Sucrose isomerase mutant with improved thermal stability and construction method thereof

Abstract

The invention discloses a sucrose isomerase mutant with improved thermal stability and a construction method thereof, and belongs to the technical field of gene engineering and enzyme engineering. According to the sucrose isomerase mutant, the thermal stability and the expression quantity are simultaneously improved, the mutant is used for preparing isomaltulose, the half-life period of the single mutant V447E at the temperature of 40 DEG C is prolonged by 1.38 times, the optimum temperature is increased to 35 DEG C from 30 DEG C, and the expression quantity is increased to 33.06 U * mL <-1 > from 18.89 U * mL <-1 >; other mutations are superposed on the basis of the mutant V447E, and the thermal stability of the constructed double mutant V447E / D232P is further improved. Compared with a wild type, the V447E / D232P has the advantages that the optimal temperature is increased by 5 DEG C, the half-life period at 40 DEG C is prolonged by 1.52 times, and the expression quantity is increased from 18.89 U.mL <-1 > to 30.73 U.mL <-1 >.

Description

Technical Field

[0001] The present invention relates to a sucrose isomerase mutant with improved thermal stability and a method for constructing the same, belonging to the technical fields of genetic engineering and enzyme engineering. Background Art

[0002] Isomaltulose, namely 6-O-α-D-glucopyranosyl-1,6-D-fructose, also known as palatinose, is an isomer of sucrose and has similar physical properties and taste to sucrose. Isomaltulose has the characteristics of a low glycemic index and anti-caries, and is very suitable for consumption by obese people, diabetes patients and athletes, and has great application potential in the fields of functional foods and medicine. Sucrose isomerase (EC 5.4.99.11), also known as isomaltose synthase and sucrose α-glucosyltransferase, can catalyze the rearrangement of the α-1,2-glycosidic bond in the sucrose molecule into an α-1,6-glycosidic bond, thereby generating isomaltulose (6-O-α-D-glucopyranosyl-D-fructose). Many sucrose isomerases have poor thermal stability at high temperatures and their activities decline rapidly, especially above 40 °C, and irreversible inactivation occurs above 50 °C. For example, the half-life of the sucrose isomerase from Klebsiella sp. LX3 at 50 °C is only 1.8 min. The wild-type enzyme of Erwinia rhapontici NX-5 has a half-life of 5 min at 60 °C, and the recombinant mutant has no activity after being cultured at the same temperature for 30 min. The isoenzyme from Enterobacter sp. Ejp617 only retains 1.7% of its maximum activity after being incubated at 50 °C for 1 h. Currently, the enzymatic synthesis of isomaltulose is usually carried out in the temperature range of 30 °C to 40 °C. Among them, CN104059901A discloses a sucrose isomerase mutant of Serratia plymuthica, and the conversion rate of the sucrose isomerase prepared under the conditions of a temperature of 30 °C, a pH of 6.0, an enzyme addition amount of 20 U·g -1 sucrose, and a sucrose concentration of 400 g·L -1 is 88% - 89% (Master's thesis of Cheng Sheng in 2015, "Recombinant Expression, Thermal Stability Improvement and Application Research of Sucrose Isomerase from Serratia plymuthica"). The sucrose isomerase from Pantoea dispersa under the conditions of a temperature of 30 °C, a pH of 6.0, an enzyme addition amount of 20 U·g -1 sucrose, and a sucrose concentration of 400 g·L -1Under the conditions, the conversion rate of sucrose isomerase is 91%, and there is a higher conversion rate of sucrose isomerase (Master's thesis of Zhao Wenchong in 2023, "Expression of Pantoea dispersa Sucrose Isomerase in Bacillus subtilis and Preparation of Isomaltulose"). Therefore, it is of great significance to modify and improve the thermal stability of Pantoea dispersa sucrose isomerase on this basis to reduce the enzyme cost and enhance its industrial application ability. Summary of the Invention

[0003] Aiming at the deficiencies of the prior art, the present invention provides mutants with improved thermal stability of sucrose isomerase, which are more suitable for the industrial production of isomaltulose.

[0004] The present invention provides a sucrose isomerase mutant, which is obtained by mutating the 232nd and / or 447th amino acids of the sucrose isomerase with the amino acid sequence shown in SEQ ID NO.1.

