A sucrose isomerase recombinant and its construction method and application

Abstract

The present invention relates to the fields of genetic engineering and immobilized enzymes, and particularly to a sucrose isomerase recombinant and its construction method and application. The present invention performs molecular modification on the wild-type sucrose isomerase (Slase) derived from Pantoea dispersa, and after screening for affinity peptide ligands, performs fusion expression. The specific enzyme activity of the obtained sucrose isomerase recombinant is increased by 88% compared to 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 the molecular modification of other enzymes. Moreover, the sucrose isomerase recombinant has good thermal stability. Finally, research on optimizing the embedding system for one-step purification and immobilization of SIase is carried out to prepare an affinity-directed immobilized enzyme microsphere with relatively high hardness and strength, and which can effectively reduce the leakage or loss of enzyme molecules, significantly improving the reusability of the sucrose isomerase recombinant.

Description

Technical Field

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

[0002] Isomaltulose is a reducing disaccharide formed by binding glucose and fructose through an α-1,6 glycosidic bond. It naturally exists 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 those of sucrose, but its sweetness is only half of that of sucrose; the viscosities of their aqueous solutions are relatively close, but isomaltulose is more stable under acidic conditions. Isomaltulose has excellent properties such as low sweetness, easy absorption, non-cariogenicity, and low calories. Moreover, the cleavage rate of isomaltulose is not as fast as that of sucrose, which provides a healthier choice for patients with obesity, diabetes, or cardiovascular diseases. Isomaltulose is a multifunctional new sweetener and is also a generally recognized safe sucrose substitute internationally, having broad application prospects in industries such as medicine and food. Sucrose isomerase is the key enzyme for the enzymatic production of isomaltulose, and the increasing demand for it has promoted the research on the immobilization of sucrose isomerase (SIase).

[0003] Immobilization of enzymes is a method applicable to various on-site processes. Enzymes can be immobilized on solid carriers through a variety of physical and chemical methods, such as adsorption, encapsulation, cross-linking, and covalent attachment. Generally speaking, compared with the use of free enzymes, the immobilization of enzymes provides higher stability, easier separation, and reusability in batch and continuous processes. The adsorption method fixes enzyme molecules around the carrier through non-specific physical adsorption, biospecific adsorption, ion adsorption, affinity adsorption, and hydrophobic interactions generated between the structure of the enzyme molecule itself and the carrier, thus successfully preparing a rich variety of immobilized enzymes. This method is simple to operate, but the disadvantage is that the force between the enzyme and the carrier is weak, making the resulting compound less stable compared to compounds produced by other technologies. However, investigations show that adsorption is still the fastest and most common immobilization method. Adsorbing enzymes on insoluble carriers 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, adjustable pore size, etc., they are often used as carriers for immobilized enzymes. However, the non-directed direct interaction between proteins and carriers 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 pathway of protein immobilization based on non-specific adsorption usually has a negative impact on protein function. In contrast, directed immobilization enables the enzyme protein to be immobilized on the surface of the carrier in an ordered manner, and its active site faces the outside of the solid surface, which can maintain the native conformation of the enzyme protein. This is conducive to the entry of the substrate into the catalytic active site, thus significantly increasing the activity of the immobilized enzyme. This limitation can be overcome by fusing a bioaffinity tag, thereby achieving one-step purification and directed immobilization of the crude enzyme, reducing the cost of separation and purification in the reaction medium, and making this effective method 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. Usually, SBPs bind to solid surfaces through multiple non-covalent interactions. Various factors can define their binding strength, such as surface topography and solution conditions. However, the key aspect of binding to solid surfaces lies in the amino acid composition and structural diversity of SBPs. SBPs exhibit binding affinity and selectivity for the surfaces of solid materials, including carrier materials for biocatalysis, silica, zeolites, glass, metals, and polymers. They strongly bind to their homologous solids by adopting specific conformations that support many non-covalent interactions (such as electrostatic bonding) between key amino acids and the solid surface, usually resulting in binding affinities in the sub-micromolar and nanomolar ranges. Studies have shown that SBPs direct the oriented immobilization of enzymes onto solid supports, retaining the native enzyme structure and catalytic activity. In addition, SBPs can be easily integrated into the permissive regions (such as the 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 for immobilizing 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 designable and has a clear purpose, and is expected to design and develop an efficient immobilized enzyme preparation.

[0005] However, there is currently no research on the application of the solid-binding peptide immobilized enzyme technology to the immobilization of SIase and its catalytic formation of isomaltulose. Existing methods for preparing isomaltulose have many drawbacks. Therefore, it is of great practical significance to study an immobilized SIase that is cost-effective, easy 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, its construction method and application 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 enzymes and affinity-oriented immobilized enzyme microspheres, which have high catalytic activity and the advantages of low cost, easy operation, and industrially producible immobilized sucrose isomerase.

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

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

[0009] The present invention provides a method for constructing the above-mentioned sucrose isomerase recombinant, which includes the step of connecting an affinity polypeptide to the C-terminus of the sucrose isomerase derived from Pantoea dispersa through a flexible linker to obtain the sucrose isomerase recombinant;

[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 as shown in SEQ ID NO.7;

[0011] The amino acid sequence of the affinity polypeptide is any one of the sequences shown in SEQ ID NO.4 - 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 any one of the sequences shown in SEQ ID NO.8 - SEQ ID NO.10.

[0014] The present invention provides a biological material containing the above-mentioned gene, and the biological material includes a recombinant expression vector and an engineered bacterium.

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

[0016] The basic bacterium of the engineered bacterium is Escherichia coli.

