Sucrose isomerase mutants with improved thermal stability and methods for constructing the same

Abstract

The application discloses a sucrose isomerase mutant with improved thermal stability and a construction method thereof, and belongs to the technical field of genetic engineering and enzyme engineering. The application constructs a sucrose isomerase mutant with improved thermal stability and expression amount, and uses the mutant for preparation of isomaltulose, wherein a single mutant V447E has a half-life at 40 DEG C prolonged by 1.38 times, an optimum temperature increased from 30 DEG C to 35 DEG C, and an expression amount increased from 18.89 U.mL ‑1 -1 to 33.06 U.mL ‑1 -1; a double mutant V447E / D232P with other mutations superimposed on the basis of the mutant V447E has further improved thermal stability; compared with a wild type, the double mutant V447E / D232P has an optimum temperature increased by 5 DEG C, a half-life at 40 DEG C prolonged by 1.52 times, and an expression amount increased from 18.89 U.mL ‑1 -1 to 30.73 U.mL ‑1 -1.

Description

Technical Field

[0001] This invention relates to a thermostable sucrose isomerase mutant and its construction method, belonging to the fields of genetic engineering and enzyme engineering technology. Background Technology

[0002] Isomaltulose, also known as 6-O-α-D-glucopyranosyl-1,6-D-fructose or palaginose, is an isomer of sucrose, sharing similar physical properties and taste. Isomaltulose has a low glycemic index and anti-caries properties, making it ideal for obese individuals, diabetics, and athletes, and it holds great potential for application in functional foods and the medical field. Sucrose isomerase (EC 5.4.99.11), also called isomaltose synthase or sucrose α-glucosyltransferase, catalyzes the rearrangement of α-1,2-glycosidic bonds in sucrose molecules to α-1,6-glycosidic bonds, thereby generating isomaltulose (6-O-α-D-glucopyranosyl-D-fructose). Many sucrose isomerases exhibit poor thermal stability at high temperatures, with activity rapidly decreasing, especially above 40°C; irreversible inactivation occurs above 50°C. For example, the sucrose isomerase of Klebsiella sp. LX3 has a half-life of only 1.8 min at 50 °C. The wild-type enzyme of Erwinia rhapontici NX-5 has a half-life of 5 min at 60 °C, and the recombinant mutant showed no activity after 30 min of culture at the same temperature. The isoenzyme from Enterobacter sp. Ejp617 retained only 1.7% of its maximum activity after incubation 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, which can be synthesized at 30 °C, pH 6.0, and enzyme dosage of 20 U·g. -1 Sucrose, sucrose concentration 400 g·L -1 The conversion rate of sucrose isomerase prepared under the following conditions was 88%-89% (Cheng Sheng's 2015 master's thesis, "Recombinant Expression, Thermostability Modification and Application Research of Serratia plymuthica Sucrose Isomerase"). Sucrose isomerase derived from Pantoea dispersa was prepared at 30℃, pH 6.0, and an enzyme dosage of 20 U / g. -1 Sucrose, sucrose concentration 400 g·L -1The conversion rate of sucrose isomerase prepared under the specified conditions was 91%, which is even higher than the conversion rate of sucrose isomerase (Master's thesis by Zhao Wenchong, 2023, "Expression of Pantoea dispersa sucrose isomerase in Bacillus subtilis and preparation of isomaltulose"). Therefore, it is of great significance to modify Pantoea dispersa sucrose isomerase to improve its thermostability, thereby reducing enzyme costs and enhancing its industrial application capabilities. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a mutant that improves the thermal stability of sucrose isomerase, making it more suitable for the industrial production of isomaltulose.

[0004] This invention provides a sucrose isomerase mutant, which is obtained by mutating the amino acids at positions 232 and / or 447 of the sucrose isomerase as shown in SEQ ID NO.1.

