2-keto-inositol isomerase mutant and application thereof
By mutating specific amino acid sites of 2-keto-inositol isomerase, a mutant with high enzyme activity and heat resistance was prepared, which solved the problems of insufficient enzyme activity and heat resistance, and improved the yield and reaction conversion rate of D-chiral inositol.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHUCHENG HAOTIAN PHARMA CO LTD
- Filing Date
- 2025-12-29
- Publication Date
- 2026-04-28
Smart Images
Figure CN121931090A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering technology, and in particular to a 2-keto-inositol isomerase mutant and its applications. Background Technology
[0002] D-chiro-inositol (DCI) is one of the nine isomers of inositol, naturally found in certain plants, buckwheat, legumes, and human cells. As a precursor to the insulin signaling pathway's "second messenger," it helps improve insulin sensitivity and has therefore been extensively studied for its role in improving ovulation, androgen levels, and glucose metabolism in patients with polycystic ovary syndrome (PCOS). Furthermore, DCI possesses potential benefits such as antioxidant activity and reduction of advanced glycation end products (AGEs), and is being explored for use in individuals with metabolic syndrome, prediabetes, and mild hyperglycemia.
[0003] In existing technologies, the reaction of muscle inositol to D-chiral inositol can be catalyzed by using inositol dehydrogenase and 2-keto-inositol isomerase in combination. However, the yield of D-chiral inositol is low due to the influence of the enzyme activity and heat resistance of 2-keto-inositol isomerase. Summary of the Invention
[0004] In view of this, the purpose of the present invention is to provide a 2-keto-inositol isomerase mutant and its application, in order to overcome the problems of low enzyme activity, poor heat resistance and short half-life of 2-keto-inositol isomerase in the prior art.
[0005] In a first aspect, the present invention provides a 2-keto-inositol isomerase mutant, wherein the 2-keto-inositol isomerase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1 through at least one of the following mutations: (1) The 63rd amino acid is mutated from L to A; (2) The amino acid at position 155 is mutated from E to R; (3) The 210th amino acid is mutated from G to R.
[0006] Compared with existing technologies, the 2-keto-inositol isomerase mutant obtained after the above mutation has higher enzyme activity, better heat resistance and longer half-life. When combined with inositol dehydrogenase to prepare D-chiral inositol, it improves the conversion rate and the yield of D-chiral inositol.
[0007] In a second aspect, the present invention provides a nucleic acid molecule that encodes the above-mentioned 2-keto-inositol isomerase mutant.
[0008] Optionally, the gene sequence encoding the 2-keto-inositol isomerase mutant L63A is shown in SEQ ID NO.3.
[0009] The gene sequence encoding the 2-keto-inositol isomerase mutant E155R is shown in SEQ ID NO.5.
[0010] The gene sequence encoding the 2-keto-inositol isomerase mutant G210R is shown in SEQ ID NO.7.
[0011] The gene sequence encoding the 2-keto-inositol isomerase mutant E155R / G210R is shown in SEQ ID NO.9.
[0012] Thirdly, the present invention provides an expression vector containing the above-mentioned nucleic acid molecules.
[0013] Fourthly, the present invention provides a recombinant strain containing the aforementioned nucleic acid molecule or expression vector.
[0014] Fifthly, the present invention provides a method for preparing a 2-keto-inositol isomerase mutant, which is used to prepare the above-mentioned 2-keto-inositol isomerase mutant, comprising the following steps: The above recombinant strains were cultured in a culture medium at 35-40℃ and 120-220rpm for 12-16h to obtain seed liquid. The seed culture was inoculated into another culture medium at an inoculum volume of 1% to 5% and cultured at 35 to 40°C and 120 to 220 rpm until OD reached. 600 The value was 0.6~0.8, then the temperature was lowered to 16~25℃ for induction culture, and the culture was induced to reach OD. 600 The value was 2.0~5.0, and a fermentation broth containing the 2-keto-inositol isomerase mutant was obtained; Centrifuge the fermentation broth, collect the bacterial resuspension, break the bacterial cells, centrifuge again, and the resulting supernatant is the crude enzyme solution of the 2-keto-inositol isomerase mutant.
