An enzyme preparation for catalytic synthesis of scyllo-inositol and a preparation method of scyllo-inositol

Abstract

The present invention discloses an enzyme preparation for catalytic synthesis of scyllo-inositol and a preparation method of scyllo-inositol, belonging to the technical field of genetic engineering. The enzyme preparation has the enzyme activity of catalyzing the reaction of myo-inositol to generate scyllo-inositol, and includes inositol dehydrogenase and scyllo-inositol dehydrogenase. The amino acid sequence of the inositol dehydrogenase is shown as 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 as SEQ ID NO.7. In the present invention, specific sites of wild-type inositol dehydrogenase and wild-type scyllo-inositol dehydrogenase are respectively mutated. After the 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 present invention relates to the technical field of genetic engineering, and particularly relates to an enzyme preparation for catalyzing the synthesis of scyllo-inositol and a preparation method of scyllo-inositol. Background Art

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

[0003] Currently, the most likely industrialized path for the biosynthesis 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 the NAD + / NADH coenzyme recycling system to convert myo-inositol into scyllo-inositol. However, this method has the following disadvantages: First, the conversion rate of scyllo-inositol is relatively low, resulting in too high costs; Second, in the reaction system after the conversion is completed, in addition to containing scyllo-inositol, there are also structurally similar myo-inositol and scyllo-inositol monoketone, resulting in difficult separation of scyllo-inositol; Third, boric acid needs to be used in this preparation process, 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 catalyzing the synthesis of scyllo-inositol and a preparation method of scyllo-inositol, which are used to overcome the problems of low conversion rate of scyllo-inositol, difficult separation, and possible environmental impact caused by the use of boric acid in the preparation of scyllo-inositol in the prior art.

[0005] In the first aspect, the present invention provides an enzyme preparation for catalyzing the synthesis of scyllo-inositol. This enzyme preparation has the enzyme activity of catalyzing the reaction of myo-inositol to generate scyllo-inositol. This enzyme preparation includes inositol dehydrogenase and scyllo-inositol dehydrogenase. The amino acid sequence of inositol dehydrogenase is shown as any one of the following:

[0006] (1) The amino acid sequence shown in SEQ ID NO.3;

[0007] (2) The amino acid sequence shown in SEQ ID NO.4;

[0008] (3) The amino acid sequence shown in SEQ ID NO.5;

[0009] (4) The amino acid sequence shown in SEQ ID NO.6;

[0010] The amino acid sequence of scyllo-inositol dehydrogenase is shown as SEQ ID NO.7.

[0011] Compared with the prior art, specific sites of wild-type inositol dehydrogenase (amino acid sequence as shown in SEQ ID NO.1) and wild-type shark inositol dehydrogenase (amino acid sequence as shown in SEQ ID NO.2) are mutated respectively. After the mutation, the enzyme activity of inositol dehydrogenase is higher, and the applicable pH range of shark inositol dehydrogenase is wider.

[0012] Furthermore, the enzyme activity ratio of inositol dehydrogenase to shark inositol dehydrogenase is (10 - 16)∶(10 - 16).

[0013] The above technical solution further defines the enzyme activity ratio of inositol dehydrogenase to shark inositol dehydrogenase. Within this enzyme activity ratio range, the two enzymes cooperate with each other to further improve the conversion rate of shark inositol.

[0014] Furthermore, the method for modifying the enzyme activity of inositol dehydrogenase includes the following methods:

[0015] (1) The amino acid sequence shown in SEQ ID NO.3 is obtained by mutating the 123rd amino acid of the amino acid sequence shown in SEQ ID NO.1 from N to Q;

[0016] (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;

[0017] (3) The amino acid sequence shown in SEQ ID NO.5 is obtained by mutating the 277th amino acid of the amino acid sequence shown in SEQ ID NO.1 from F to P;

[0018] (4) The amino acid sequence shown in SEQ ID NO.6 is obtained by mutating the 123rd amino acid of the amino acid sequence shown in SEQ ID NO.1 from N to Q and simultaneously mutating the 277th amino acid from F to P.