[0005] In one embodiment, the mutant is obtained by mutating the aspartic acid at the 232nd position of the sucrose isomerase with the amino acid sequence shown in SEQ ID NO.1 to proline, named D232P, and its amino acid sequence is shown in SEQ ID NO.3.

[0006] In one embodiment, the mutant is obtained by mutating the valine at the 447th position of the sucrose isomerase with the amino acid sequence shown in SEQ ID NO.1 to glutamic acid, named V447E, and its amino acid sequence is shown in SEQ ID NO.4.

[0007] In one embodiment, the mutant is obtained by mutating the aspartic acid at the 232nd position of the sucrose isomerase with the amino acid sequence shown in SEQ ID NO.1 to proline and mutating the valine at the 447th position to glutamic acid, named D232P / V447E, and its amino acid sequence is shown in SEQ ID NO.5.

[0008] The present invention provides a method for preparing the above mutant, and the specific steps of the method are as follows:

[0009] I: Using the nucleotide sequence shown in SEQ ID NO.2 as a template, according to the rationally designed sites, designing site-directed mutagenesis primers, performing PCR amplification to obtain a gene containing the mutation site, and then constructing a vector containing the gene encoding the mutant;

[0010] II: Transforming the gene vector containing the encoding mutant into a host cell;

[0011] III: Screen and verify the recombinant strain constructed in the previous step to obtain positive clones, and then produce enzymes through culture and fermentation. Centrifuge to collect the fermentation supernatant to obtain a crude enzyme solution containing the sucrose isomerase mutant.

[0012] The present invention also provides a gene encoding the sucrose isomerase mutant.

[0013] The present invention also provides a recombinant vector carrying the gene.

[0014] In one embodiment, pHY300PLK is used as the expression vector.

[0015] The present invention also provides a recombinant cell carrying the gene or the recombinant vector.

[0016] In one embodiment, the recombinant cell uses Bacillus subtilis as the expression host.

[0017] In one embodiment, the Bacillus subtilis is Bacillus subtilis WS9C, which has been disclosed in the paper "Identification and optimization of genes potentially related to protein expression for enhancing α - amylase production in Bacillus subtilis".

[0018] The present invention provides a method for improving the thermal stability of sucrose isomerase. The method is to mutate the amino acid at position 232 and / or position 447 of the sucrose isomerase with the amino acid sequence shown in SEQ ID NO.1.

[0019] The present invention also provides the application of the recombinant vector or the recombinant cell in the preparation of sucrose isomerase.

[0020] In one embodiment, in the application, sucrose is used as the substrate, and the sucrose isomerase reacts in an environment of 30 - 45 °C and pH 6.0 - 6.5 for at least 8 - 12 h.

[0021] The present invention also provides the application of the sucrose isomerase mutant, or the gene encoding the mutant, or the recombinant vector, or the recombinant cell in the conversion of sucrose to produce isomaltulose or products containing isomaltulose.

[0022] Beneficial effects:

[0023] (1) Based on natural sucrose isomerase, through computer-aided design and homologous sequence alignment, combined with site-directed mutagenesis biotechnology to modify the molecular structure of sucrose isomerase, the influence of mutated residues on the thermal stability of the enzyme was analyzed, and finally six single-point mutant strains with improved stability, namely WS9C-M62E, WS9C-V105I, WS9C-N109H, WS9C-D232P, WS9C-V447E, and WS9C-S481M, and an iterative combined mutant strain WS9C-D232P / V447E with further improved thermal stability were obtained.

[0024] (2) The half-life of the sucrose isomerase mutant V447E provided by the present invention at 40 °C is 51.3 min, which is 1.38 times that of the wild-type sucrose isomerase (37.1 min); the half-life of D232P / V447E at 40 °C is 56.4 min, which is 1.56 times that of the wild-type sucrose isomerase.

[0025] (3) The thermal stability and expression level of the sucrose isomerase mutants provided by the present invention are both improved. The optimal temperature of V447E is increased from 30 °C to 35 °C, and the expression level is increased from 18.89 U·mL -1 to 33.06 U·mL -1 . The optimal temperature of D232P / V447E is increased from 30 °C to 35 °C, the half-life at 40 °C is extended by 1.52 times, and the expression level is increased from 18.89 U·mL -1 to 30.73 U·mL -1 . After heat treatment at 40 °C for 20 min, the sucrose isomerase mutants V447E and D232P / V447E retain 74.82% and 78.67% of the residual enzyme activity respectively, while the wild-type retains 69.56% of the residual enzyme activity.