[0015] In a sixth aspect, the present invention provides the application of the above-mentioned 2-keto-inositol isomerase mutant, nucleic acid molecule, expression vector or recombinant strain in the preparation of D-chiral inositol.
[0016] Compared with the prior art, the 2-keto-inositol isomerase mutant of the present invention has higher enzyme activity and thermal stability, and a longer half-life. Therefore, when combined with inositol dehydrogenase to prepare D-chiral inositol, it can effectively increase the yield of D-chiral inositol.
[0017] In a seventh aspect, the present invention provides a method for preparing D-chiral inositol, using inositol dehydrogenase and the above-mentioned 2-keto-inositol isomerase mutant to catalyze the reaction of muscle inositol and NAD. +The reaction produces D-chiral inositol.
[0018] Compared with existing technologies, the use of inositol dehydrogenase and the aforementioned 2-keto-inositol isomerase mutant catalyzes the reaction of muscle inositol and NAD. + The reaction produces D-chiral inositol, which further improves the conversion rate and the yield of D-chiral inositol.
[0019] Furthermore, the preparation method for the reaction system to generate D-chiral inositol is as follows: Add muscle inositol and NAD to the reaction system + Inositol dehydrogenase, crude enzyme solution of 2-keto-inositol isomerase mutant, metal ions, and phosphate buffer were added to make the concentration of muscle inositol in the reaction system 5-15 mg / mL, and NAD+. + The concentrations of the enzymes were 1-5 mg / mL, the concentrations of inositol dehydrogenase were 1-10 mg / mL, the concentrations of crude 2-keto-inositol isomerase mutant were 1-10 mg / mL, the concentrations of metal ions were 0.5-3 mM, and the concentrations of phosphate buffer were 50-150 mM.
[0020] Furthermore, the metal ion is a magnesium ion.
[0021] Furthermore, the reaction temperature is 30~40℃, and the pH value is 7.0~8.0. Attached Figure Description
[0022] Figure 1 The relative enzyme activity of the 2-keto-inositol isomerase mutant E155R / G210R in Example 7 at 50°C.
[0023] Figure 2 The relative enzyme activity of the 2-keto-inositol isomerase mutant E155R / G210R in Example 7 at 60°C. Detailed Implementation
[0024] To make the technical problems to be solved, the technical solutions, and the beneficial effects of 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 for explaining the present invention and are not intended to limit the present invention.
[0025] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.
[0026] Example 1 Constructing plasmid pET28a-iolI Wild-type 2-keto-inositol isomerase from *Bacillus atrophaeus* was selected, and its amino acid sequence is shown in SEQ ID NO.1. Codon optimization was performed on the gene sequence encoding this wild-type 2-keto-inositol isomerase to obtain the optimized 2-keto-inositol isomerase gene sequence (iolI gene fragment), the nucleotide sequence of which is shown in SEQ ID NO.2. The iolI gene fragment was artificially synthesized. Using the iolI gene fragment as a template, primers F1 and F2 were designed for PCR amplification to obtain the iolI target gene fragment with homologous arms. The PCR amplification reaction system is shown in Table 1, and the reaction procedure is shown in Table 2.
[0027] F1: atgggtcgcggatccgaattcATGAAACTGTGCTTCAACGAAGC (SEQ ID NO. 11); F2: ctcgagtgcggccgcaagcttTCACTGCATTTTGAAGTACTTGGAG (SEQ ID NO. 12).
[0028] Table 1 Table 2 It should be understood that the above-mentioned pre-denaturation, final extension, and storage steps do not participate in the cycle, and the entire PCR process is performed only once.
[0029] After the above PCR amplification reaction was completed, the reaction product was recovered by gel extraction to obtain the iolI target gene fragment with high purity.
