.0% of the PPD was converted in the ordered co-immobilization array, but in contrast, only 51.0% of the PPD was converted in the random co-immobilization, and only 73.7% of the PPD was converted in the free self-assembly system.

Compared to random co-immobilization and free MENC systems, the ordered co-immobilization array showed the highest initial rate of Rh2 synthesis, especially at low concentrations of UDPG (less than 400 μ M) (fig. 12B). These results indicate that ordered co-immobilized UGTm-SUS arrays not only enhance the catalytic efficacy of the enzyme cascade, but also result in a 3-fold reduction in UDPG loading compared to random co-immobilization.

Ordered co-immobilized UGTm-SUS arrays, randomly co-immobilized UGTm/SUS and free UGTm-SUS MENC have all been prepared and stored at 4 ℃. These biocatalytic systems were used to detect enzyme activity on day 0, then every two days, for a total of eight days. The storage stability of the ordered co-immobilized UGTm-SUS array was higher than the random co-immobilized array and free MENC counterpart. By day 8, free UGTm-SUS MENCs and random co-immobilized arrays retained only 25% and 36% of their original activity, respectively, while ordered co-immobilized UGTm-SUS arrays retained about 51% of their original activity under the same conditions

Various immobilized enzymes can be easily separated from the reaction solution by filtration. The reusability of the fixed UGTm-SUS biocatalytic cascade is evidenced by its observed activity in each successive test round. In each round, the immobilized UGTm-SUS biocatalytic cascade was incubated with 5mM PPD, 0.5mM UDP, 50mM Tris-HCl (pH 8.0), 400mM sucrose and 1% Tween 80(v/v) for 3 hours at 35 ℃. The ordered immobilized UGTm-SUS array showed stable and continuous operation stability, and remained over 75.1 + -4.4% of the initial activity even after 8 rounds of reaction

Compared with the prior art, the invention has the following main advantages:

(1) in the construction of the multi-enzyme co-immobilized array catalytic material, the self-assembly body is utilized to carry a plurality of His labels. In the process of realizing immobilization, the Ni-NTA functionalized substrate can be directly combined with the surface of the Ni-NTA functionalized substrate without introducing other additional modification. The fixing method is simple and direct, has few operation steps, and is beneficial to keeping the high activity of the biocatalyst. Moreover, a population effect is formed by a plurality of His labels on the utilized assembly, and compared with other single fusion protein His label mediated immobilization, the method has strong binding force, high obvious immobilization efficiency and strong stability, and is not easy to separate from a substrate material.

(2) In the multi-enzyme co-immobilized array catalytic material, the His labels fused on each subunit of the oligomerase are utilized for substrate combination, so that the oligomerase is combined with the substrate at multiple points, and the inactivation of the oligomerase caused by the dissociation of the common subunits in the application of the oligomerase can be effectively avoided, so that the overall stability of the oligomerase catalytic material is improved, and the cyclic reuse of the catalytic material is facilitated.

(3) In the multi-enzyme co-immobilized array catalytic material, the first enzyme (such as SUS) and the second enzyme (such as UGTm) are orderly, directionally and stably arranged and combined on the solid phase substrate, when the multi-enzyme co-immobilized array catalytic material is applied to biochemical synthesis reaction, a product of the first enzyme after catalysis can be immediately contacted by the second enzyme in the system to be used as a substrate for conversion, common substrate inhibition effect in enzyme catalysis is eliminated, the cooperativity among cascade multi-enzymes is improved, and the overall catalytic efficiency of the multi-enzyme cascade system can be greatly promoted by the orderly array arrangement among the multi-enzymes.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples are generally carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.

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. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Experimental part

Material

Primers STAR Max DNA polymerase, restriction endonucleases and Polymerase Chain Reaction (PCR) reagents were purchased from TaKaRa (Chinese Dalian). Protein assay kits were purchased from Sangon (shanghai, china), plasmids, gels and PCR purification kits were purchased from general (shanghai, china). Pentanediol (PPD), UDP-glucose, UDP and ginsenoside Rh2 standards were purchased from Sigma-Aldrich (St. Louis, Mo.). Isopropyl- β -D-thiogalactopyranoside (IPTG), sucrose and nickel Nitrite Triacetate (NTA) functionalized polystyrene-based microspheres were purchased from Raygood Biotech (shanghai, china). All other reagents were the highest purity available.