[0017] The present invention provides a sucrose isomerase mutant, and the sucrose isomerase mutant is obtained by any one of the following site mutations on SEQ ID NO.1:

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

[0019] (b) The amino acid corresponding to site 232 on 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 site 264 on SEQ ID NO.1 is mutated from alanine (F) to tyrosine (Y);

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

[0022] (e) The amino acid corresponding to position 388 on 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 on SEQ ID NO.1 is mutated from glutamic acid (E) to aspartic acid (D), glutamine (Q);

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

[0025] (h) The amino acid corresponding to position 401 on 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 on SEQ ID NO.1 is mutated from asparagine (N) to glutamic acid (E), serine (S), or glutamine (Q);

[0027] (j) The amino acid corresponding to position 412 on 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 on 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 on SEQ ID NO.1 is mutated from alanine (A) to glutamic acid (E).

[0030] The present invention provides a gene encoding the above-mentioned sucrose isomerase mutant, and the nucleotide sequence of the gene is shown as SEQ ID NO.8 or is mutated on the basis of the degenerate sequence based on the sequence shown in SEQ ID NO.8.

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

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

[0033] The basic bacterium of the engineered bacterium 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-directed immobilized enzyme or affinity-directed immobilized enzyme microspheres.

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

[0036] (1) After fermenting and culturing the engineering bacteria containing the above-mentioned gene, collecting the bacterial cells;

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

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

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

[0040] The particle size of the nano-silica spheres is 20 nm, 50 nm or 500 nm.

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

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

[0043] (1) After fermenting and culturing the engineering bacteria containing the above-mentioned gene, collecting the bacterial cells;

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

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

[0046] (4) Mixing the primary mixture and the immobilized enzyme material to obtain a secondary mixture;

[0047] (5) Dropwise adding the secondary mixture to a CaCl2 solution for solidification reaction to obtain the affinity-directed immobilized enzyme microspheres.

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

[0049] The particle size of the nano-silica spheres is 20 nm, 50 nm or 500 nm.

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

[0051] When the immobilized enzyme material is a mixed solution of sodium alginate and sodium carboxymethyl cellulose, the mass percentage of sodium alginate in the mixed solution of sodium alginate and sodium carboxymethyl cellulose 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 mixed solution of sodium alginate and sodium carboxymethyl cellulose, the mass percentage of sodium alginate in the mixed solution of sodium alginate and sodium carboxymethyl cellulose is 3%, and the mass percentage of sodium carboxymethyl cellulose is 0.75%.

[0053] More 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] More preferably, the time of the curing reaction is 1-5 h, and the temperature is room temperature.

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

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

[0058] More preferably, after the curing reaction, it further includes washing the product obtained from the curing reaction.

[0059] More preferably, the washing includes repeatedly washing with a citric acid-disodium hydrogen phosphate (pH 6.0, 10 mM) buffer solution to remove the 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-directed immobilized enzyme microsphere prepared by the above preparation method.

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

[0062] The present invention provides a method for synthesizing isomaltulose, comprising the following steps: in a system containing the substrate sucrose, adding a mediator phosphate buffer solution and affinity-directed immobilized enzyme microspheres, 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 content of sucrose in the substrate is 67%;

[0064] Further preferably, the pH of the adjusted reaction system is 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 conducts molecular modification on the wild-type sucrose isomerase (Slase) derived from Pantoea dispersa, performs fusion expression after screening the affinity peptide ligand, and the specific enzyme activity of the obtained sucrose isomerase recombinant is increased by 88% compared with that of the sucrose isomerase, significantly improving its enzyme activity level and improving its specific catalytic efficiency for the sucrose substrate; it provides a reference for the molecular modification of other laccases. And this sucrose isomerase recombinant has good thermal stability. Finally, research on optimizing the embedding system for one-step purification and immobilization of SIase is carried out, and an affinity-directed immobilized enzyme microsphere (gel microsphere) with relatively high hardness and strength and capable of effectively reducing the leakage or loss of enzyme molecules is prepared, significantly improving the reusability of this sucrose isomerase recombinant. The immobilized sucrose isomerase prepared by this method can still maintain a sucrose conversion rate of 83.38% after being continuously reused 40 times. The present invention also improves the efficiency of preparing isomaltulose by the enzymatic method, reduces the production cost and the difficulty of separation and purification, is suitable for large-scale continuous production, and has great significance for promoting the application of isomaltulose in the food field and improving the technical level of producing isomaltulose in China.

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

[0069] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained according to these drawings.

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

[0071] Figure 2 SDS-PAGE analysis of the fermentation expression and purification of wild-type and Slase-Tag at 16 °C and 30 °C; among them, M is the protein Marker; lanes 1 and 5 are SI expressed at 16 °C and 30 °C respectively; lanes 2 and 6 are SI-P1 expressed at 16 °C and 30 °C respectively; lanes 3 and 7 are SI-P3 expressed at 16 °C and 30 °C respectively; lanes 4 and 8 are SI-P4 expressed at 16 °C and 30 °C 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; among them, A is random immobilization; B is affinity directional immobilization;

[0074] Figure 5 Schematic diagram of the affinity directional immobilization model of the present invention;

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

[0076] Figure 7 Adsorption curve of equal amount of SNs-50 in enzyme solutions with different concentrations;

[0077] Figure 8 Effect of sodium alginate concentration on the enzyme activity recovery rate during the silica sphere affinity-embedding process;

[0078] Figure 9 Effect of the addition amount of sodium carboxymethyl cellulose on the enzyme activity recovery rate during the silica sphere affinity-embedding process;

[0079] Figure 10 Effect of the concentration of CaCl2 solution on the enzyme activity recovery rate during the silica sphere affinity-embedding process;

[0080] Figure 11 Effect of the curing time on the enzyme activity recovery rate during the silica sphere affinity-embedding process;

[0081] Figure 12 Effect of the addition ratio of buffer solution to composite solution on the enzyme activity recovery rate during the silica sphere affinity-embedding process;

[0082] Figure 13Graph of the affinity of silica spheres - sodium alginate - sodium carboxymethyl cellulose - entrapped immobilized enzyme for continuous multiple catalytic reactions;

[0083] Figure 14 Graph of the specific enzyme activity comparison between wild - type sucrose isomerase and single - site mutants of sucrose isomerase. Detailed implementation mode

[0084] Now, the various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0085] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0086] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can 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 related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0087] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation modes of the present invention's specification, which are obvious to those skilled in the art. Other implementation modes obtained from the present invention's specification are also obvious to those skilled in the art. The present invention's specification and examples are only exemplary.