[0030] The expression vector pET28a was double-digested with restriction endonucleases EcoRI and HindIII at 37°C for 10 min to obtain the linearized vector pET28a. The double digestion system is shown in Table 3.
[0031] Table 3 After double enzyme digestion, the linearized vector pET28a of the digestion product was ligated to the iolI target gene fragment obtained by PCR amplification. The ligation was carried out at 37℃ for 30 min, allowing the iolI target gene fragment to be ligated between the EcoRI and HindIII restriction sites of the expression vector pET28a, resulting in plasmid pET28a-iolI. The ligation system is shown in Table 4.
[0032] Table 4 After ligation, the ligation product was transformed into E. coli DH5α competent cells using chemical transformation. Single cells were then picked for plasmid extraction, and the extracted plasmids were sequenced for DNA.
[0033] Example 2 Constructing single-point mutant plasmids Using the plasmid pET28a-iolI as a template, site-directed mutagenesis was performed by reverse PCR to construct a mutant plasmid of the iolI gene.
[0034] Using plasmid pET28a-iolI as a template, reverse PCR was performed with L63A-F as the upstream primer and L63A-R as the downstream primer to obtain the mutant plasmid pET28a-iolI. L63A .
[0035] L63A-F: TAAACCGgccGCCTTAAACGCACTTGTTTTTCTT (SEQ ID NO. 13); L63A-R: TTAAGGCggcCGGTTTAATGTGATGGGTTTGA (SEQ ID NO. 14).
[0036] Using plasmid pET28a-iolI as a template, reverse PCR was performed with E155R-F as the upstream primer and E155R-R as the downstream primer to obtain the mutant plasmid pET28a-iolI. E155R .
[0037] E155R-F: TACGTTTcgcCAGGCATATGACATCATCAATACTGT (SEQ ID NO. 15); E155R-R:ATGCCTGgcgAAACGTATTGACTGTACATTGCGG (SEQ ID NO. 16).
[0038] Using plasmid pET28a-iolI as a template, reverse PCR was performed with G210R-F as the upstream primer and G210R-R as the downstream primer to obtain the mutant plasmid pET28a-iolI. G210R .
[0039] G210R-F: CCCGATCcgcTTCTTAACGGATGAAGATCGCG (SEQ ID NO. 17); G210R-R: TTAAGAAgcgGATCGGGAAATCTTCCGTATCG (SEQ ID NO. 18).
[0040] The reaction systems for each of the above reverse PCR methods are shown in Table 5, and the reaction procedures are shown in Table 6.
[0041] Table 5 Table 6 It should be understood that the above steps of 94℃ pre-denaturation and 4℃ storage do not participate in the cycling process, and the entire reverse PCR process is performed only once.
[0042] Template elimination: After each of the above reverse PCR reactions was completed, three reaction solutions were obtained. 2 μL of restriction endonuclease DpnⅠ was added to each of the three reaction solutions. The reaction solutions were gently pipetted to mix the enzyme and the reaction solution thoroughly. The solutions were then incubated at 37°C for 1 h to obtain three different enzyme digestion solutions.
[0043] Self-circularization of reverse PCR products: Prepare reaction solutions according to Table 7 for the three enzyme digestion solutions obtained above, mix gently, and react at 16℃ for 1 hour.
[0044] Table 7 After the reverse PCR product self-circulation was completed, the circularized product was transformed into E. coli DH5α competent cells using chemical transformation. Single cells were then picked for plasmid extraction, and the extracted plasmids were sequenced to obtain three mutant plasmids containing the 2-keto-inositol isomerase mutant gene: pET28a-iolI. L63A pET28a-iolI E155R pET28a-iolI G210R .
[0045] Mutant plasmid pET28a-iolI L63A It contains a gene encoding the 2-keto-inositol isomerase mutant L63A, the nucleotide sequence of which is shown in SEQ ID NO.3; the amino acid sequence of the 2-keto-inositol isomerase mutant L63A is shown in SEQ ID NO.4.