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

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

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

[0008] This invention provides a method for preparing the above-mentioned mutant, the specific steps of which are as follows:

[0009] I: Using the nucleotide sequence shown in SEQ ID NO.2 as a template, based on rationally designed sites, site-directed mutagenesis primers were designed, and PCR amplification was performed to obtain the gene containing the mutation site. Then, a vector containing the gene encoding the mutant was constructed.

[0010] II: Transform a gene vector containing the coding mutant into a host cell;

[0011] III: Screen and verify the recombinant strain constructed in the previous step to obtain positive clones, then culture and ferment to produce enzymes, centrifuge to collect the fermentation supernatant, and obtain crude enzyme solution containing 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 said gene.

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

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

[0016] In one embodiment, the recombinant cells use 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 thermostability of sucrose isomerase, wherein the method comprises mutating the amino acid at position 232 and / or position 447 of the sucrose isomerase as shown in SEQ ID NO.1.

[0019] The present invention also provides the use of the recombinant vector, or the recombinant cells, in the preparation of sucrose isomerase.

[0020] In one embodiment, the application uses sucrose as a substrate and reacts the sucrose isomerase at 30–45°C and pH 6.0–6.5 for at least 8–12 hours.

[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, this invention combines computer-aided design and homologous sequence alignment with site-directed mutagenesis to modify the molecular structure of sucrose isomerase, analyzes the effect of mutated residues on enzyme thermostability, and finally obtains six mutant strains with improved single-point mutation stability: WS9C-M62E, WS9C-V105I, WS9C-N109H, WS9C-D232P, WS9C-V447E and WS9C-S481M, as well as an iterative combination mutant strain WS9C-D232P / V447E with further improved thermostability.

[0024] (2) The sucrose isomerase mutant V447E provided by the present invention has a half-life of 51.3 min at 40 °C, 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 thermostability and expression level of the sucrose isomerase mutant provided by this invention are improved. The optimal temperature of V447E increased from 30℃ to 35℃, and the expression level increased from 18.89 U·mL. -1 Increased to 33.06 U·mL -1 The optimal temperature for D232P / V447E increased from 30℃ to 35℃, and the half-life at 40℃ was prolonged by 1.52-fold, with the expression level increasing from 18.89 U·mL. -1 Increased to 30.73 U·mL -1 After heat treatment at 40℃ for 20 min, the sucrose isomerase mutants V447E and D232P / V447E retained 74.82% and 78.67% of the residual enzyme activity, respectively, while the wild type retained 69.56% of the residual enzyme activity.

[0026] (4) The sucrose isomerase mutant obtained in this invention is more suitable for catalyzing the production of isomaltulose from sucrose than the wild type, and is more conducive to the flexibility of the production process. Attached Figure Description

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

[0028] Figure 2 Comparison of key amino acid sequences of sucrose isomerases from different sources.

[0029] Figure 3 The relative expression levels of wild-type and single mutant sucrose isomerase and the residual enzyme activity after incubation at 40°C and pH 6.0 for 20 min were determined.

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

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

[0032] Figure 6 Temperature stability of sucrose isomerase activity in wild-type and mutant forms.

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

[0034] Figure 8 pH stability of sucrose isomerase wild-type and mutant enzyme activities. Detailed Implementation

[0035] (I) Culture medium

[0036] LB liquid culture medium (g·L) -1 ): Peptone 10g, yeast extract 5g, NaCl 10g; LB solid medium requires the addition of 15g·L⁻¹ to LB liquid medium. -1 Agar powder.