[0088] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open - ended terms, meaning including but not limited to.

[0089] Example 1 Simulation design of affinity peptide ligands

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

[0091] Docking was performed using Auto Dock, and MD simulations were carried out using the GROMACS 2020 software to analyze the affinity between the affinity short peptide ligand and sucrose isomerase. Mutants SI-P1 (amino acid sequence shown in SEQ ID NO.1, specifically: MATNIQKSADFPIWWKQAVFYQIYPRSFKDSNGDGIGD IPGIIEKLDYLKMLGVDAIWINPHYESPNTDNGYDISDYRKIMKEYGSMADFDRLVAEMNKRGMRLMIDIVINHTSDRHRWFVQSRSGKDNPYRDYYFWRDGKQGQAPNNYPSFFGGSAWQLDKQTDQYYLHYFAPQQPDLNWDNPKVRAELYDILRFWLDKGVSGLRFDTVATFSKIPGFPDLSKAQLKNFAEAYTEGPNIHKYIHEMNRQVLSKYNVATAGEIFGVPVSAMPDYFDRRREELNIAFTFDLIRLDRYPDQRWRRKPWTLSQFRQVISQTDRAAGEFGWNAFFLDNHDNPRQVSHFGDDSPQWRERSAKALATLLLTQRATPFIFQGAELGMTNYPFKNIEEFDDIEVKGFWNDYVASGKVNAAEFLQEVRMTSRDNSRTPMQWNDSVNAGFTQGKPWFHLNPNYKQINAAREVNKPDSVFSYYRQLINLRHQIPALTSGEYRDLDPQNNQVYAYTRILDNEKYLVVVNFKPEQLHYALPDNLTIASSLLENVHQPSLQENASTLTLAPWQAGIYKLNGSAGSAAGSGVDNKFNKERRRARREIRHLPNLNREQRRAFIRSLRDDPSQSANLLAEAKKLNDAQAPKHHHHHH), SI-P3 (amino acid sequence as SEQ ID NO.As shown in 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 coding gene for the affinity peptide ligand P1 is shown in SEQ ID NO.11: GTGGATAACAAATTTAATAAAGAACGCCGCCGCGCACGCCGCGAAATTCGTCATTTACCAAATCTGAATCGCGA ACAGCGCCGCGCGTTTATTCGCAGCTTACGCGATGATCCGAGCCAGAGCGCGAATCTGC TGGCGGAAGCGAAGAAACTGAATGATGCGCAGGCGCCGAAA;

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

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

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

[0099] The online server of the ATB website (https: / / atb.uq.edu.au) was used to generate the topological file of the affinity short peptide ligand. The complex structure with the highest docking score and reliable geometry was carried out with 100 ns MD simulation at 340K. In addition, the Gromos 9654A7 united atom force field was adopted to study the interaction relationship between the affinity short peptide ligand and sucrose isomerase. The Gromos 9654A7 force field was applied to the protein, and the SPC water model was used for solvation. The system was equilibrated through 100 ps NVT and 100 ps NPT simulation ensembles, and then 100 ns MD simulation was carried out. The LINCS algorithm was used to constrain all covalent bonds with hydrogen atoms; afterwards, molecular docking and molecular dynamics simulation analysis were performed on sucrose isomerase.

[0100] This example studied the conformational changes of SIase at 340K. The root mean square deviation (RMSD) analysis at 340K depicted that the RMSD fluctuations of the recombinant were greater than those of SI. The results showed that the increase in temperature led to conformational changes in the recombinant protein. The four sucrose isomerase recombinants were relatively stable during the 100 ns simulation. Among them, SI-P1 reached the equilibrium state faster during the simulation, and the overall conformation of the recombinant remained rigid and showed greater structural stability. The root mean square fluctuation (RMSF) analysis can monitor which regions of the enzyme become rigid and flexible upon mutation. During the interaction with the ligand, the affinity peptide of SI-P1 had the smallest fluctuations, indicating that the C-terminal domain of the SI-P1 recombinant was more stable than those of other sucrose isomerase recombinants, which was consistent with the results of the later adsorption stability experiment of SI-P1.

[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 is from the preservation of the previous research results of the research group and was published in the article "Construction and Catalytic Performance Study of Affinity-Directed Photo-Crosslinked Immobilized Enzymes". At the same time, appropriate degeneracy optimization was carried out in combination with the innovative requirements of this experiment (predict and delete its own signal peptide), 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 successfully introduce an affinity tag (Tag) at the C-terminus of sucrose isomerase to ensure the effective exertion of its function. This process involves applying specific primers to the gene with the added affinity polypeptide fragment and performing PCR amplification. The system and conditions are shown in Tables 2 and 3. Reverse PCR amplification uses the pET22b-SI-His plasmid vector as a template and inserts the affinity fusion tag through the primers shown in Table 1. The obtained PCR products are detected by agarose gel electrophoresis. The next step involves ligating the intercepted fragment into the host bacterium through homologous recombination. The reaction system is shown in Table 4. After connecting in a 55 °C water bath for 15 min, it is transformed into E. coli JM109, and the transformed single colonies are verified by colony PCR. The transformants with PCR bands consistent with the expected target size are used to extract plasmids to verify the correctness. The base sequence results obtained by sequencing are compared and verified with the known target gene sequence using the biological software DNAMAN. After correct sequencing, the recombinant plasmid is transformed into E. coli BL21 in the same steps to obtain 4 kinds of 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 °C refrigerator.

