Enzyme preparation for catalytic synthesis of scyllo-inositol and preparation method of scyllo-inositol

Abstract

The invention discloses an enzyme preparation for catalytic synthesis of scyllo-inositol and a preparation method of scyllo-inositol, and belongs to the technical field of genetic engineering. The enzyme preparation has enzyme activity of catalyzing myo-inositol reaction to generate scyllo-inositol and comprises inositol dehydrogenase and scyllo-inositol dehydrogenase, and the amino acid sequence of the inositol dehydrogenase is shown as any one of (1) the amino acid sequence shown as SEQ ID NO.3; (2) an amino acid sequence as shown in SEQ ID NO.4; (3) an amino acid sequence as shown in SEQ ID NO.5; (4) an amino acid sequence as shown in SEQ ID NO.6; the amino acid sequence of the scyllo-inositol dehydrogenase is as shown in SEQ ID NO. 7. The specific sites of the wild type inositol dehydrogenase and the wild type scyllo-inositol dehydrogenase are mutated respectively, after mutation, the enzyme activity of the inositol dehydrogenase is higher, and the applicable pH range of the scyllo-inositol dehydrogenase is wider.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to an enzyme preparation for catalytic synthesis of scyllo-inositol and a method for preparing scyllo-inositol. Background Art

[0002] Scyllo-inositol is a cyclohexanehexol, one of the nine isomers of inositol. It has multiple biological activities. It not only has a therapeutic effect on Alzheimer's disease, but also has multiple functions such as anti-oxidation, regulating metabolism, and maintaining skin health. In addition, scyllo-inositol has also been widely used in clinical trials of behavioral and mental diseases, protein aggregation diseases, anti-cancer drug compositions, etc., and it still has huge development potential in the future.

[0003] At present, the most likely industrialized biosynthesis route of scyllo-inositol is to use myo-inositol as a substrate and utilize inositol dehydrogenase (IDH) and scyllo-inositol dehydrogenase (SID) with the help of NAD + / NADH coenzyme cycle system, converting myo-inositol into scyllo-inositol. However, this method has the following disadvantages: first, the conversion rate of scyllo-inositol is low, resulting in high cost; second, after the conversion, the reaction system contains not only scyllo-inositol, but also myo-inositol and scyllo-inositol monoketone with similar structures, which makes it difficult to separate scyllo-inositol; third, the preparation process requires the use of boric acid, which may have a certain impact on the environment. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide an enzyme preparation for catalytic synthesis of scyllo-inositol and a method for preparing scyllo-inositol, so as to overcome the problems in the prior art of low conversion rate of scyllo-inositol, difficulty in separation and possible environmental impact of using boric acid when preparing scyllo-inositol.

[0005] In a first aspect, the present invention provides an enzyme preparation for catalyzing the synthesis of scyllo-inositol, wherein the enzyme preparation has an enzyme activity for catalyzing the reaction of myo-inositol to generate scyllo-inositol, and the enzyme preparation comprises inositol dehydrogenase and scyllo-inositol dehydrogenase, and the amino acid sequence of inositol dehydrogenase is shown in any one of the following: (1) the amino acid sequence shown in SEQ ID NO.3; (2) the amino acid sequence shown in SEQ ID NO.4; (3) the amino acid sequence shown in SEQ ID NO.5; (4) the amino acid sequence shown in SEQ ID NO.6; The amino acid sequence of scyllo-inositol dehydrogenase is shown in SEQ ID NO.7.

[0006] Compared with the prior art, the present invention mutates specific sites of wild-type inositol dehydrogenase (amino acid sequence as shown in SEQ ID NO.1) and wild-type scyllo-inositol dehydrogenase (amino acid sequence as shown in SEQ ID NO.2), and after mutation, the enzyme activity of inositol dehydrogenase is higher and the applicable pH range of scyllo-inositol dehydrogenase is wider.

[0007] Furthermore, the enzyme activity ratio of inositol dehydrogenase to scyllo-inositol dehydrogenase is (10~16):(10~16).