[0046] Mutant plasmid pET28a-iolI E155R It contains a gene encoding the 2-keto-inositol isomerase mutant E155R, the nucleotide sequence of which is shown in SEQ ID NO.5; the amino acid sequence of the 2-keto-inositol isomerase mutant E155R is shown in SEQ ID NO.6.
[0047] Mutant plasmid pET28a-iolI G210RIt contains a gene encoding the 2-keto-inositol isomerase mutant G210R, the nucleotide sequence of which is shown in SEQ ID NO.7; the amino acid sequence of the 2-keto-inositol isomerase mutant G210R is shown in SEQ ID NO.8.
[0048] Example 3 Preparation of crude enzyme solution The plasmid pET28a-iolI constructed in Example 1 and the mutant plasmid pET28a-iolI constructed in Example 2 were used. L63A pET28a-iolI E155R pET28a-iolI G210R The cells were chemically transformed into BL21(DE3) competent cells, and positive transformants were screened to obtain one recombinant strain containing the plasmid pET28a-iolI and three strains containing the mutant plasmid pET28a-iolI. L63A pET28a-iolI E155R pET28a-iolI G210R mutant strains.
[0049] The recombinant strain and three mutant strains were seed cultured separately in a culture medium, for example, liquid LB medium, at 37°C and 120 rpm for 14 h to obtain four seed cultures. The four seed cultures were then inoculated into four other culture media at a 2% (v / v) inoculum for fermentation. For example, liquid LB medium could be used again, and the cultures were cultured at 37°C and 120 rpm until OD (dose retardation). 600 The value was 0.7, then the temperature was lowered to 18℃, and induction culture was performed separately. For example, the induction culture can be performed under the following conditions: IPTG with a final concentration of 0.5mM is added for induction culture, and the culture is induced to OD. 600 With a value of 3.0, fermentation broths containing wild-type 2-keto-inositol isomerase, fermentation broths containing the 2-keto-inositol isomerase mutant L63A, fermentation broths containing the 2-keto-inositol isomerase mutant E155R, and fermentation broths containing the 2-keto-inositol isomerase mutant G210R were obtained.
[0050] The four fermentation broths obtained above were centrifuged at 4000 rpm for 15 min at 4℃, and the cell bodies were collected separately. The cell bodies were resuspended in 100 mM phosphate buffer (pH 7.5), and the cells were then disrupted by sonication at 400 W for 15 min (3 s sonication followed by a 2 s pause). After sonication, the cells were centrifuged at 4000 rpm for 15 min at 4℃, and the precipitate was discarded, yielding four supernatants, which were the four crude enzyme solutions: crude enzyme solution of wild-type 2-keto-inositol isomerase, crude enzyme solution of 2-keto-inositol isomerase mutant L63A, crude enzyme solution of 2-keto-inositol isomerase mutant E155R, and crude enzyme solution of 2-keto-inositol isomerase mutant G210R.
[0051] Example 4 Enzyme activity measurement Reaction System 1: Add muscle inositol and NAD to the reaction system. + The reaction mixture consisted of crude enzyme solution of inositol dehydrogenase and wild-type 2-keto-inositol isomerase, magnesium chloride, and phosphate buffer, making the total volume of the reaction system 10 mL. The concentration of muscle inositol in the reaction system was 20 mM, and NAD+ was also present. + The concentrations of the following were: 1.5 mM of α-aminobutyric acid (API), 0.5 mg / mL of inositol dehydrogenase, 0.5 mg / mL of crude wild-type 2-keto-inositol isomerase, 1 mM of magnesium chloride, 100 mM of phosphate buffer, and pH 7.5.
[0052] Reaction System 2: Add muscle inositol and NAD to the reaction system. + The reaction mixture consisted of inositol dehydrogenase, crude enzyme solution of 2-keto-inositol isomerase mutant L63A, magnesium chloride, and phosphate buffer, to a total volume of 10 mL. The concentration of muscle inositol in the reaction mixture was 20 mM, and NAD+ was also present. + The concentrations of the following were: 1.5 mM of iodine dehydrogenase, 0.5 mg / mL of 2-keto-inositol isomerase mutant L63A crude enzyme, 1 mM of magnesium chloride, 100 mM of phosphate buffer, and pH 7.5.