Construction of fusion genes

It has previously been reported that E.coli codon optimized genes encoding Saccharomyces cerevisiae UGT51 and Arabidopsis SUS were synthesized and used as templates for constructing variants for use in this study. UGTm (S81A/L82A/V84A/K92A/E96K/S129A/N172D), S129A FP/RP and N172D FP/RP were amplified by PCR using SQuick Quick with primers S81A FP/RP, L82A FP/RP, V84A FP/RP, K92A FP/RP, E96K FP/RP. The fragment of UGTm was subsequently cloned into pET28a-SpyCatcher vector to generate plasmid pET28-SpyCatcherUGTm with an N-terminal SpyCatcher and a C-terminal 6XHis tag using ligation-independent cloning. The SUS coding sequence was subcloned into pET28a-SpyTag vector using SUS FP/RP primers to generate plasmid pET28-SpyTagSUS with an N-terminal 6XHis tag and SpyTag. The pRSFDuet-1 vector (Novagen, Inc.) with two Multiple Cloning Sites (MCS) was used to clone and co-express the fusion protein. A co-expression plasmid for pACYDuet-6XHis-SpyCatcherUGTm-SpyTagSUS was also constructed by standard molecular cloning procedures. In this expression vector, the SpyCatcherUGTm gene inserted into MCS-1 can produce a recombinant protein having a-6 XHis tag at the N-terminus. The recombinant protein SpyTagSUS corresponding to the gene located in MCS-II does not have a fusion affinity tag. Table S1 lists the primers and amino acid sequences used for plasmid construction. All plasmids were verified by sequencing and then transformed into e.coli BL21(DE3) for recombinant protein expression.

Protein expression and purification

The positively transformed E.coli BL21(DE3) strain was grown in LB liquid medium supplemented with 50. mu.g/mL kanamycin at 37 ℃. When the optical density at 600nm reached 0.8, protein expression was induced by 0.5mM IPTG after 16h incubation at 20 ℃. Cells were harvested by centrifugation at 8,000rpm for 15 minutes and resuspended in 20mM Tris-HCl buffer, pH 8.0. The resuspended cells were lysed with a homogenizer at 4 ℃ and centrifuged at 12,000rpm for 30 minutes. The supernatant was filtered using a 0.44 μm filter and then applied to a Ni-NTA agarose column (GE, USA). After washing with 20mM Tris-HCl pH 8.0, 500mM NaCl and 30mM imidazole buffer, the recombinantly expressed protein was eluted with 20mM Tris-HCl (pH 8.0), 500mM NaCl and 200mM buffer and concentrated by centrifugation at 4 ℃ using Amicon Ultra-15 tubes (Millipore, USA). Gel filtration chromatography was performed by using a Superdex200 increment-10/300 column on an AKTA FPLC system (GE, USA). Protein concentration was determined according to the Broadford method. For in vivo co-expression and testing of self-assembly, the co-expression plasmid of pACYDuet-6XHis-SpyCatcherUGTm-SpyTagSUS was also transformed into E.coli BL21(DE3) using standard procedures in the presence of chloramphenicol (34 mg/L). For protein expression and purification.

Enzyme Activity assay

UGTm and SUS enzyme activities were detected by High Performance Liquid Chromatography (HPLC). The standard assay mixture for UGTm activity contained 50 Tris-HCl buffer (50mM pH 8.0), PPD (0.5mM), UDP-glucose (5mM), 1% (v/v) Tween 80, and 0.25mg/mL purified protein, and for SUS enzyme assay, the mixture contained 50mM Tris-HCl (pH 8.0), 0.5mM UDP, and 500mM sucrose, 0.25mg/mL protein. After initiation of the reaction by addition of 0.25mg/ml enzyme, the reaction mixture was sealed and incubated at 37 ℃ for 2 hours. The enzymatic reaction was quenched by addition of 300. mu.l of n-butanol (300. mu.l), the precipitated protein was removed by centrifugation at 13,000 g for 10 min, and the n-butanol layer was carefully removed. The reaction mixture was extracted once more with a second 300. mu.l volume of n-butanol. The combined n-butanol layers were concentrated by rotary evaporation and the residue was redissolved in 100. mu.l methanol and analyzed by HPLC using an Agilent Eclipse XDB-C18(5 μm, 4.6X 250mm) column. Sample separation was achieved using the following gradient: 0-14 minutes, 100% B in 14-18 minutes, 45% B in 18-20 minutes, and the flow rate is kept at 0.5 mL/min. SUS activation was performed at 35 ℃ for 1 hour, and the assay mixture was boiled for 3 minutes to terminate the reaction. The reaction mixture was centrifuged at 13,000 Xg for 20 minutes. The supernatant was used for HPLC analysis and the Eclipse XDB-C18 column was pre-equilibrated with 100mM Na2HPO4/NaH2PO4 pH 6.5 and 10mM tetrabutylammonium bromide. The generated UDPG is assigned according to a standard retention time. All measurements were repeated three times.