[0103] The specific steps for cloning the transformed and cloned circularized products of the plasmid in the Escherichia coli cloning host bacterium JM109 are as follows:

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

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

[0106] ① Activate the original engineered E. coli JM109 strain and culture it overnight at 37°C;

[0107] ② Culture a single colony in a 5 mL LB test tube and shake it at 37°C for about 12 h;

[0108] ③ According to an inoculation amount of 2%, pipette 1 mL of the bacterial solution into 50 mL of LB medium, shake it at 37°C for 1 h, and then measure OD 600 to make the cells reach the logarithmic phase (OD 600 = about 0.4 - 0.5);

[0109] ④ Place a 50 mL centrifuge tube sterilized at 121°C on ice for pre-cooling, pour the bacterial solution into the centrifuge tube, and ice-bath for 10 min;

[0110] ⑤ Centrifuge at a low speed of 400 r / min for 10 min, and pour off the supernatant;

[0111] ⑥ Pipette pre-cooled 0.1 M CaCl2 sterilized at 121°C to wash the cells, and slowly blow and suck the bacterial cells until they are completely dissolved in the solution; after ice-bathing for 40 min, centrifuge at 4000 r / min and pour off the supernatant. Wash the bacterial cells twice with 0.1 M CaCl2;

[0112] ⑦ Add 1.5 mL or 2 mL of 0.1 M CaCl2 (15% glycerol) solution stored at low temperature to the bacterial cells, mix the bacterial cells thoroughly, aliquot 50 μL per portion, and store at -80°C.

[0113] (2) Use the prepared competent cells to perform the transformation of the ligation product. The transformation process is as follows:

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

[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, Isolation, and Purification of Sucrose Isomerase

[0124] For the four recombinant sucrose isomerase engineering bacteria stored in an -80°C refrigerator in Example 2, ferment and express Slase, and then conduct studies on shake-flask fermentation, one-step purification and immobilization of the mutated Slase, verification of specific enzyme activity of the purified protein, and determination of the conversion rate for positive mutations.

[0125] 1. Cultivation of Recombinant Sucrose Isomerase Engineering Bacteria

[0126] Streak the four recombinant sucrose isomerase engineering bacteria strains stored at -80°C in Example 2 on a resistant LB plate (Amp) and incubate at 37°C for 12 h. Pick a single colony on the plate and inoculate it into a 5 mL test tube containing 50 μg / mL ampicillin, and incubate at 37°C overnight to obtain a seed solution.

[0127] Transfer the prepared seed solution to 250 mL of LB liquid medium (containing Amp) at an inoculation amount of 2% (v / v) and incubate at 37°C for 2 - 3 h. When the OD 600 reaches 0.6 - 0.8, add 0.5 mM IPTG, transfer to a shaker at 16°C or 30°C, and culture at 120 r / min for 14 h. After fermentation, centrifuge at 8000 r / min and 4°C for 15 min to collect the bacterial cells.

[0128] 2. Disruption of Recombinant Sucrose Isomerase Engineering Bacteria Cells

[0129] Slase is a protein expressed intracellularly in E. coli BL21. Therefore, the cells need to be disrupted and the supernatant after disruption should be collected. The specific steps for cell disruption 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 oscillator.

[0131] (2) Add lysozyme (final concentration of 50 μg / mL) and protease inhibitor PMSF (final concentration of 1 mM), place in an ice-water mixture, and stir thoroughly with a magnetic stirrer for 30 min.

[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 min 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 analysis results of the above-mentioned high-purity target proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are as follows Figure 2 shown. The results show that there is an obvious band at around 66 kDa, proving the expression of the affinity ligand-mediated sucrose isomerase. And the content of the protein expressed at low temperature (shaking incubator at 16 °C) is significantly better than that at 30 °C shaking incubator. Therefore, 16 °C low temperature is selected to induce protein production in the follow-up.

[0143] Example 4 Determination of the enzyme activity of sucrose isomerase

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

[0145] Method for determining the enzyme activity of sucrose isomerase

[0146] Pipette 100 μL of the enzyme solution with a concentration of 0.5 mg / mL into a 2 mL EP tube, and then mix it with 400 μL of 25% sucrose solution (containing 10 mmol / L citrate-sodium dihydrogen phosphate buffer, pH 6.0) evenly.

[0147] Put the mixture into a water bath and incubate it at a constant temperature of 35 °C for 10 min. After the reaction is completed, quickly add 1 mL of DNS reagent, and quickly transfer it to a boiling water bath and boil for 5 min to stop the enzyme action. Then immediately place it in ice water to cool for 2 min. Take 200 μL from the reaction mixture and place it in a 96-well plate, and measure its absorbance at a wavelength of 540 nm using an enzyme-labeling instrument, with the enzyme solution inactivated by boiling as a control.

[0148] Accurately weigh 20 mg of isomaltulose standard product, completely dissolve it with citrate-sodium dihydrogen phosphate buffer at pH 6.0, and then make the volume up to 10 mL to prepare a standard solution of isomaltulose with a concentration of 2 mg / mL. Further dilute the standard solution of isomaltulose to obtain a series of standard solutions of isomaltulose with different concentrations, ranging from 0.2 - 1.6 mg / mL.