[0008] The above technical solution further limits the enzyme activity ratio of inositol dehydrogenase and scyllo-inositol dehydrogenase. Within the enzyme activity ratio range, the two enzymes cooperate with each other to further improve the conversion rate of scyllo-inositol.

[0009] Furthermore, the method for modifying the enzymatic activity of inositol dehydrogenase includes the following method: (1) The amino acid sequence shown in SEQ ID NO.3 is obtained by mutating the amino acid at position 123 of the amino acid sequence shown in SEQ ID NO.1 from N to Q; (2) The amino acid sequence shown in SEQ ID NO.4 is obtained by mutating the 154th amino acid of the amino acid sequence shown in SEQ ID NO.1 from D to A; (3) The amino acid sequence shown in SEQ ID NO.5 is obtained by mutating the amino acid at position 277 of the amino acid sequence shown in SEQ ID NO.1 from F to P; (4) The amino acid sequence shown in SEQ ID NO.6 is obtained by mutating the amino acid at position 123 from N to Q and the amino acid at position 277 from F to P in the amino acid sequence shown in SEQ ID NO.1.

[0010] The amino acid sequences shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 in the above technical scheme are respectively the amino acid sequences of inositol dehydrogenase mutants N123Q, D154A, F277P and N123Q / F277P.

[0011] Preferably, the gene sequences of the inositol dehydrogenase mutants N123Q, D154A, F277P and N123Q / F277P are shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13 respectively.

[0012] Furthermore, the method for modifying the enzymatic activity of scyllo-inositol dehydrogenase includes the following method: The amino acid sequence shown in SEQ ID NO.7 is obtained by mutation of the 279th amino acid in the amino acid sequence shown in SEQ ID NO.2 from Q to Y.

[0013] The amino acid sequence shown in SEQ ID NO.7 in the above technical solution is the amino acid sequence of the scyllo-inositol dehydrogenase mutant Q279Y. Preferably, the gene sequence of the scyllo-inositol dehydrogenase mutant Q279Y is shown in SEQ ID NO.14.

[0014] Further, the preparation of inositol dehydrogenase comprises the following method: 1) Providing a gene encoding the above-mentioned inositol dehydrogenase; 2) transforming host cells with the above gene; 3) obtaining a host cell producing inositol dehydrogenase; 4) recovering inositol dehydrogenase; The preparation of scyllo-inositol dehydrogenase comprises the following method: 1) Providing a gene encoding the above-mentioned scyllo-inositol dehydrogenase; 2) transforming host cells with the above gene; 3) obtaining a host cell that produces scyllo-inositol dehydrogenase; 4) Recovering scyllo-inositol dehydrogenase.

[0015] Specifically, the following method can be used to prepare the inositol dehydrogenase enzyme solution or the scyllo-inositol dehydrogenase enzyme solution: The expression vectors containing the inositol dehydrogenase gene or the scyllo-inositol dehydrogenase gene are respectively transferred into the host strain to obtain the recombinant strain containing the inositol dehydrogenase gene or the recombinant strain containing the scyllo-inositol dehydrogenase gene; The above-mentioned recombinant strain containing the inositol dehydrogenase gene or the recombinant strain containing the scyllo-inositol dehydrogenase gene was inoculated into the fermentation medium for fermentation culture, and the bacterial biomass was grown to OD 600 The temperature was set at 70-90°C, then cooled to 25-30°C, and L-arabinose with a final concentration of 1-2 g / L was added to start induction culture until the biomass of the above strains stopped growing, and fermentation broths were obtained respectively; The two fermentation broths are centrifuged separately, the bacterial cells are collected separately, and the two bacterial cells are resuspended separately, the cells are homogenized and broken, and the supernatants are collected separately to obtain the inositol dehydrogenase enzyme solution or the scyllo-inositol dehydrogenase enzyme solution.

[0016] During fermentation culture, the temperature was controlled at 35~40°C, the pH value was 6.8~7.2, and the DO value was 20%~30%.

[0017] Furthermore, when preparing scyllo-inositol dehydrogenase, the fermentation medium contains PQQ and ammonium chloride; the concentration of PQQ in the fermentation medium is 0.015-0.025 mmol / L, and the concentration of ammonium chloride is 1-2 g / L.