Self-assembly and SDS-PAGE analysis of multiple enzymes

After determining the concentration of the freshly purified proteins, SpyCatcherUGTm and SpyTagSUS with different molarity were mixed in a buffer containing 50mM Tris-HCl pH 7.0 to 8.5, 100mM NaCl for 1h at room temperature, and the assembly was then subjected to 10% SDS-PAGE analysis. For detailed analysis of the gels, the intensity of each band was quantified by densitometry using a Tanon GIS digital image analysis system (Tanon, shanghai, china).

Immobilization assay

Mixing Ni²⁺: NTA-functionalized polystyrene microspheres were equilibrated in 50mM Tris-HCl buffer, 30mM imidazole, 100mM NaCl, pH 7.6 for 30 min. For ordered co-immobilization, spycatchergtm and SpyTagSUS were first mixed at equal subunit molar ratios. After 1 hour of reaction at room temperature, the formation of isopeptide bonds was confirmed by SDS-PAGE. Subsequently, the enzyme self-assembly solution was mixed with 300mg Ni²⁺: NTA-polystyrene microspheres were incubated at 20 ℃ with gentle continuous shaking. After 1 hour of incubation, the microspheres were filtered through a paper filter and washed 5 times in 50mM Tris-HCl buffer, pH 7.6, 30mM imidazole, 100mM NaCl and 1% Tween. Similarly, 3mg/mL of an equimolar mixture of 6XHis-UGTm and 6XHis-SUS was mixed with 300mg of Ni2 +: NTA polystyrene microspheres were incubated at 20 ℃ for 1h to generate random immobilizations as controls. The randomly immobilized material was also washed and collected using the same procedure as described above. Fixation was confirmed using Broadford staining and examination under light microscopy. The loading capacity of the immobilized microspheres was calculated by determining the amount of protein recovered after 20mM EDTA treatment. For immobilization rate analysis, the amount of immobilized biocatalyst was measured by mass balance between the initial solution and the collected fractions at different time points. The protein was quantified by directly measuring the absorbance at 280nm using a NanoDrop 2000 spectrophotometer (Thermo Scientific, usa).

FE-SEM, TEM and AFM experiments

For FE-SEM, the material of the immobilized UGTm-SUS was lyophilized on a 10N lyophilizer (Kryster, USA). Images were collected on a FEI Sirion 200 scanning electron microscope (FEI, usa) operating at 10 kV. To reduce the charging effect, the sample was sputter coated with nanogold prior to analysis. For TEM, the samples were compared with 1.0% phosphotungstic acid. TEM was performed using a Tecnai G2 spirit Biotwin microscope (FEI, USA). For AFM, samples were prepared by dropping 10 μ L of immobilized MENC onto cleaved mica for 10 minutes, washed with deionized water and dried in air. Images were collected in the air using a scanning probe microscope (AFM/Multimode nanoscope iia, Bruker, GER) operated in tapping mode. Image analysis was performed using the Nanoscope v.5.30 software package.

Catalytic activity of co-immobilized biocatalytic cascades

The overall enzyme activity of the ordered co-immobilized UGTm-SUS array, randomly co-immobilized enzymes and their unbound free MENC counterparts was determined by monitoring PPD substrate conversion, which was obtained by HPLC analysis. The assay was performed in the following mixture: 50mM Tris-HCl buffer (pH 8.0), 2mM PPD (in ethanol), 500mM sucrose and 1% (v/v) Tween 80 were mixed with ordered co-immobilized UGTm-SUS array, free MENC or enzyme mixture and pre-incubated for 5 min before initiating the reaction by adding various concentrations of UDP. Subsequently, the reaction was quenched with the same volume of n-butanol, filtered through a 0.22 μm filter, and directly analyzed by HPLC according to the enzyme activity assay. All measurements were repeated three times.

Stability and reusability assays

The storage stability C of the immobilized UGTm-SUS MENC and free enzyme mixture was evaluated by measuring the activity of Rh2 synthesized enzyme after storage in 50mM Tris-HCl pH 8.0 buffer after 0, 2, 4, 6 and 8 days at 25 ℃. The initial activity was defined as 100%. Reusability of ordered co-immobilized UGTm-SUS arrays was investigated through multiple cycles of filters. The activity of the ordered co-immobilized UGTm-SUS array after each cycle was normalized to the initial value.

Discussion of results and analysis

FIG. 1 is a schematic diagram of the design of a cascade enzyme catalytic array of ordered co-immobilized dimeric glycosyltransferase UGTm and tetrameric sucrose synthase SUS; A) firstly, SpyCatcher/SpyTag is fused to the subunit ends of UGTm and SUS, and a stable covalent isopeptide bond is spontaneously formed between SpyCatcherUGTm and SpyTagSUS and the multi-enzyme nano self-assembly is formed by oligomerization of multi-subunit enzyme. Further utilizing a plurality of polyhistidine tags carried on the constitutive enzymes in the multienzyme nano self-assembly to be further fixed on Ni2⁺: a co-immobilized biological catalytic material with ordered multi-enzyme array arrangement is created on the NTA surface functionalized microspheres. B) An artificial cascade catalytic system consisting of glycosyltransferase UGTm and sucrose synthase SUS; in the constructed ordered co-immobilized multi-enzyme array of UGTm-SUS, the conversion of ginsenoside protopanaxadiol PPD into rare ginsenoside Rh2 and the regeneration of expensive sugar donor UDPG can be promoted.

