Sucrose isomerase recombinant as well as construction method and application thereof

Abstract

The invention relates to the field of genetic engineering and immobilized enzymes, in particular to a sucrose isomerase recombinant as well as a construction method and application thereof. Wild type sucrose isomerase (Sase) from dispersed pantoea is subjected to molecular modification, fusion expression is performed after screening of affinity peptide ligands, the specific enzyme activity of the obtained sucrose isomerase recombinant is 88% higher than that of sucrose isomerase, the enzyme activity level of the sucrose isomerase recombinant is remarkably improved, and the specific catalysis efficiency of the sucrose isomerase recombinant on a sucrose substrate is improved; and reference is provided for improving molecular modification of other enzymes. The sucrose isomerase recombinant has good thermal stability, and finally, research on embedding system optimization is performed on one-step purification and immobilization of SIase, so that the affinity directional immobilized enzyme microspheres which have high hardness and strength and can effectively reduce leakage or loss of enzyme molecules are prepared. The reusability of the sucrose isomerase recombinant is obviously improved.

Description

Technical Field

[0001] The invention relates to the fields of genetic engineering and immobilized enzymes, and in particular to a sucrose isomerase recombinant and a construction method and application thereof. Background Art

[0002] Isomaltulose is a reducing disaccharide formed by the combination of glucose and fructose with α-1,6 glycosidic bonds. It exists naturally in honey, sugarcane juice and its industrial products, molasses and syrup, but the content is very small. As an isomer of sucrose, its taste and physical properties are very similar to sucrose, but its sweetness is only half of that of sucrose; the viscosity of their aqueous solutions is close, but isomaltulose is more stable under acidic conditions. Isomaltulose has excellent characteristics such as low sweetness, easy absorption, no caries, and low calories. Moreover, the cleavage rate of isomaltulose is not as fast as sucrose, which provides a healthier choice for patients with obesity, diabetes or cardiovascular disease. Isomaltulose is a new multifunctional sweetener and a sucrose substitute that is generally recognized as safe internationally. It has broad application prospects in the pharmaceutical and food industries. Sucrose isomerase is the key enzyme for the enzymatic production of isomaltulose. Its growing demand has promoted the research on the immobilization of sucrose isomerase (SIase).

[0003] Enzyme immobilization is a method applicable to various field processes. Enzymes can be immobilized on solid supports by a variety of physical and chemical methods, such as adsorption, encapsulation, cross-linking and covalent bonding. In general, compared with the use of free enzymes, enzyme immobilization provides higher stability, easy separation and reusability in batch and continuous processes. Adsorption is a method that immobilizes enzyme molecules around the support through nonspecific physical adsorption, biospecific adsorption, ion adsorption, affinity adsorption and hydrophobic interaction between the structure of the enzyme molecules themselves and the support, thereby successfully preparing a wide variety of immobilized enzymes. This method is simple to operate, but the disadvantage is that the interaction between the enzyme and the support is weak, making the resulting compounds unstable compared to compounds produced by other technologies, but a survey shows that adsorption is still the fastest and most common immobilization method. Adsorption of enzymes on insoluble supports is a very simple and widely used method. Among them, porous materials are often used for enzyme immobilization, such as organic polymers, silica, nanomaterials and ceramics. Due to their low density, high specific surface area and adjustable pore size, they are often used as carriers for immobilized enzymes. However, the non-directional direct interaction between the protein and the carrier may lead to undesirable conformational effects and steric hindrance, which distort the active site and thus reduce the catalytic activity of the enzyme. The classical approach to protein immobilization based on nonspecific adsorption usually has a negative impact on protein function. Directed immobilization enables the enzyme protein to be fixed on the carrier surface in an orderly manner, and its active site faces the outside of the solid surface, which can maintain the natural conformation of the enzyme protein, which is conducive to the entry of the substrate into the catalytic active site, thereby significantly improving the activity of the immobilized enzyme. This limitation can be overcome by fusing bioaffinity tags, thereby achieving one-step purification and directed immobilization of the crude enzyme, reducing the cost of separation and purification in the reaction medium, so that this effective method is more cost-effective,

[0004] Solid binding peptides (SBPs) are short amino acid sequences that can specifically recognize and bind to a variety of solid surfaces. Typically, SBPs bind to solid surfaces through a variety of non-covalent interactions. Various factors can define their binding strength, such as surface morphology and solution conditions. However, the key aspects of binding to solid surfaces are the amino acid composition and structural diversity of SBPs. SBPs exhibit binding affinity and selectivity to the surfaces of solid materials, including support materials for biocatalysis, silica, zeolites, glass, metals and polymers. They strongly bind to their cognate solids by adopting specific conformations that support many non-covalent interactions (such as electrostatic bonding) between key amino acids and solid surfaces, usually resulting in binding affinities in the submicromolar and nanomolar range. Studies have shown that SBPs guide enzymes to be directional fixed to solid supports, retaining native enzyme structure and catalytic activity. In addition, SBPs can be easily integrated into permissive regions (such as N or C termini) of proteins using genetic engineering techniques to produce bifunctional fusion proteins. Therefore, SBPs are often used as molecular linkers and / or anchoring molecules to immobilize functional proteins onto solid surfaces in a single step without the need for any complex chemical reactions or physical treatments. This type of immobilization technology is highly designed and has a clear purpose, and is expected to design and develop an efficient immobilized enzyme preparation.

[0005] However, there is no research on the application of solid-bound peptide immobilized enzyme technology to the immobilization of SIase and its catalytic formation of isomaltulose. The existing methods for preparing isomaltulose have many drawbacks. Therefore, it is of great practical significance to study an immobilized SIase that is cost-effective, simple to operate, has high catalytic activity and can be industrially produced. Summary of the invention

[0006] The object of the present invention is to provide a sucrose isomerase recombinant and a construction method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The sucrose isomerase recombinant provided by the present invention can be used to prepare affinity-oriented immobilized enzyme and affinity-oriented immobilized enzyme microspheres, which have high catalytic activity and have the advantages of low cost, simple operation and industrial production of immobilized sucrose isomerase.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a recombinant sucrose isomerase, wherein the amino acid sequence of the recombinant sucrose isomerase is any one of SEQ ID NO.1 to SEQ ID NO.3.

[0009] The present invention provides a method for constructing the above-mentioned recombinant sucrose isomerase, comprising the steps of connecting an affinity polypeptide to the C-terminus of a sucrose isomerase derived from a dispersed Pantoea via a flexible linker to obtain the recombinant sucrose isomerase;

[0010] The flexible linker includes but is not limited to the decapeptide flexible linkers GSA, NL, G8 and GSA; in a specific embodiment of the present invention, the amino acid sequence of the flexible linker is shown in SEQ ID NO.7;

[0011] The amino acid sequence of the affinity polypeptide is any one of SEQ ID NO.4 to SEQ ID NO.6;

[0012] The accession number of the sucrose isomerase is: AAP57083.1.

[0013] The present invention provides a gene encoding the above-mentioned sucrose isomerase recombinant, and the nucleotide sequence of the gene is shown in any one of SEQ ID NO.8 to SEQ ID NO.10.

[0014] The present invention provides a biological material containing the above gene, and the biological material comprises a recombinant expression vector and an engineering bacterium.

[0015] Further preferably, the basic plasmid of the recombinant expression vector is pET-22b(+);

[0016] The basic bacteria of the engineering bacteria is Escherichia coli.

[0017] The present invention provides a sucrose isomerase mutant, wherein the sucrose isomerase mutant is obtained by mutation of any of the following sites on SEQ ID NO.1:

[0018] (a) the amino acid corresponding to position 213 in SEQ ID NO. 1 is mutated from phenylalanine (F) to leucine (L);

[0019] (b) the amino acid corresponding to position 232 of SEQ ID NO. 1 is mutated from glutamic acid (E) to aspartic acid (D), tyrosine (Y) or tryptophan (W);

[0020] (c) the amino acid corresponding to position 264 in SEQ ID NO. 1 is mutated from alanine (F) to tyrosine (Y);

[0021] (d) the amino acid corresponding to position 299 of SEQ ID NO. 1 is mutated from glutamine (Q) to glutamic acid (E);

[0022] (e) the amino acid corresponding to position 388 of SEQ ID NO. 1 is mutated from isoleucine (I) to glutamic acid (E), tyrosine (Y) or tryptophan (W);

[0023] (f) the amino acid corresponding to position 389 in SEQ ID NO.1 is mutated from glutamic acid (E) to aspartic acid (D) or glutamine (Q);

[0024] (g) the amino acid corresponding to position 392 in SEQ ID NO. 1 is mutated from aspartic acid (D) to glycine (G);

[0025] (h) the amino acid corresponding to position 401 in SEQ ID NO. 1 is mutated from asparagine (N) to glutamic acid (E), tyrosine (Y), aspartic acid (D) or tryptophan (W);

[0026] (i) the amino acid corresponding to position 410 in SEQ ID NO. 1 is mutated from asparagine (N) to glutamic acid (E), serine (S) or glutamine (Q);

[0027] (j) the amino acid at position 412 of SEQ ID NO. 1 is mutated from alanine (A) to glutamic acid (E), asparagine (N) or aspartic acid (D);

[0028] (k) the amino acid corresponding to position 416 in SEQ ID NO. 1 is mutated from glutamine (Q) to phenylalanine (F), tryptophan (W) or tyrosine (Y);

[0029] (l) The amino acid corresponding to position 459 in SEQ ID NO. 1 is mutated from alanine (A) to glutamic acid (E).

