Enzyme preparation for catalytic synthesis of D-chiro-inositol and preparation method of D-chiro-inositol

Abstract

The invention discloses an enzyme preparation for catalytic synthesis of D-chiro-inositol and a preparation method of D-chiro-inositol, and belongs to the technical field of genetic engineering. The enzyme preparation for catalytic synthesis of D-chiral inositol has enzyme activity for catalyzing the reaction of muscle inositol and NADP < + > to generate D-chiral inositol, and comprises inositol dehydrogenase and keto isomerase, the amino acid sequence of the inositol dehydrogenase is shown as any one of SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, and the amino acid sequence of the keto isomerase is shown as any one of SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8. The invention further discloses a preparation method of the enzyme preparation for catalytic synthesis of D-chiral inositol. The inositol dehydrogenase and the keto isomerase in the enzyme preparation are mutated on the basis of wild types, so that the enzyme activity of the inositol dehydrogenase and the keto isomerase is improved, and the conversion rate of D-chiral inositol is further improved.

Description

Technical Field

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

[0002] D-chiro-inositol (DCI), a stereoisomer of inositol, belongs to the cyclohexanehexol family of compounds and has important physiological functions in vivo. It participates in the insulin signaling pathway, promoting cellular glucose uptake and utilization, improving insulin resistance, and assisting in blood sugar control and metabolic disorders in patients with type 2 diabetes and polycystic ovary syndrome (PCOS).

[0003] D-chiro-inositol can be extracted from plants such as buckwheat. However, its concentration in plants is low, making its isolation and extraction costly. D-chiro-inositol can also be produced through organic synthesis, but this involves complex steps, difficult byproduct separation, and the potential for toxic residues, which can affect product quality.

[0004] The biosynthesis of D-chiro-inositol from starch involves numerous enzymatic conversion steps with low enzyme activity. If a single enzyme fails, the entire synthesis process can be limited. Alternatively, gene editing techniques can be used to knock out endogenous genes in a strain, replace related elements, and introduce genes expressing enzymes such as inositol monophosphatase to create genetically engineered strains capable of synthesizing D-chiro-inositol from glucose. However, these engineered strains often exhibit growth inhibition and exhibit low expression and activity of exogenous enzymes. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide an enzyme preparation for catalyzing the synthesis of D-chiro-inositol and a method for preparing D-chiro-inositol, thereby improving the enzymatic activities of inositol dehydrogenase and ketoisomerase and further improving the conversion rate of D-chiro-inositol.

[0006] In the first aspect, the present invention provides an enzyme preparation for catalyzing the synthesis of D-chiro-inositol, which has the function of catalyzing the synthesis of myo-inositol and NADP. + The enzyme activity of the reaction to generate D-chiro-inositol, the enzyme preparation comprising inositol dehydrogenase and ketoisomerase, the amino acid sequence of inositol dehydrogenase being any of the following: The amino acid sequence shown in SEQ ID NO.3; The amino acid sequence shown in SEQ ID NO.4; The amino acid sequence shown in SEQ ID NO.5; The amino acid sequence of ketoisomerase is shown as any one of the following: The amino acid sequence shown in SEQ ID NO.6; The amino acid sequence shown in SEQ ID NO.7; The amino acid sequence shown in SEQ ID NO.8.

[0007] Compared with the wild-type inositol dehydrogenase and wild-type ketoisomerase in the prior art, the enzyme activity of inositol dehydrogenase and ketoisomerase in the enzyme preparation of the present invention is higher. + When D-chiro-inositol is synthesized as a substrate, the conversion rate of D-chiro-inositol can be further improved.

[0008] Furthermore, the mass ratio of inositol dehydrogenase to ketoisomerase is (1.8-2.2):1.

[0009] Furthermore, the method for modifying the enzymatic activity of inositol dehydrogenase includes the following: The amino acid sequence shown in SEQ ID NO.3 is obtained by mutating the amino acid at position 236 of the amino acid sequence shown in SEQ ID NO.1 from G to A, and is an inositol dehydrogenase mutant G236A; The amino acid sequence shown in SEQ ID NO.4 is obtained by mutating the 141st amino acid of the amino acid sequence shown in SEQ ID NO.1 from T to Y, and is an inositol dehydrogenase mutant T141Y; The amino acid sequence shown in SEQ ID NO.5 is obtained by mutating the 236th amino acid of the amino acid sequence shown in SEQ ID NO.1 from G to A and the 141st amino acid from T to Y, which is the inositol dehydrogenase mutant G236A / T141Y.