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

[0150] Calculation formula for the enzyme activity of sucrose isomerase: Enzyme activity (U / mL) = (A + b) × 1000 × N × 5 × V1 / (a × 342.3 × Δt × V2). Where A is the absorbance of the solution at 540 nm; 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 produced by the recombinant sucrose isomerase engineering bacteria carrying the wild-type SI protein gene, the recombinant sucrose isomerase engineering bacteria carrying the SI-P1 protein gene, the recombinant sucrose isomerase engineering bacteria carrying the SI-P3 protein gene, and the recombinant sucrose isomerase engineering bacteria carrying the SI-P4 protein gene were respectively detected for sucrose isomerase activity by the DNS method under the conditions of 35 °C and pH = 6. The results are as Figure 3 shown. The results show that compared with the recombinant sucrose isomerase engineering bacteria strain carrying the wild-type SI protein gene, the free SI-P1 enzyme activity obtained by fusing the linker gene and the silica affinity polypeptide P1 gene to the C-terminus of sucrose isomerase is 88% higher than that of free SI. The enzyme activity of free SI-P3 obtained by fusing the linker gene and the silica affinity polypeptide P3 gene to the C-terminus of sucrose isomerase is 44% higher than that of free SI. The enzyme activity of free SI-P4 obtained by fusing the linker gene and the silica affinity polypeptide P4 gene to the C-terminus of sucrose isomerase is 78% higher than that of free SI. The results show that the recombinant fusion biological affinity tag effectively improves the enzyme activity of sucrose isomerase.

[0152] Example 5 One-step purification and directed immobilization process of sucrose isomerase mediated by silica affinity polypeptide

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

[0154] The schematic diagram of the affinity-directed immobilization model of the present invention is as shown in Figure 5 As shown. The specific steps of affinity-directed immobilization are as follows: Take 10 mg of silica nanoparticles (particle size 50 nm) and disperse them separately and fully in 1.6 mL of the 4 kinds of crude enzyme solutions (10 mg / mL) in Example 3, so that the mass concentration of the silica nanoparticles is 6.25 mg / mL, and the ratio of silica nanoparticles to the crude enzyme solution is 1.25:1. Shake and adsorb for reaction at 16 °C and 120 rpm for 10 h, then centrifuge at 12,000 rpm for 5 min to remove the supernatant, and wash repeatedly with buffer to obtain four kinds of immobilized enzymes, namely SNs-SI (without adding an affinity tag), SNs-SI-P1 (adding the silicon affinity polypeptide gene P1), SNs-SI-P3 (adding the silicon affinity polypeptide gene P3), and SNs-SI-P4 (adding the silicon affinity polypeptide gene P4). The enzyme activity recovery rates of these four kinds of immobilized enzymes are 39.5%, 77.2%, 58.7%, and 68.67% 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 relatively high enzyme activity recovery rate is obtained.

[0155] Experimental comparison of enzyme loading and enzyme activity of immobilized enzymes on different silicon nanomaterials in Example 6

[0156] The catalytic performance of the immobilized enzyme was studied by measuring the enzyme loading and enzyme activity. Silica nanoparticles (SNs, silica nanomaterials, particle sizes 20 nm, 50 nm, 500 nm), various micron-sized SIO2 materials (SMs, silica micron materials, particle sizes 200 μm, 500 μm, 800 μm), and mesoporous silica (MS, mesoporous silica material, particle size 200 μm) materials and other silicon-based materials were used to purify and immobilize the 4 kinds of crude enzyme solutions in Example 3 in one step to explore the immobilization effect of the silicon affinity polypeptide-mediated sucrose isomerase on different silicon nanomaterials. Using the same method as in Example 5 to prepare the immobilized enzyme, the enzyme loading and enzyme activity of the immobilized enzyme were measured, and the results are as shown in 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 the silicon affinity polypeptide-mediated sucrose isomerase (SNs-SI-P1, SNs-SI-P3, and SNs-SI-P4) to silicon nanoparticles was better than that of SNs-SI. Among them, in the experiment of immobilizing sucrose isomerase with different particle sizes of silica nanoparticles, the small particle size silica nanoparticles (50 nm) had more advantages in enzyme loading during the adsorption immobilization process and could better exert the adsorption ability of the silicon affinity polypeptide-mediated sucrose isomerase. Unexpectedly, based on the fact that the SI activity after immobilization of the silicon affinity polypeptide P1 was better than that of the tags based on polypeptide P3 and polypeptide P4 or the tagless wild-type enzyme system, 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, starting from an in-depth understanding of the substructure, mutagenize single amino acid positions or the entire range of amino acid sequences near the active site of SI-P1, and perform specific screening and selection strategies on the resulting library. After reconstructing the ancestral sequence of sucrose isomerase through the FireProtASR website, use MEGA for multiple sequence alignment. During the alignment process, consider the points with a higher diversity of amino acid types at the same position as residues with lower conservation, and use this to select non-conserved residues. The preliminary results determined the catalytic residues (F213, A225, K228, E232, E236, F264, Q299, E324, F325, R320, A321, I388, E389, D392, N401, A405, N410, A412, and Q416). Analyze the positions of the above sites, select the sites far from the catalytic center, and finally select the mutation sites E232, I388, E389, D392, N401, N410, A412, and Q416 as potential mutation points. Next, use DDMut to perform saturation mutagenesis on the selected sites, calculate the Gibbs free energy (ΔΔG) of the mutated protein, and select the points with enhanced stability after mutation for experimental verification. The results are the 12 potential mutation sites in Table 5 below.

[0159] Using the wild-type plasmid pET-22b(+) containing the wild-type sucrose isomerase encoding gene as a template, site-directed mutagenesis was performed at the following sites of the wild-type sucrose isomerase, and mutations were introduced at the mutation sites. The following primers (the underlined parts are the mutant site sequences) were used to amplify by two-step PCR with KOD plus enzyme to obtain the single-site mutant plasmid:

[0160] Table 5 Primer Sequence Table

[0161]

[0162]

[0163] Note: The underlined parts are the mutant bases.