[0019] In the above technical solution, the amino acid sequences shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 are the amino acid sequences of inositol dehydrogenase mutants N123Q, D154A, F277P, and N123Q / F277P respectively.

[0020] Preferably, the gene sequences of inositol dehydrogenase mutants N123Q, D154A, F277P, and N123Q / F277P are as shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13 respectively.

[0021] Furthermore, the method for modifying the enzyme activity of shark inositol dehydrogenase includes the following methods:

[0022] The amino acid sequence shown in SEQ ID NO.7 is obtained by mutating the 279th amino acid of the amino acid sequence shown in SEQ ID NO.2 from Q to Y.

[0023] In the above technical solution, the amino acid sequence shown in SEQ ID NO.7 is the amino acid sequence of the shark inositol dehydrogenase mutant Q279Y. Preferably, the gene sequence of the shark inositol dehydrogenase mutant Q279Y is as shown in SEQ ID NO.14.

[0024] Furthermore, the preparation of inositol dehydrogenase includes the following methods:

[0025] 1) Provide the gene encoding the above inositol dehydrogenase;

[0026] 2) Transform the host cell with the above gene;

[0027] 3) Obtain the host cell producing inositol dehydrogenase;

[0028] 4) Recover inositol dehydrogenase;

[0029] The preparation of shark inositol dehydrogenase includes the following methods:

[0030] 1) Provide the gene encoding the above shark inositol dehydrogenase;

[0031] 2) Transform the host cell with the above gene;

[0032] 3) Obtain the host cell producing shark inositol dehydrogenase;

[0033] 4) Recover shark inositol dehydrogenase.

[0034] Specifically, the following methods can be selected to prepare the inositol dehydrogenase enzyme solution or the shark inositol dehydrogenase enzyme solution:

[0035] Transfer the expression vectors containing the inositol dehydrogenase gene or the shark inositol dehydrogenase gene into the host strains respectively to obtain the recombinant strains containing the inositol dehydrogenase gene or the recombinant strains containing the shark inositol dehydrogenase gene;

[0036] Inoculate the above recombinant strains containing the inositol dehydrogenase gene or the recombinant strains containing the shark inositol dehydrogenase gene into the fermentation medium for fermentation culture. When the cell biomass grows to OD 600 of 70-90, cool down to 25-30 °C and add L-arabinose with a final concentration of 1-2 g / L to start induction culture until the cell biomass of each strain no longer increases, and obtain the fermentation broth respectively;

[0037] Centrifuge the above two fermentation broths separately, collect the bacterial cells respectively, resuspend the above two kinds of bacterial cells respectively, homogenize and break the cells, and then centrifuge to collect the supernatant, which is the inositol dehydrogenase enzyme solution or the scyllo-inositol dehydrogenase enzyme solution.

[0038] Among them, during fermentation culture, the temperature is controlled at 35-40 °C, the pH value is 6.8-7.2, and the DO value is 20%-30%.

[0039] 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.

[0040] When the recombinant strain containing the scyllo-inositol dehydrogenase gene is fermented and cultured in the present invention, a specific concentration of PQQ and ammonium chloride is added to the fermentation medium. PQQ can increase the biomass of the recombinant strain, and ammonium chloride can further increase the expression level of scyllo-inositol dehydrogenase in the recombinant strain.

[0041] In the second aspect, the present invention provides a method for preparing scyllo-inositol, which uses the above enzyme preparation to catalyze the reaction of muscle inositol to generate scyllo-inositol.

[0042] Compared with the prior art, using the enzyme preparation including specific inositol dehydrogenase and scyllo-inositol dehydrogenase of the present invention to catalyze the reaction of muscle inositol to generate scyllo-inositol can effectively improve the conversion rate of scyllo-inositol.

[0043] Furthermore, the concentrations of each component in the reaction system for catalyzing the reaction of muscle inositol to generate scyllo-inositol are as follows: muscle inositol 50-100 g / L, inositol dehydrogenase 10-16 U / mL, scyllo-inositol dehydrogenase 10-16 U / mL, dipotassium hydrogen phosphate 10-50 mM.

