Multifunctional protein tag for enhancing thermal stability and immobilization rate of protein and application of multifunctional protein tag

Abstract

The invention discloses a multifunctional protein tag for enhancing the thermal stability and immobilization rate of protein and application of the multifunctional protein tag, and belongs to the field of enzyme engineering. The multifunctional protein tag is composed of a polymerization promoting tag and an immobilized tag, the amino acid sequence of the polymerization promoting tag is as shown in SEQ ID NO.1, or any one or more of the following amino acid sites corresponding to the amino acid sequence as shown in SEQ ID NO.1 are mutated: the second site, the third site and / or the sixteenth site; the amino acid sequence of the immobilized tag is DDD, EEE or any one of SEQ ID NO. 2-5. After the bifunctional tag is fused with D-psicose 3-epimerase (DAE), the thermal stability of the fusion protein and the immobilized enzyme activity yield are remarkably improved. The label has universality for improving the thermal stability of DAE from different sources, and an efficient and stable immobilized enzyme solution is provided for preparing psicose by an enzyme method.

Description

Technical Field

[0001] The present invention belongs to the field of enzyme engineering, and in particular relates to a multifunctional protein label for enhancing protein thermal stability and immobilization rate and application thereof. Background Art

[0002] D-psicose (allulose), the C-3 diastereomer of D-fructose, has a sweetness and taste similar to sucrose, yet contains only 0.4 kcal / g of energy. As a low-calorie functional sweetener, allulose is an ideal substitute for table sugars such as sucrose and is widely used in food. Currently, several countries and regions, including the United States, Japan, South Korea, Singapore, Mexico, Colombia, Chile, Costa Rica, Australia, and New Zealand, have approved allulose for use as a food ingredient or sweetener. Due to its extremely low calorie content, the US Food and Drug Administration (FDA) has removed allulose from the "added sugars" and "total sugars" labels, further enhancing its market appeal as a healthy sweetener. Allulose also exhibits numerous unique physiological benefits, including hypoglycemic and hypolipidemic effects, anti-inflammatory, anti-caries, weight management, and free radical scavenging. Studies have shown that allulose can significantly reduce serum cholesterol, triglycerides, and low-density lipoprotein levels, offering potential applications in the prevention and treatment of obesity and diabetes. In addition, it can promote the release of glucagon-like peptide-1 (GLP-1) receptors and activate vagal afferent signals, thereby reducing food intake and achieving weight control. As a bulk sweetener, D-psicose can also enhance food flavor through the Maillard reaction and significantly improve food gelling properties, showing broad application prospects in food processing.

[0003] Currently, allulose is mainly produced on a large scale through bioconversion, using ketose 3-epimerase to catalyze the conversion of fructose into allulose. D-psicose 3-epimerase (DAE) is the most common ketose 3-epimerase used in the bioproduction of allulose. It is derived from rumen bacteria. Ruminococcus sp., Clostridium Clostridium bolteae Agrobacterium tumefaciens Agrobacterium tumefaciens DAE from microorganisms such as β-actin has been used in the large-scale production of allulose. To improve the catalytic activity and thermal stability of DAE, researchers have molecularly modified the enzyme using methods such as directed evolution and rational design, achieving significant results. However, in the actual production of allulose, the amount of enzyme added is still generally high, which not only increases production costs but also places a heavy burden on downstream separation and purification of the product.

[0004] The development of immobilized enzyme technology is of significant significance for the enzymatic preparation of allulose. Immobilizing DAE enzymes significantly improves enzyme stability and reuse, while facilitating enzyme recovery and simplifying product purification. Reported DAE immobilization methods primarily include adsorption, embedding, crosslinking, and covalent immobilization. Enzymes immobilized using adsorption and embedding methods typically use calcium alginate or chitosan as supports, which have poor mechanical strength and thus cannot be used for long periods of time. Covalent immobilization, which forms a covalent bond between the enzyme and the support, offers greater stability and robustness. However, covalent binding sites often exhibit nonspecific binding properties. Covalent binding of key sites of the enzyme molecule to the support can cause structural changes in the enzyme, leading to reduced or even inactivation of its activity. This not only reduces the yield of immobilized enzyme activity but also wastes resources. The immobilization efficiency of enzymes can be greatly improved through label modification technology. For example, patent CN112831489A (publication date: 2021.05.21) improves the immobilization rate of DAE and epoxy resin by adding an AKAKAKAKAK tag rich in basic amino acids.