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

[0038] (II) Testing methods:

[0039] In 1.8 mL of citrate-disodium hydrogen phosphate buffer (50 mmol·L⁻¹) -1 Add 200 μL of appropriately diluted enzyme solution to the sucrose solution (pH 6.0) to achieve a final sucrose concentration of 200 g·L⁻¹. -1 The reaction mixture was thoroughly mixed by shaking and then incubated in a 30°C water bath for 15 min. The reaction was terminated by heating in a boiling water bath for 10 min. The reaction mixture was then centrifuged at 12,000 rpm for 10 min at 40°C, and the supernatant was filtered through a 0.22 μm syringe filter. The concentration of isomaltose in the filtered solution was quantified using an AllianceiS high-performance liquid chromatography (HPLC) system (Waters) equipped with a refractive index (RI) detector and a Syncronis Amino column (4.6 mm × 25 mm × 5 mm, Thermo Scientific, USA). After injection of the sample (10 μL), elution was performed with 80% (v / v) acetonitrile aqueous solution at a flow rate of 0.8 mL / min, while maintaining the column temperature at 40°C.

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

[0041] Example 1: Preparation of sucrose isomerase mutant

[0042] (1) Construction of mutants

[0043] Proteins with amino acid sequences as shown in GenBank accessions AAP57085.1, ACF42098.1, AAP57084.1, ADJ56407.2, EKF64560.1, AAP57083.1, AGT14432.1, ABC33903.1, and AAK82938.1 were sequence aligned using the online server Multiple Sequence Alignment by CLUSTALW, and the alignment results were then 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 template was used for analysis in PyMOL software.

[0045] Based on the protein structure of sucrase isomerase, PROSS and FireProt, two computational prediction online servers used to improve protein thermostability and expression levels, were designed, and common mutation sites predicted by them were selected. Simultaneously, based on the three-dimensional structural model of sucrase isomerase, obviously unreasonable sites were eliminated, and primers were designed for the remaining candidate sites for subsequent mutagenesis. The selection criteria are as follows:

[0046] (1) The mutated residues cannot increase the disruption of hydrogen bonds or salt bridge interactions;

[0047] (2) The mutated amino acid cannot be located inside the protein to destroy the protein structure;

[0048] (3) Eliminate all sites that are too close to the catalytic active center and select sites located in the α-helical region of the TIM barrel and other structural domains around the TIM barrel.

[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 mutation sites were screened out.

[0050] The gene encoding sucrose isomerase (nucleotide sequence shown in Sequence 2) was synthesized and ligated into pET24a to obtain the recombinant plasmid pET24a-WT. Using this plasmid as a template, site-directed mutagenesis of sucrose isomerase was performed using a two-step PCR method with primers listed in Table 1. The mutagenesis 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, along with 1 μL of QuickCut Dpn I and 1 μL of Quickcut Green Buffer. The mixture was incubated at 37°C 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 then added to the competent cells, and the transformation buffer was plated onto a plate containing 25 μg / mL of Dpn I solution. -1 Kan r The mutant was placed on LB agar plates and incubated at 37°C for 10–12 h. The coding sequence of the mutant was sequenced to confirm successful mutation. 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. This plasmid was then introduced into B. subtilis WS9C (published 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 the primers in Table 1 as templates, site-directed mutagenesis was performed on WS9C-V447E using a two-step PCR method. The mutagenesis PCR system and reaction procedure, as well as the construction method of the pHY300PLK-sim iterative combination mutant plasmid, were the same as those for the single-point mutants described above. The obtained iterative combination mutant gene plasmid was transformed into B. subtilis WS9C for expression, resulting in five sucrose isomerase iterative combination mutants: WS9C-M62E / V447E, WS9C-V105I / V447E, WS9C-N109H / V447E, WS9C-D232P / V447E, and WS9C-V447E / S481M.

[0052] Table 1 Primers used in Example 1

[0053]

[0054]

[0055] Table 2 Two-step PCR reaction system

[0056]

[0057] Table 3. Reaction Procedure for the First Round of the Two-Step Method

[0058]

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

[0060] Table 4. Two-step reaction procedure for the second round.