[0018] In the present invention, when fermenting and culturing a recombinant strain containing a scyllo-inositol dehydrogenase gene, PQQ and ammonium chloride at specific concentrations are added to the fermentation medium, wherein PQQ can increase the biomass of the recombinant strain, and ammonium chloride can further increase the expression amount of the scyllo-inositol dehydrogenase by the recombinant strain.

[0019] In a second aspect, the present invention provides a method for preparing scyllo-inositol, using the above enzyme preparation to catalyze the reaction of myo-inositol to generate scyllo-inositol.

[0020] Compared with the prior art, the use of the enzyme preparation comprising specific inositol dehydrogenase and scyllo-inositol dehydrogenase of the present invention to catalyze the reaction of myo-inositol to generate scyllo-inositol can effectively improve the conversion rate of scyllo-inositol.

[0021] Furthermore, the concentrations of the components in the reaction system for catalyzing the reaction of myo-inositol to generate scyllo-inositol are as follows: myo-inositol 50-100 g / L, inositol dehydrogenase 10-16 U / mL, scyllo-inositol dehydrogenase 10-16 U / mL, and dipotassium hydrogen phosphate 10-50 mM.

[0022] Furthermore, the reaction temperature of the reaction system is 35-60° C., and the pH value is 8-10.

[0023] Furthermore, the preparation method further comprises: after the reaction is completed, the reaction system is maintained at a temperature of 115-130°C for 0.5-1h to convert the intermediate product scyllo-inositol monoketone into scyllo-inositol, and then a bacterial cell expressing inositol oxidase is added to make the concentration of the bacterial cell in the reaction system 10-50 OD 600 , the remaining myo-inositol in the reaction system is converted into glucuronic acid, and scyllo-inositol and glucuronic acid are separated.

[0024] The above technical solution first converts the intermediate product scyllo-inositol monoketone into scyllo-inositol by high temperature, eliminates the intermediate product scyllo-inositol monoketone, and then uses inositol oxidase to convert the remaining myo-inositol in the reaction system into glucuronic acid, and obtains a mixed solution of scyllo-inositol and glucuronic acid, and separates the scyllo-inositol and glucuronic acid in the mixed solution to obtain high-purity scyllo-inositol and glucuronic acid. Compared with the prior art, the above-mentioned method for preparing scyllo-inositol converts scyllo-inositol monoketone and myo-inositol, which are similar in structure to scyllo-inositol, into scyllo-inositol and glucuronic acid, which is more conducive to the separation and extraction of scyllo-inositol after the reaction is completed. In addition, the above-mentioned method also eliminates the use of boric acid, making the preparation process more environmentally friendly.

[0025] Furthermore, the reaction temperature when converting the remaining myo-inositol in the reaction system into glucuronic acid is 35-40° C. and the pH value is 7.8-8.2. DETAILED DESCRIPTION

[0026] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Example 1

[0027] The wild-type scyllo-inositol dehydrogenase (SID, named PT) and wild-type inositol dehydrogenase (IDH, named GT) were mutated and site prediction was performed using AlphaFold, and saturation mutation screening was performed on the predicted key sites. The PT mutant Q279Y and GT mutants N123Q, D154A, and F277P were screened, and then the GT mutants were combined and mutated to obtain GT mutants N123Q / D154A, D154A / F277P, N123Q / F277P, and N123Q / D154A / F277P.

[0028] The specific method for constructing a plasmid containing the above-mentioned GT mutant gene or PT mutant gene is as follows: The wild-type inositol dehydrogenase gene (SEQ ID NO.8) and the wild-type scyllo-inositol dehydrogenase gene (SEQ ID NO.9) were cloned into the vector pYB1s by restriction endonucleases BamHI / SpeI, respectively, to obtain plasmids pYB1s-GT and pYB1s-PT.

[0029] N123Q mutant plasmid: Using N123QF and N123QR as primers and pYB1s-GT as template, a mutant plasmid containing the mutant N123Q gene was obtained by PCR amplification, which is plasmid 1.

[0030] D154A mutant plasmid: Using D154AF and D154AR as primers and pYB1s-GT as template, a mutant plasmid containing the mutant D154A gene was obtained by PCR amplification, which is plasmid 2.