In the invention, a novel facial strategy is developed by taking self-assembled polymerase nanoclusters (MENCs) as a design principle, and is used for orderly co-immobilizing polymerase and promoting substrate channeling, so that the overall performance of a biocatalytic cascade reaction is improved. In the construction, dimeric UGT51 glycosyltransferase mutants from Saccharomyces cerevisiae (UGTm, S81A/L82A/V84A/K92A/E96K/S129A/N172D) and tetrameric sucrose synthase (SUS) from Arabidopsis were selected as model enzymes. The multimeric enzyme first undergoes hierarchical self-assembly driven by multimeric oligomerization, where the SpyTag/SpyCatcher covalent pairing reaction produces a multimeric enzyme nanocluster assembly. Subsequently, the MENC module can be further immobilized on suitable Ni by polyhistidine-tagging of MENC constitutive enzymes²⁺: NTA coated microspheres or microplates (fig. 1A). The established polymerase co-immobilization defines an ordered biocatalytic array with multiple enzymes in spatially adjacent tissues, and is expected to provide substrate channels for different biocatalysts so as to realize better cascade performance. In the cascade unit, the rare ginsenoside Rh2 having pharmacological activity can be synthesized by UGTm glycosylation of the main ginsenoside protopanaxadiol (PPD) with concomitant UDP-glucose (UDPG) to UDP. UDP intermediates can be directly transported to neighboring SUS enzymes in the presence of high sucrose concentrationsIn this case, the enzyme catalyzes the regeneration of the expensive sugar donor UDPG (fig. 1B). The design and implementation of co-immobilized UGT-SUS arrays shows the potential to produce valuable glycosides in an efficient and economical manner.

To avoid compromising enzyme activity, we first planed out the quaternary structure of the UGTm-SUS cascade constituting the enzyme. Both UGTm and SUS have N-termini directed outward from the molecular surface of each protein (fig. 2). SUS is a homotetramer composed of monomers with a molecular weight of 92 kDa. Therefore, we fused a relatively small SpyTag (. about.1.5 kDa) and 6XHis peptide to the N-terminus of each SUS monomer. The UGTm is a homodimeric molecule with a 45kDa monomer, on which we fused a SpyCatcher (-9.5 kDa) peptide to the N-terminus and 6 histidine tags at the C-terminus of each UGTm monomer (fig. 3A, B). These enzymes with SpyTag and SpyCatcher reaction sequences can be expressed and purified for bioconjugation (fig. 3C). To retain sufficient rotational flexibility for enzyme-substrate interactions, a flexible ([ (AG)3PEG ]5 linker was introduced between the corresponding Spycatcher/SpyTag and the peptide in the fusion construct (FIG. 3).

FIG. 2 shows the quaternary structure of the multi-subunit enzyme used for construction. (A) The structure of UGT51 (PDB: 5GL5) from Saccharomyces cerevisiae. (B) The structure of SUS, a sucrose synthase derived from Arabidopsis thaliana (PDB: 3S 28). The N and C termini are labeled as red and blue chains, respectively.

FIG. 3 is a construct expressing a SpyTag/SpyCatcher fusion enzyme and the spontaneous covalent binding reaction between SpyCatcher and SpyTag. (A) pET28a-SpyCatcherUGT plasmid. SpyC: a SpyCatcher domain; LL: (GGGGS)2 junction element. (B) pET28a-SpyTagSUS plasmid. SpyT: a SpyTag domain; LL: [ (AG)3PEG ]5 junction element. (C) Structure and molecular mechanism of SpyCatcher/SpyTag chemical pairing.

The fused SpyCatcherUGTm and SpyTagSUS, and the corresponding enzyme without fusion were expressed and purified in E.coli. Gel filtration chromatography analysis of the purified proteins showed that spycatchergtm and SpyCatcherSUS showed similar retention curves to the unfused corresponding enzymes (fig. 4). Furthermore, analysis of UDPG conversion showed that the fused spycatchergtm and SpyTagSUS exhibited similar enzymatic activity to the non-fused counterpart (fig. 5), indicating that the SpyTag or SpyCatcher fusion did not significantly compromise the structural and functional integrity design of these enzymes.

FIG. 4 is an elution profile of SpyCatcherUGTm and unfused UGTm of the fused or unfused enzyme by gel filtration chromatography (A). (B) Elution profiles of SpyTagSUS and unfused SUS counterparts.