[0030] The present invention provides a gene encoding the above sucrose isomerase mutant, wherein the nucleotide sequence of the gene is shown in SEQ ID NO.8 or is obtained by mutation based on a degenerate sequence of the sequence shown in SEQ ID NO.8.

[0031] The present invention provides a biological material containing the above gene, and the biological material comprises a recombinant expression vector and an engineering bacterium.

[0032] Further preferably, the basic plasmid of the recombinant expression vector is pET-22b(+);

[0033] The basic bacteria of the engineering bacteria is Escherichia coli.

[0034] The present invention provides the use of the above-mentioned sucrose isomerase recombinant, the above-mentioned gene, the above-mentioned biological material or the above-mentioned sucrose isomerase mutant in the preparation of affinity-oriented immobilized enzyme or affinity-oriented immobilized enzyme microspheres.

[0035] The present invention provides a method for preparing an affinity-oriented immobilized enzyme, comprising the following steps:

[0036] (1) fermenting and culturing the engineered bacteria containing the above-mentioned gene and collecting the bacteria;

[0037] (2) subjecting the bacterial cells to ultrasonic disruption to obtain a crude enzyme solution containing the above-mentioned recombinant sucrose isomerase;

[0038] (3) Mixing the crude enzyme solution, the immobilization carrier material and the buffer to obtain the affinity-oriented immobilized enzyme.

[0039] Further preferably, the immobilized carrier material comprises nano-silica spheres (SNs), micron-sized (SMs) SiO2 particles and mesoporous silica (MS);

[0040] The particle size of the nano silicon dioxide sphere is 20nm, 50nm or 500nm.

[0041] The present invention provides an affinity-oriented immobilized enzyme prepared by the above-mentioned preparation method.

[0042] The present invention provides a method for preparing affinity-oriented immobilized enzyme microspheres, comprising the following steps:

[0043] (1) fermenting and culturing the engineered bacteria containing the above-mentioned gene and collecting the bacteria;

[0044] (2) subjecting the bacterial cells to ultrasonic disruption to obtain a crude enzyme solution containing the above-mentioned recombinant sucrose isomerase;

[0045] (3) mixing the crude enzyme solution, the immobilized carrier material and the buffer to obtain a primary mixed solution;

[0046] (4) mixing the primary mixed solution and the immobilized enzyme material to obtain a secondary mixed solution;

[0047] (5) adding the secondary mixed solution dropwise into a CaCl2 solution to carry out a solidification reaction to obtain the affinity-oriented immobilized enzyme microspheres.

[0048] Further preferably, the immobilized carrier material comprises nano-silica spheres (SNs), micron-sized (SMs) SiO2 particles and mesoporous silica (MS);

[0049] The particle size of the nano silicon dioxide sphere is 20nm, 50nm or 500nm.

[0050] Further preferably, the immobilized enzyme material is a mixture of sodium alginate and sodium carboxymethyl cellulose or a mixture of sodium alginate and carboxymethyl chitosan;

[0051] When the immobilized enzyme material is a mixture of sodium alginate and sodium carboxymethyl cellulose, the mass percentage of sodium alginate in the mixture is 1.5%-3.5%, and the mass percentage of sodium carboxymethyl cellulose is 0.5%-1.5%.

[0052] More preferably, when the immobilized enzyme material is a mixture of sodium alginate and sodium carboxymethyl cellulose, the mass percentage of sodium alginate in the mixture of sodium alginate and sodium carboxymethyl cellulose is 3%, and the mass percentage of sodium carboxymethyl cellulose is 0.75%.

[0053] Further preferably, the mass percentage of CaCl2 in the CaCl2 solution is 1.5%-3.5%

[0054] More preferably, the mass percentage of CaCl2 in the CaCl2 solution is 2%.

[0055] Further preferably, the curing reaction time is 1-5 hours and the temperature is room temperature.

[0056] More preferably, the curing reaction time is 4 hours.

[0057] Further preferably, the volume ratio of the immobilized carrier material to the buffer is 1.5:1.

[0058] More preferably, after the curing reaction, the product obtained by the curing reaction is washed.

[0059] Further preferably, the washing comprises the step of repeatedly washing with a citric acid-disodium hydrogen phosphate (pH 6.0, 10 mM) buffer solution to remove residual calcium chloride solution on the surface of the gel microspheres until no enzyme activity is detected in the washing solution.

[0060] The present invention provides an affinity-oriented immobilized enzyme microsphere prepared by the above-mentioned preparation method.

[0061] The present invention provides the use of the above-mentioned sucrose isomerase recombinant, the above-mentioned gene, the above-mentioned biological material, the above-mentioned sucrose isomerase mutant, the above-mentioned affinity-oriented immobilized enzyme or the above-mentioned affinity-oriented immobilized enzyme microsphere in catalytic synthesis of isomaltulose.

[0062] The invention provides a method for synthesizing isomaltulose, comprising the following steps: adding a mediator phosphate buffer solution and affinity-oriented immobilized enzyme microspheres into a system containing substrate sucrose, adjusting the pH of the reaction system to 5.0-8.0, and performing a catalytic reaction at 30-35°C to synthesize isomaltulose.

[0063] Further preferably, the mass percentage of sucrose in the substrate is 67%;

[0064] Further preferably, the pH of the reaction system is adjusted to 6.0.

[0065] Further preferably, the temperature of the catalytic reaction is 35°C.

[0066] The present invention discloses the following technical effects:

[0067] The present invention molecularly transforms wild-type sucrose isomerase (Slase) from Pantoea dispersa, and performs fusion expression after screening affinity peptide ligands. The specific enzyme activity of the obtained sucrose isomerase recombinant is 88% higher than that of the sucrose isomerase, significantly improving its enzyme activity level and improving its specific catalytic efficiency for sucrose substrates; it provides a reference for improving the molecular transformation of other laccases. In addition, the sucrose isomerase recombinant has good thermal stability. Finally, the one-step purification and immobilization of SIase are studied to optimize the embedding system, and an affinity-oriented immobilized enzyme microsphere (gel microsphere) with high hardness and strength and capable of effectively reducing the leakage or loss of enzyme molecules is prepared, which significantly improves the reusability of the sucrose isomerase recombinant. After the immobilized sucrose isomerase prepared by this method is reused for 40 times, the sucrose conversion rate can still be maintained at 83.38%. The present invention also improves the efficiency of preparing isomaltulose by enzymatic method, reduces the production cost and the difficulty of separation and purification, is suitable for large-scale continuous production, and is of great significance for promoting the application of isomaltulose in the food field and improving the technical level of isomaltulose production in my country.