[0010] Furthermore, the method for modifying the enzymatic activity of ketoisomerase includes the following: The amino acid sequence shown in SEQ ID NO.6 is obtained by mutating the 35th amino acid of the amino acid sequence shown in SEQ ID NO.2 from D to I, which is the ketoisomerase mutant D35I; The amino acid sequence shown in SEQ ID NO.7 is obtained by mutating the 139th amino acid of the amino acid sequence shown in SEQ ID NO.2 from G to R, which is the ketoisomerase mutant G139R; The amino acid sequence shown in SEQ ID NO.8 is obtained by mutating the 35th amino acid of the amino acid sequence shown in SEQ ID NO.2 from D to I and the 139th amino acid from G to R, which is the ketoisomerase mutant D35I / G139R.

[0011] Furthermore, the preparation method of the above enzyme preparation comprises the following steps: Constructing a recombinant expression vector containing both the gene of inositol dehydrogenase and the gene of ketoisomerase, transferring the recombinant expression vector into a host strain to obtain a recombinant strain, and fermenting and culturing the recombinant strain to obtain a fermentation broth containing both the inositol dehydrogenase and the ketoisomerase; The fermentation broth is centrifuged to collect the bacterial cells, the bacterial cells are resuspended and then the cells are broken, and the supernatant obtained by centrifugation is the crude enzyme solution containing both inositol dehydrogenase and ketoisomerase.

[0012] Compared with the existing technology, the recombinant expression vector constructed by the present invention contains both the inositol dehydrogenase gene and the ketoisomerase gene, avoiding the need to construct the gene fragments of different enzymes into different plasmids and then transfer them into the host strain separately, which would cause the host strain to be overburdened, growth to be inhibited, and thus affect the expression of the enzyme.

[0013] Furthermore, fermentation culture of the recombinant strain includes the following steps: Pick a single colony of the recombinant strain and place it in LB medium. Cultivate the culture medium at a stirring speed of 120 rpm to 220 rpm and a temperature of 35°C to 40°C for 12 to 16 hours to obtain a bacterial solution. Inoculate the bacterial solution into another LB medium with an inoculum volume of 1%~5% of the volume of the other LB medium. Cultivate the culture at a stirring speed of 120r / min~220r / min and a temperature of 35℃~40℃ until the bacterial OD reaches 600 When the value reaches 0.6~0.8, add IPTG inducer and continue to induce culture until OD 600 The value reaches 6~10.

[0014] Furthermore, the induction culture temperature is 16°C to 25°C, and the concentration of the IPTG inducer is 0.02 mM to 0.06 mM.

[0015] In the second aspect, the present invention provides a method for preparing D-chiro-inositol, using the above enzyme preparation to catalyze the reaction of myo-inositol and NADP + The reaction produces D-chiro-inositol.

[0016] Compared with the prior art, the enzyme preparation of the present invention includes inositol dehydrogenase and ketoisomerase, which are mutated on the basis of their respective wild types, have higher enzyme activity, and can further improve the conversion rate of the reaction product D-chiro-inositol.

[0017] Further, catalyzes the conversion of myo-inositol and NADP + Reactions to produce D-chiro-inositol include: Add myo-inositol and NADP to 50mM~120mM phosphate buffer + , and a crude enzyme solution containing both inositol dehydrogenase and ketoisomerase, reacting to produce D-chiro-inositol; In the reaction system, the concentration of myo-inositol is 10mM~25mM, NADP + The concentration is 0.5mM~1.5mM, and the total protein concentration in the crude enzyme solution is 3mg / mL~5mg / mL.

[0018] Furthermore, the reaction temperature of the reaction system is 30° C. to 37° C., and the pH value is 7.0 to 8.0. DETAILED DESCRIPTION

[0019] 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 intended to limit the present invention.

[0020] It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified.