FIG. 5 is a graph comparing the relative enzyme activities of SpyCatcherUGTm, SpyTagSUS and non-fusion enzymes. All measurements were taken at least 3 times and the error bars represent the standard error.

Next, we investigated whether mixing these fused SpyCatcher/SpyTag enzymes would result in the formation of covalently cross-linked enzyme complexes. SDS-PAGE showed protein bands corresponding to molecular weights above 150kDa, which is consistent with the theoretically calculated molecular weight of the covalently coupled 166kDa SpyCatcherUGTm/SpyTagSUS subunit (FIG. 6A). The SpyCatcher/SpyTag pair has been shown to self-assemble and form covalent isopeptide bonds between different polypeptides. Notably, this crosslinking reaction was pH sensitive (fig. 7), supporting SpyCatcher/SpyTag mediated covalent binding. When the fusion enzyme is expressed as a 1: when mixed at a 1 subunit molar ratio (50mM Tris-HCl buffer (pH 7.6), incubated at room temperature for 1 hour), the SpyCatcherUGTm was found to bind to SpyTagSUS at least > 90% (densitometric analysis).

FIG. 6 is a SDS-PAGE analysis of (A) SpyCatcher/SpyTag mediated UGTm-SUS self-assembly. The purified enzyme and the assembled UGTm-SUS multienzyme nanoclusters were subjected to 10% SDS-PAGE operation and stained with coomassie brilliant blue. Lane M, protein molecular weight; lane 1, purified recombinant SpyTagSUS protein; lane 2, purified recombinant spycatchereugtm protein; lane 3, assembled SpyCatcherUGTm-SpyTagSUS protein. (B) The purity of the assembled SpyCatcherUGTm-SpyTagSUS protein was determined by densitometry and was 91%.

FIG. 7 is a graph of SpyCatcher/SpyTag mediated irreversible covalent binding at various pH. Lane M: protein markers (kDa), lane 1: SpyTagSUS, lane 2: spycatcheergtm, lane 3: self-assembly in Tris-HCl buffer pH 7.0, lane 4: self-assembly in Tris-HCl buffer pH 7.5, lane 5: self-assembly in Tris-HCl buffer pH 8.0, lane 6: self-assembly in Tris-HCl buffer pH 8.5.

We also examined whether co-expression in E.coli BL21(DE3) cells could be used to obtain self-assembled UGTm-SUS functional units for immobilization. Indeed, after in vivo self-assembly, the co-expression construct containing the vector from pRSFDuet-1, i.e., the covalently bound 6 XHis-SpyCatcherUGTm/SpyTagSUS product, was generated directly and purified by affinity chromatography (FIG. 8B). These initial co-expression results indicate that assembled products can be formed in vivo. However, in view of the high assembly efficiency of the in vitro mixing procedure, we used the in vitro method for the subsequent immobilization experiments.

FIG. 8, (A) constructs of pACYDuet-6XHis-SpyCatcherUGTm-SpyTagSUS for in vivo protein co-expression and self-assembly (B) vs. Ni⁺Analysis of self-assembled 6XHis-SpyCatcherUGTm-SpyTagSUS in crude E.coli lysate obtained by NTA affinity chromatography by 10% SDS-PAGE. Lane M: protein markers (kDa), lane 1: cell lysate, lane 2: supernatant, lane 3: washed samples, lane 4: eluted sample.

For fast and efficient immobilization of UGTm-SUS components, we used commercially available polystyrene-based microspheres (about 100 to 150 μm in diameter) with Ni²⁺: the NTA complex is surface functionalized. The functionalized microspheres were incubated and gently shaken with UGTm-SUS assembly solution at 20 ℃ for 1 hour to achieve immobilization. After thorough washing with 30mM imidazole, 50mM Tris-HCl buffer (pH 7.6) and 1% Tween to remove non-specific protein adsorption, microspheres with immobilization requirements were collected for Broadford assay analysis. A visible blue change of Broadford was observed only in the assembly of UGTm-SUS immobilized microspheres, indicating that the protein can be rapidly loaded onto the support (fig. 9A). Unlike the smooth surface of the unfixed microspheres, the UGTm-SUS component-immobilized microspheres showed a distinct heterogeneous dimple surface morphology under light microscopy (fig. 9B and 10).

FIG. 9 is a representation of the UGTm-SUS components immobilized on functionalized microspheres. A) Analyzing the immobilized material by a Broadford method; after thorough washing of the incubated microspheres in the enzyme solution, the support was examined for attached proteins using Bradford solution; blank control: a Broadford solution; non-immobilized material: freshly prepared microspheres in a Broadford solution; immobilization: the treated microspheres were immobilized in Broadford solution. (B) Optical microscope images of immobilized or non-immobilized treated functionalized microspheres. Examples of convex-concave surface morphologies are indicated by blue arrows. The non-immobilized functionalized microspheres showed a smooth surface (inset).