[0053] Reaction System 3: Add muscle inositol and NAD to the reaction system. + The reaction mixture consisted of inositol dehydrogenase, crude enzyme solution of 2-keto-inositol isomerase mutant E155R, magnesium chloride, and phosphate buffer, to a total volume of 10 mL. The concentration of muscle inositol in the reaction mixture was 20 mM, and NAD+ was also present. +The concentrations of the following were: 1.5 mM of iodine dehydrogenase, 0.5 mg / mL of 2-keto-inositol isomerase mutant E155R crude enzyme, 1 mM of magnesium chloride, 100 mM of phosphate buffer, and pH 7.5.
[0054] Reaction system 4: Add muscle inositol and NAD to the reaction system. + The reaction mixture consisted of inositol dehydrogenase, crude enzyme solution of 2-keto-inositol isomerase mutant G210R, magnesium chloride, and phosphate buffer, to a total volume of 10 mL. The concentration of muscle inositol in the reaction mixture was 20 mM, and NAD+ was also present. + The concentrations of the following were: 1.5 mM of iodine dehydrogenase, 0.5 mg / mL of 2-keto-inositol isomerase mutant G210R crude enzyme, 1 mM of magnesium chloride, 100 mM of phosphate buffer, and pH 7.5.
[0055] Each of the above reaction systems was reacted at 35°C for 5 min to obtain a reaction solution. The relative enzyme activity of each 2-keto-inositol isomerase mutant was calculated with the wild-type 2-keto-inositol isomerase activity as 100%. The results are shown in Table 8.
[0056] Enzyme activity is defined as the amount of enzyme required to produce 1 μM of D-chiral inositol per unit time (1 min).
[0057] Table 8 As can be seen from the above results, compared with wild-type 2-keto-inositol isomerase, the 2-keto-inositol isomerase mutants L63A, E155R, and G210R obtained by mutation in this invention have all shown further improved enzyme activity, among which mutants E155R and G210R have significantly improved enzyme activity.
[0058] Example 5 Combination mutation Based on the enzyme activity test results of Example 4 above, the mutation sites in the mutants with better effects were selected for combined mutation, and the specific process is as follows: The mutant plasmid pET28a-iolI constructed in Example 2 E155R Using the template, with primer G210R-F as the upstream primer and primer G210R-R as the downstream primer, a reverse PCR amplification reaction was performed to obtain the mutant plasmid pET28a-iolI. E155R / G210R The reaction system is shown in Table 9, and the reaction procedure is shown in Table 6.
[0059] G210R-F: CCCGATCcgcTTCTTAACGGATGAAGATCGCG (SEQ ID NO. 17); G210R-R: TTAAGAAgcgGATCGGGAAATCTTCCGTATCG (SEQ ID NO. 18).
[0060] Table 9 Template elimination: After the above reverse PCR reaction is completed, the reaction solution is obtained. 2 μL of restriction endonuclease DpnⅠ is added to the above reaction solution. The reaction solution is gently blown with a pipette to mix the enzyme and the reaction solution thoroughly. The solution is then placed at 37℃ for 1 h to obtain the enzyme digestion solution.
[0061] Reverse PCR product self-circularization: Prepare the reaction solution according to Table 7 using the above enzyme digestion solution, mix gently, and incubate at 16℃ for 1 hour.
[0062] After the reverse PCR product was self-circulated, the circularized product was transformed into E. coli DH5α competent cells using chemical transformation. Single cells were then picked for plasmid extraction. The extracted plasmids were sequenced to obtain the mutant plasmid pET28a-iolI containing the 2-keto-inositol isomerase mutant E155R / G210R gene. E155R / G210R .