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

[0165] Take 2 μL of the PCR product and detect it by 0.8% agarose gel electrophoresis. Use DpnⅠ enzyme to digest and remove the template plasmid in the mutant plasmid library, add the treated sample and reagents according to the components and volumes in Table 6 below, and then carry out a 1 h circular ligation reaction at 16 °C. Then use T4 DNA ligase to self-ligate the linear fragment amplified by PCR to form a circular plasmid. Transform the ligation product into E. coli BL21(DE3), and spread it on an LB solid plate containing 50 μg / mL ampicillin resistance to screen for mutant strains containing the single-site mutant plasmid. At the same time, investigate the specific enzyme activities of sucrose isomerase and its single-site mutants towards the substrate sucrose, and the investigation results are shown in Table 7 and Figure 14 as follows.

[0166] Table 6 Linear Plasmid Cyclization System

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

[0168] Table 7 Specific Enzyme Activities of Sucrose Isomerase and Its Single-Site Mutants towards the 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 Figure 14As can be seen from the records, among the 12 laccase single-site mutants, those with significantly higher specific enzyme activity than the wild type are I388W, D392G, N401W, and Q416W, and the specific enzyme activities are 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 is 838.91 U / mg, which is not only significantly higher than that of the wild-type laccase but also higher than that of the other 11 single-site mutants. Therefore, the D392G mutant was used for subsequent experiments.

[0171] Example 8 SI Directed Affinity-Embedding and Re-Immobilization Process

[0172] Directed affinity-embedding and re-immobilization process: Take 80 mL of the SI-P1 mutant D392G crude enzyme solution (10 mg / mL) prepared in Example 3 and Example 7 respectively, and add a certain amount of hydroxylated nano-silica spheres (manufacturer: Shanghai Macklin Biochemical Co., Ltd., CAS No.: 60676-86-0; particle size: 50 nm) to make its final concentration 6.25 mg / mL. React with shaking at 16 °C and 220 rpm for 10 h, centrifuge to remove the supernatant, then add an appropriate amount of citrate-disodium hydrogen phosphate (pH 6.0, 10 mM) buffer solution to resuspend, so that the mass concentration of the silica nano-sphere carrier is 50 mg / mL, and add it to a mixed solution of sodium alginate and sodium carboxymethylcellulose with different concentrations. Stir and react at 4 °C and 220 rpm for 15 min for homogenization and defoaming. Use a syringe and gradually drop its content into a CaCl2 solution with a specific concentration, ensure that the needle tip is at a certain distance from the liquid surface and is vertically aligned, and then carry out a 5-h solidification reaction (stand still at room temperature) under the environmental condition of 4 °C to promote the formation of microspheres with a diameter of about 2.0 mm. Subsequently, filter it and wash it twice with phosphate buffer solution to obtain the final directed affinity-embedded immobilized enzyme, and store the silica micro-gel beads at 4 °C for later use.

[0173] Example 9 SI Directed Affinity-Embedding and Re-Immobilization Process

[0174] Preparation of the mixed solution of sodium alginate and sodium carboxymethylcellulose: Preparation of a 3% sodium alginate and 1.5% sodium carboxymethylcellulose mixed solution (50 mL): Weigh 1.5 g of sodium alginate and 0.75 g of sodium carboxymethylcellulose, dissolve them in 40 mL of filtered ultrapure water, and then make up the volume to 50 mL. Prepare it freshly before use.

[0175] 80 mL of the crude enzyme solution of SI-P1 mutant D392G prepared in Example 7 (10 mg / mL, the preparation method of this crude enzyme solution is the same as that in Example 3) was taken and added with a certain amount of hydroxylated nano-silica spheres (SNs-50) to make the initial protein and carrier ratio concentrations be 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 reaction was carried out with shaking at 16 °C and 220 rpm for 10 h. After centrifuging to remove the supernatant, an appropriate amount of citrate-disodium hydrogen phosphate (pH 6.0, 10 mM) buffer solution was added to resuspend it to make the carrier mass concentration be 50 mg / mL, and it was added to the sodium alginate solution with a volume 1 time that of the buffer solution. The mass percentage content of sodium alginate in this mixed solution was 3 wt.%, and the mass percentage content of sodium carboxymethylcellulose was 0.75 wt.%.

[0176] The reaction was stirred at 4 °C and 220 rpm for 15 min for homogenization and defoaming. Then, a syringe was taken and its content was gradually dropped into the 2 wt.% CaCl2 solution, and then the curing reaction (stood at room temperature) was carried out for 1 h. It was washed twice with the citrate-disodium hydrogen phosphate (pH 6.0, 10 mM) buffer solution to obtain the oriented affinity-embedded immobilized enzyme microspheres. The immobilized enzyme loading is as Figure 7 shown. Among them, SNs-SI-P1 showed a more significant immobilization effect, which was 1.4 times that of the SNs-SI enzyme loading. Specifically, the enzyme loading of SNs-SI-P1 was 360.96 μg protein / mg carrier, while that of SI was only 257.28 μg protein / mg carrier, with a ratio of 1.4 times. Therefore, SNs-50 nano-silica spheres and a crude enzyme addition amount 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 Oriented Affinity-Embedding and Re-Immobilization Process

[0178] Other conditions were the same as those in Example 9, and the sodium alginate concentration was changed. Specifically:[[]]END]]

[0179] 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 its final concentration 6.25 mg / mL. The reaction was carried out with shaking at 16 °C and 220 rpm for 10 h. After centrifugation to remove the supernatant, an appropriate amount of citrate-disodium hydrogen phosphate (pH 6.0, 10 mM) buffer was added to resuspend it to make the carrier mass concentration 50 mg / mL. It was added to a mixed solution of sodium alginate and sodium carboxymethylcellulose with a volume 1 time that of the buffer. The mass percentage of sodium alginate in this mixed solution was 1.5 wt.%, 2 wt.%, 2.5 wt.%, 3 wt.%, 3.5 wt.% respectively, and the mass percentage of sodium carboxymethylcellulose was 0.75 wt.%.