[0068] In summary, the present invention provides a one-step purification and directed enzyme immobilization method that is cost-effective, simple to operate, has excellent performance and can be industrialized for production, and can further improve the catalytic efficiency of sucrose isomerase, which has very important research significance and practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] 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 will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0070] Figure 1 The three-dimensional structure diagram of wild-type sucrose isomerase and Slase-Tag; a is SI; b is SI-P1; c is SI-P3; d is SI-P4;

[0071] Figure 2 The SDS-PAGE analysis of wild-type expression and Slase-Tag purification at 16℃ and 30℃ fermentation; M is a protein marker; lanes 1 and 5 are SI expressed at 16℃ and 30℃, respectively; lanes 2 and 6 are SI-P1 expressed at 16℃ and 30℃, respectively; lanes 3 and 7 are SI-P3 expressed at 16℃ and 30℃, respectively; lanes 4 and 8 are SI-P4 expressed at 16℃ and 30℃, respectively;

[0072] Figure 3 Determination of the relative activities of wild-type sucrose isomerase and Slase-Tag;

[0073] Figure 4 Schematic diagram of one-step directional immobilization; A is random immobilization; B is affinity directional immobilization;

[0074] Figure 5 It is a schematic diagram of the affinity directional immobilization model of the present invention;

[0075] Figure 6 Comparison of enzyme loading of SI and SI-SBPs on different silicon-based materials; a is a silica nanomaterial; b is a silica micromaterial; c is a mesoporous silica material;

[0076] Figure 7 is the adsorption curve of equal amount of SNs-50 in enzyme solutions of different concentrations;

[0077] Figure 8 The effect of sodium alginate concentration on the recovery rate of enzyme activity during the affinity-encapsulation process of silica spheres;

[0078] Fig. 9 The effect of the amount of sodium carboxymethylcellulose added on the recovery rate of enzyme activity during the affinity-embedding process of silica spheres;

[0079] Fig.10 The effect of CaCl2 solution concentration on enzyme activity recovery during silica sphere affinity-embedding process;

[0080] Fig.11 The effect of solidification time on enzyme activity recovery rate during silica sphere affinity-embedding process;

[0081] Fig.12 The effect of the ratio of buffer solution to composite solution on the recovery rate of enzyme activity during the affinity-embedding process of silica spheres;

[0082] Fig.13The diagram of the silica ball affinity-sodium alginate-sodium carboxymethyl cellulose encapsulation of immobilized enzyme for multiple consecutive catalytic reactions;

[0083] Fig.14 The figure is a comparison of the enzyme activities of wild-type sucrose isomerase and single-site mutants of sucrose isomerase. DETAILED DESCRIPTION

[0084] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0085] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0086] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0087] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0088] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0089] Example 1 Simulation design of affinity peptide ligand

[0090] The wild-type sucrose isomerase (also known as SIase, SI for short) protein gene (accession number: AAP57083.1) was fused with different affinity peptide ligands (affinity peptide ligand P1, amino acid sequence as shown in SEQ ID NO.4, specifically: VDNKFNKERRRARREIRHLPNLNREQRRAFIRSLRDDPSQSANLLAEAKKLNDAQAPK; affinity peptide ligand P3, amino acid sequence as shown in SEQ ID NO.5, specifically: SSRSSSHRRHDHHDHRRGS; affinity peptide ligand P4, amino acid sequence as shown in SEQ ID NO.6, specifically: KAAKIFKGKSGK) at the C-terminus through a flexible peptide linker GSA (amino acid sequence as shown in SEQ ID NO.7, specifically: GSAGSAAGSG) to construct a recombinant SIase, and the results were shown in FIG. Figure 1 The three-dimensional structure of the protein is shown.

[0091] Auto Dock was used for docking, and GROMACS2020 software was used for MD simulation to analyze the affinity between the affinity short peptide ligand and sucrose isomerase. Pymol (https: / / www.pymol.com) was used to construct mutants SI-P1 (amino acid sequence as shown in SEQ ID NO.1, specifically: MATNIQKSADFPIWWKQAVFYQIYPRSFKDSNGDGIGD ), SI-P3 (amino acid sequence as shown in SEQ ID NO.As shown in Figure 2, specifically: MATNIQKSADFPIWWKQAVFYQIYPRSFKD SNGDGIGDIPGIIEKLDYLKMLGVDAIWINPHYESPNTDNGYDISDYRKIMKEYGSMADFDRLVAEMNKRGMRLMIDIVINHTSDRHRWFVQSRSGKDNPYRDYYFWRDGKQGQAPNNYPSFFGGSAWQLDKQTDQYYLHYFAPQQPDLNWDNPKVRAELYDILRFWLDKGVSGLRFDTVATFSKIPGFPDLSKAQLKNFAEAYTEGPNIHKYIHEMNRQVLSKYNVATAGEIFGVPVSAMPDYFDRRREELNIAFTFDLIRLDRYPDQRWRRKPWTLSQFRQVISQTDRAAGEFGWNAFFLDNHDNPRQVSHFGDDSPQWRERSAKALATLLLTQRATPFIFQGAELGMTNYPFKNIEEFDDIE VKGFWNDYVASGKVNAAEFLQEVRMTSRDNSRTPMQWNDSVNAGFTQGKPWFHLNPNYKQINAAREVNKPDSVFSYYRQLINLRHQIPALTSGEYRDLDPQNNQVYAYTRILDNEKYLVVVNFKPEQLHYALPDNLTIASSLLENVHQPSLQENASTLTLAPWQAGIYKLNGSAGSAAGSGSSRSSSHRRHDHHDHRRGSHHHHHH) and SI-P4 (amino acid sequence as SEQ ID NO.As shown in Figure 3, specifically: the 3D structure of MATNIQKSADFPIWWKQAVFYQIYPRSFKDSNGDGIGDIPGIIEKLDYLKMLGVDAIWINPHYESPNTDNGYDISDYRKIMKEYGSMADFDRLVAEMNKRGMRLMIDIVINHTSDRHRWFVQSRSGKDNPYRDYYFWRDGKQGQAPNNYPSFFGGSAWQLDKQTDQYYLHYFAPQQPDLNWDNPKVRAELYDILRFWLDKGVSGLRFDTVATFSKIPGFPDLSKAQLKNFAEAYTEGPNIHKYIHEMNRQVLSKYNVATAGEIFGVPVSAMPDYFDRRREELNIAFTFDLIRLDRYPDQRWRRKPWTLSQFRQVISQTDRAAGEFGWNAFFLDNHDNPRQVSHFGDDSPQWRERSAKALATLLLTQRATPFIFQGAELGMTNYPFKNIEEFDDIEVKGFWNDYVASGKVNAAEFLQEVRMTSRDNSRTPMQWNDSVNAGFTQGKPWFHLNPNYKQINAAREVNKPDSVFSYYRQLINLRHQIPALTSGEYRDLDPQNNQVYAYTRILDNEKYLVVVNFKPEQLHYALPDNLTIASSLLENVHQPSLQENASTLTLAPWQAGIYKLNGSAGSAAGSGKAAKIFKGKSGKHHHHHH).

[0092]

[0093]

[0094]

[0095] The nucleotide sequence of the gene encoding the affinity peptide ligand P1 is shown in SEQ ID NO.11: GTGGATAACAAATTTAATAAAGAACGCCGCCGCGCACGCCGCGAAATTCGTCATTTACCAAATCTGAATCGCGA ACAGCGCCGCGCGTTTATTCGCAGCTTACGCGATGATCCGAGCCAGAGCGCGAATCTGC TGGCGGAAGCGAAGAAACTGAATGATGCGCAGGCGCCGAAA;

[0096] The nucleotide sequence of the gene encoding the affinity peptide ligand P3 is shown in SEQ ID NO.12: AGTAGTCGTAGTAGTAGTCATCGCCGTCATGATCATCATGATCATCGCCGCGGCAGC;

[0097] The nucleotide sequence of the gene encoding the affinity peptide ligand P4 is shown in SEQ ID NO.13: AAAGCAGCGAAAATTTTTAAAGGCAAAAGCGGCAAA;

[0098] The nucleotide sequence of the gene encoding the flexible peptide linker GSA is shown in SEQ ID NO.14: GGTAGCGCGGGCAGCGCGGCGGGTAGCGGT.

[0099] The ATB website (https: / / atb.uq.edu.au) online server was used to generate affinity peptide ligand topology files, and the complex structures with the highest docking scores and reliable geometries were simulated with 100ns MD at 340K. In addition, the Gromos 9654A7 united atom force field was used to study the interaction between the affinity peptide ligand and sucrose isomerase. The Gromos 9654A7 force field was applied to the protein, the SPC water model was used for solvation, and the system was equilibrated by 100ps NVT and 100ps NPT simulation ensembles, followed by 100ns MD simulations. The LINCS algorithm was used to constrain all covalent bonds to hydrogen atoms; molecular docking and molecular dynamics simulations of sucrose isomerase were then performed.

[0100] This embodiment studies the conformational changes of SIase at 340K, and the root mean square deviation (RMSD) analysis at 340K depicts that the RMSD fluctuations of the recombinant are greater than the RMSD fluctuations of SI. The results show that the increase in temperature leads to conformational changes in the recombinant protein. The four sucrose isomerase recombinants are relatively stable in the 100ns simulation, among which SI-P1 reaches equilibrium state faster during the simulation process, and the overall conformation of the recombinant remains rigid and shows greater structural stability. The root mean square fluctuation (RMSF) analysis can monitor which region of the enzyme becomes rigid and flexible when mutated. In the process of interacting with the ligand, the affinity peptide of SI-P1 fluctuates the least, and the C-terminal domain of the SI-P1 recombinant is more stable than other sucrose isomerase recombinants, which is consistent with the adsorption stability experimental results of SI-P1 in the later period.