[0061]

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

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

[0064] Select the monoclonal antibodies obtained in step (1) and inoculate them into a solution containing 25 μg·mL⁻¹ -1 Tet r 10 mL of LB liquid medium was used, with B. subtilis WS9C recombinant bacteria containing plasmid pHY300PLK-sim as a control. The cultures were incubated at 37℃ and 200 rpm for 12 h. The seed culture was then transferred at a 5% inoculum volume to a medium containing 25 μg / mL of LB liquid medium. -1 Tet r 50 mL of liquid TB fermentation medium was incubated at 37°C and 200 rpm for 2 h. The fermentation temperature was then changed to 33°C and incubated for another 48 h.

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

[0066] Example 2: Thermostability of Sucrose Isomerase Improves Mutant Screening

[0067] Preliminary screening method for thermal stability: Fermentation supernatant of wild type and mutant was prepared according to the method of Example 1. The fermentation supernatant of wild type and mutant was incubated at 40℃ for 20 min and its enzyme activity was measured. The enzyme activity of fermentation supernatant without incubation treatment was taken as 100%. Each group of experiments was performed in triplicate. The residual enzyme activity was calculated. Mutants with residual enzyme activity higher than that of wild type were selected for subsequent experiments.

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

[0069] To further improve stability, other mutants with improved thermostability were superimposed on the mutant V447E, which had the highest residual enzyme activity. Following the mutation introduction method in Example 1, double mutants (M62E / V447E, N105I / V447E, N109H / V447E, D232P / V447EP, and V447E / S481M) were obtained and cultured under the same conditions as in Example 1. Residual enzyme activity was then measured according to the method described in this example. Figure 4 As shown, with wild-type expression level as 100%, the expression levels of the double mutants M62E / V447E, N109H / V447E, D232P / V447E, and V447E / S481M were increased by 22.50%, 48.76%, 62.68%, and 85.50%, respectively. Furthermore, the expression levels of all double mutants were lower than that of V447E. The residual enzyme activity of D232P / V447E reached 78.67% compared to V447E, while the thermostability of the other combined mutants was reduced to varying degrees.

[0070] Example 3: Enzymatic properties of wild-type sucrose isomerase and mutants V447E and D232P / V447E

[0071] (1) Optimal temperature and temperature stability

[0072] Optimal temperature: Sucrose isomerase and its mutants were prepared into enzyme proteins according to the method in Example 1. After dilution by a certain factor, they were cultured for 20 min in a temperature gradient water bath (20–45 °C in 5 °C increments). The enzyme protein was then incubated in citrate-disodium hydrogen phosphate buffer (50 mmol·L⁻¹). -1 Prepare a solution with a final concentration of 200 g·L⁻¹ in pH 6.0. -1 Sucrose solutions were cultured at different temperatures, and then enzyme solutions were added. The enzyme activities of sucrose isomerase and its mutants were measured, and the optimal temperature for sucrose isomerase was determined. The highest enzyme activity was taken as 100%, and the residual enzyme activity was calculated accordingly. Results are as follows: Figure 5 As shown, the enzyme activities of WT, V447E, and D232P / V447E were measured at 20–45℃. The optimal temperature for V447E and D232P / V447E was 35℃, which is 5℃ higher than the optimal temperature for WT. Within the temperature range of 20–45℃, 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 40°C constant temperature water bath. Samples were collected periodically, and enzyme activity was measured at 30°C and pH 6.0. The highest enzyme activity was taken as 100%, and the residual enzyme activity was calculated accordingly. The time required for the initial enzyme activity to halve was defined as t. 1 / 2 The assay method involves incubating the purified enzyme at 40°C for a period of time, periodically taking equal portions of the enzyme solution and placing them on ice. Then, the residual enzyme activity and the first-order rate constant (k) are measured. d ) is ln (residual enzyme activity / incubation time), t 1 / 2 The calculation formula is t 1 / 2 =ln2 / k d The result is as follows Figure 6 As shown, the t of WT, V447E and D232P / V447E 1 / 2 The values ​​were 37.1, 51.3, and 56.4 min, respectively, and the t values ​​for the two mutants were... 1 / 2 These values ​​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, their ability to maintain enzyme activity is significantly stronger than that of the wild type.