[0005] Therefore, developing DAE recombinant proteins that help improve thermal stability and immobilization rate through enzyme engineering technology to prepare immobilized enzymes with industrial application performance is of significant significance for the industrial production of allulose. Summary of the Invention

[0006] Based on the problems existing in the prior art, the present invention discloses a multifunctional protein tag, which includes a promoting aggregation tag and an immobilization tag. The amino acid sequence of the promoting aggregation tag is as shown in SEQ ID NO.1, or there is a mutation at any one or several (2 or 3) of the following amino acid sites corresponding to the amino acid sequence shown in SEQ ID NO.1: position 2, position 3 and / or position 16; the amino acid sequence of the immobilization tag is DDD, EEE or as shown in any one of SEQID NO.2-5.

[0007] In one embodiment, the mutation exists at the second amino acid site corresponding to the amino acid sequence shown in SEQ ID NO.1. Furthermore, based on the mutation at the second amino acid site, the mutation also includes mutations at any one or any two amino acid sites of the third or 16 amino acid sites.

[0008] In one embodiment, the mutation exists at the 3rd amino acid site corresponding to the amino acid sequence shown in SEQ ID NO.1. Furthermore, based on the mutation at the 3rd amino acid site, the mutation also includes mutations at any one or any two amino acid sites at the 2nd or 16th amino acid sites.

[0009] In one embodiment, there is a mutation at the 16th amino acid site corresponding to the amino acid sequence shown in SEQ ID NO.1. Furthermore, based on the mutation at the 16th amino acid site, it also includes mutations at any one or any two amino acid sites at the 2nd or 3rd amino acid sites.

[0010] In one embodiment, the amino acid at position 2, position 3 or position 16 corresponding to the amino acid sequence shown in SEQ ID NO. 1 is mutated.

[0011] In one embodiment, the amino acids at positions 2 and 3 corresponding to the amino acid sequence shown in SEQ ID NO. 1 are mutated simultaneously.

[0012] In one embodiment, the amino acids at positions 2, 3 and 16 corresponding to the amino acid sequence shown in SEQ ID NO. 1 are mutated simultaneously.

[0013] In one embodiment, the amino acid at position 2 is mutated to a non-S amino acid, such as A, R, K, F, W, H, L, I, M, E, D, T, G, Y, C, Q, P, V, N; preferably, C or E.

[0014] In one embodiment, the amino acid at position 3 is mutated to a non-S amino acid, such as A, R, K, F, W, H, L, I, M, E, D, T, G, Y, C, Q, P, V, N; preferably, E or T.

[0015] In one embodiment, the amino acid at position 16 is mutated to a non-R amino acid, such as A, K, F, W, H, L, I, M, E, S, T, G, Y, C, Q, P, V, N; preferably, D, K or Y.

[0016] Specifically, the aggregation-promoting tag is used to enhance the thermal stability of the protein, and the immobilization tag is used to increase the immobilization rate of the protein and the carrier.

[0017] Specifically, the immobilization tag is connected to the N-terminus of the aggregation-promoting tag, specifically, directly connected or connected through a linker.

[0018] The present invention also provides a fusion protein, which includes the multifunctional protein tag and a target protein, and the target protein is D-psicose 3-epimerase.

[0019] Specifically, the source of the D-psicose 3-epimerase is rumen bacteria ( Ruminococcus sp.), Arthrobacter sphaeroides ( Arthrobacter globiformis ), Clostridium boulardii ( Clostridium bolteae ) Thermophilic Bacillus ( Novibacillus thermophilus), Pseudomonas chicory ( Pseudomonas cichorii ), preferably rumen bacteria.

[0020] Specifically, the amino acid sequence of the D-psicose 3-epimerase is shown in any one of Genbank accession numbers ZP_04858451.1, AAK88700.1, AB981957.1, EDP19602.1, WP_077721022.1, or BAA24429.1.

[0021] Specifically, the multifunctional protein tag is located at the N-terminus of D-psicose 3-epimerase.

[0022] The present invention also provides a method for preparing an immobilized enzyme, wherein the method comprises immobilizing the fusion protein and a carrier through covalent bonding, wherein the immobilization is achieved by bonding the amino groups of the carrier with the acidic amino acid groups of the fusion protein.

[0023] Specifically, the carrier is an amino macroporous resin.