[0031] F277P mutant plasmid: Using F277PF and F277PR as primers and pYB1s-GT as template, a mutant plasmid containing the mutant F277P gene was obtained by PCR amplification, which is plasmid 3.

[0032] N123Q / D154A mutant plasmid: Using N123Q / D154AF and N123Q / D154AR as primers and plasmid 1 as template, a mutant plasmid containing the mutant N123Q / D154A gene was obtained by PCR amplification, which is plasmid 4.

[0033] D154A / F277P mutant plasmid: Using D154A / F277PF and D154A / F277PR as primers and plasmid 2 as a template, a mutant plasmid containing the mutant D154A / F277P gene was obtained by PCR amplification, which is plasmid 5.

[0034] N123Q / F277P mutant plasmid: Using D154A / F277PF and D154A / F277PR as primers and plasmid 1 as template, a mutant plasmid containing the mutant N123Q / F277P gene was obtained by PCR amplification, which is plasmid 6.

[0035] N123Q / D154A / F277P mutant plasmid: Using D154A / F277PF and D154A / F277PR as primers and plasmid 4 as a template, a mutant plasmid containing the mutant N123Q / D154A / F277P gene was obtained by PCR amplification, which is plasmid 7.

[0036] Q279Y mutant plasmid: Using Q279YF and Q279YR as primers and pYB1s-PT as a template, a mutant plasmid containing the mutant Q279Y gene was obtained by PCR amplification, which is plasmid 8.

[0037] The above PCR system is shown in Table 1, and the primers used in PCR are shown in Table 2.

[0038] Table 1 PCR system (10 μL)

[0039] PCR reaction program: pre-denaturation: 98°C denaturation for 3 min; amplification cycle: 98°C denaturation for 10 s, 55°C annealing for 30 s, 72°C extension for 4.5 min, 32 cycles; fill-in: 72°C extension for 10 min.

[0040] Table 2 Primers

[0041] After the PCR reaction was completed, the template was digested with restriction endonuclease DpnI. The digestion reaction system was: 1 μL of 10× buffer, 8 μL of PCR reaction product, and 1 μL of DpnI. The digestion reaction was carried out at 37°C for 1 hour.

[0042] After the digestion reaction is completed, take 10 μL of digestion solution and add it to E. coli DH5α competent cells, place on ice for 30 minutes, heat shock in 42℃ water bath for 90 seconds, and stand on ice for 5 minutes. Add it to 600 μL LB liquid culture medium, and then spread it on LB plate (containing 50 μg / mL streptomycin sulfate) until the bacterial solution is completely absorbed. Invert the plate, culture at 37℃ for 14 hours, and extract the plasmid and send it to BGI for sequencing verification. Example 2

[0043] Preparation of expression strains Take 1 μL of plasmids 1 to 8 in Example 1 and add them to E. coli BW25113 competent cells, place them on ice for 30 minutes, heat shock them in a 42°C water bath for 90 seconds, and place them on ice for 5 minutes. Add them to 8 600 μL LB liquid culture media and spread them on 8 LB plates (containing 50 μg / mL streptomycin sulfate) until the bacterial liquid is completely absorbed. Invert the plates and culture them at 37°C for 14 hours to obtain 8 colonies. The successfully transformed strains were named BW25113::pYB1s-GT-N123Q, BW25113::pYB1s-GT-D154A, BW25113::pYB1s-GT-F277P, BW25113::pYB1s-GT-N123Q / D154A, BW25113::pYB1s-GT-D154A / F277P, BW25113::pYB1s-GT-N123Q / F277P, BW25113::pYB1s-GT-N123Q / D154A / F277P, and BW25113::pYB1s-PT-Q279Y, and were stored in a -80°C refrigerator until use. Example 3

[0044] Preparation of crude enzyme solution The 8 strains successfully transformed in Example 2 were inoculated with a volume ratio of 5% into the fermentation medium in 8 fermentation tanks, the temperature was controlled at 37°C, the pH value was controlled to 7.0 with 50% volume percentage of ammonia water, the tank pressure was 0.05Mpa, the initial speed was 300rpm, the dissolved oxygen was 20% serial speed, and the DO value was controlled between 20% and 30%. When the substrate sugar in the fermentation medium was exhausted, feed medium was added for feeding, and the feed amount was adjusted according to the dissolved oxygen, and the DO value was always controlled between 20% and 30%. When the bacterial biomass grew to OD 600 After the value reached 70, the temperature was lowered to 30°C and L-arabinose with a final concentration of 2 g / L was added to start induction culture. After the biomass of the bacteria stopped increasing, the culture was placed in a tank and 8 kinds of fermentation broth were obtained.