[0026] (4) The sucrose isomerase mutants obtained in the present invention are more suitable for the application of catalyzing sucrose to produce isomaltulose than the wild-type, which is more conducive to the flexibility of the production process. Description of the Drawings

[0027] Figure 1 is the three-dimensional structure of sucrose isomerase.

[0028] Figure 2 is the amino acid sequence comparison of the key regions of sucrose isomerases from different sources.

[0029] Figure 3 is the relative expression level of the wild-type and single mutants of sucrose isomerase, and the residual enzyme activity after incubation at 40 °C and pH 6.0 for 20 min.

[0030] Figure 4The relative expression levels of wild-type and double mutant sucrose isomerase, and the residual enzyme activity after incubation at 40 °C and pH 6.0 for 20 min.

[0031] Figure 5 The optimal temperature for the enzyme activity of wild-type and mutant sucrose isomerase.

[0032] Figure 6 The temperature stability of the enzyme activity of wild-type and mutant sucrose isomerase.

[0033] Figure 7 The optimal pH for the enzyme activity of wild-type and mutant sucrose isomerase.

[0034] Figure 8 The pH stability of the enzyme activity of wild-type and mutant sucrose isomerase. Detailed implementation

[0035] (I) Culture medium

[0036] LB liquid medium (g·L -1 ): Tryptone 10, yeast extract 5, NaCl 10; LB solid medium needs to add 15 g·L -1 of agar powder.

[0037] TB fermentation medium (g·L -1 ): Tryptone 12, yeast extract 24, glycerol 5, K2HPO4·3H2O 16.43 (anhydrous K2HPO4 12.54), KH2PO4 2.31.

[0038] (II) Detection method:

[0039] Add 200 μL of appropriately diluted enzyme solution to 1.8 mL of citrate-disodium hydrogen phosphate buffer (50 mmol·L -1 , pH 6.0) to make the final concentration of sucrose reach 200 g·L -1 . Vigorously shake to thoroughly mix the reaction mixture, and then incubate in a 30 °C water bath for 15 min. Heat in a boiling water bath for 10 min to terminate the reaction. Then, centrifuge the reaction mixture at 12,000 rpm for 10 min at 40 °C, and filter the supernatant with a 0.22 μm syringe filter. Quantify the concentration of isomaltose in the filtered solution using an Alliance iS high-performance liquid chromatography system (Waters) equipped with a refractive index (RI) detector and a Syncronis Amino chromatographic column (4.6 mm × 25 mm × 5 mm, Thermo Scientific, USA). After injecting the sample (10 μL), elute with an 80% (v / v) aqueous acetonitrile solution at a flow rate of 0.8 mL / min, and keep the column temperature at 40 °C.

[0040] The enzyme activity unit of sucrose isomerase is defined as: under the conditions of 30 °C and pH 6.0, the amount of enzyme required to produce 1 μmol of isomaltulose within 1 minute is 1 enzyme activity unit (1 U).

[0041] Example 1: Preparation of sucrose isomerase mutants

[0042] (1) Construction of mutants

[0043] The proteins with amino acid sequences shown in Genbank accession numbers: AAP57085.1, ACF42098.1, AAP57084.1, ADJ56407.2, EKF64560.1, AAP57083.1, AGT14432.1, ABC33903.1 and AAK82938.1 were analyzed by sequence alignment using the online server Multiple Sequence Alignment by CLUSTALW, and then the alignment results were processed using the online server Easy Sequencing in PostScript 3.0.

[0044] Protein structure analysis: The three-dimensional structure of sucrose isomerase was predicted using AlphaFold3, and the analysis was carried out using this template on PyMOL software.

[0045] Based on the protein structure of sucrose isomerase, two online servers for computational prediction design, PROSS and FireProt, which are used to improve protein thermal stability and expression level, were used to select the common mutant sites predicted by them. At the same time, based on the three-dimensional structure model of sucrose isomerase, obviously unreasonable sites were excluded, and primers were designed for the remaining candidate sites and used for subsequent mutagenesis. The screening principles are as follows:

[0046] (1) The mutated residues should not greatly disrupt hydrogen bond or salt bridge interactions;

[0047] (2) The mutated amino acids should not be located inside the protein to disrupt the protein structure;

[0048] (3) All sites too close to the catalytic active center were excluded, and sites located in the α-helix region of the TIM barrel and other domains outside the TIM barrel were selected.

