Preparation method of 2-keto-inositol isomerase mutant and D-chiral inositol

Abstract

The invention discloses a preparation method of a 2-keto-inositol isomerase mutant and D-chiral inositol, and belongs to the technical field of genetic engineering. The 2-keto-inositol isomerase mutant is obtained by mutating the 37th amino acid of the amino acid sequence as shown in SEQ ID NO.4 from aspartic acid to tryptophan. The invention also discloses a preparation method of the 2-keto-inositol isomerase mutant. The 2-keto-inositol isomerase mutant D37W has higher enzyme activity and higher catalytic efficiency, and when the 2-keto-inositol isomerase mutant D37W is combined with inositol dehydrogenase to catalyze muscle inositol reaction to generate D-chiral inositol, the yield of D-chiral inositol is further increased.

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 a method for preparing D-chiral inositol. Background Technology

[0002] D-Chiral Inositol is one of the nine isomers of inositol that is optically active. In nature, it is commonly found in compound form in plants such as legumes and buckwheat, as well as in insects. In the pharmaceutical and health product fields, D-chiral inositol can be used as an ingredient in diabetes management preparations and adjunctive treatment products for polycystic ovary syndrome. In the food industry, D-chiral inositol can be added to beverages and dairy products as a nutritional fortifier, and can also be formulated into dietary supplements such as capsules and tablets. In the cosmetics industry, D-chiral inositol can be used in functional skincare products due to its antioxidant properties.

[0003] The fermentation method for preparing D-chiral inositol from glucose is highly promising in terms of cost, but it faces significant challenges due to its complex pathway and heavy metabolic burden. To reduce the complexity of initial research, a simplified biocatalytic system was constructed: inositol dehydrogenase, which catalyzes this core step, was co-expressed with 2-keto-inositol isomerase, using inositol as a direct substrate to synthesize D-chiral inositol. However, the yield of D-chiral inositol is limited by the activity 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 a method for preparing D-chiral inositol, in order to overcome the problem that the enzyme activity of 2-keto-inositol isomerase is not high enough in the prior art, so as to further improve the yield of D-chiral inositol.

[0005] In a first aspect, the present invention provides a 2-keto-inositol isomerase mutant, which is obtained by mutating the 37th amino acid of the amino acid sequence shown in SEQ ID NO.4 from aspartic acid to tryptophan.

[0006] Compared with the prior art, the present invention mutates the wild-type 2-keto-inositol isomerase to obtain the 2-keto-inositol isomerase mutant D37W. The mutant D37W has higher enzyme activity, specifically higher catalytic efficiency. When the 2-keto-inositol isomerase mutant D37W and inositol dehydrogenase are combined to catalyze the reaction of muscle inositol to produce D-chiral inositol, the yield of D-chiral inositol is further increased.

[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 nucleotide sequence of the nucleic acid molecule encoding the above-mentioned 2-keto-inositol isomerase mutant is shown in SEQ ID NO. 6.

[0009] Thirdly, the present invention provides an expression vector containing the above-mentioned nucleic acid molecules.

[0010] Fourthly, the present invention provides a recombinant strain containing the above-mentioned nucleic acid molecule or the above-mentioned expression vector.

[0011] Fifthly, the present invention provides the use of the above-mentioned 2-keto-inositol isomerase mutant, the above-mentioned nucleic acid molecule, the above-mentioned expression vector or the above-mentioned recombinant strain in the preparation of D-chiral inositol.

[0012] In a sixth aspect, the present invention provides a method for preparing D-chiral inositol, using a crude enzyme solution containing both inositol dehydrogenase and a 2-keto-inositol isomerase mutant to catalyze the reaction of muscle inositol and NAD. + The reaction produces D-chiral inositol.

[0013] Compared with existing technologies, the above-mentioned crude enzyme solution contains both inositol dehydrogenase and a 2-keto-inositol isomerase mutant. This 2-keto-inositol isomerase mutant exhibits higher enzyme activity and catalytic efficiency, thereby improving its ability to catalyze the reaction of muscle inositol and NAD+. + The reaction produces D-chiral inositol, which further improves the reaction conversion rate and increases the yield of D-chiral inositol.

[0014] Furthermore, the method for preparing the crude enzyme solution containing both inositol dehydrogenase and the 2-keto-inositol isomerase mutant includes the following steps: The inositol dehydrogenase gene and the 2-keto-inositol isomerase mutant gene were simultaneously transferred into the host strain to obtain recombinant strain 1. The recombinant strain 1 was cultured in LB medium at 35℃~40℃ and 120r / min~220r / min for 12h~16h to obtain seed culture; The seed culture was inoculated into another LB medium at an inoculation rate of 1% to 5% by volume, and cultured with shaking at 35℃ to 40℃ and 120 r / min to 220 r / min until OD. 600 Once the concentration reaches 0.6–0.8, add IPTG to a final concentration of 0.1–1.0 mM for induction culture, and incubate at 16–30℃ and 120–220 r / min until OD reaches 0.6–0.8. 600 The fermentation broth containing both inositol dehydrogenase and 2-keto-inositol isomerase mutants was obtained by reaching a pH of 2.0~6.0. Centrifuge the fermentation broth, collect the cells, resuspend them, break the cells, centrifuge again, and the resulting supernatant is the crude enzyme solution containing both inositol dehydrogenase and 2-keto-inositol isomerase mutants.