[0044] Furthermore, the reaction temperature of the reaction system is 35-60 °C, and the pH value is 8-10.

[0045] Furthermore, the preparation method further includes: after the reaction is completed, maintaining the reaction system at a temperature of 115-130 °C for 0.5-1 h to convert the intermediate scyllo-inositol monoketone into scyllo-inositol, and then adding the bacterial cells expressing inositol oxidase to make the concentration of the bacterial cells in the reaction system 10-50 OD 600 , converting the remaining muscle inositol in the reaction system into glucuronic acid, and separating to obtain scyllo-inositol and glucuronic acid.

[0046] In the above technical solution, the intermediate product scyllo-inosose monoketone is first converted into scyllo-inositol by high temperature to eliminate the intermediate product scyllo-inosose monoketone, and then inositol oxidase is used to convert the remaining myo-inositol in the reaction system into glucuronic acid, obtaining a mixed solution of scyllo-inositol and glucuronic acid. The scyllo-inositol and glucuronic acid in the mixed solution are separated to obtain high-purity scyllo-inositol and glucuronic acid. Compared with the prior art, the above method for preparing scyllo-inositol converts scyllo-inosose monoketone and myo-inositol, which have structures similar to scyllo-inositol, into scyllo-inositol and glucuronic acid, which is more conducive to the separation and extraction of scyllo-inositol after the reaction. In addition, the above method also eliminates the use of boric acid, making the preparation process more environmentally friendly.

[0047] Further, when converting the remaining myo-inositol in the reaction system into glucuronic acid, the reaction temperature is 35-40 °C and the pH value is 7.8-8.2. Specific embodiments

[0048] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention will be further described in detail below with reference to 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

[0049] The wild-type scyllo-inositol dehydrogenase (SID, named PT) and wild-type inositol dehydrogenase (IDH, named GT) were subjected to mutagenesis and transformation. Site prediction was carried out by AlphaFold, and saturation mutagenesis screening was carried out on the predicted key sites. The PT mutant Q279Y and the GT mutants N123Q, D154A, and F277P were obtained. Then, the GT mutants were subjected to combinatorial mutagenesis to obtain the GT mutants N123Q / D154A, D154A / F277P, N123Q / F277P, and N123Q / D154A / F277P.

[0050] Plasmids containing the above GT mutant genes or PT mutant genes were constructed as follows:

[0051] 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 the plasmids pYB1s-GT and pYB1s-PT.

[0052] N123Q mutant plasmid: Using N123QF and N123QR as primers and pYB1s-GT as a template, a mutant plasmid containing the mutant N123Q gene was obtained by PCR amplification, which was plasmid 1.

[0053] D154A mutant plasmid: Using D154AF and D154AR as primers and pYB1s-GT as a template, a mutant plasmid containing the mutant D154A gene was obtained by PCR amplification, which is plasmid 2.

[0054] F277P mutant plasmid: Using F277PF and F277PR as primers and pYB1s-GT as a template, a mutant plasmid containing the mutant F277P gene was obtained by PCR amplification, which is plasmid 3.

[0055] N123Q / D154A mutant plasmid: Using N123Q / D154AF and N123Q / D154AR as primers and plasmid 1 as a template, a mutant plasmid containing the mutant N123Q / D154A gene was obtained by PCR amplification, which is plasmid 4.

[0056] 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.

[0057] N123Q / F277P mutant plasmid: Using D154A / F277PF and D154A / F277PR as primers and plasmid 1 as a template, a mutant plasmid containing the mutant N123Q / F277P gene was obtained by PCR amplification, which is plasmid 6.

[0058] 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.

[0059] 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.

[0060] The above PCR system is shown in Table 1, and the primers used in PCR are shown in Table 2.

[0061] Table 1 PCR system (10 μL)

[0062]

[0063] PCR reaction program: Pre-denaturation: Denature at 98 °C for 3 min; Amplification cycle: Denature at 98 °C for 10 s, anneal at 55 °C for 30 s, extend at 72 °C for 4.5 min, cycle 32 times; Final extension: Extend at 72 °C for 10 min.