[0021] Example 1 Construction of recombinant plasmid pET28a-IDH-KMI After codon optimization of the wild-type inositol dehydrogenase gene sequence, sequence A (shown as SEQ ID NO. 11) was synthesized. Using sequence A as a template and primers F1 and R1, PCR amplification was performed to obtain the target fragment IDH with homology arms at both ends. The PCR amplification system and conditions are shown in Table 1 and Table 2, respectively. After amplification, the target fragment IDH was recovered by agarose gel extraction to obtain a highly pure fragment.

[0022] F1:5'-cagcaaatgggtcgcggatccATGGCACTGACTGTAGGCGTTA-3' (SEQ ID NO. 21).

[0023] R1: 5'-tgcggccgcaagcttgtcgacTTAAATCGCGGCCGCTGG-3' (SEQ ID NO. 22).

[0024] Table 1

[0025] Table 2

[0026] Sequence B (SEQ ID NO. 12) was synthesized after codon optimization of the wild-type ketoisomerase gene sequence. Using sequence B as a template and primers F2 and R2, PCR amplification was performed to obtain the target fragment KMI with homology arms at both ends. The PCR amplification system and conditions are shown in Table 3 and Table 4, respectively. After amplification, the target fragment KMI was recovered by agarose gel extraction to obtain a highly pure fragment.

[0027] F2: 5'-tccgtcgacaagcttgcggccgcATGAAGACTACTCTGAACCACATGACC-3' (SEQ ID NO. 23).

[0028] R2: 5'-gtggtggtggtggtgctcgagAGCAGCACGTGCCTGCAG-3' (SEQ ID NO. 24).

[0029] Table 3

[0030] Table 4

[0031] The expression vector pET28a was double-digested with restriction endonucleases BamHI and SalI. The linearized vector was then ligated with the IDH fragment amplified by PCR using the recombinase Exnase II. The ligation temperature was 37°C for 30 minutes to obtain the recombinant plasmid pET28a-IDH. The double-digestion system is shown in Table 5, and the ligation system is shown in Table 6.

[0032] Table 5

[0033] Table 6

[0034] The recombinant plasmid pET28a-IDH was double-digested with restriction endonucleases Not Ⅰ and Xho Ⅰ, respectively. The linearized vector pET28a-IDH after enzyme digestion was ligated with the target fragment KMI obtained by the above PCR amplification using the recombinase Exnase II. The ligation temperature was 37°C and the ligation time was 30 min to obtain the recombinant plasmid pET28a-IDH-KMI.

[0035] The double enzyme digestion system is shown in Table 7, and the ligation system is shown in Table 8.

[0036] Table 7

[0037] Table 8

[0038] After the ligation was completed, the ligation product was transformed into E. coli DH5α competent cells using the chemical transformation method, and a single colony was picked for sequencing.

[0039] Example 2 Using the recombinant plasmid pET28a-IDH-KMI verified by sequencing as a template, the mutant plasmid was constructed by inverse PCR: 1. Using the recombinant plasmid pET28a-IDH-KMI as a template and F3 and R3 as primers, reverse PCR amplification was performed to obtain the mutant plasmid pET28a-IDH G236A -KMI.

[0040] 2. Using the recombinant plasmid pET28a-IDH-KMI as a template and F4 and R4 as primers, reverse PCR amplification was performed to obtain the mutant plasmid pET28a-IDH T141Y -KMI.

[0041] 3. Using the recombinant plasmid pET28a-IDH-KMI as a template and F5 and R5 as primers, reverse PCR amplification was performed to obtain the mutant plasmid pET28a-IDH D271A -KMI.

[0042] 4. Using the recombinant plasmid pET28a-IDH-KMI as a template and F6 and R6 as primers, reverse PCR amplification was performed to obtain the mutant plasmid pET28a-IDH G236A / T141Y -KMI D35I .

[0043] 5. Using the recombinant plasmid pET28a-IDH-KMI as a template and F7 and R7 as primers, reverse PCR amplification was performed to obtain the mutant plasmid pET28a-IDH G236A / T141Y -KMI G139R .

[0044] 6. Using the recombinant plasmid pET28a-IDH-KMI as a template and F8 and R8 as primers, reverse PCR amplification was performed to obtain the mutant plasmid pET28a-IDH G236A / T141Y -KMI H202R .