[0045] The eight fermentation broths obtained above were centrifuged at 5000 rpm for 10 min, the supernatant was discarded, the bacterial cells were collected, and the bacterial cells were resuspended in 20 mM potassium hydrogen phosphate buffer to make the bacterial cell concentration in the resuspended liquid 200 OD 600 The 8 resuspensions obtained were homogenized under a pressure of 80 MPa to break the cells, and then centrifuged at 10000 rpm and 4°C for 10 min. The precipitates were discarded to obtain crude enzyme solutions containing inositol dehydrogenase mutants N123Q, D154A, F277P, N123Q / D154A, D154A / F277P, N123Q / F277P, N123Q / D154A / F277P, and crude enzyme solutions containing scyllo-inositol dehydrogenase mutant Q279Y, respectively.

[0046] When preparing inositol dehydrogenase (GT) enzyme solution, the fermentation medium in the fermentation tank is: citric acid 2 g / L, potassium dihydrogen phosphate 14 g / L, dipotassium hydrogen phosphate 4.5 g / L, ammonium sulfate 4 g / L, glucose 20 g / L, magnesium sulfate 0.6 g / L, yeast powder 1 g / L, defoamer 0.1 g / L, 100× trace elements - 10 g / L.

[0047] The feed medium was: glucose 600 g / L, magnesium sulfate 2 g / L, yeast powder 10 g / L, 100× trace elements II 10 mL / L.

[0048] When preparing scyllo-inositol dehydrogenase (PT) enzyme solution, the fermentation medium in the fermentation tank was: 2 g / L citric acid, 14 g / L potassium dihydrogen phosphate, 4.5 g / L potassium hydrogen phosphate, 4 g / L ammonium sulfate, 20 g / L glucose, 0.6 g / L magnesium sulfate, 1 g / L yeast powder, 0.1 g / L defoamer, 0.02 mmol / L PQQ, 1.5 g / L ammonium chloride, and 100× trace elements - 10 g / L.

[0049] The feed medium was: glucose 600 g / L, magnesium sulfate 2 g / L, yeast powder 10 g / L, 100× trace elements II 10 mL / L.

[0050] The concentrations of the components of the above 100× trace elements one are: CoCl2 6H2O 25mg / L, MnCl2 4H2O 150mg / L, CuCl2 2H2O 15mg / L, H3BO3 30mg / L, Na2MoO4 2H2O 25mg / L, ZnSO4 7H2O 130mg / L, and ferric citrate 1g / L.

[0051] The concentrations of the components of the above 100× trace elements II are: CoCl2 6H2O 40mg / L, MnCl2 4H2O 24mg / L, CuCl2 2H2O 25mg / L, H3BO3 50mg / L, Na2MoO4 2H2O 40mg / L, ZnSO4 7H2O 160mg / L, and ferric citrate 0.4g / L.

[0052] The inventors found that when preparing scyllo-inositol dehydrogenase (PT) enzyme solution, when the fermentation medium contains a small amount of PQQ, the biomass of strain BW25113::pYB1s-PT-Q279Y can be increased by 12.8%. When the fermentation medium contains a certain amount of PQQ and ammonium chloride at the same time, the expression level of scyllo-inositol dehydrogenase can be doubled. Example 4

[0053] Determination of enzyme activity GT enzyme activity determination: The concentrations of the components in the reaction system are as follows: myo-inositol 20 mM, NAD(P) + 1 mM, GT enzyme crude enzyme solution 14U / mL, wild-type PT enzyme crude enzyme solution 14U / mL, PBS buffer 50 mM. The above reaction system was reacted at pH 8.0 and temperature 37°C for 8h to obtain a conversion solution, and the conversion rate of scyllo-inositol was detected by high performance liquid chromatography. The test results are shown in Table 3.