[0180] The reaction was stirred at 4 °C and 220 rpm for 15 min for homogenization and defoaming. Then a syringe was taken and its content was gradually dropped into 2 wt.% CaCl2 solution, followed by a curing reaction for 1 h. It was washed twice with phosphate buffer solution to obtain the oriented affinity-embedded immobilized enzyme microspheres. The enzyme activity recovery rate was as Figure 8 shown. The enzyme activity recovery rates were 94.12% (when the mass percentage of sodium alginate was 1.5 wt.%), 95.87% (when the mass percentage of sodium alginate was 2 wt.%), 96.99% (when the mass percentage of sodium alginate was 2.5 wt.%), 100% (when the mass percentage of sodium alginate was 3 wt.%), 96.66% (when the mass percentage of sodium alginate was 3.5 wt.%). Therefore, subsequent studies were carried out with 3 wt.% sodium alginate.

[0181] Example 11 SI Oriented Affinity-Embedding and Re-Immobilization Process

[0182] Other conditions were the same as those in Example 9, and the concentration of sodium carboxymethylcellulose 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 (with a particle size of SNs-50) was added to make its final concentration 6.25 mg / mL. The reaction was carried out with shaking at 16 °C and 220 rpm for 10 h. After centrifugation to remove the supernatant, an appropriate amount of citrate-disodium hydrogen phosphate (pH 6.0, 10 mM) buffer was added to resuspend it to make the carrier mass concentration 50 mg / mL. It was added to a mixed solution of sodium alginate and sodium carboxymethylcellulose with a volume 1 time that of the buffer. The mass percentage of sodium alginate in this mixed solution was 3 wt.%, and the mass percentage of sodium carboxymethylcellulose was 0.5 wt.%, 0.75 wt.%, 1 wt.%, 1.25 wt.%, 1.5 wt.%.

[0184] The reaction was stirred at 4 °C and 220 rpm for 15 min for homogenization and defoaming. Then, a syringe was taken and its content was gradually dropped into 2 wt.% CaCl2 solution, followed by a curing reaction for 1 h. It was washed twice with phosphate buffer solution to obtain the oriented affinity-embedded immobilized enzyme microspheres. The enzyme activity recovery rate was as Figure 9 shown. The enzyme activity recovery rates were 87.02% (sodium carboxymethyl cellulose mass percentage content was 0.5 wt.%), 100% (sodium carboxymethyl cellulose mass percentage content was 0.75 wt.%), 91.07% (sodium carboxymethyl cellulose mass percentage content was 1 wt.%), 83.76% (sodium carboxymethyl cellulose mass percentage content was 1.25 wt.%), 77.43% (sodium carboxymethyl cellulose mass percentage content was 1.5 wt.%). Therefore, subsequent studies were carried out with 0.75 wt.% sodium carboxymethyl cellulose.

[0185] Example 12 SI Oriented Affinity-Embedding Re-Immobilization Process

[0186] Other conditions were the same as those in Example 9, and the CaCl2 concentration was changed, specifically:

[0187] 80 mL of the crude enzyme solution (10 mg / mL) of 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 its final concentration 6.25 mg / mL. The reaction was oscillated at 16 °C and 220 rpm for 10 h, and the supernatant was removed by centrifugation. Then, an appropriate amount of citrate-disodium hydrogen phosphate (pH 6.0, 10 mM) buffer solution was added to resuspend it to make the carrier mass concentration 50 mg / mL. It was added to the mixed solution of sodium alginate and sodium carboxymethyl cellulose with 1.5 times the volume of the buffer solution. The mass percentage content of sodium alginate in this mixed solution was 3 wt.%, and the mass percentage content of sodium carboxymethyl cellulose was 0.75 wt.%.

[0188] The reaction was stirred at 4 °C and 220 rpm for 15 min for homogenization and defoaming. Then, a syringe was taken and its content was gradually dropped into 1.5 wt.%, 2 wt.%, 2.5 wt.%, 3 wt.%, 3.5 wt.% CaCl2 solutions, followed by a curing reaction for 3 h. It was washed twice with phosphate buffer solution to obtain the oriented affinity-embedded immobilized enzyme microspheres. The enzyme activity recovery rate was as Figure 10 shown. The enzyme activity recovery rates were 95.69% (1.5 wt.% CaCl2 solution), 100% (2 wt.% CaCl2 solution), 87.33% (2.5 wt.% CaCl2 solution), 85.1% (3 wt.% CaCl2 solution), 84.89% (3.5 wt.% CaCl2 solution). Therefore, subsequent studies were carried out with a CaCl2 concentration of 2%.

[0189] Example 13 SI Directed Affinity-Embedding and Re-Immobilization Process

[0190] Other conditions were the same as in Example 9, and the curing time of the immobilized microspheres was changed. Specifically:

[0191] 80 mL of the crude enzyme solution (10 mg / mL) of SI-P1 mutant D392G prepared in Example 7 was taken and added with a certain amount of hydroxylated nano-silica spheres (SNs-50) to make its final concentration 6.25 mg / mL. The reaction was carried out by shaking at 16 °C and 220 rpm for 10 h. After centrifuging to remove the supernatant, it was resuspended with an appropriate amount of citrate-disodium hydrogen phosphate (pH 6.0, 10 mM) buffer to make the carrier mass concentration 50 mg / mL, and it was added to a mixed solution of sodium alginate and sodium carboxymethylcellulose with a volume 1.5 times that of the buffer. The mass percentage content of sodium alginate in this mixed solution was 3 wt.%, and the mass percentage content of sodium carboxymethylcellulose was 0.75 wt.%.