[0045] 7. Using the recombinant plasmid pET28a-IDH-KMI as a template and F9 and R9 as primers, reverse PCR amplification was performed to obtain the mutant plasmid pET28a-IDH G236A / T141Y -KMI.

[0046] 8. Using the recombinant plasmid pET28a-IDH-KMI as a template and F10 and R10 as primers, reverse PCR amplification was performed to obtain the mutant plasmid pET28a-IDH G236A / T141Y -KMI D35I / G139R .

[0047] The primer sequences used for the above-mentioned reverse PCR amplification are shown in Table 9.

[0048] Table 9

[0049] Among them, the mutant plasmid pET28a-IDH G236A -KMI contains both the inositol dehydrogenase mutant G236A gene (shown in SEQ ID NO. 13) and the wild-type ketoisomerase gene.

[0050] Mutant plasmid pET28a-IDH T141Y -KMI contains both the inositol dehydrogenase mutant T141Y gene (shown in SEQ ID NO. 14) and the wild-type ketoisomerase gene.

[0051] Mutant plasmid pET28a-IDH D271A -KMI contains both the inositol dehydrogenase mutant D271A gene (shown in SEQ ID NO. 15) and the wild-type ketoisomerase gene.

[0052] Mutant plasmid pET28a-IDH G236A / T141Y -KMI D35I The strain contains both the inositol dehydrogenase mutant G236A / T141Y gene (shown in SEQ ID NO.16) and the ketoisomerase mutant D35I gene (shown in SEQ ID NO.17).

[0053] Mutant plasmid pET28a-IDH G236A / T141Y -KMI G139R It also contains the inositol dehydrogenase mutant G236A / T141Y gene and the ketoisomerase mutant G139R gene (shown in SEQ ID NO. 18).

[0054] Mutant plasmid pET28a-IDH G236A / T141Y -KMI H202R It also contains the inositol dehydrogenase mutant G236A / T141Y gene and the ketoisomerase mutant H202R gene (shown in SEQ ID NO.19).

[0055] Mutant plasmid pET28a-IDH G236A / T141Y-KMI contains both the inositol dehydrogenase mutant G236A / T141Y gene and the wild-type ketoisomerase gene.

[0056] Mutant plasmid pET28a-IDH G236A / T141Y -KMI D35I / G139R It also contains the inositol dehydrogenase mutant G236A / T141Y gene and the ketoisomerase mutant D35I / G139R gene (shown in SEQ ID NO. 20).

[0057] Example 3 The recombinant plasmid pET28a-IDH-KMI constructed in Example 1 was transformed into E. coli BW25113 by electroporation to obtain the recombinant strain BW25113-IDH-KMI.

[0058] The eight mutant plasmids constructed in Example 2 were transformed into E. coli BW25113 by electroporation to obtain mutant strain 1, mutant strain 2, mutant strain 3, mutant strain 4, mutant strain 5, mutant strain 6, mutant strain 7, and mutant strain 8, respectively.

[0059] The recombinant strain BW25113-IDH-KMI and mutant strains 1 to 8 were fermented and cultured to obtain crude enzyme solution 1 containing both wild-type inositol dehydrogenase and wild-type ketoisomerase, crude enzyme solution 2 containing both inositol dehydrogenase mutant G236A and wild-type ketoisomerase, crude enzyme solution 3 containing both inositol dehydrogenase mutant T141Y and wild-type ketoisomerase, crude enzyme solution 4 containing both inositol dehydrogenase mutant D271A and wild-type ketoisomerase, crude enzyme solution 5 containing both inositol dehydrogenase mutant G236A / T141Y and wild-type ketoisomerase, and crude enzyme solution 6 containing both inositol dehydrogenase mutant D271A and wild-type ketoisomerase. The crude enzyme solution 5 contains the ketoisomerase mutant D35I, the crude enzyme solution 6 contains the inositol dehydrogenase mutant G236A / T141Y and the ketoisomerase mutant G139R, the crude enzyme solution 7 contains the inositol dehydrogenase mutant G236A / T141Y and the ketoisomerase mutant H202R, the crude enzyme solution 8 contains the inositol dehydrogenase mutant G236A / T141Y and the wild-type ketoisomerase, and the crude enzyme solution 9 contains the inositol dehydrogenase mutant G236A / T141Y and the ketoisomerase mutant D35I / G139R.