[0024] Specifically, the immobilization step includes: S1. Resin activation: Soak the macroporous resin in potassium phosphate buffer at pH 7.5-8.5 for 2-4 hours, and filter to obtain the activated resin; S2. Enzyme preparation: heterologously expressing the fusion protein according to any one of claims 4 to 7 using a host strain such as Escherichia coli, and purifying the enzyme using Ni affinity chromatography; S3. Immobilization: Add the activated resin to the pure enzyme solution for immobilization reaction at a temperature of 20-40°C for 12-16 hours. S4. Rinsing: The immobilized particles are obtained by filtration and rinsed 2-3 times with potassium phosphate buffer at pH 7.5-8.5 to obtain the immobilized enzyme.

[0025] The present invention also provides an immobilized enzyme prepared by the method.

[0026] The present invention also provides the use of the multifunctional protein tag, the fusion protein, or the immobilized enzyme obtained by the immobilized enzyme preparation method in the preparation of psicose.

[0027] Those skilled in the art can identify the amino acids in the protein tags of the present invention using methods known in the art, such as site-directed mutagenesis, protein evolution, or bioinformatics analysis. The catalytic domain, active site, or other functional domains of a protein can also be determined by physical structural analysis, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with mutations of amino acids at putative key sites.

[0028] In the present invention, amino acid residues can be represented by single letters or three letters, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamine (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), arginine (Arg, R).

[0029] The term "AxxB" means that the amino acid A at position xx is changed to amino acid B, for example, S2E means that the S at position 2 is mutated to E. When multiple amino acid sites are mutated simultaneously, they can be expressed in a similar form as "S2C / S3T" or "S2C / S3T / R16Y". For example, "S2C / S3T" means that the S at position 2 is mutated to C and the S at position 3 is mutated to T. Preferably, the aggregation-promoting tag in the present invention can be a single mutant, a double mutant or a triple mutant, the single mutant being S2C, S2E, S3E, S3T, R16D, R16K or R16Y, the double mutant being 2C / S3T or S2E / S3T, the triple mutant being S2C / S3T / R16D, S2C / S3T / R16Y or S2E / S3T / R16K, and preferably the triple mutant S2C / S3T / R16Y.

[0030] This study addresses the poor thermal stability and low immobilization efficiency of DAE by developing a novel bifunctional protein tag. Using this tag-modified DAE fusion protein, an ultrastable immobilized enzyme was prepared. The thermal denaturation temperature (Tm) of the fusion protein was increased by over 6.9°C compared to the wild-type, and the immobilized enzyme activity yield was increased by 7.6%. The immobilized enzyme maintained a relative conversion rate of over 90% after 520 consecutive reactions. This tag offers advantages such as high stability, high reuse, and low cost, and has potential for large-scale production of allulose. This tag is universally applicable to improving the thermal stability of DAE from diverse sources, providing a highly efficient and stable immobilized enzyme solution for the enzymatic production of allulose. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The thermal stability of the aggregation-promoting tag modified enzyme is compared with that of the wild-type enzyme.

[0032] Figure 2 The effect of bifunctional tags on the thermal stability of D-psicose 3-epimerase from different sources.

[0033] Figure 3 is the relative conversion rate of immobilized enzyme batch reaction to produce psicose. DETAILED DESCRIPTION

[0034] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications or replacements shall fall within the scope of protection of the present invention.

[0035] The enzyme activity of the free D-psicose 3-epimerase in the present invention is determined as follows: D-fructose with a final concentration of 10 g / L, 1 mmol / L MnCl2, and an appropriate amount of free enzyme are added to a 10 mmol / L PBS buffer (pH 7.5), reacted in a 55°C water bath for 10 minutes, and then boiled at 100°C for 10 minutes to terminate the reaction.

[0036] The enzyme activity of the immobilized D-psicose 3-epimerase in the present invention is determined as follows: D-fructose with a final concentration of 50 g / L, 1 mmol / L MnCl2, and an appropriate amount of free enzyme are added to 10 mmol / L PBS buffer (pH 7.5), reacted in a 55°C water bath for 10 minutes, and then boiled at 100°C for 10 minutes to terminate the reaction.

[0037] The psicose content in the reaction products was determined by high-performance liquid chromatography, and the enzymatic activities of the free and immobilized enzymes were calculated. One unit (U) of enzyme activity was defined as the amount of enzyme required to catalyze the production of 1 μmol of D-psicose per minute.