[0049] Based on the above amino acid sequence alignment and computer-aided design results, key sites that may affect catalytic activity were identified and excluded, and 14 mutant sites were screened out.

[0050] The gene encoding sucrose isomerase (nucleotide sequence shown in SEQ ID NO: 2) was synthesized and linked to pET24a to obtain the recombinant plasmid pET24a-WT. Using this as a template, the primers in Table 1 were used to perform site-directed mutagenesis of sucrose isomerase using a two-step PCR method. The mutation PCR system is shown in Table 2, and the reaction procedures are shown in Tables 3 and 4. 8 μL of PCR product was added to a PCR tube, and 1 μL of QuickCut Dpn I and 1 μL of Quickcut Green Buffer were added and mixed. The tube was placed in a 37°C constant temperature water bath and kept warm for 2 hours to digest the template DNA. The Dpn I-treated PCR product and E. coli JM109 competent cells were placed on ice for 10 minutes, the PCR product was added to the competent cells, and the transformation solution was spread on a plate containing 25 μg mL -1 Kan r The mutant was plated on a solid LB plate and incubated in a 37°C incubator for 10-12 hours. The coding sequence of the mutant was sequenced to confirm that the mutation was successful. The mutant gene was ligated into the pHY300PLK expression vector to obtain a recombinant plasmid with the nucleotide sequence shown in SEQ ID NO.6, which was then introduced into B. subtilis WS9C (disclosed in the paper "Identification and optimization of genes potentially related to protein expression for enhancing α-amylase production in Bacillus subtilis") for expression, resulting in 14 single-point mutants of sucrose isomerase, namely WS9C-P51R, WS9C-M62E, WS9C-V105I, WS9C-N109H, WS9C-Q153D, WS9C-D232P, WS9C-H256A, WS9C-A333L, WS9C-Q377M, WS9C-V447E, WS9C-Q453T, WS9C-S481M, WS9C-T497V and WS9C-Q507D.

[0051] pET-24a-sim V447EUsing as a template and the primers in Table 1, site-directed mutagenesis was carried out on the basis of WS9C-V447E by two-step PCR. The mutation PCR system, reaction program, and the construction method of the pHY300PLK-sim iterative combined mutant plasmid were the same as those of the above single-point mutant. The obtained iterative combined mutant gene plasmid was transferred into B. subtilis WS9C for expression, and five sucrose isomerase iterative combined mutants WS9C-M62E / V447E, WS9C-V105I / V447E, WS9C-N109H / V447E, WS9C-D232P / V447E, and WS9C-V447E / S481M were obtained.

[0052] Table 1 Primers used in Example 1

[0053]

[0054]

[0055] Table 2 Two-step PCR reaction system

[0056]

[0057] Table 3 First-round reaction program of two-step method

[0058]

[0059] *: The number of cycles of denaturation, annealing, and extension steps is 5.

[0060] Table 4 Second-round reaction program of two-step method

[0061]

[0062] *: The number of cycles of denaturation, annealing, and extension steps is 18.

[0063] (2) Expression of wild-type sucrose isomerase and its mutants

[0064] Pick the monoclonal obtained in step (1) and inoculate it into 10 mL of LB liquid medium containing 25 μg·mL -1 Tet r Using the recombinant B. subtilis WS9C strain containing the plasmid pHY300PLK-sim as a control, culture at 37 °C and 200 rpm for 12 h, and transfer the seed liquid into 50 mL of liquid TB fermentation medium containing 25 μg·mL -1 Tet r at an inoculation amount of 5%, culture at 37 °C and 200 rpm for 2 h, and then change the fermentation temperature to 33 °C and continue to culture for 48 h.

[0065] The recombinant strain was fermented in a shake flask. The fermentation broth was centrifuged at 12,000 rpm for 15 min, and the fermentation supernatant was collected. First, the expression level was detected. Taking the enzyme activity in the fermentation broth of wild-type sucrose isomerase as 100%, the results are as Figure 3 shown. The enzyme activities of some mutants decreased compared with the wild type, while the enzyme activities of M62E, V105I, N109H, Q153D, H256A, Q333L, V447E, Q453T, and S481M increased to varying degrees.