[0064] Table 2 Primers

[0065]

[0066] After the PCR reaction was completed, the template was digested with the restriction endonuclease DpnI. The digestion reaction system was as follows: 1 μL of 10× buffer, 8 μL of the PCR reaction product, and 1 μL of DpnI. The digestion reaction was carried out at 37 °C for 1 h.

[0067] After the digestion reaction was completed, 10 μL of the digested solution was added to the competent cells of Escherichia coli DH5α, incubated on ice for 30 min, heat-shocked in a 42 °C water bath for 90 s, and allowed to stand on ice for 5 min. It was added to 600 μL of LB liquid medium and then spread on an LB plate (containing 50 μg / mL streptomycin sulfate) until the bacterial solution was completely absorbed. The plate was inverted and cultured at 37 °C for 14 h, and then the plasmid was extracted and sent to BGI for sequencing verification. Example 2

[0068] Preparation of expression strains

[0069] 1 μL of each of the plasmids 1 - 8 in Example 1 was respectively added to the competent cells of Escherichia coli BW25113, incubated on ice for 30 min respectively, heat-shocked in a 42 °C water bath for 90 s respectively, and allowed to stand on ice for 5 min respectively. They were respectively added to 8 tubes of 600 μL of LB liquid medium and spread on 8 LB plates (containing 50 μg / mL streptomycin sulfate) respectively until the bacterial solutions were completely absorbed. The plates were inverted and cultured at 37 °C for 14 h to obtain 8 colonies. The successfully transformed strains were respectively 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, BW25113::pYB1s-PT-Q279Y, and stored in a -80 °C refrigerator for future use. Example 3

[0070] Preparation of crude enzyme solution

[0071] The 8 strains successfully transformed in Example 2 above were respectively inoculated into the fermentation medium in 8 fermenters at an inoculation amount of 5% by volume. The temperature was controlled at 37°C, the pH value was controlled at 7.0 with ammonia water with a volume percentage of 50%, the tank pressure was 0.05 Mpa, the starting rotation speed was 300 rpm, and the dissolved oxygen was 20% in series with the rotation speed, and the DO value was controlled between 20% and 30%. When the bottom sugar in the fermentation medium was exhausted, a feeding medium was added for feeding. The feeding amount was adjusted according to the dissolved oxygen, and the DO value was always controlled between 20% and 30%. When the cell biomass grew to OD 600 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 the induction culture. After the cell biomass no longer increased, the fermentation broth was taken out of the tank, and 8 kinds of fermentation broths were obtained respectively.

[0072] The 8 kinds of fermentation broths obtained above were centrifuged at 5000 rpm for 10 min respectively, the supernatant was discarded, the cells were collected respectively, and the cells were resuspended with 20 mM potassium hydrogen phosphate buffer to make the cell concentration in the resuspended solution 200 OD 600 , and the 8 resuspended solutions obtained were homogenized and crushed at a pressure of 80 MPa respectively, and then centrifuged at 10000 rpm and 4°C for 10 min respectively. The precipitate was discarded, and crude enzyme solutions containing inositol dehydrogenase mutants N123Q, D154A, F277P, N123Q / D154A, D154A / F277P, N123Q / F277P, N123Q / D154A / F277P, and a crude enzyme solution containing the shark inositol dehydrogenase mutant Q279Y were obtained respectively.

[0073] The fermentation medium in the fermenter for preparing the inositol dehydrogenase (GT) enzyme solution was: citric acid 2 g / L, potassium dihydrogen phosphate 14 g / L, potassium 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, antifoaming agent 0.1 g / L, 100×trace element I 10 g / L.

[0074] The feeding medium was: glucose 600 g / L, magnesium sulfate 2 g / L, yeast powder 10 g / L, 100×trace element II 10 mL / L.