[0063] Mutant plasmid pET28a-iolI E155R / G210R It contains a gene encoding the 2-keto-inositol isomerase mutant E155R / G210R, the nucleotide sequence of which is shown in SEQ ID NO.9; the amino acid sequence of the 2-keto-inositol isomerase mutant E155R / G210R is shown in SEQ ID NO.10.
[0064] Example 6 The mutant plasmid pET28a-iolI obtained in Example 5 above E155R / G210R The enzyme was transformed into BL21(DE3) competent cells according to the method in Example 3, and crude enzyme solution of 2-keto-inositol isomerase mutant E155R / G210R was prepared.
[0065] Using the method described in Example 4 above, with the enzyme activity of wild-type 2-keto-inositol isomerase as 100%, the relative enzyme activity of the 2-keto-inositol isomerase mutant E155R / G210R was calculated, and the results are shown in Table 10.
[0066] Table 10 As can be seen from the above results, the enzyme activity of the 2-keto-inositol isomerase mutant E155R / G210R obtained by combining mutation sites E155R and G210R in this invention is further improved.
[0067] Example 7 The half-life and thermostability of the 2-keto-inositol isomerase mutant E155R / G210R were determined using the following steps: 1. The crude enzyme solutions of wild-type 2-keto-inositol isomerase and the crude enzyme solution of the 2-keto-inositol isomerase mutant E155R / G210R were incubated at 50°C. Samples were taken after 0h, 3h, 6h, 9h, 12h, and 15h of incubation. The enzyme activity at 0h of incubation was taken as 100%, and the enzyme activity was tested according to the method in Example 4. The thermostability and half-life of wild-type 2-keto-inositol isomerase and the 2-keto-inositol isomerase mutant E155R / G210R at 50°C were obtained. The results are as follows: Figure 1 As shown.
[0068] 2. The crude enzyme solutions of wild-type 2-keto-inositol isomerase and the crude enzyme solution of the 2-keto-inositol isomerase mutant E155R / G210R were incubated at 60°C. Samples were taken after 0h, 3h, 6h, 9h, 12h, and 15h of incubation. The enzyme activity at 0h of incubation was taken as 100%, and the enzyme activity was tested according to the method in Example 4. The thermostability and half-life of wild-type 2-keto-inositol isomerase and the 2-keto-inositol isomerase mutant E155R / G210R at 60°C were obtained. The results are as follows: Figure 2 As shown.
[0069] Depend on Figure 1 It can be seen that, compared with the wild-type 2-keto-inositol isomerase, the 2-keto-inositol isomerase mutant E155R / G210R has higher thermostability at 50℃, and its half-life is extended from 8h to 13.5h.
[0070] Depend on Figure 2 It can be seen that, compared with the wild-type 2-keto-inositol isomerase, the 2-keto-inositol isomerase mutant E155R / G210R has higher thermostability at 60℃, and its half-life is extended from 6.5h to 10h.
[0071] Example 8 Preparation of D-chiral inositol Add muscle inositol and NAD to the reaction system + Inositol dehydrogenase, crude wild-type 2-keto-inositol isomerase, magnesium chloride, and phosphate buffer were prepared into a 10 mL reaction system. The concentration of muscle inositol in this reaction system was 10 mg / mL. +The concentrations of the enzymes were 2 mg / mL, inositol dehydrogenase 5 mg / mL, wild-type 2-keto-inositol isomerase crude enzyme 5 mg / mL, magnesium chloride 2 mM, phosphate buffer 100 mM, and pH 7.5.
[0072] Add muscle inositol and NAD to the reaction system + Inositol dehydrogenase, crude enzyme solution of 2-keto-inositol isomerase mutant E155R / G210R, magnesium chloride, and phosphate buffer were prepared into a 10 mL reaction system. The concentration of muscle inositol in this reaction system was 10 mg / mL, and NAD+ was also present. + The concentrations of the enzymes were 2 mg / mL, 5 mg / mL, 5 mg / mL, 5 mg / mL, 2 mM, 100 mM, and pH 7.5.