[0101] Example 2 Construction of recombinant sucrose isomerase engineering bacteria

[0102] In this experiment, the pET22b-SI plasmid with His tag preserved in the laboratory was selected (the basic plasmid is pET22b-SI-His, which comes from the preservation of the previous research results of the research group and is disclosed in the article "Construction and catalytic performance of affinity-oriented photocrosslinking immobilized enzyme". At the same time, appropriate degeneracy optimization (prediction and deletion of its own signal peptide) was carried out in combination with the innovative needs of this experiment. The insertion site is the mature protein binding domain, and the inserted sequence is the nucleotide sequence shown in SEQ ID NO.11-14). The purpose of using this plasmid is to smoothly introduce an affinity tag (Tag) at the C-terminus of sucrose isomerase to ensure that its function can be effectively exerted. This process involves applying specific primers to the gene to which the affinity polypeptide fragment is added, and performing PCR amplification. The system and conditions are shown in Tables 2 and 3. The pET22b-SI-His plasmid vector is used as a template for reverse PCR amplification, and the affinity fusion tag is inserted by the primers shown in Table 1. The obtained PCR product is detected by agarose gel electrophoresis. The next step involves inserting the intercepted fragment into the host bacteria through homologous recombination. The reaction system is shown in Table 4. After connecting in a 55℃ water bath for 15 minutes, the transformed E.coli JM109 is transformed, and the transformed single colony is verified by colony PCR. The obtained PCR band is consistent with the expected target size. The transformant is extracted and the plasmid is verified for correctness. The base sequence results obtained by sequencing are compared and verified with the known target gene sequence using the biological software DNAMAN. After the sequencing is correct, the recombinant plasmid is transformed into E.coli BL21 in the same steps to obtain 4 recombinant sucrose isomerase engineering bacteria (recombinant sucrose isomerase engineering bacteria with wild-type SI protein gene, recombinant sucrose isomerase engineering bacteria with SI-P1 protein gene, recombinant sucrose isomerase engineering bacteria with SI-P3 protein gene, and recombinant sucrose isomerase engineering bacteria with SI-P4 protein gene), and they are stored in a -80℃ refrigerator.

[0103] The specific steps of plasmid transformation and cloning circularized products in E. coli cloning host strain JM109 are as follows:

[0104] (1) The specific preparation method of the competent cells E. coli JM109 used in this experiment is as follows:

[0105] Before preparation, prepare 0.1M CaCl2 solution and 0.1M CaCl2 (15% glycerol) solution (preparation method: dissolve 1.11g CaCl2 in 100mL deionized water to obtain 0.1M CaCl2 solution; measure 8.5mL of 0.1M CaCl2 in a test tube, add 1.5mL of glycerol to make 0.1M CaCl2 (15% glycerol)); avoid bacterial contamination during the preparation of competent cells;

[0106] ① Activate the original engineered bacteria of E.coli JM109 and culture at 37℃ overnight;

[0107] ② Culture a single colony in a 5 mL LB test tube and culture at 37°C with shaking for about 12 hours;

[0108] ③According to the inoculation volume of 2%, take 1 mL of bacterial solution into 50 mL of LB medium, shake and culture at 37°C for 1 hour, and then measure the OD 600 The cells were allowed to reach the logarithmic phase (OD 600 = about 0.4-0.5);

[0109] ④ Place the 50mL centrifuge tube sterilized at 121℃ on ice for precooling, pour the bacterial solution into the centrifuge tube, and place it in an ice bath for 10 minutes;

[0110] ⑤ Centrifuge at low temperature at 400r / min for 10min and discard the supernatant;

[0111] ⑥Pull the pre-cooled 121℃ sterilized 0.1M CaCl2 to wash the cells, slowly blow the bacteria until they are completely dissolved in the solution; after ice bathing for 40 minutes, centrifuge at 4000r / min and discard the supernatant. Wash the bacteria twice with 0.1M CaCl2;

[0112] ⑦ Add 1.5mL or 2mL of 0.1M CaCl2 (15% glycerol) solution stored at low temperature to the bacteria, mix the bacteria thoroughly, divide into 50μL portions, and store at -80℃.

[0113] (2) Using the prepared competent cells, the ligation product is transformed. The transformation process is as follows:

[0114] Place competent cells E.coli JM109 in an ice box, add 20 μL of ligation product to the clean bench, and gently pipette to mix. After ice bathing the ligation mixture for 30 minutes, place it in a 42°C water bath for 90 seconds, immediately ice bath for 2 minutes, pour in 1 mL of LB recovery solution, place it in a shaker (37°C, 220 rpm) for 1 hour of recovery culture, centrifuge at 4000 r / min for 5 minutes to collect the bacteria, remove part of the supernatant, leave about 100 μL of the supernatant and gently pipette to mix, draw the bacterial solution onto LB solid culture medium containing 50 μg / mL ampicillin, spread it evenly with a spreader, and invert it in a 37°C incubator for overnight culture.

[0115] Table 1 Primers for SI-P1 / P3 / P4 gene amplification (5'→3')

[0116]

[0117] Table 2 PCR system

[0118]

[0119] Table 3 PCR reaction conditions

[0120]

[0121] Table 4 Homologous recombination system

[0122]

[0123] Example 3 Fermentation expression, separation and purification of sucrose isomerase

[0124] The four recombinant sucrose isomerase engineering bacteria stored in a -80°C refrigerator in Example 2 were fermented to express Slase, and then the mutated Slase was subjected to shaking fermentation and one-step purification and immobilization of the crude enzyme, and the purified protein was measured for enzyme activity verification and conversion rate determination to determine positive mutations.

[0125] 1. Cultivation of recombinant sucrose isomerase engineering bacteria

[0126] The four recombinant sucrose isomerase engineered bacterial strains stored at -80°C in Example 2 were streaked onto resistant LB plates (Amp) and cultured at 37°C for 12 hours. A single colony on the plate was picked and inoculated into a 5 mL test tube containing 50 μg / mL ampicillin and cultured at 37°C overnight to obtain seed solution.

[0127] The prepared seed solution was transferred to 250 mL LB liquid medium (containing Amp) at an inoculum volume of 2% (v / v) and cultured at 37°C for 2-3 hours. 600 When the pH value is 0.6-0.8, add 0.5 mM IPTG and culture at 16°C or 30°C shaker at 120 r / min for 14 h. After the fermentation, centrifuge at 8000 r / min and 4°C for 15 min to collect the bacterial cells.

[0128] 2. Disintegration of recombinant sucrose isomerase engineering bacteria

[0129] Slase is a protein expressed in E.coli BL21 cells, so the cells need to be broken and the supernatant collected. The specific steps for breaking the cells are as follows:

[0130] (1) Add 30 mL of Lysis Buffer to the centrifuge cup containing the bacterial cells and suspend the cells using a vortex shaker.

[0131] (2) Lysozyme (final concentration of 50 μg / mL) and protease inhibitor PMSF (final concentration of 1 mM) were added to the mixture and placed in an ice-water mixture and stirred for 30 min using a magnetic stirrer.

[0132] (3) Ultrasonic disruption of bacteria: The working power is 300 W, the ultrasound is on for 2 s and off for 4 s, and the total disruption time is 30 min.

[0133] (4) The broken bacterial liquid was placed in a 50 mL centrifuge tube and centrifuged at 12,000 r / min and 4°C for 30 min in a high-speed low-temperature centrifuge. The supernatant was collected as the crude enzyme liquid, and the precipitate was broken and retained as a sample.

[0134] 3. Purification of SI

[0135] The C-terminus of SI carries a 6×His tag, so Ni+-NTA affinity chromatography is used to purify and collect high-purity target protein. The specific steps of purification are as follows:

[0136] (1) Pipette 2 mL of Ni-NTA resin into the purification column. After the ethanol in the resin flows out, add 10 mL of ultrapure water to wash the resin. Then add 10 mL of Lysis Buffer (1 M Tris-HCl, 4 M NaCl, 20 mM imidazole, pH 6.0) to equilibrate the resin column.