FIG. 10 is an optical microscope image of functionalized microspheres of immobilized UGTm-SUS self-assemblies. An example of a relief surface morphology is indicated by blue arrows.

Field emission scanning electron microscope (FE-SEM) imaging showed the presence of a particulate nanolayer covering the support (fig. 11A). Transmission Electron Microscopy (TEM) showed a well-defined dense distribution of uniform nanoparticles, indicating that the tiny biocatalyst assembly particles are present on the surface in a highly ordered array (fig. 11B). The quaternary structure of the enzyme and the potential multipoint attachment of multimeric complexes may be responsible for this observed morphology. Atomic Force Microscopy (AFM) showed that the average height of the nanolayers of the highly uniform protein layer was about 20nm (fig. 11C). This height is consistent with the estimated geometry (about 16nm) of the UGTm-SUS assembly structure. The above results indicate that the assembly can be effectively immobilized, creating an ordered array of biocatalytic cascades.

FIG. 11 is a representative electron microscopy image of ordered co-immobilization of UGTm-SUS arrays. A) Scanning electron microscope images of immobilized UGTm-SUS self-assemblies on the surface of the support. After fixation, the samples were freeze dried and sputter coated with nanogold before analysis. The appearance of a granular structure in the immobilized material was observed under a scanning electron microscope. B) Transmission electron microscopy images of immobilized UGTm-SUS self-assemblies. Prior to analysis, the assembled samples were stained with phosphotungstic acid. The fine particles arranged orderly on the surface can be seen under a transmission electron microscope. C) Atomic force microscopy images of immobilized UGTm-SUS self-assemblies. The height of the microstructure of the immobilized multienzyme complex is about 20 nm.

Next, we subtract the residual in solution by determining the initial amount of enzymeThe protein pair was incubated for a fixed period of time to examine the fixation rate. Ordered co-immobilized UGTm-SUS arrays reach the loading plateau only within 30 minutes, while due to random immobilization, direct attachment to the same Ni²⁺: equimolar mixtures of 6XHis-UGTm and 6XHis-SUS of NTA microspheres did not reach the loading plateau for up to 60 minutes (FIG. 12A). This is probably due to the presence of abundant multivalent histidine tags in the covalent self-assembly complexes, which ensure relatively more efficient and stoichiometric immobilization, resulting in ordered immobilization showing higher immobilization rates than random immobilization. The loading capacity of UGTm-SUS MENC calculated by determining the amount of protein recovered after 20mM EDTA treatment was 376. mu.g protein/mg microsphere.

FIG. 12 is a graph of the multi-enzyme immobilization rate, UDPG regeneration efficiency, and ordered co-immobilized UGTm-SUS array storage stability and repeated utility analysis. A) UGTm-SUS self-assembly and immobilization efficiency analysis of multi-enzyme mixture on the carrier at 20 ℃ and pH 7.0; the amount of immobilized protein was determined from the difference between the total protein used for immobilization and the residual amount in the solution after immobilization. B) And (5) analyzing conversion efficiency. The conversion is shown as the percentage of PPD converted to Rh2, the final product. C) Orderly co-immobilization, random co-immobilization system and initial conversion rate of free multi-enzyme nano self-assembly under different UDP concentrations. D) Storage stability at 4 ℃. All measurements were performed at a protein concentration of 40 μ M; E) the ordered co-immobilized UGTm-SUS arrays are reusable after multiple recovery cycles; data represent the mean ± standard deviation of three measurements.

By HPLC analysis, we determined that the ordered co-immobilized UGTm-SUS biocatalytic cascade on solid support is active and useful for the biosynthesis of rare Rh2 (fig. 13). To analyze the efficiency of the biocatalyst, we examined the overall cascade enzyme activity of ordered co-immobilized UGTm-SUS arrays, randomly co-immobilized UGTm and SUS enzymes, and their unbound free MENC. The reactions were performed at different times and the corresponding ginsenoside PPD substrate conversions are summarized in fig. 12B. Under the same reaction conditions, the conversion rate of the ordered co-immobilized array in the first hour was approximately 3.8 and 2.3 times higher than that of the random co-immobilized and free MENCs systems, respectively, under the same total enzyme concentration. At 4 hours, nearly 100% of the PPD was converted in the ordered co-immobilized array, but in contrast, only 43.5% of the PPD was converted in the random co-immobilizer, and only 66.7% of the PPD in the free MENCs system. It is believed that the appropriate substrate channels between UGTm and SUS may exist in an ordered co-immobilized array compared to a randomly arranged enzyme cascade, thereby significantly improving the synergistic effect of the biocatalytic cascade. The ordered co-immobilized arrays showed higher catalytic efficiency than free MENC at all time points tested, indicating that the supramolecular structure of MENC may generate diffusion constraints for substrates and products, which can be eliminated when MENC is surface immobilized on a support. Ordered co-immobilization arrays of UGTm-SUS ensure that the substrate molecules have easy access to the active sites of the biocatalyst, thereby enhancing activity.