[0075] The fermentation medium in the fermenter for preparing the shark inositol dehydrogenase (PT) enzyme solution was: citric acid 2 g / L, potassium dihydrogen phosphate 14 g / L, potassium 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, antifoaming agent 0.1 g / L, PQQ 0.02 mmol / L, ammonium chloride 1.5 g / L, 100×trace element I 10 g / L.

[0076] The feeding medium is: glucose 600 g / L, magnesium sulfate 2 g / L, yeast powder 10 g / L, 100×trace elements II 10 mL / L.

[0077] The concentrations of each component of the above 100×trace elements I are: CoCl2·6H2O 25 mg / L, MnCl2·4H2O 150 mg / L, CuCl2·2H2O 15 mg / L, H3BO3 30 mg / L, Na2MoO4·2H2O 25 mg / L, ZnSO4·7H2O 130 mg / L, ferric citrate 1 g / L.

[0078] The concentrations of each component of the above 100×trace elements II are: CoCl2·6H2O 40 mg / L, MnCl2·4H2O 24 mg / L, CuCl2·2H2O 25 mg / L, H3BO3 50 mg / L, Na2MoO4·2H2O 40 mg / L, ZnSO4·7H2O 160 mg / L, ferric citrate 0.4 g / L.

[0079] The inventors found that when preparing the inositol dehydrogenase (PT) enzyme solution, when a small amount of PQQ is contained in the fermentation medium, the biomass of the strain BW25113::pYB1s-PT-Q279Y can be increased by 12.8%. When a certain amount of PQQ and ammonium chloride are contained in the fermentation medium at the same time, the expression level of inositol dehydrogenase can be doubled. Example 4

[0080] Determination of enzyme activity

[0081] GT enzyme activity determination: The concentrations of each component in the reaction system are as follows: myo-inositol 20 mM, NAD(P) + 1 mM, crude GT enzyme solution 14 U / mL, crude wild-type PT enzyme solution 14 U / mL, PBS buffer 50 mM. React the above reaction system at pH 8.0 and a temperature of 37 °C for 8 h to obtain a conversion solution, and detect the conversion rate of inositol by high performance liquid chromatography. The detection results are shown in Table 3.

[0082] Table 3

[0083]

[0084] As can be seen from Table 3: Compared with the wild-type inositol dehydrogenase, the enzyme activities of the inositol dehydrogenase mutants N123Q, D154A, F277P, and N123Q / F277P are increased, thus increasing the conversion rate of inositol in the reaction system.

[0085] PT enzyme activity determination: The concentrations of each component in the reaction system are as follows: myo-inositol 20 mM, NAD(P)+ 1 mM, crude wild-type GT enzyme solution at 14 U / mL, crude PT enzyme solution at 14 U / mL, and 50 mM PBS buffer. The above reaction systems were reacted at different pH values and 37 °C for 8 h to obtain conversion solutions, and the conversion rate of scyllo-inositol was detected by high performance liquid chromatography. The detection results are shown in Table 4.

[0086] Table 4

[0087]

[0088] As can be seen from Table 4: Compared with the wild-type scyllo-inositol dehydrogenase, the scyllo-inositol dehydrogenase mutant Q279Y has a wider applicable pH range on the basis of ensuring that the conversion rate of scyllo-inositol does not decrease. Example 5

[0089] Preparation of scyllo-inositol

[0090] The concentrations of each component 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 hydrogen phosphate 30 mM. The pH value was adjusted with sodium hydroxide to make the pH value of the reaction system 8, heated to 40 °C, and stirring was started for conversion. When the conversion rate of scyllo-inositol reached the maximum, the conversion was ended. The reaction system was heated to 120 °C and maintained for 40 min to inactivate the enzyme at high temperature. At the same time, the intermediate scyllo-inosose was converted into scyllo-inositol, and then the cells expressing inositol oxidase were added to make the concentration of the cells expressing inositol oxidase in the reaction system reach 30 OD 600 , and the remaining myo-inositol in the reaction system was catalyzed into glucuronic acid, and the conversion was carried out at pH 8 and 37 °C for 12 h to obtain a conversion solution.