[0137] (2) The crude enzyme solution was filtered through a water filter membrane with a pore size of 0.22 μm to remove impurities, then fully mixed with the resin and poured into a beaker equipped with a rotor. The beaker was placed in an ice box containing an ice-water mixture and stirred on a magnetic stirrer to allow the target protein to bind to the resin for 50 minutes.

[0138] (3) In a low-temperature chromatography cabinet, pour the crude enzyme solution bound to the resin into the column and let it stand for 5 minutes to allow the resin to sink to the membrane at the bottom of the column. Pull out the cover of the column to allow the liquid to flow out and keep a sample. Rinse the beaker repeatedly with the liquid and re-load the column to avoid wasting resin.

[0139] (4) Pipette 10 mL of 50 mM Wash Buffer (1 M Tris-HCl, 4 M NaCl, 50 mM imidazole, pH 6.0) and add it to the column to resuspend the resin and elute the impurities. After standing for 5 min, drain the liquid and retain the sample.

[0140] (5) Pipette 5 mL of Elution Buffer (1M Tris-HCl, 4M NaCl, 500 mM imidazole, pH 6.0) and add it to the column. Fully resuspend the resin to elute the target protein. After standing for 5 minutes, the liquid that flows out is the effluent containing the target protein. The high concentration of imidazole (500 mM) in the Elution Buffer competes with the His tag on the target protein for binding to Ni-NTA, so that the high-purity target protein is eluted from the resin.

[0141] 4. Results Analysis

[0142] The above high-purity target proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Figure 2 As shown. The results showed that there was a clear band at around 66 kDa, proving that the affinity ligand-mediated sucrose isomerase was expressed. And the protein content of low-temperature expression (16°C shaker) was significantly better than that of 30°C shaker, so 16°C low temperature was selected to induce protein production.

[0143] Example 4 Determination of Sucrose Isomerase Activity

[0144] Definition of sucrose isomerase activity: Under certain conditions, the amount of enzyme required to produce 1 μmol of isomaltulose per minute is called one enzyme activity unit, recorded as U / mL, and the specific enzyme activity is the ratio of enzyme activity to protein concentration.

[0145] Determination method of sucrose isomerase activity:

[0146] 100 μL of 0.5 mg / mL enzyme solution was pipetted into a 2 mL EP tube, and then 400 μL of 25% sucrose solution (containing 10 mmol / L citric acid-sodium dihydrogen phosphate buffer, pH 6.0) was added and mixed evenly.

[0147] The mixture was placed in a water bath and incubated at 35°C for 10 min. After the reaction was completed, 1 mL of DNS reagent was quickly added and the mixture was quickly transferred to a boiling water bath and boiled for 5 min to stop the enzyme action. The mixture was then immediately placed in ice water to cool for 2 min. 200 μL of the reaction mixture was taken and placed in a 96-well plate. The absorbance was measured at a wavelength of 540 nm using a microplate reader, and the boiled inactivated enzyme solution was used as a control.

[0148] Accurately weigh 20 mg of isomaltulose standard, dissolve it completely in pH 6.0 citric acid-sodium dihydrogen phosphate buffer, and dilute it to 10 mL to prepare a 2 mg / mL isomaltulose standard solution. The isomaltulose standard solution was further diluted to obtain a series of isomaltulose standard solutions with different concentrations, ranging from 0.2-1.6 mg / mL.

[0149] Take 500 μL of isomaltulose standard solution in a 2mL EP tube, add 1mL of DNS reagent, mix well and place in a boiling water bath to heat for 5 minutes, then quickly transfer to ice water to cool for 2 minutes. Use a pipette to take 200 μL of the solution and put it into a 96-well plate, and use a spectrophotometer to measure the absorbance at a wavelength of 540nm. When drawing the standard curve, the isomaltulose concentration is used as the x-axis and the OD value at a wavelength of 540nm is used as the y-axis. The expected absorbance value should be within the linear range of detection (absorbance value A is between 0.25-0.8). The standard equation for isomaltulose (Y=aX-b) is Y=1.80815X-0.09021; R 2 =0.9889.

[0150] The calculation formula of sucrose isomerase enzyme activity is: enzyme activity (U / mL) = (A+b)×1000×N×5×V1 / (a×342.3×Δt×V2) where A is the absorbance of the solution at 540nm; 1000 is the conversion factor (milligrams converted to micrograms); N is the dilution factor of the enzyme solution; V1 is the total volume of the reaction system; Δt is the reaction time (min); V2 is the volume of the enzyme solution added to the reaction system.

[0151] The sucrose isomerase activity of the recombinant sucrose isomerase engineered bacteria with wild-type SI protein gene, recombinant sucrose isomerase engineered bacteria with SI-P1 protein gene, recombinant sucrose isomerase engineered bacteria with SI-P3 protein gene and recombinant sucrose isomerase engineered bacteria with SI-P4 protein gene was tested by DNS method at 35℃ and pH=6. The results are as follows: Figure 3 As shown. The results showed that compared with the recombinant sucrose isomerase engineered bacteria strain with wild-type SI protein gene, the enzyme activity of free SI-P1 obtained by fusing the linker gene and silicon affinity polypeptide P1 gene to the C-terminus of sucrose isomerase was 88% higher than that of free SI, the enzyme activity of free SI-P3 obtained by fusing the linker gene and silicon affinity polypeptide P3 gene to the C-terminus of sucrose isomerase was 44% higher than that of free SI, and the enzyme activity of free SI-P4 obtained by fusing the linker gene and silicon affinity polypeptide P4 gene to the C-terminus of sucrose isomerase was 78% higher than that of free SI. The results showed that the recombinant fusion biological affinity tag effectively improved the enzyme activity of sucrose isomerase.

[0152] Example 5 One-step purification and directional immobilization of sucrose isomerase mediated by silicon affinity polypeptide

[0153] One-step purification and immobilization of enzymes utilizes the specific interaction between the carrier and the enzyme molecules to selectively immobilize the enzyme directly from the unpurified enzyme solution, such as Figure 4As shown. In enzymatic reactions, in order to reduce the generation of unnecessary by-products, people separate and purify the enzymes obtained through biological fermentation to obtain high-purity enzymes, which are then used to promote reactions. In the preparation of immobilized enzymes, the process is similar. The enzymes are first purified and then immobilized for enzymatic reactions. The directional immobilization technology is a more precise method. It determines the exact binding position of the carrier and the enzyme through selective connection, thereby ensuring that the enzyme molecules are fixed in the correct direction. The following is its one-step purification and directional immobilization process;

[0154] The schematic diagram of the affinity directional immobilization model of the present invention is as follows Figure 5 As shown, the specific steps of affinity directional immobilization are: 10 mg of nano-silica nanospheres (particle size of 50 nm) are fully dispersed in 1.6 mL of the four kinds of crude enzyme solutions (10 mg / mL) in Example 3, so that the mass concentration of nano-silica nanospheres is 6.25 mg / mL, and the ratio of nano-silica nanospheres to crude enzyme solution is 1.25:1. The adsorption reaction is carried out under 16 ° C and 120 rpm conditions for 10 hours, followed by centrifugation at 12000 rpm for 5 minutes to remove the supernatant, and four immobilized enzymes are obtained by repeated washing with buffer, namely SNs-SI, (without adding affinity tag), SNs-SI-P1 (adding silicon affinity polypeptide gene P1), SNs-SI-P3 (adding silicon affinity polypeptide gene P3) and SNs-SI-P4 (adding silicon affinity polypeptide gene P4), and the enzyme activity recovery rates of these four immobilized enzymes are 39.5%, 77.2%, 58.7% and 68.6.7%, respectively. It can be seen that the recombinant sucrose isomerase provided by the present invention can be directly non-covalently immobilized on silica nanoparticles and a higher enzyme activity recovery rate is obtained.