[0054] Table 3

[0055] It can be seen from Table 3 that compared with the wild-type inositol dehydrogenase, the enzyme activities of the inositol dehydrogenase mutants N123Q, D154A, F277P, and N123Q / F277P are improved, thereby improving the conversion rate of scyllo-inositol in the reaction system.

[0056] PT enzyme activity determination: The concentrations of the components in the reaction system are as follows: myo-inositol 20 mM, NAD(P) + 1 mM, wild-type GT enzyme crude enzyme solution 14U / mL, PT enzyme crude enzyme solution 14U / mL, PBS buffer 50 mM. The above reaction systems were reacted at different pH values ​​and 37°C for 8h to obtain conversion solutions, and the conversion rate of scyllo-inositol was detected by high performance liquid chromatography. The test results are shown in Table 4.

[0057] Table 4

[0058] It can be seen from Table 4 that compared with the wild-type scyllo-inositol dehydrogenase, the scyllo-inositol dehydrogenase mutant Q279Y has a wider applicable pH range while ensuring that the conversion rate of scyllo-inositol is not reduced. Example 5

[0059] Preparation of scyllo-inositol The concentrations of the components in the conversion system are as follows: myo-inositol 75 g / L, inositol dehydrogenase enzyme solution 12 U / mL, scyllo-inositol dehydrogenase enzyme solution 14 U / mL, dipotassium phosphate 30 mM, sodium hydroxide was used to adjust the pH value so that the pH value of the reaction system was 8, and the temperature was heated to 40°C and stirred to start the conversion. When the conversion rate of scyllo-inositol reached the maximum, the conversion was completed, and the reaction system was heated to 120°C for 40 minutes to inactivate the enzyme at high temperature, and at the same time, the intermediate product scyllo-inositol monoketone was converted into scyllo-inositol, and then the bacteria expressing inositol oxidase were added to make the concentration of the bacteria expressing inositol oxidase in the reaction system reach 30 OD 600 The remaining myo-inositol in the reaction system is catalyzed into glucuronic acid, and the conversion is carried out at a pH of 8 and a temperature of 37 for 12 hours to obtain a conversion solution.

[0060] The conversion rate of scyllo-inositol was detected by high performance liquid chromatography. The detection results are shown in Table 5.

[0061] Table 5

[0062] It can be seen from Table 5 that compared with the wild-type inositol dehydrogenase and the wild-type scyllo-inositol dehydrogenase, the use of inositol dehydrogenase mutants N123Q, D154A, F277P or N123Q / F277P, and scyllo-inositol dehydrogenase mutant Q279Y increased the conversion rate of scyllo-inositol in the reaction system. Example 6

[0063] Scyllo-Inositol Extract The above conversion solution obtained in Example 5 is filtered through a ceramic membrane with a pore size of 50nm to remove the bacteria, and the obtained filtrate is passed through a 5000Da ultrafiltration membrane at a pressure of 4MPa and a flow rate of 8L / h to remove the protein, and the clear liquid is collected. The clear liquid is passed through a cation exchange resin at a flow rate of 1BV / h to remove the cations, and the effluent is passed through an anion exchange resin at a flow rate of 1BV / h, and the effluent is a scyllo-inositol solution. The scyllo-inositol solution is passed through a 150Da nanofiltration membrane at a pressure of 3MPa for nanofiltration concentration, concentrated to a solid content of 13%, and then concentrated to a solid content of 30% at 65°C by heat, 0.5 times the volume of ethanol is added, and the temperature is reduced to 20°C at a rate of 5°C / h for crystallization to obtain scyllo-inositol. The anion exchange resin is resolved with sodium hydroxide at a flow rate of 1BV / h, and the resolved liquid is a sodium glucuronide solution, which can be used to prepare glucuron lactone.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An enzyme preparation for catalyzing the synthesis of scyllo-inositol, characterized in that: The enzyme preparation has the enzyme activity of catalyzing the reaction of myo-inositol to generate scyllo-inositol, and the enzyme preparation comprises inositol dehydrogenase and scyllo-inositol dehydrogenase, and the amino acid sequence of the inositol dehydrogenase is shown in any one of the following: (1) the amino acid sequence shown in SEQ ID NO.3; (2) the amino acid sequence shown in SEQ ID NO.4; (3) the amino acid sequence shown in SEQ ID NO.5; (4) the amino acid sequence shown in SEQ ID NO.6; The amino acid sequence of the scyllo-inositol dehydrogenase is shown in SEQ ID NO.