[0038] Example 1: Enhancement of the thermal stability of D-psicose 3-epimerase by polymerization-promoting tags Ruminococcus Ruminococcus sp., was fused with a 19-amino acid protein tag at the amino terminus. The amino acid sequence of the tag was GSSHHHHHHSSGLVPRGSH (as shown in SEQ ID NO.1) to construct the tag-modified enzyme M0. To test the effect of the tag on the thermal stability of RDAE, WT and M0 were incubated at 55°C, and the enzyme activity after different incubation times was measured. The residual enzyme activity was calculated with the enzyme activity without incubation as 100%, and the residual enzyme activity curve was drawn. Figure 1As shown in the figure, after incubation at 55°C for 24 hours, the residual enzyme activity of WT was less than 20%, while that of M0 was approximately 75%. This indicates that the addition of this tag significantly improves the thermal stability of RDAE. This tag enhances the interactions between RDPE subunit interfaces, stabilizing the tetramer conformation and thus improving the thermal stability of the enzyme. Therefore, this tag is named a pro-aggregation tag.

[0039] Example 2: Molecular modification of aggregation-promoting tags Alanine scanning of amino acid residues other than histidine within the aggregation-promoting tag was performed to determine the effect of each tag position on thermal stability. After incubation at 55°C for 8 hours, the residual enzyme activity of each mutant was measured. The relative residual enzyme activity of each mutant was calculated, with the untagged WT residual enzyme activity as 100%. The relative thermal stability of each mutant was defined as 120.17%. The relative thermal stabilities of the M0 single mutants S2A, S3A, and R16A were significantly lower than those of M0, reaching 88.63%, 90.15%, and 83.38%, respectively, all falling below 110%. This result indicates that these three positions in the aggregation-promoting tag significantly influence thermal stability.

[0040] Site-directed saturation mutagenesis was performed at Ser2, Ser3, and Arg16 in the aggregation-promoting tag (shown in SEQ ID NO. 1). The single mutants with improved enzymatic activity and thermostability are shown in Table 1, with the relative enzymatic activity and thermostability of M0, which carries the unmutated aggregation-promoting tag, taken as 100%. The results showed that when Ser at position 2 of the aggregation-promoting tag was mutated to Cys or Glu, the enzymatic activity and thermostability of the single mutants M1 (S2C) and M2 (S2E) were significantly improved compared to M0. When Ser at position 3 of the aggregation-promoting tag was mutated to Glu or Thr, the enzymatic activity and thermostability of the single mutants M3 (S3E) and M4 (S3T) were significantly improved compared to M0. When Arg at position 16 was mutated to Asp (R16D), Lys (R16K), or Tyr (R16Y), the enzymatic activity and thermostability of the single mutants M5, M6, or M7 were significantly improved compared to M0.

[0041] Table 1 Effects of single mutants of aggregation-promoting tags on the enzymatic activity and thermal stability of RDAE

[0042] Double mutants were constructed by combining mutations at positions 2 and 3. The changes in enzyme activity and thermostability of the double mutants are shown in Table 2. M8 exhibited a significant decrease in enzyme activity, while M10 exhibited a moderate decrease in thermostability compared to M0. While M9 and M10 exhibited slightly decreased enzyme activity, their thermostability was significantly improved. Therefore, triple mutants were constructed based on the double mutants M9 (S2C / S3T) and M11 (S2E / S3T). The changes in enzyme activity and thermostability of the triple mutants are shown in Table 2. Among them, the thermostability of M12 (S2C / S3T / R16D), M14 (S2C / S3T / R16Y) and M16 (S2E / S3T / R16K) was significantly improved, and the enzyme activity did not show a significant decrease. The thermostability and enzyme activity of M15 (S2E / S3T / R16D) were slightly improved. The enzyme activity of M13 (S2C / S3T / R16K) and M17 (S2E / S3T / R16Y) was slightly decreased, but the thermostability was improved to a certain extent.