[0155] Example 6 Comparative experiment on enzyme loading and enzyme activity of immobilized enzyme on different silicon nanomaterials

[0156] The catalytic performance of the immobilized enzyme was studied by measuring the enzyme loading and enzyme activity. Various silicon-based materials such as silica nanospheres (SNs, silica nanomaterials, particle sizes of 20nm, 50nm, 500nm), various micron-sized SIO2 materials (SMs, silica micron materials, particle sizes of 200μm, 500μm, 800μm) and mesoporous silica (MS, mesoporous silica materials, particle size of 200μm) were used to one-step purify and immobilize the four crude enzyme solutions in Example 3 to explore the immobilization effect of sucrose isomerase mediated by silicon affinity polypeptides on different silicon nanomaterials. The same method as in Example 5 was used to prepare the immobilized enzyme, and the enzyme loading and enzyme activity of the immobilized enzyme were measured. The results are as follows: Figure 6As shown. The results showed that compared with SNs-SI, the recombinant sucrose isomerase was selectively loaded onto the silica surface from the crude enzyme solution. The specific non-covalent adsorption ability of sucrose isomerase mediated by silicon affinity peptide (SNs-SI-P1, SNs-SI-P3 and SNs-SI-P4) on silicon nanoparticles was better than that of SNs-SI. In the experiment comparing the immobilization of sucrose isomerase on nano-silica nanospheres with different particle sizes, the enzyme loading capacity of small-size nano-silica nanospheres (50nm) was more advantageous in the adsorption and immobilization process, and could better exert the adsorption ability of sucrose isomerase mediated by silicon affinity peptide. Unexpectedly, the SI activity based on the silicon affinity peptide P1 after immobilization was superior to that of the tagged or untagged wild-type enzyme systems based on peptide P3 and peptide P4, among which SNs-SI-P1 showed a more significant immobilization effect with a maximum enzyme loading of 361.28 μg / mg, which was 3 times that of the micron material (122 μg / mg) and 2.3 times that of the mesoporous material (154.88 μg / mg).

[0157] Example 7 Preparation of single-site mutants of sucrose isomerase

[0158] Using a rational design strategy guided by conservation and stability, we started with a deep understanding of the substructure, mutated single amino acid positions or the entire range of amino acid sequences for the amino acid positions near the active site of SI-P1, and performed specific screening and selection strategies on the resulting library. After the ancestral sequence of sucrose isomerase was reconstructed through the FireProtASR website, multiple sequence alignment was performed using MEGA. During the alignment process, points with high diversity of amino acid types at the same site were regarded as residues with low conservation, so as to select non-conserved residues. Preliminary results identified the catalytic residues (F213, A225, K228, E232, E236, F264, Q299, E324, F325, R320, A321, I388, E389, D392, N401, A405, N410, A412, and Q416). The locations of the above sites were analyzed, and sites far from the catalytic center were selected. Finally, mutation sites E232, I388, E389, D392, N401, N410, A412 and Q416 were selected as potential mutation sites. Next, DDMut was used to perform saturation mutation on the selected sites, calculate the Gibbs free energy (ΔΔG) of the mutated protein, and select the sites with enhanced stability after mutation for experimental verification. The results are shown in Table 5 below for the 12 potential mutant sites.

[0159] Using the wild-type plasmid pET-22b(+) containing the wild-type sucrose isomerase encoding gene as a template, the following sites of the wild-type sucrose isomerase were selected for site-directed mutagenesis, mutations were introduced at the mutation sites, and amplification was performed by two-step PCR using the following primers (underlined is the mutation site sequence) and KOD plus enzyme to obtain a single-site mutant plasmid:

[0160] Table 5 Primer sequence list

[0161]

[0162]

[0163] Note: The underlined part is the mutated base.

[0164] The PCR amplification system was 50 μL: 25 μL PrimeSTAR Max enzyme, 2 μL upstream and downstream primers and 2 μL plasmid template, and the rest was made up with 19 μL sterile ultrapure water; the PCR amplification program was: 94°C pre-denaturation for 2 min, followed by 7 cycles (95°C denaturation for 10 s, 60°C annealing for 7 min, 72°C extension for 40 s), and insulation at 4°C after the cycle.

[0165] Take 2 μL of PCR product and detect it by 0.8% agarose gel electrophoresis. Use DpnⅠ to remove the template plasmid in the mutant plasmid library, add the treated samples and reagents according to the components and volumes in Table 6, and then place them at 16°C for 1 hour for circular ligation reaction. Then use T4 DNA ligase to self-ligate the linear fragment amplified by PCR to form a circular plasmid. The ligation product was transformed into E.coli BL21 (DE3) and spread on an LB solid plate containing 50 μg / mL ampicillin resistance to screen for mutant strains containing single-site mutant plasmids. At the same time, the specific enzymatic activity of sucrose isomerase and its single-site mutants on the substrate sucrose was investigated. The results are shown in Tables 7 and Fig.14 shown.

[0166] Table 6 Linear plasmid circularization system

[0167] Components Volume (μL) Purification of the product after DpnI treatment 2 Ligationhigh 5 T4 DNA ligase 1 <![CDATA[ddH2O]]> 7

[0168] Table 7 Specific enzyme activity of sucrose isomerase and its single-site mutants on substrate sucrose

[0169] Sucrose isomerase WT E232W I388W E389D D392G N401E N401W Specific enzyme activity (U / mg) 508.39 380.57 561.83 234.34 838.91 401.43 571.42 Sucrose isomerase N410Q A412N A412D A412E Q416W Q416Y Specific enzyme activity (U / mg) 77.94 294.62 289.57 223.96 687.77 153.01

[0170] From Table 7 and Fig.14It is recorded that among the 12 laccase single-site mutants, the enzyme activities of I388W, D392G, N401W, and Q416W were significantly higher than those of the wild type, and the enzyme activities of sucrose isomerase were increased by 10.51%, 65.01%, 12.40%, and 35.28%, respectively, compared with the wild-type laccase; among them, the specific enzyme activity of D392G was 838.91 U / mg, which was not only significantly higher than the specific enzyme activity of the wild-type laccase, but also higher than the specific enzyme activities of the other 11 single-site mutants, so the subsequent experiments were conducted on the D392G mutant.

[0171] Example 8 SI directional affinity-embedding and immobilization process

[0172] Affinity-encapsulation and immobilization process: 80 mL of the SI-P1 mutant D392G crude enzyme solution (10 mg / mL) prepared in Example 3 and Example 7 were added with a certain amount of hydroxylated nano-silica spheres (manufacturer: Shanghai McLean Biochemical Technology Co., Ltd., CAS No.: 60676-86-0; particle size 50 nm) to a final concentration of 6.25 mg / mL. The mixture was shaken at 16°C and 220 rpm for 10 h, centrifuged to remove the supernatant, and then an appropriate amount of citric acid-disodium hydrogen phosphate (pH 6.0, 10 mM) buffer was added to resuspend the mixture to a mass concentration of 50 mg / mL of the silica nanosphere carrier, which was added to a mixture of sodium alginate and sodium carboxymethyl cellulose of different concentrations, stirred at 4°C and 220 rpm for 15 min, and homogenized and defoamed. Take a syringe and gradually drip its contents into a CaCl2 solution of a specific concentration, ensuring that the needle is kept at a certain distance from the liquid surface and aligned vertically, and then perform a solidification reaction for 5 hours at 4°C (standing at room temperature) to promote the formation of microspheres with a diameter of approximately 2.0 mm. Subsequently, it is filtered and washed twice with a phosphate buffer solution to obtain the final directional affinity-embedded immobilized enzyme, and the silica microgel beads are stored at 4°C for use.

[0173] Example 9 SI directional affinity-embedding and immobilization process

[0174] The preparation of the mixed solution of sodium alginate and sodium carboxymethyl cellulose: The preparation of the mixed solution of 3% sodium alginate and 1.5% sodium carboxymethyl cellulose (50 mL): weigh 1.5 g sodium alginate and 0.75 g sodium carboxymethyl cellulose, add them into 40 mL of ultrapure water, dissolve them, and then adjust the volume to 50 mL, prepare them before use.

[0175] 80 mL of the crude enzyme solution (10 mg / mL, the crude enzyme solution was prepared in the same manner as in Example 3) of the SI-P1 mutant D392G prepared in Example 7 was added with a certain amount of hydroxylated nano-silica spheres (SNs-50) to make the initial protein and carrier ratio concentrations 1.28 mg / mg, 2.56 mg / mg, 3.84 mg / mg, 5.12 mg / mg, 6.40 mg / mg and 7.68 mg / mg. The mixture was shaken at 16°C and 220 rpm for 10 h, centrifuged to remove the supernatant, and then an appropriate amount of citric acid-disodium hydrogen phosphate (pH 6.0, 10 mM) buffer was added to resuspend the mixture to make the carrier mass concentration 50 mg / mL, which was added to a sodium alginate solution of 1 times the volume of the buffer solution. The mass percentage of sodium alginate in the mixture was 3 wt.%, and the mass percentage of sodium carboxymethyl cellulose was 0.75 wt.%.