[0073] After reacting the two reaction systems at 35℃ for 90 min, the yield of D-chiral inositol in the reaction solution was detected by high performance liquid chromatography. The results are shown in Table 11.
[0074] Detection method: The chromatographic column is a 4.6 × 250 mm, 5 μm amino column; Mobile phase: Acetonitrile: 50 mM ammonium acetate aqueous solution = 75: 25 (volume ratio); The column temperature was set to 30℃ and the flow rate to 1.0 mL / min.
[0075] Conversion rate = Amount of D-chiral inositol produced / Initial amount of muscle inositol.
[0076] Table 11 The results above show that, compared with wild-type 2-keto-inositol isomerase, the mutant 2-keto-inositol isomerase E155R / G210R obtained by the present invention has higher enzyme activity, higher thermal stability and longer half-life. Therefore, it further improves the conversion rate and yield of D-chiral inositol in the preparation of D-chiral inositol.
[0077] The above description is merely a preferred embodiment of the present invention and is 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 make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A 2-keto-inositol isomerase mutant, characterized in that, The 2-keto-inositol isomerase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1 through at least one of the following mutations: (1) The 63rd amino acid is mutated from L to A; (2) The amino acid at position 155 is mutated from E to R; (3) The 210th amino acid is mutated from G to R.
2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the 2-keto-inositol isomerase mutant of claim 1.
3. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 2.
4. A recombinant bacterial strain, characterized in that, The recombinant strain contains the nucleic acid molecule of claim 2 or the expression vector of claim 3.
5. A method for preparing a 2-keto-inositol isomerase mutant, used to prepare the 2-keto-inositol isomerase mutant according to claim 1, characterized in that, Includes the following steps: The recombinant strain described in claim 4 was cultured in a culture medium at 35-40°C and 120-220 rpm for 12-16 h to obtain a seed solution. The seed culture was inoculated into another culture medium at an inoculum volume of 1% to 5% and cultured at 35 to 40°C and 120 to 220 rpm until OD reached. 600 The value was 0.6~0.8, then the temperature was lowered to 16~25℃ for induction culture, and the culture was induced to reach OD. 600 The value was 2.0~5.0, and a fermentation broth containing the 2-keto-inositol isomerase mutant was obtained; Centrifuge the fermentation broth, collect the bacterial resuspension, break the bacterial cells, centrifuge again, and the resulting supernatant is the crude enzyme solution of the 2-keto-inositol isomerase mutant.
6. The use of the 2-keto-inositol isomerase mutant of claim 1, the nucleic acid molecule of claim 2, the expression vector of claim 3, or the recombinant strain of claim 4 in the preparation of D-chiral inositol.
7. A method for preparing D-chiral inositol, characterized in that, Using inositol dehydrogenase and the 2-keto-inositol isomerase mutant of claim 1, catalysis of muscle inositol and NAD5 is performed. + The reaction produces D-chiral inositol.
8. The preparation method according to claim 7, characterized in that, The preparation method of the reaction system for generating D-chiral inositol is as follows: Add muscle inositol and NAD to the reaction system + Inositol dehydrogenase, crude enzyme solution of 2-keto-inositol isomerase mutant, metal ions, and phosphate buffer were added to make the concentration of muscle inositol in the reaction system 5-15 mg / mL, and NAD+. + The concentrations of the enzymes were 1-5 mg / mL, the concentrations of inositol dehydrogenase were 1-10 mg / mL, the concentrations of crude 2-keto-inositol isomerase mutant were 1-10 mg / mL, the concentrations of metal ions were 0.5-3 mM, and the concentrations of phosphate buffer were 50-150 mM.
9. The preparation method according to claim 8, characterized in that, The metal ion is a magnesium ion.
10. The preparation method according to any one of claims 7 to 9, characterized in that, The reaction temperature is 30~40℃, and the pH value is 7.0~8.0.