[0060] Wherein, the amino acid sequence of wild-type inositol dehydrogenase is shown in SEQ ID NO.1; The amino acid sequence of the wild-type ketoisomerase is shown in SEQ ID NO. 2; The amino acid sequence of the inositol dehydrogenase mutant G236A is shown in SEQ ID NO. 3; The amino acid sequence of the inositol dehydrogenase mutant T141Y is shown in SEQ ID NO. 4; The amino acid sequence of the inositol dehydrogenase mutant G236A / T141Y is shown in SEQ ID NO. 5; The amino acid sequence of the ketoisomerase mutant D35I is shown in SEQ ID NO.6; The amino acid sequence of the ketoisomerase mutant G139R is shown in SEQ ID NO.7; The amino acid sequence of the ketoisomerase mutant D35I / G139R is shown in SEQ ID NO.8; The amino acid sequence of the inositol dehydrogenase mutant D271A is shown in SEQ ID NO.9; The amino acid sequence of the ketoisomerase mutant H202R is shown in SEQ ID NO.10.

[0061] The fermentation culture methods of the above-mentioned recombinant strain BW25113-IDH-KMI and mutant strains 1 to 8 are the same, and the specific steps are as follows: Pick a single colony and transfer it to 10 mL of liquid LB medium. Incubate at 120 r / min and 37°C for 14 h. Inoculate the resulting bacterial suspension into another liquid LB medium at a volume of 1% of the volume of the other LB medium. Incubate at 200 r / min and 37°C until the bacterial OD reaches 0. 600 The value reached 0.6, and then IPTG inducer with a final concentration of 0.05mM was added, and the induction culture was continued at 20℃ for 14h. At this time, the OD 600 A value of 10 indicates that the fermentation broth contains both inositol dehydrogenase and ketoisomerase.

[0062] The fermentation broth was centrifuged at 4°C and 4000 rpm for 15 minutes to collect the cells. The cells were then resuspended in 100 mM phosphate buffer (pH 7.5). The resulting suspension was homogenized at 750 bar for 25 minutes using a high-pressure homogenizer to disrupt the cells. After disruption, the suspension was centrifuged at 4°C and 8000 rpm for 1 hour to obtain the supernatant, which was the crude enzyme solution.

[0063] Example 4 Myo-inositol and NADP were added to 100 mM phosphate buffer at pH 7.5. + , and the crude enzyme solution 1 in Example 3, so that the concentration of myo-inositol in the reaction system is 20mM, NADP +The concentration of 1 mM was 1 mM, and the total protein concentration in crude enzyme solution 1 was 4 mg / mL. The reaction system was reacted at 35°C for 6 hours to obtain a reaction solution. The concentration of D-chiro-inositol in the reaction solution was determined by high performance liquid chromatography, and the conversion rate was calculated. The results are shown in Table 10.

[0064] Myo-inositol and NADP were added to 100 mM phosphate buffer at pH 7.5. + , and the crude enzyme solution 2 in Example 3, so that the concentration of myo-inositol in the reaction system is 20mM, NADP + The concentration of was 1 mM, and the total protein concentration in crude enzyme solution 2 was 4 mg / mL. The reaction system was reacted at 35° C. for 6 h to obtain a reaction solution. The concentration of D-chiro-inositol in the reaction solution was determined by high performance liquid chromatography, and the conversion rate was calculated. The results are shown in Table 10.

[0065] Myo-inositol and NADP were added to 100 mM phosphate buffer at pH 7.5. + , and the crude enzyme solution 3 in Example 3, so that the concentration of myo-inositol in the reaction system is 20mM, NADP + The concentration of was 1 mM, and the total protein concentration in crude enzyme solution 3 was 4 mg / mL. The reaction system was reacted at 35° C. for 6 h to obtain a reaction solution. The concentration of D-chiro-inositol in the reaction solution was determined by high performance liquid chromatography, and the conversion rate was calculated. The results are shown in Table 10.