[0176] The mixture was stirred at 4°C and 220 rpm for 15 min, homogenized and defoamed, and then the syringe was used to gradually drip the contents into a 2 wt.% CaCl2 solution, followed by a solidification reaction (standing at room temperature) for 1 h, and then washed twice with a citric acid-sodium hydrogen phosphate (pH 6.0, 10 mM) buffer solution to obtain directional affinity-embedded immobilized enzyme microspheres. The immobilized enzyme loading was as follows: Figure 7 As shown, SNs-SI-P1 showed a more significant immobilization effect, which was 1.4 times the enzyme loading of SNs-SI. Specifically, the enzyme loading of SNs-SI-P1 was 360.96 μg protein / mg carrier, while SI was only 257.28 μg protein / mg carrier, which was 1.4 times. Therefore, SNs-50 nanosilica spheres and crude enzyme addition of 6.40 mg protein / mg SNs-50 carrier (crude enzyme solution 10 mg / mL) were selected as the optimal conditions for subsequent research.

[0177] Example 10 SI directional affinity-embedding and immobilization process

[0178] The other conditions were the same as those in Example 9, except that the concentration of sodium alginate was changed, specifically:

[0179] 80 mL of the crude enzyme solution (10 mg / mL) of the SI-P1 mutant D392G prepared in Example 7 was taken and added with a certain amount of hydroxylated nano-silica spheres (SNs-50) to a final concentration of 6.25 mg / mL. The mixture was shaken at 16°C and 220 rpm for 10 h, centrifuged to remove the supernatant, and then resuspended in an appropriate amount of citric acid-sodium hydrogen phosphate (pH 6.0, 10 mM) buffer to make the carrier mass concentration 50 mg / mL, which was added to a mixed solution of sodium alginate and sodium carboxymethyl cellulose with 1 volume of buffer, wherein the mass percentage of sodium alginate in the mixed solution was 1.5 wt.%, 2 wt.%, 2.5 wt.%, 3 wt.%, and 3.5 wt.%, respectively, and the mass percentage of sodium carboxymethyl cellulose was 0.75 wt.%.

[0180] The mixture was stirred at 4°C and 220 rpm for 15 min, homogenized and defoamed, and then the syringe was used to gradually drip the contents into a 2 wt.% CaCl2 solution. The mixture was then solidified for 1 h and washed twice with a phosphate buffer solution to obtain directional affinity-embedded immobilized enzyme microspheres. The enzyme activity recovery rate was as follows: Figure 8 The enzyme activity recovery rates were 94.12% (sodium alginate mass percentage of 1.5wt.%), 95.87% (sodium alginate mass percentage of 2wt.%), 96.99% (sodium alginate mass percentage of 2.5wt.%), 100% (sodium alginate mass percentage of 3wt.%), and 96.66% (sodium alginate mass percentage of 3.5wt.%), so 3wt.% sodium alginate was used for subsequent research.

[0181] Example 11 SI directional affinity-embedding and immobilization process

[0182] The other conditions were the same as those in Example 9, except that the concentration of sodium carboxymethyl cellulose was changed, specifically:

[0183] 80 mL of the crude enzyme solution (10 mg / mL) of the SI-P1 mutant D392G prepared in Example 7 was taken and a certain amount of hydroxylated nano-silica spheres (particle size SNs-50) were added to make the final concentration 6.25 mg / mL. The mixture was shaken at 16°C and 220 rpm for 10 h, centrifuged to remove the supernatant, and then an appropriate amount of citric acid-disodium hydrogen phosphate (pH 6.0, 10 mM) buffer was added to resuspend the mixture to make the carrier mass concentration 50 mg / mL, and the mixture was added to a mixed solution of sodium alginate and sodium carboxymethyl cellulose with 1 volume of buffer, wherein the mass percentage of sodium alginate in the mixed solution was 3 wt.%, and the mass percentage of sodium carboxymethyl cellulose was 0.5 wt.%, 0.75 wt.%, 1 wt.%, 1.25 wt.%, and 1.5 wt.%.

[0184] The mixture was stirred at 4°C and 220 rpm for 15 min, homogenized and defoamed, and then the syringe was used to gradually drip the contents into a 2 wt.% CaCl2 solution. The mixture was then solidified for 1 h and washed twice with a phosphate buffer solution to obtain directional affinity-embedded immobilized enzyme microspheres. The enzyme activity recovery rate was as follows: Fig. 9 The enzyme activity recovery rates were 87.02% (the mass percentage of sodium carboxymethyl cellulose was 0.5wt.%), 100% (the mass percentage of sodium carboxymethyl cellulose was 0.75wt.%), 91.07% (the mass percentage of sodium carboxymethyl cellulose was 1wt.%), 83.76% (the mass percentage of sodium carboxymethyl cellulose was 1.25wt.%), and 77.43% (the mass percentage of sodium carboxymethyl cellulose was 1.5wt.%), so 0.75wt.% sodium carboxymethyl cellulose was used for subsequent research.

[0185] Example 12 SI directional affinity-embedding and immobilization process

[0186] Other conditions are consistent with those in Example 9, except that the concentration of CaCl2 is changed, specifically:

[0187] 80 mL of the crude enzyme solution (10 mg / mL) of the SI-P1 mutant D392G prepared in Example 7 was taken and a certain amount of hydroxylated nano-silica spheres (SNs-50) was added to make the final concentration 6.25 mg / mL. The mixture was shaken at 16°C and 220 rpm for 10 h, centrifuged to remove the supernatant, and then an appropriate amount of citric acid-disodium hydrogen phosphate (pH 6.0, 10 mM) buffer was added to resuspend the carrier to a mass concentration of 50 mg / mL, which was added to a mixture of sodium alginate and sodium carboxymethyl cellulose with a volume of 1.5 times the buffer, wherein the mass percentage of sodium alginate in the mixture was 3 wt.%, and the mass percentage of sodium carboxymethyl cellulose was 0.75 wt.%.

[0188] The mixture was stirred at 4°C and 220 rpm for 15 min, homogenized and defoamed, and then the syringe was used to gradually drip the contents into 1.5 wt.%, 2 wt.%, 2.5 wt.%, 3 wt.%, and 3.5 wt.% CaCl2 solutions, followed by curing for 3 h, and washed twice with phosphate buffer solution to obtain directional affinity-embedded immobilized enzyme microspheres. The enzyme activity recovery rate was as follows: Fig.10 As shown, the enzyme activity recoveries were 95.69% (1.5wt.% CaCl2 solution), 100% (2wt.% CaCl2 solution), 87.33% (2.5wt.% CaCl2 solution), 85.1% (3wt.% CaCl2 solution), and 84.89% (3.5wt.% CaCl2 solution), respectively. Therefore, a CaCl2 concentration of 2% was used for subsequent studies.

[0189] Example 13 SI directional affinity-embedding and immobilization process

[0190] The other conditions were the same as those in Example 9, except that the curing time of the immobilized microspheres was changed, specifically:

[0191] 80 mL of the crude enzyme solution (10 mg / mL) of the SI-P1 mutant D392G prepared in Example 7 was taken and a certain amount of hydroxylated nano-silica spheres (SNs-50) was added to make the final concentration 6.25 mg / mL. The mixture was shaken at 16°C and 220 rpm for 10 h, centrifuged to remove the supernatant, and then an appropriate amount of citric acid-disodium hydrogen phosphate (pH 6.0, 10 mM) buffer was added to resuspend the carrier to a mass concentration of 50 mg / mL, which was added to a mixture of sodium alginate and sodium carboxymethyl cellulose with a volume of 1.5 times the buffer, wherein the mass percentage of sodium alginate in the mixture was 3 wt.%, and the mass percentage of sodium carboxymethyl cellulose was 0.75 wt.%.

[0192] The mixture was stirred at 4°C and 220 rpm for 15 min, homogenized and defoamed, and then the syringe was used to gradually drip the contents into a 2 wt.% CaCl2 solution. The mixture was then cured for 1 h, 2 h, 3 h, 4 h or 5 h, and washed twice with a phosphate buffer solution to obtain directional affinity-embedded immobilized enzyme microspheres. The enzyme activity recovery rate was as follows: Fig.11 The enzyme activity recovery rates were 84.21% (curing reaction 1h), 90.14% (curing reaction 2h), 100% (curing reaction 3h), 98.66% (curing reaction 4h), and 91.08% (curing reaction 5h), respectively, so the subsequent research was carried out with a curing time of 3h.