[0066] Example 2: Screening of mutants with improved thermal stability of sucrose isomerase

[0067] Primary screening method for thermal stability: Prepare the fermentation supernatant of the wild type and mutants according to the method of Example 1. The fermentation supernatants of the wild type and mutants were incubated at 40 °C for 20 min and then their enzyme activities were measured. Taking the enzyme activity of the fermentation supernatant without incubation treatment as 100%, each group of experiments was performed in triplicate, and the residual enzyme activity was calculated. Mutants with residual enzyme activity higher than that of the wild type were selected for subsequent experiments.

[0068] After incubation at 40 °C for 20 min, the residual enzyme activity of the wild type was 69.56%. The thermal stabilities of mutants M62E, V105I, N109H, D232P, V447E, and S481M were improved, and the residual enzyme activities were 70.35%, 72.47%, 72.58%, 73.68%, 74.82%, and 71.91% respectively, among which the residual enzyme activity of V447E was the highest.

[0069] To further improve the stability, other mutants with improved thermal stability were superimposed on the mutant V447E with the highest residual enzyme activity. According to the method of introducing mutations in Example 1, double mutants (M62E / V447E, N105I / V447E, N109H / V447E, D232P / V447EP, and V447E / S481M) were obtained respectively, and they were cultured under the same conditions as in Example 1, and the residual enzyme activity was measured according to the method of this example. As Figure 4 shown, taking the expression level of the wild type as 100%, the expression levels of double mutants M62E / V447E, N109H / V447E, D232P / V447E, and V447E / S481M increased by 22.50%, 48.76%, 62.68%, and 85.50% respectively. In addition, the expression levels of all double mutants were lower than that of V447E. The residual enzyme activity of D232P / V447E reached 78.67%, compared with V447E, while the thermal stabilities of other combined mutants decreased to varying degrees.

[0070] Example 3: Detection of Enzymatic Properties of Wild-Type Sucrose Isomerase and Mutants V447E and D232P / V447E

[0071] (1) Optimal Temperature and Temperature Stability

[0072] Optimal temperature: The enzyme proteins of sucrose isomerase and its mutants were prepared according to the method of Example 1. After dilution by a certain multiple, they were incubated for 20 min in a temperature-gradient water bath (20 - 45 °C, with an increment of 5 °C). A sucrose solution with a final concentration of 200 g·L -1 was prepared in a citrate-disodium hydrogen phosphate buffer (50 mmol·L -1 , pH 6.0). After incubation at different temperatures, the enzyme solution was added. The enzyme activities of sucrose isomerase and its mutants were measured, and the optimal temperature of sucrose isomerase was determined. Taking the highest enzyme activity as 100%, the residual enzyme activity was calculated accordingly. The results are as Figure 5 shown. The enzyme activities of WT, V447E, and D232P / V447E were measured at 20 - 45 °C. The optimal temperatures of V447E and D232P / V447E were 35 °C, which was 5 °C higher than that of WT. In the temperature range of 20 - 45 °C, the residual activities of the mutant enzymes V447E and D232P / V447E were similar to those of the wild type.

[0073] Temperature stability: Sucrose isomerase and its mutants were placed in a constant-temperature water bath at 40 °C. Samples were collected regularly, and the enzyme activity was measured at 30 °C and pH 6.0. Taking the highest enzyme activity as 100%, the residual enzyme activity was calculated accordingly. The time required for the initial enzyme activity to be halved was defined as t 1 / 2 . The measurement method was to incubate the purified enzyme at 40 °C for a period of time, and aliquots of the enzyme solution were taken regularly and placed on ice. Then the residual enzyme activity was measured. The first-order rate constant (k d ) was ln(residual enzyme activity / incubation time), and the calculation formula for t 1 / 2 was t 1 / 2 = ln2 / k d . The results are as Figure 6 shown. The t 1 / 2 values of WT, V447E, and D232P / V447E were 37.1, 51.3, and 56.4 min, respectively. The t 1 / 2 values of the two mutants were 1.38 and 1.52 times that of the wild type, respectively. This indicates that when these two mutants undergo irreversible denaturation at 40 °C, the ability of the mutants to maintain enzyme activity is significantly stronger than that of the wild type.