FIG. 13 is an HPLC analysis of the final product Rh2 formed from PPD by sequential fixed UGTm-SUS cascade concerted catalysis.

We further analyzed the UDPG regeneration efficiency of the biocatalytic cascade by measuring the initial rate of Rh2 production using various concentrations of UDP. In contrast to the random co-immobilized and free MENCs systems, the ordered co-immobilized arrays showed the highest initial rate of Rh2 synthesis, especially at low concentrations of UDP (less than 0.4mM) (FIG. 12C). Ordered immobilized UGTm-SUS arrays showed the highest initial rate of Rh2 synthesis at 0.4mM UDP level. However, random co-immobilization of multiple enzymes gave the highest synthesis rate at 1.2mM UDP. These results indicate that ordered co-immobilized UGTm-SUS arrays not only enhance the catalytic efficacy of enzyme cascade reactions, but also result in a 3-fold reduction in UDP usage compared to random co-immobilization. Substrate channeling facilitates transport of intermediates, avoids UDP inhibition often observed in glycosyltransferase reactions, and substantially stimulates Rh2 synthesis. Furthermore, since SUS catalysis is easily reversed, the rapid consumption and UDP transport of UDPG shifts the SUS-mediated equilibrium of reactions towards UDPG synthesis, thereby facilitating the entire enzymatic cascade.

When talking about the importance of immobilization for bioproduction applications, storage stability and reusability are important parameters. Ordered co-immobilized UGTm-SUS arrays, randomly co-immobilized UGTm/SUS and free UGTm-SUS MENC have all been prepared and stored at 4 ℃. These biocatalytic systems were used to test for enzyme activity starting on day zero and then every second day for eight days. The storage stability of the ordered co-immobilized UGTm-SUS array was higher than the random co-immobilized array and free MENCs counterpart. By day 8, the random co-immobilized UGTm and SUS biocatalysts and free MENC retained only 19.1% and 26.6% of their original activities, respectively, while the ordered co-immobilized UGTm-SUS array retained about 52.6% of their original activities under the same conditions. Condition (fig. 12D). This enhanced storage stability may result from the formation of a precise supramolecular structure that retains the structure of the enzyme during long-term storage.

Furthermore, it was demonstrated that various immobilized enzymes can be easily separated from the reaction solution by filtration. The reusability of the immobilized UGTm-SUS biocatalytic cascade is evidenced by its observed activity in each successive test round. In each round, the immobilized UGTm-SUS biocatalytic cascade was incubated with 5mM PPD, 400. mu.M UDP, 50mM Tris-HCl (pH 8.0), 400mM sucrose and 1% Tween 80(v/v) at 35 ℃. For 3 hours. The ordered immobilized UGTm-SUS array showed stable and continuous operational stability, remaining over 73.1 ± 3.2% of the initial activity even after 8 rounds of reaction (fig. 12E). The simple isolation and excellent reusability I observe will significantly reduce the operating costs of practical application of stationary cascade reactions in bioconversion.

In summary, this study has developed a new strategy for the ordered co-localization of different biocatalytic components on solid phase supports by using self-assembling polymerase nanoclusters as building blocks and protein hexahistidine tag affinity technology. The developed topologically well-defined co-immobilization system may activate the enzyme cascade better than the random, non-organized co-immobilization system. Likewise, recent advances in self-assembly indicate that other novel self-reactive bioorthogonal reaction chemical pairs can be used to design different cascades of organismsCombination of the catalyst. The site-specific introduction of these bio-orthogonal groups into proteins and the organization of three or more enzyme cascades, or the topological self-assembly of fixed cascade reaction centers, represent exciting future opportunities. Histidine tags selectively introduced at certain positions of the enzyme may also be used in combination with Ni²⁺: the NTA supports fine control of orientation and catalytic performance after surface binding, thereby promoting uniform orientation of the immobilized protein layer. Overall, these results indicate that the method is a rapid, simple method that allows for accurate design and control of co-immobilization of multiple enzymes in a well-defined, ordered fashion.

FIG. 14 is an SEM picture of microspheres before loading.

FIG. 15 is a scanning electron micrograph of the catalytic material obtained after random immobilization of the multiple enzymes.

FIG. 16 is a scanning electron micrograph of microspheres obtained by sequential immobilization of multiple enzymes.