[0193] Example 14 SI directional affinity-embedding and immobilization process

[0194] The other conditions were the same as those in Example 9, except that the addition ratio of the buffer solution to the mixed solution of sodium alginate and sodium carboxymethyl cellulose was changed to:

[0195] 80 mL of the crude enzyme solution (10 mg / mL) of the SI-P1 mutant D392G prepared in Example 7 were added with a certain amount of hydroxylated nano-silica spheres (SNs-50) to a final concentration of 6.25 mg / mL. The mixture was shaken at 16°C and 220 rpm for 10 h, centrifuged to remove the supernatant, and then resuspended in an appropriate amount of citric acid-sodium hydrogen phosphate (pH 6.0, 10 mM) buffer to a carrier mass concentration of 50 mg / mL, which was added to a mixture of sodium alginate and sodium carboxymethyl cellulose with a volume of 1, 1.5, 2, 2.5 or 3 times the volume of the buffer, wherein the mass percentage of sodium alginate in the mixture was 3 wt.%, and the mass percentage of sodium carboxymethyl cellulose was 0.75 wt.%.

[0196] The mixture was stirred at 4°C and 220 rpm for 15 min, homogenized and defoamed, and then the syringe was used to gradually drip the contents into a 2 wt.% CaCl2 solution. The mixture was then solidified for 3 h and washed twice with a phosphate buffer solution to obtain directional affinity-embedded immobilized enzyme microspheres. The enzyme activity recovery rate was as follows: Fig.12 The enzyme activity recovery rates were 90.98% (added to a mixture of sodium alginate and sodium carboxymethyl cellulose with 1 volume of buffer), 100% (added to a mixture of sodium alginate and sodium carboxymethyl cellulose with 1.5 volume of buffer), 94.89% (added to a mixture of sodium alginate and sodium carboxymethyl cellulose with 2 volume of buffer), 94.42% (added to a mixture of sodium alginate and sodium carboxymethyl cellulose with 2.5 volume of buffer), and 90.60% (added to a mixture of sodium alginate and sodium carboxymethyl cellulose with 3 volume of buffer), so the subsequent study was carried out with the addition ratio of buffer solution to sodium alginate and sodium carboxymethyl cellulose being 1:1.5.

[0197] Example 15 Reusability test of directional affinity-embedded immobilized enzyme

[0198] The reusability of the immobilized enzyme was studied by high performance liquid chromatography. Isomaltulose was prepared by using the optimized process in Examples 8-14, and the isomerization of sucrose was catalyzed in a constant temperature shaker at 35°C and 120pm for continuous cyclic reaction. Specifically, 25mL of a high concentration sucrose substrate solution dissolved in a mediator phosphate buffer (the mass percentage of sucrose in the system was 67%) and the affinity-oriented immobilized enzyme microspheres prepared in Examples 8-14 (the SI-P1 mutant D392G affinity-oriented immobilized enzyme microspheres used in the present invention) were added to the system, the pH of the reaction system was adjusted to 6, and the catalytic reaction was carried out at 35°C to synthesize isomaltulose. After the reaction cycle was completed, all the supernatant was taken out and immediately boiled at high temperature and its conversion effect was determined by HPLC. The immobilized enzyme was washed with citric acid-disodium hydrogen phosphate citric acid buffer solution (pH 6.0, 10mM) to remove unreacted substrate and product. The washed immobilized enzyme was re-added to the substrate for the next catalytic reaction. The above operation was repeated 40 times, and the sucrose conversion rate of the immobilized enzyme was continuously detected after multiple cycles. Fig.13 After the immobilized sucrose isomerase prepared by this process was reused for 40 times, the sucrose conversion rate could still be maintained at 83.38%, indicating that the affinity-encapsulation and immobilization in the present invention better retained the activity of the enzyme, reduced the leakage of enzyme molecules in the reaction system, improved the efficiency of enzymatic preparation of isomaltulose, and was suitable for large-scale continuous industrial production.

[0199] The above results preliminarily indicate that rational structure-based design of single-site mutations can screen out sucrose isomerase mutants to improve the specific enzyme activity of the protein.

[0200] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A recombinant sucrose isomerase, characterized in that: The amino acid sequence of the recombinant sucrose isomerase is any one of SEQ ID NO.1 to SEQ ID NO.

3.

2. The method for constructing a recombinant sucrose isomerase according to claim 1, characterized in that: The method comprises the steps of connecting the affinity polypeptide to the C-terminus of the sucrose isomerase derived from Pantoea dispersed through a flexible linker to obtain the recombinant sucrose isomerase; The amino acid sequence of the flexible linker is shown in SEQ ID NO.7; The amino acid sequence of the affinity polypeptide is any one of SEQ ID NO.4 to SEQ ID NO.6; The accession number of the sucrose isomerase is: AAP57083.

1.

3. The gene encoding the recombinant sucrose isomerase according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in any one of SEQ ID NO.8-SEQ ID NO.

10.

4. A biological material containing the gene according to claim 4, characterized in that: The biological material includes a recombinant expression vector and an engineering bacterium.

5. A sucrose isomerase mutant, characterized in that: The sucrose isomerase mutant is obtained by mutation of any of the following sites on SEQ ID NO.1: (a) the amino acid corresponding to position 213 of SEQ ID NO. 1 is mutated from phenylalanine to leucine; (b) the amino acid corresponding to position 232 in SEQ ID NO. 1 is mutated from glutamic acid to aspartic acid, tyrosine or tryptophan; (c) the amino acid corresponding to position 264 of SEQ ID NO. 1 is mutated from alanine to tyrosine; (d) the amino acid corresponding to position 299 of SEQ ID NO. 1 is mutated from glutamine to glutamic acid; (e) the amino acid corresponding to position 388 of SEQ ID NO.1 is mutated from isoleucine to glutamic acid, tyrosine or tryptophan; (f) the amino acid at position 389 corresponding to SEQ ID NO.1 is mutated from glutamic acid to aspartic acid or glutamine; (g) the amino acid corresponding to position 392 in SEQ ID NO.1 is mutated from aspartic acid to glycine; (h) the amino acid corresponding to position 401 in SEQ ID NO. 1 is mutated from asparagine to glutamic acid, tyrosine, aspartic acid or tryptophan; (i) the amino acid corresponding to position 410 in SEQ ID NO. 1 is mutated from asparagine to glutamic acid, serine or glutamine; (j) the amino acid at position 412 of SEQ ID NO. 1 is mutated from alanine to glutamic acid, asparagine or aspartic acid; (k) the amino acid corresponding to position 416 in SEQ ID NO. 1 is mutated from glutamine to phenylalanine, tryptophan or tyrosine; (l) The amino acid corresponding to position 459 in SEQ ID NO. 1 is mutated from alanine to glutamic acid.

6. Use of the recombinant sucrose isomerase according to claim 1, the gene according to claim 3, the biomaterial according to claim 4 or the sucrose isomerase mutant according to claim 5 in the preparation of affinity-oriented immobilized enzyme or affinity-oriented immobilized enzyme microspheres.

7. A method for preparing an affinity-oriented immobilized enzyme, characterized in that: The following steps are involved: (1) fermenting and culturing the engineered bacteria containing the gene of claim 4, and collecting the bacteria; (2) subjecting the bacterial cells to ultrasonic disruption to obtain a crude enzyme solution containing the recombinant sucrose isomerase according to claim 1; (3) Mixing the crude enzyme solution, the immobilization carrier material and the buffer to obtain the affinity-oriented immobilized enzyme.

8. An affinity-oriented immobilized enzyme prepared by the preparation method according to claim 7.

9. A method for preparing affinity-oriented immobilized enzyme microspheres, characterized in that: The following steps are involved: (1) fermenting and culturing the engineered bacteria containing the gene of claim 4, and collecting the bacteria; (2) subjecting the bacterial cells to ultrasonic disruption to obtain a crude enzyme solution containing the recombinant sucrose isomerase according to claim 1; (3) mixing the crude enzyme solution, the immobilized carrier material and the buffer to obtain a primary mixed solution; (4) mixing the primary mixed solution and the immobilized enzyme material to obtain a secondary mixed solution; (5) adding the secondary mixed solution dropwise into a CaCl2 solution to carry out a solidification reaction to obtain the affinity-oriented immobilized enzyme microspheres.

10. Affinity-oriented immobilized enzyme microspheres prepared by the preparation method according to claim 9.

11. Use of the recombinant sucrose isomerase according to claim 1, the gene according to claim 3, the biomaterial according to claim 4, the sucrose isomerase mutant according to claim 5, the affinity-oriented immobilized enzyme according to claim 7 or the affinity-oriented immobilized enzyme microspheres according to claim 9 in catalytic synthesis of isomaltulose.

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