[0074] (2) Optimal pH and pH Stability

[0075] Optimal pH value: Prepare a sucrose solution with a pH value of 4.0 - 8.0 (citric acid - disodium hydrogen phosphate buffer) and a final concentration of 200 g·L -1 -1. Dilute sucrose isomerase with buffers of different pH values by a certain multiple and add it to the substrates with corresponding pH values, and then measure the enzyme activity at 30 °C to determine the optimal pH value of sucrose isomerase and its mutants. Take the highest enzyme activity as 100%, and calculate the residual enzyme activity accordingly. The results are as Figure 7 shown. The enzyme activities of WT, V447E, and D232P / V447E were measured at pH values of 4.0 - 8.0. The optimal pH values of WT, V447E, and D232P / V447E are 6.0, 6.5, and 6.5, respectively. At these optimal pH levels, the enzyme activities were normalized to 100%. At pH 4.0, the enzyme activities of WT, V447E, and D232P / V447E were only 5.77%, 6.69%, and 5.98%, respectively. At pH values of 5.5 - 7.0, the enzyme activity of sucrose isomerase remained above 80%.

[0076] pH stability: To further study the pH stability of V447E and D232P / V447E under conditions close to room temperature, dilute sucrose isomerase and its mutants with citric acid - disodium hydrogen phosphate buffer (50 mmol·L -1 ) at pH values of 4.0 - 8.0 by a certain number of times, and then incubate them in a water bath at 25 °C for 24 h. Take the highest enzyme activity as 100%, and calculate the residual enzyme activity accordingly. When measuring the above - mentioned enzyme characteristics, set the highest enzyme activity as 100%, and the ratio of the enzyme activity under the remaining conditions to the highest enzyme activity is the residual enzyme activity. The results are as Figure 8 shown. After incubating in the buffer at pH 4.0 for 24 h, the enzyme activities of WT, V447E, and D232P / V447E were zero. WT had the best stability in the buffer at pH 6.0, while V447E and D232P / V447E had the best stability in the buffer at pH 6.5 Figure 8 . WT, V447E, and D232P / V447E maintained relatively high residual enzyme activities in the buffer at pH values of 5.5 - 8.0, indicating that these enzymes had good stability within a relatively wide pH range.

[0077] Example 4: Application of sucrose isomerase mutants in the preparation of isomaltulose

[0078] Prepare the sucrose isomerase mutant V447E according to the method of Example 1. Using sucrose at pH 6.0 and 400 g·L -1 -1 as the substrate, and the addition amount of the recombinant enzyme is 20 U·g -1 -1 sucrose. When converting at 30 °C for 10 h, the yield of isomaltulose reached 91.25%.

[0079] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Any person skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Sucrose isomerase mutant, characterized in that, It is obtained by mutating the amino acids at positions 232 and / or 447 of the sucrose isomerase with the amino acid sequence shown in SEQ ID NO.

1.

2. The sucrose isomerase mutant according to claim 1, wherein Based on the sucrose isomerase with the amino acid sequence shown in SEQ ID NO.1, it has any one of the following mutations (a) to (c): (a) Mutating the aspartic acid at position 232 to proline; (b) Mutating the valine at position 447 to glutamic acid; (c) Mutating the aspartic acid at position 232 to proline and mutating the valine at position 447 to glutamic acid.

3. A gene encoding the sucrose isomerase mutant according to claim 1 or 2.

4. A recombinant plasmid carrying the gene according to claim 3.

5. The recombinant plasmid according to claim 4, characterized in that, The plasmid is pHY300PLK.

6. A recombinant microorganism expressing the sucrose isomerase mutant according to claim 1 or 2.

7. Recombinant Bacillus subtilis, characterized in that, Using pHY300PLK as an expression vector, it expresses the sucrose isomerase mutant according to claim 1 or 2.

8. A method for improving the thermal stability of sucrose isomerase, characterized in that, Based on the sucrose isomerase with the amino acid sequence shown in SEQ ID NO.1, mutating the aspartic acid at position 232 to proline, and / or mutating the valine at position 447 to glutamic acid.

9. A method for preparing isomaltulose, characterized in that, Using sucrose as a substrate and the sucrose isomerase mutant according to claim 1 or 2 as a catalyst, reacting at 30 - 45 °C for at least 8 - 12 h.

10. Use of the sucrose isomerase mutant according to claim 1 or 2, or the gene according to claim 3, or the recombinant plasmid according to claim 4 or 5, or the recombinant microorganism according to claim 6, or the recombinant Bacillus subtilis according to claim 7, or any one of the methods according to claims 8 - 9 in the preparation of isomaltulose or a product containing isomaltulose.

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