7.

2. The enzyme preparation according to claim 1, characterized in that The enzyme activity ratio of the inositol dehydrogenase to the scyllo-inositol dehydrogenase is (10-16): (10-16).

3. The enzyme preparation according to claim 1, characterized in that The method for modifying the enzyme activity of inositol dehydrogenase comprises the following method: (1) The amino acid sequence shown in SEQ ID NO.3 is obtained by mutating the amino acid at position 123 of the amino acid sequence shown in SEQ ID NO.1 from N to Q; (2) The amino acid sequence shown in SEQ ID NO.4 is obtained by mutating the 154th amino acid of the amino acid sequence shown in SEQ ID NO.1 from D to A; (3) The amino acid sequence shown in SEQ ID NO.5 is obtained by mutating the amino acid at position 277 of the amino acid sequence shown in SEQ ID NO.1 from F to P; (4) The amino acid sequence shown in SEQ ID NO.6 is obtained by mutating the amino acid at position 123 from N to Q and the amino acid at position 277 from F to P in the amino acid sequence shown in SEQ ID NO.

1.

4. The enzyme preparation according to claim 1, characterized in that The method for modifying the enzymatic activity of scyllo-inositol dehydrogenase comprises the following method: The amino acid sequence shown in SEQ ID NO.7 is obtained by mutation of the 279th amino acid in the amino acid sequence shown in SEQ ID NO.2 from Q to Y.

5. The enzyme preparation according to claim 1, characterized in that The preparation of the inositol dehydrogenase comprises the following method: 1) Providing a gene encoding the inositol dehydrogenase according to any one of claims 1 to 4; 2) transforming a host cell with the gene; 3) obtaining a host cell producing the inositol dehydrogenase; 4) recovering the inositol dehydrogenase; The preparation of the scyllo-inositol dehydrogenase comprises the following method: 1) Providing a gene encoding the scyllo-inositol dehydrogenase according to any one of claims 1 to 4; 2) transforming a host cell with the gene; 3) obtaining a host cell producing the scyllo-inositol dehydrogenase; 4) Recovering the scyllo-inositol dehydrogenase.

6. A method for preparing scyllo-inositol, characterized in that: The enzyme preparation according to any one of claims 1 to 5 is used to catalyze the reaction of myo-inositol to produce scyllo-inositol.

7. The preparation method according to claim 6, characterized in that: The concentrations of the components in the reaction system for catalyzing the reaction of myo-inositol to generate scyllo-inositol are as follows: myo-inositol 50-100 g / L, inositol dehydrogenase 10-16 U / mL, scyllo-inositol dehydrogenase 10-16 U / mL, and dipotassium hydrogen phosphate 10-50 mM.

8. The preparation method according to claim 7, characterized in that: The reaction temperature of the reaction system is 35-60° C., and the pH value is 8-10.

9. The preparation method according to claim 6, characterized in that: The preparation method further comprises: after the reaction is completed, the reaction system is kept at a temperature of 115-130° C. for 0.5-1 hour to convert the intermediate product scyllo-inositol monoketone into scyllo-inositol, and then a bacterial cell expressing inositol oxidase is added to make the concentration of the bacterial cell in the reaction system be 10-50 OD 600 , the remaining myo-inositol in the reaction system is converted into glucuronic acid, and scyllo-inositol and glucuronic acid are separated.

10. The preparation method according to claim 9, characterized in that: The reaction temperature when converting the remaining myo-inositol in the reaction system into glucuronic acid is 35-40° C. and the pH value is 7.8-8.2.

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