[0074] (2) Optimal pH and pH stability

[0075] Optimal pH: Prepare a citrate-disodium hydrogen phosphate buffer solution with a pH of 4.0–8.0 and a final concentration of 200 g / L. -1 Sucrose isomerase was diluted with buffer solutions of different pH values ​​and then added to substrates of corresponding pH values. Enzyme activity was then measured at 30°C to determine the optimal pH for sucrose isomerase and its mutants. The highest enzyme activity was taken as 100%, and residual enzyme activity was calculated accordingly. Results are as follows: Figure 7 As shown, the enzyme activities of WT, V447E, and D232P / V447E were measured at pH values ​​ranging from 4.0 to 8.0. The optimal pH values ​​for WT, V447E, and D232P / V447E were 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. Between pH values ​​of 5.5 and 7.0, the enzyme activity of sucrose isomerase remained above 80%.

[0076] pH stability: To further investigate the pH stability of V447E and D232P / V447E under near-room temperature conditions, sucrose isomerase and its mutants were buffered in citrate-disodium hydrogen phosphate buffer (50 mmol / L) with a pH range of 4.0–8.0. -1 The enzyme was diluted a certain number of times and then incubated in a water bath at 25°C for 24 hours. The highest enzyme activity was taken as 100%, and the residual enzyme activity was calculated accordingly. When determining the above enzyme characteristics, the highest enzyme activity was set to 100%, and the ratio of the enzyme activity under remaining conditions to the highest enzyme activity is the residual enzyme activity. Results are as follows... Figure 8 As shown, after culturing in a buffer with a pH of 4.0 for 24 h, the enzyme activities of WT, V447E, and D232P / V447E were zero. WT showed the best stability in a buffer with a pH of 6.0, while V447E and D232P / V447E showed the best stability in a buffer with a pH of 6.5. Figure 8 WT, V447E, and D232P / V447E maintained high residual enzyme activity in buffer solutions with pH values ​​ranging from 5.5 to 8.0, indicating that these enzymes have good stability over a wide pH range.

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

[0078] The sucrose isomerase mutant V447E was prepared according to the method in Example 1, and was subjected to pH 6.0 and 400 g·L⁻¹. -1 Using sucrose as the substrate, the amount of recombinase added was 20 U·g. -1 When sucrose is converted at 30℃ for 10 hours, the yield of isomaltulose reaches 91.25%.

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

Claims

1. A sucrose isomerase mutant, characterized in that, Based on the sucrose isomerase shown in SEQ ID NO.1, it has any of the following mutations (a) to (c): (a) Mutate the aspartic acid at position 232 to proline; (b) Mutate valine at position 447 to glutamic acid; (c) Mutate aspartic acid at position 232 to proline and valine at position 447 to glutamic acid.

2. The gene encoding the sucrose isomerase mutant of claim 1.

3. A recombinant plasmid carrying the gene described in claim 2.

4. The recombinant plasmid according to claim 3, characterized in that, The plasmid is pHY300PLK.

5. A recombinant microorganism expressing the sucrose isomerase mutant of claim 1.

6. Recombinant Bacillus subtilis, characterized in that, The sucrose isomerase mutant described in claim 1 was expressed using pHY300PLK as the expression vector.

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

8. A method for preparing isomaltulose, characterized in that, Using sucrose as a substrate and the sucrose isomerase mutant described in claim 1 as a catalyst, the reaction was carried out at 30-45°C for 8-12 h.

9. The use of the sucrose isomerase mutant of claim 1, or the gene of claim 2, or the recombinant plasmid of any one of claims 3-4, or the recombinant microorganism of claim 5, or the recombinant Bacillus subtilis of claim 6, or the method of claim 8 in the preparation of isomaltulose or products containing isomaltulose.

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