[0066] Myo-inositol and NADP were added to 100 mM phosphate buffer at pH 7.5. + , and the crude enzyme solution 4 in Example 3, so that the concentration of myo-inositol in the reaction system is 20mM, NADP + The concentration of was 1 mM, and the total protein concentration in crude enzyme solution 4 was 4 mg / mL. The reaction system was reacted at 35° C. for 6 h to obtain a reaction solution. The concentration of D-chiro-inositol in the reaction solution was determined by high performance liquid chromatography, and the conversion rate was calculated. The results are shown in Table 10.

[0067] Myo-inositol and NADP were added to 100 mM phosphate buffer at pH 7.5. + , and the crude enzyme solution 5 in Example 3, so that the concentration of myo-inositol in the reaction system is 20mM, NADP + The concentration of was 1 mM, and the total protein concentration in the crude enzyme solution 5 was 4 mg / mL. The reaction system was reacted at 35° C. for 6 h to obtain a reaction solution. The concentration of D-chiro-inositol in the reaction solution was determined by high performance liquid chromatography, and the conversion rate was calculated. The results are shown in Table 10.

[0068] Myo-inositol and NADP were added to 100 mM phosphate buffer at pH 7.5. + , and the crude enzyme solution 6 in Example 3, so that the concentration of myo-inositol in the reaction system is 20mM, NADP + The concentration of was 1 mM, and the total protein concentration in the crude enzyme solution 6 was 4 mg / mL. The reaction system was reacted at 35° C. for 6 h to obtain a reaction solution. The concentration of D-chiro-inositol in the reaction solution was determined by high performance liquid chromatography, and the conversion rate was calculated. The results are shown in Table 10.

[0069] Myo-inositol and NADP were added to 100 mM phosphate buffer at pH 7.5. + , and the crude enzyme solution 7 in Example 3 above, so that the concentration of myo-inositol in the reaction system is 20mM, NADP + The concentration of was 1 mM, and the total protein concentration in the crude enzyme solution 7 was 4 mg / mL. The reaction system was reacted at 35° C. for 6 h to obtain a reaction solution. The concentration of D-chiro-inositol in the reaction solution was determined by high performance liquid chromatography, and the conversion rate was calculated. The results are shown in Table 10.

[0070] Myo-inositol and NADP were added to 100 mM phosphate buffer at pH 7.5. + , and the crude enzyme solution 8 in Example 3, so that the concentration of myo-inositol in the reaction system is 20mM, NADP + The concentration of was 1 mM, and the total protein concentration in the crude enzyme solution 8 was 4 mg / mL. The reaction system was reacted at 35° C. for 6 h to obtain a reaction solution. The concentration of D-chiro-inositol in the reaction solution was determined by high performance liquid chromatography, and the conversion rate was calculated. The results are shown in Table 10.

[0071] Myo-inositol and NADP were added to 100 mM phosphate buffer at pH 7.5. + , and the crude enzyme solution 9 in Example 3, so that the concentration of myo-inositol in the reaction system is 20mM, NADP + The concentration of was 1 mM, and the total protein concentration in the crude enzyme solution 9 was 4 mg / mL. The reaction system was reacted at 35° C. for 6 h to obtain a reaction solution. The concentration of D-chiro-inositol in the reaction solution was determined by high performance liquid chromatography, and the conversion rate was calculated. The results are shown in Table 10.

[0072] Table 10

[0073] From the above results, it can be seen that compared with the crude enzyme solution 1 containing both wild-type inositol dehydrogenase and wild-type ketoisomerase, the inositol dehydrogenase mutants G236A, T141Y, G236A / T141Y, and the ketoisomerase mutants D35I, G139R, D35I / G139R of the present invention can improve the yield and conversion rate of D-chiro-inositol.

[0074] 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 aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. An enzyme preparation for catalyzing the synthesis of D-chiro-inositol, characterized in that The enzyme preparation has the function of catalyzing the conversion of myo-inositol to NADP + The enzyme activity of the reaction to generate D-chiro-inositol, the enzyme preparation comprises inositol dehydrogenase and ketoisomerase, and the amino acid sequence of the inositol dehydrogenase is shown in any one of the following: The amino acid sequence shown in SEQ ID NO.3; The amino acid sequence shown in SEQ ID NO.4; The amino acid sequence shown in SEQ ID NO.5; The amino acid sequence of the ketoisomerase is shown in any one of the following: The amino acid sequence shown in SEQ ID NO.6; The amino acid sequence shown in SEQ ID NO.7; The amino acid sequence shown in SEQ ID NO.