The above results show that: the multienzyme orderly co-immobilization forms stable loading on the surface of the microsphere, and the microsphere is loaded after simply mixing the multienzyme to generate random immobilization; after the microspheres were washed with the buffer solution after the same immobilization was completed, the layer structure was damaged and lost under an electron microscope (see fig. 15), and the ordered immobilized surface was clearly immobilized and intact (see fig. 16). This comparative result indicates that the direct his-tag mediated generation of randomly immobilized multienzymes is relatively unstable.

The sequence of the invention is shown as follows:

TABLE S1 primers used in plasmid constructs

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it will be appreciated that various changes or modifications may be made by those skilled in the art after reading the above teachings of the invention, and such equivalents will fall within the scope of the invention as defined in the appended claims.

Sequence listing

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Claims (10)

1. A catalytic material of a multi-enzyme co-immobilized array, comprising:

1) a multienzyme nano self-assembly body comprises an element enzyme and a mate enzyme of more than two cascade catalytic systems and a histidine tag fused on the element enzyme and/or the mate enzyme, wherein,

the element enzyme is an oligomeric enzyme molecule, and a self-reaction peptide segment or a self-reaction chemical pair is fused on a subunit of the oligomeric enzyme molecule, and a histidine tag is optionally fused;

the pairing enzyme is a functional enzyme molecule, and a self-reaction peptide segment or a self-reaction chemical pair is fused on a subunit of the functional enzyme molecule, and a histidine tag is optionally fused;

the multienzyme nano self-assembly is obtained by spontaneous linking reaction between the element enzyme and the pairing enzyme through respective fused self-reaction peptide fragments or self-reaction chemical pairs; and

2)Ni²⁺: NTA surface-functionalized solid substrate, Ni²⁺Binding to the surface of the solid phase substrate through NTA;

the multi-enzyme nano self-assembly is connected with the Ni through the histidine tag²⁺Coordinate bonding to said Ni²⁺: the immobilization and combination of the NTA surface functionalized solid phase substrate;

in the catalytic material, 10-100% mole of the element enzyme is fused with the histidine tag; and/or

10-100% moles of said companion enzyme is fused with said histidine tag.

2. The catalytic material of claim 1 wherein the elemental enzyme is selected from the group consisting of: sucrose synthase SUS, glucose dehydrogenase, or a combination thereof; and/or

The pairing enzyme is selected from the group consisting of: a glycosyltransferase UGTm, a cytochrome oxidase P450m, or a combination thereof.

3. The catalytic material of claim 1, wherein in the multi-enzyme nano self-assembly, the molar ratio of subunits or monomers of the element enzyme to the histidine tag is 1: 3-15; and/or

The molar ratio of the subunit or monomer of the pairing enzyme to the histidine tag is 1: 3-15.

4. The catalytic material of claim 1 wherein 30-100 mole% of the elementary enzymes in the catalytic material are fused with the histidine tag; and/or

30-100% moles of said companion enzyme is fused with said histidine tag.

5. The catalytic material of claim 1 wherein the solid phase substrate is a shape functional shape and is selected from the group consisting of: resin microspheres, agarose microspheres, magnetic bead microspheres, or a combination thereof.

6. A method of preparing the catalytic material of claim 1, comprising the steps of:

1) providing a first reaction solution, a second reaction solution and a third reaction solution, wherein the first reaction solution contains an element enzyme, the second reaction solution contains a pairing enzyme, and the third reaction solution contains Ni²⁺: a solid phase substrate with NTA surface functionalized, wherein,

the element enzyme is a tetrameric molecule, and the element enzyme is fused with a SpyTag and an optional histidine tag;

the mate enzyme is a dimer molecule, and the mate enzyme is fused with a SpyCatcher and an optional histidine tag;

the element enzyme and/or the companion enzyme are fused with histidine tags;

2) mixing the first reaction solution and the second reaction solution to obtain a fourth reaction solution, and carrying out self-assembly reaction to obtain a multienzyme nano self-assembly;

3) and (3) mixing the product obtained in the step 2) with the third reaction solution to obtain a fifth reaction solution, and reacting to obtain the multienzyme co-immobilized array catalytic material.

7. The method according to claim 6, wherein the pH of the fourth reaction solution is 4.5 to 8.5.

8. Use of the catalytic material of the multi-enzyme co-immobilized array of claim 1 for a use selected from the group consisting of:

1) used for catalyzing the synthesis of saponin compounds;

2) for self-cycling to produce UDP-glucose (UDPG) as a sugar donor.

9. A method for synthesizing saponin compounds is characterized by comprising the following steps:

1) reacting sucrose with UDP by using the multi-enzyme co-immobilized array catalytic material of claim 1 as an enzyme to obtain UDPG;

2) directly taking the product obtained in the step 1) as a raw material, and reacting the product with protopanaxadiol PPD to obtain Uridine Diphosphate (UDP) and saponin Rh 2.

10. The method of claim 9, wherein the reaction efficiency of the method is 50-100%.

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