[0192] The reaction was stirred at 4 °C and 220 rpm for 15 min for homogenization and defoaming. Then a syringe was taken and its content was gradually dropped into 2 wt.% CaCl2 solution, and then the curing reaction was carried out for 1 h, 2 h, 3 h, 4 h or 5 h, and it was washed twice with phosphate buffer solution to obtain directed affinity-embedded immobilized enzyme microspheres. The enzyme activity recovery rates were as Figure 11 shown. The enzyme activity recovery rates were 84.21% (curing reaction for 1 h), 90.14% (curing reaction for 2 h), 100% (curing reaction for 3 h), 98.66% (curing reaction for 4 h), 91.08% (curing reaction for 5 h), respectively. Therefore, the subsequent research was carried out with a curing time of 3 h.

[0193] Example 14 SI Directed Affinity-Embedding and Re-Immobilization Process

[0194] Other conditions were the same as in Example 9, and the addition ratio of the buffer to the mixed solution of sodium alginate and sodium carboxymethylcellulose was changed. Specifically:

[0195] 80 mL each of the crude enzyme solution (10 mg / mL) 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 its final concentration 6.25 mg / mL. The mixture was subjected to oscillating reaction at 16 °C and 220 rpm for 10 h, and then centrifuged to remove the supernatant. Subsequently, it was resuspended with an appropriate amount of citrate-disodium hydrogen phosphate (pH 6.0, 10 mM) buffer to make the carrier mass concentration 50 mg / mL, and then added to the mixed solution of sodium alginate and sodium carboxymethylcellulose with 1-fold, 1.5-fold, 2-fold, 2.5-fold or 3-fold volume of the buffer. The mass percentage content of sodium alginate in the mixed solution was 3 wt.%, and the mass percentage content of sodium carboxymethylcellulose was 0.75 wt.%.

[0196] The mixture was stirred at 4 °C and 220 rpm for 15 min for homogenization and defoaming. Then, a syringe was taken and its content was gradually dropped into 2 wt.% CaCl2 solution, followed by a curing reaction for 3 h. After being washed twice with phosphate buffer solution, the oriented affinity-embedded immobilized enzyme microspheres were obtained, and the enzyme activity recovery rates were as Figure 12 shown. The enzyme activity recovery rates were 90.98% (added to the mixed solution of sodium alginate and sodium carboxymethylcellulose with 1-fold volume of the buffer), 100% (added to the mixed solution of sodium alginate and sodium carboxymethylcellulose with 1.5-fold volume of the buffer), 94.89% (added to the mixed solution of sodium alginate and sodium carboxymethylcellulose with 2-fold volume of the buffer), 94.42% (added to the mixed solution of sodium alginate and sodium carboxymethylcellulose with 2.5-fold volume of the buffer), and 90.60% (added to the mixed solution of sodium alginate and sodium carboxymethylcellulose with 3-fold volume of the buffer). Therefore, the subsequent research was carried out with the addition ratio of the added buffer solution to sodium alginate and sodium carboxymethylcellulose being 1:1.5.

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

[0198] The reusability of the immobilized enzyme was studied by high performance liquid chromatography (HPLC) determination method. Isomaltulose was prepared using the optimized process in Examples 8 - 14, and the isomerization of sucrose was catalyzed in a constant temperature shaker under the conditions of 35 °C and 120 rpm for continuous cyclic reactions. Specifically, a high - concentration sucrose substrate solution (the mass percentage of sucrose in the system was 67%) dissolved in 25 mL of mediator phosphate buffer and the affinity - directed immobilized enzyme microspheres prepared in Examples 8 - 14 (the SI - P1 mutant D392G affinity - directed immobilized enzyme microspheres were 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 ended, 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 citrate - disodium hydrogen phosphate citrate buffer solution (pH 6.0, 10 mM) to remove the unreacted substrates and products. The washed immobilized enzyme was re - added to the substrate for the next catalytic reaction. The above operations were repeated 40 times, and the sucrose conversion rate of the immobilized enzyme after multiple cycles was continuously detected. The results are as Figure 13 . After the immobilized sucrose isomerase prepared by this process was continuously reused 40 times, the sucrose conversion rate could still be maintained at 83.38%; it was shown that the affinity - embedding re - immobilization in the present invention better retained the enzyme activity, reduced the leakage of enzyme molecules in the reaction system, improved the efficiency of preparing isomaltulose by the enzymatic method, and was suitable for large - scale continuous industrial production.

[0199] The above results preliminarily showed that rational design of single - site mutations based on structure could screen sucrose isomerase mutants to improve the specific enzyme activity of proteins.

[0200] The above - described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A sucrose isomerase mutant, characterized in that, The sucrose isomerase mutant is obtained by any one of the following site mutations on the amino acid sequence as described in SEQ ID NO.1: (a) The amino acid at position 388 is mutated from isoleucine to tryptophan; (b) The amino acid at position 392 is mutated from aspartic acid to glycine; (c) The amino acid at position 401 is mutated from asparagine to tryptophan; (d) The amino acid at position 416 is mutated from glutamine to tryptophan.

2. Use of the sucrose isomerase mutant according to claim 1 in the preparation of an affinity-directed immobilized enzyme.

3. Use of the sucrose isomerase mutant according to claim 1 in the preparation of affinity-directed immobilized enzyme microspheres.

4. Use of the sucrose isomerase mutant according to claim 1 in the catalytic synthesis of isomaltulose.

Images