[0043] Table 2 Effects of aggregation-promoting tag combination mutants on RDAE enzyme activity and thermal stability

[0044] Example 3: Screening of immobilized tags and construction of dual-function tags RDAE was immobilized on a commercially available amino-based macroporous resin with high mechanical strength. Immobilization was achieved by covalently linking the active amino groups on the support with free carboxyl groups in the protein. Amino acid sequence analysis revealed that Asp and Glu, which carry free carboxyl groups, predominate in RDAE. Nonspecific binding to the support at sites closely associated with enzyme activity can lead to significant loss of activity in the immobilized enzyme. Therefore, six immobilization tags rich in Asp or Glu were designed. By enhancing the binding probability of the acidic amino acids within the tags to the support and reducing nonspecific binding, they achieved improved immobilization efficiency and enzyme activity. The amino acid sequences of these six immobilization tags are: DDD, ADADAD (SEQ ID NO. 2), PDPDPD (SEQ ID NO. 3), EEE, AEAEAE (SEQ ID NO. 4), and PEPEPE (SEQ ID NO. 5). The immobilization tag was attached to the N-terminus of the aggregation-promoting tag (shown in SEQ ID NO. 1) to form a bifunctional tag. The bifunctional tag was then fused to the N-terminus of RDAE to form a bifunctional tag-modified fusion protein. Fusion proteins containing the six immobilization tags and RDAE were constructed, and the yield of immobilized enzyme activity using the fusion proteins and amino resin was measured. The results showed that the use of Glu in the immobilization tag significantly improved the yield of immobilized enzyme activity. The three immobilization tags (EEE, PEPEPE, and AEAEAE) increased the yield of enzyme activity by 30.69%, 24.98%, and 19.77%, respectively, compared to the yield without an immobilized tag.

[0045] The EEE immobilization tag that significantly improved the yield of immobilized enzyme activity was fused with the best-modified aggregation-promoting tag (i.e., the aggregation-promoting tag in M14, which contains S2C / S3T / R16Y mutations relative to the sequence of SEQ ID NO.1) to construct a dual-functional tag. This dual-functional tag was fused to the N-terminus of RDAE, and the fusion protein was named M18. In order to evaluate the effect of the dual-functional tag on the thermal stability of RDAE, the thermal denaturation temperature (T m The results showed that the T m The values ​​were 64.9, 70.4, 71.8, and 70.4 °C, respectively. Among them, the T m The T value of RDAE fusion protein M18 containing dual functional tags was the highest, which was 6.9℃ higher than that of WT without tag. m The value is slightly lower, but still much higher than the T of WT mThe above experimental data on the thermal stability and immobilization efficiency of RDAE modified with a bifunctional tag confirmed that the bifunctional tag has the dual effect of improving thermal stability and the yield of immobilized enzyme activity.

[0046] Example 4: Effect of dual-functional tags on the thermal stability of DAE from different sources In order to test whether the bifunctional tag is universal for enzymes such as DAE, several common DAEs from different sources were selected and fused with bifunctional tags (the bifunctional tag type carried by M18, the amino acid sequence of the aggregation-promoting tag is S2C / S3T / R16Y relative to the sequence of SEQ ID NO.1, and the amino acid sequence of the immobilized tag is EEE). The selected DAEs were derived from Agrobacterium tumefaciens ( Agrobacterium tumefaciens ), Arthrobacter sphaeroides ( Arthrobacter globiformis ), Clostridium boulardii ( Clostridium bolteae ) Thermophilic Bacillus ( Novibacillus thermophilus ), Pseudomonas chicory ( Pseudomonas cichorii )、Rumen bacteria( Ruminococcus sp.), respectively referred to as ATDAE (Genbank No. AAK88700.1), AGDAE (Genbank No. AB981957.1), CBDAE (Genbank No. EDP19602.1), NTDAE (Genbank No. WP_077721022.1), PCDAE (Genbank No. BAA24429.1), and RDAE (Genbank No. ZP_04858451.1). The fusion proteins of the dual-functional tag and the above DAEs were incubated at 55°C for 8 hours, and the residual enzyme activities were measured and the relative thermal stability was calculated. The results are shown in Figure 2. Figure 2 As shown in the figure, the thermal stability of the selected DAEs was improved to varying degrees after adding the dual-functional tags, indicating that the dual-functional tags have a certain universality in improving the thermal stability of DAEs.

[0047] Example 5: Development of immobilized enzyme using DAE modified with a bifunctional tag The dual-functional tag-modified RDAE recombinant protein M18 was immobilized using a commercial amino-type macroporous resin. The immobilization method includes the following steps: (1) Resin activation: The macroporous resin was placed in potassium phosphate buffer at pH 8.0 for 3 h, and the activated resin was obtained by filtration; (2) Enzyme preparation: Use host strains such as Escherichia coli to heterologously express the bifunctional tag-modified recombinant protein, and purify the enzyme using Ni affinity chromatography; (3) Immobilization: Add the activated resin to the pure enzyme solution for immobilization reaction at a temperature of 30°C for 14 h. (4) Rinsing: The immobilized particles were obtained by filtration and rinsed three times with potassium phosphate buffer at pH 8.0 to obtain the immobilized enzyme.