8.

2. The enzyme preparation according to claim 1, characterized in that The mass ratio of the inositol dehydrogenase to the ketoisomerase is (1.8-2.2):

1.

3. The enzyme preparation according to claim 1, characterized in that The method for modifying the enzymatic activity of the inositol dehydrogenase comprises the following steps: The amino acid sequence shown in SEQ ID NO.3 is obtained by mutating the amino acid at position 236 of the amino acid sequence shown in SEQ ID NO.1 from G to A; The amino acid sequence shown in SEQ ID NO.4 is obtained by mutating the 141st amino acid of the amino acid sequence shown in SEQ ID NO.1 from T to Y; The amino acid sequence shown in SEQ ID NO.5 is obtained by mutating the amino acid at position 236 from G to A and the amino acid at position 141 from T to Y 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 the ketoisomerase comprises the following: The amino acid sequence shown in SEQ ID NO.6 is obtained by mutating the amino acid at position 35 of the amino acid sequence shown in SEQ ID NO.2 from D to I; The amino acid sequence shown in SEQ ID NO.7 is obtained by mutating the 139th amino acid of the amino acid sequence shown in SEQ ID NO.2 from G to R; The amino acid sequence shown in SEQ ID NO.8 is obtained by mutating the amino acid at position 35 of the amino acid sequence shown in SEQ ID NO.2 from D to I, and simultaneously mutating the amino acid at position 139 from G to R.

5. The enzyme preparation according to claim 1, characterized in that The preparation method of the enzyme preparation comprises the following steps: Constructing a recombinant expression vector containing both the gene of the inositol dehydrogenase and the gene of the ketoisomerase, transferring the recombinant expression vector into a host strain to obtain a recombinant strain, and fermenting and culturing the recombinant strain to obtain a fermentation broth containing both the inositol dehydrogenase and the ketoisomerase; The fermentation broth is centrifuged to collect bacterial cells, which are then resuspended and then broken into cells. The supernatant obtained by centrifugation is a crude enzyme solution containing both inositol dehydrogenase and ketoisomerase.

6. The enzyme preparation according to claim 5, characterized in that The fermentation culture of the recombinant strain comprises the following steps: Pick a single colony of the recombinant strain and place it in LB medium. Cultivate it for 12 h to 16 h at a stirring speed of 120 r / min to 220 r / min and a temperature of 35°C to 40°C to obtain a bacterial solution. The bacterial solution was inoculated into another LB medium with an inoculum volume of 1% to 5% of the volume of the other LB medium, and cultured at a stirring speed of 120 r / min to 220 r / min and a temperature of 35°C to 40°C until the bacterial OD 600 When the value reaches 0.6~0.8, add IPTG inducer and continue to induce culture until OD 600 The value reaches 6~10.

7. The enzyme preparation according to claim 6, characterized in that The induction culture temperature is 16° C. to 25° C., and the concentration of the IPTG inducer is 0.02 mM to 0.06 mM.

8. A method for preparing D-chiro-inositol, characterized in that: The enzyme preparation according to any one of claims 1 to 7 is used to catalyze the conversion of myo-inositol to NADP. + The reaction produces D-chiro-inositol.

9. The preparation method according to claim 8, characterized in that The catalytic activity of myo-inositol and NADP + Reactions to produce D-chiro-inositol include: Add myo-inositol and NADP to 50mM~120mM phosphate buffer + , and a crude enzyme solution containing both inositol dehydrogenase and ketoisomerase, reacting to produce D-chiro-inositol; In the reaction system, the concentration of myo-inositol is 10mM~25mM, the NADP + The concentration of the crude enzyme is 0.5 mM to 1.5 mM, and the total protein concentration in the crude enzyme solution is 3 mg / mL to 5 mg / mL.

10. The preparation method according to claim 9, characterized in that The reaction temperature of the reaction system is 30° C. to 37° C., and the pH value is 7.0 to 8.0.

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