[0091] The conversion rate of scyllo-inositol was detected by high performance liquid chromatography. The detection results are shown in Table 5.

[0092] Table 5

[0093]

[0094] As can be seen from Table 5: Compared with the wild-type inositol dehydrogenase and the wild-type scyllo-inositol dehydrogenase, after using the inositol dehydrogenase mutants N123Q, D154A, F277P or N123Q / F277P, and the scyllo-inositol dehydrogenase mutant Q279Y, the conversion rate of scyllo-inositol in the reaction system was increased. Example 6

[0095] Scyllo-inositol extraction

[0096] The above-mentioned conversion solution obtained in Example 5 was filtered through a ceramic membrane with a pore size of 50 nm to remove the thalli. The obtained filtrate was passed through a 5000 Da ultrafiltration membrane at a flow rate of 8 L / h under a pressure of 4 MPa to remove the proteins. The clear liquid was collected and passed through a cation exchange resin at a flow rate of 1 BV / h to remove the cations. The effluent was then passed through an anion exchange resin at a flow rate of 1 BV / h, and the effluent was the inositol solution. The inositol solution was subjected to nanofiltration concentration through a 150 Da nanofiltration membrane at a pressure of 3 MPa until the solid content of the solution reached 13%. Then, it was thermally concentrated at 65 °C until the solid content reached 30%. 0.5 times the volume of ethanol was added, and the temperature was lowered to 20 °C at a rate of 5 °C / h for crystallization to obtain inositol. The anion exchange resin was eluted with sodium hydroxide at a flow rate of 1 BV / h, and the eluate was the sodium glucuronate solution, which can be used to prepare glucurone.

[0097] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, 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 catalytic synthesis of scyllo-inositol, characterized in that, The enzyme preparation has the enzyme activity of catalyzing the reaction of muscle inositol to generate scyllo-inositol. The enzyme preparation includes inositol dehydrogenase and scyllo-inositol dehydrogenase. The amino acid sequence of the inositol dehydrogenase is shown as 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 and the scyllo-inositol dehydrogenase is (10-16):(10-16).

3. The enzyme preparation according to claim 1, characterized in that, The preparation of the inositol dehydrogenase includes the following method: 1) Provide a gene encoding the inositol dehydrogenase described in any one of claims 1-2; 2) Transform a host cell with the gene; 3) Obtain a host cell that produces the inositol dehydrogenase; 4) Recover the inositol dehydrogenase; The preparation of the scyllo-inositol dehydrogenase includes the following method: 1) Provide a gene encoding the scyllo-inositol dehydrogenase described in any one of claims 1-2; 2) Transform a host cell with the gene; 3) Obtain a host cell that produces the scyllo-inositol dehydrogenase; 4) Recover the scyllo-inositol dehydrogenase.

4. A method for preparing shark inositol, characterized in that, Use the enzyme preparation described in any one of claims 1-3 to catalyze the reaction of muscle inositol to generate scyllo-inositol.

5. The preparation method according to claim 4, wherein The concentrations of the components in the reaction system for catalyzing the reaction of muscle inositol to generate scyllo-inositol are as follows: muscle inositol 50-100 g / L, inositol dehydrogenase 10-16 U / mL, scyllo-inositol dehydrogenase 10-16 U / mL, dipotassium hydrogen phosphate 10-50 mM.

6. The preparation method according to claim 5, wherein, The reaction temperature of the reaction system is 35-60 °C, and the pH value is 8-10.

7. The preparation method according to claim 4, characterized in that, The preparation method further includes: after the reaction is completed, maintaining the reaction system at a temperature of 115-130 °C for 0.5-1 h to convert the intermediate product scyllo-inosose into scyllo-inositol, and then adding the bacterial cells expressing inositol oxidase so that the concentration of the bacterial cells in the reaction system is 10-50 OD 600 , converting the remaining myo-inositol in the reaction system into glucuronic acid, and separating to obtain scyllo-inositol and glucuronic acid.

8. The preparation method according to claim 7, wherein, The reaction temperature for converting the remaining muscle 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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