[0048] In order to verify the actual application effect of RDAE recombinant protein M18 immobilized enzyme, a high concentration fructose solution was used as a substrate for immobilized enzyme batch conversion reaction. The reaction temperature was set to 55 ° C, and the single reaction time was 2h. After the reaction, the immobilized enzyme was recovered from the system, and a new fructose solution was added for a new round of reaction. The conversion rate of allulose in each reaction was determined, and the relative conversion rate of each conversion was calculated with the conversion rate of the first reaction as 100%. The results showed that the conversion rate of allulose in the first reaction was 30.2%. After 520 consecutive reactions, the immobilized enzyme still maintained a relative conversion rate of more than 90%. Figure 3 shown.

[0049] This study uses a dual-functional tag modification strategy to improve the thermal stability and immobilization efficiency of DAE enzyme, developing a novel, ultrastable immobilized enzyme with superior recycling capabilities. This immobilization method is simple, low-cost, and suitable for industrial production.

Claims

1. A multifunctional protein tag, characterized in that the multifunctional protein tag comprises an aggregation-promoting tag and an immobilization tag, the amino acid sequence of the aggregation-promoting tag is as shown in SEQ ID NO.1, or there is a mutation at any one or several of the following amino acid sites corresponding to the amino acid sequence shown in SEQ ID NO.1: position 2, position 3 and / or position 16; the amino acid sequence of the immobilization tag is DDD, EEE or as shown in any one of SEQ ID NO.2-5.

2. The multifunctional protein tag according to claim 1, characterized in that The amino acid at position 2 is mutated into a non-S amino acid, the amino acid at position 3 is mutated into a non-S amino acid, and the amino acid at position 16 is mutated into a non-R amino acid; Preferably, the amino acid mutation at position 2 is mutated to C or E, preferably, the amino acid mutation at position 3 is mutated to E or T, preferably, the amino acid mutation at position 16 is mutated to D, K or Y.

3. The multifunctional protein tag according to claim 2, characterized in that The immobilization tag is connected to the N-terminus of the aggregation-promoting tag, specifically, directly connected or connected through a linker.

4. A fusion protein, characterized in that The fusion protein comprises the multifunctional protein tag according to any one of claims 1 to 3 and a target protein. Preferably, the target protein is D-psicose 3-epimerase.

5. The fusion protein according to claim 4, characterized in that The D-psicose 3-epimerase is derived from Ruminococcus, Arthrobacter sphaeroides, Clostridium boulardii, Neobacillus thermophilus, and Pseudomonas chicory, preferably, Ruminococcus.

6. The fusion protein according to claim 5, characterized in that The Genbank accession number of the amino acid sequence of the D-psicose 3-epimerase is any one of ZP_04858451.1, AAK88700.1, AB981957.1, EDP19602.1, WP_077721022.1, or BAA24429.

1.

7. The fusion protein according to claim 6, characterized in that The multifunctional protein tag is located at the N-terminus of the target protein.

8. A method for preparing an immobilized enzyme, characterized in that: The method comprises immobilizing the fusion protein according to any one of claims 4 to 7 by covalently binding to a carrier, wherein the immobilization is achieved by binding the amino groups of the carrier to the acidic amino acid groups of the fusion protein. Preferably, the carrier is an amino-type macroporous resin.

9. The preparation method according to claim 8, characterized in that The immobilization step comprises: S1. Resin activation: Soak the macroporous resin in potassium phosphate buffer at pH 7.5-8.5 for 2-4 hours, and filter to obtain the activated resin; S2. Enzyme preparation: heterologously expressing the fusion protein according to any one of claims 4 to 7 using a host strain such as Escherichia coli, and purifying the enzyme using Ni affinity chromatography; S3. Immobilization: Add the activated resin to the pure enzyme solution for immobilization reaction at a temperature of 20-40°C for 12-16 hours. S4. Rinsing: The immobilized particles are obtained by filtration and rinsed 2-3 times with potassium phosphate buffer at pH 7.5-8.5 to obtain the immobilized enzyme.

10. Use of the multifunctional protein tag according to any one of claims 1 to 3, or the fusion protein according to any one of claims 4 to 7, or the immobilized enzyme obtained by the preparation method according to any one of claims 8 to 9 in the preparation of psicose.

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