[0015] Furthermore, in the crude enzyme solution containing both inositol dehydrogenase and 2-keto-inositol isomerase mutants, the concentration ratio of inositol dehydrogenase to 2-keto-inositol isomerase mutant is (0.5 mg / mL~3 mg / mL): (0.5 mg / mL~3 mg / mL).

[0016] 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 + The reaction system contains crude enzyme solution containing both inositol dehydrogenase and 2-keto-inositol isomerase mutants, metal ions, and phosphate buffer, to achieve a muscle inositol concentration of 25-35 mM and NAD+ concentration. + The concentrations of the enzyme were 1-2 mM, the total crude enzyme protein concentration was 0.25-3 mg / mL, the metal ion concentration was 0.5-2 mM, and the phosphate buffer concentration was 50-150 mM.

[0017] Furthermore, the reaction temperature is 35℃~40℃, and the pH value is 7.0~8.0; and / or, The metal ions are derived from magnesium chloride, cobalt chloride, or manganese chloride. Detailed Implementation

[0018] 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.

[0019] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.

[0020] Example 1 Based on the codon bias of Escherichia coli BL21(DE3), the encoding gene (idh gene) of inositol dehydrogenase from Geobacillus thermonitrificans was optimized to obtain the optimized idh gene (as shown in SEQ ID NO.1).

[0021] The optimized idh gene was artificially synthesized, with an EcoRI restriction site introduced at the 5' end and a SalRI restriction site introduced at the 3' end. The synthesized gene sequence was verified by sequencing and used in subsequent examples. The amino acid sequence of the inositol dehydrogenase derived from *Bacillus thermophilus* is shown in SEQ ID NO. 2.

[0022] Based on the codon bias of Escherichia coli BL21(DE3), the encoding gene (iolI gene) of wild-type 2-keto-inositol isomerase from Bacillus atrophaeus was optimized to obtain the optimized iolI gene (as shown in SEQ ID NO.3).

[0023] The optimized iolI gene was artificially synthesized, with a BglⅡ restriction site introduced at the 5' end and an XhoⅠ restriction site introduced at the 3' end. The synthesized gene sequence was verified by sequencing and used in subsequent examples. The amino acid sequence of the wild-type 2-keto-inositol isomerase from *Bacillus atrophus* is shown in SEQ ID NO. 4.

[0024] Based on wild-type 2-keto-inositol isomerase derived from Bacillus atrophus, mutations were made at relevant sites in its three-dimensional structure and catalytic mechanism. The 37th amino acid of wild-type 2-keto-inositol isomerase was mutated from aspartic acid (D) to tryptophan (W) to obtain the 2-keto-inositol isomerase mutant D37W. The amino acid sequence of mutant D37W is shown in SEQ ID NO.5.

[0025] Based on the gene sequence described in SEQ ID NO.3, the three nucleotides encoding aspartic acid at position 37 were replaced with codons encoding tryptophan, and then artificially synthesized to obtain the 2-keto-inositol isomerase mutant D37W. Gene (as shown in SEQ ID NO. 6). During artificial synthesis, this gene... A BglⅡ restriction site was introduced at the 3' end, and an XhoⅠ restriction site was introduced at the 3' end. The synthesized gene sequence was verified by sequencing and used in subsequent examples.

[0026] Example 2 Preparation of recombinant strains and mutant strains The optimized idh gene and expression vector pETDuet-1, synthesized artificially in Example 1, were double-digested using restriction endonucleases EcoRI and SalRI, respectively, at 37°C for 20 min. The digested idh gene and expression vector pETDuet-1 were then ligated using T4 DNA ligase at 16°C for 2 h. The ligation product was transformed into *E. coli* DH5α competent cells, and positive clones were screened on LB agar plates. Plasmids were extracted, and those correctly sequenced were identified as the intermediate plasmid pETDuet-idh. The double digestion system is shown in Table 1, and the ligation system is shown in Table 2.

[0027] Table 1 Table 2 The optimized iolI gene and intermediate plasmid pETDuet-idh, synthesized artificially in Example 1, were double-digested using restriction endonucleases BglII and XhoI, respectively, at 37°C for 20 min. The digested iolI gene and the digested intermediate plasmid pETDuet-idh were then ligated using T4 DNA ligase at 16°C for 2 h. The ligation product was transformed into *E. coli* DH5α competent cells, and positive clones were screened on LB agar plates. Plasmids were extracted, and those correctly sequenced were identified as the recombinant plasmid pETDuet-idh-iolI. The double digestion system is shown in Table 3, and the ligation system is shown in Table 4.

[0028] Table 3 Table 4 The artificially synthesized [product name] in Example 1 was treated with restriction endonucleases BglⅡ and XhoⅠ, respectively. The gene and the intermediate plasmid pETDuet-idh were double-digested with enzymes at 37°C for 20 min. The digested products were then... The gene and the enzyme-digested intermediate plasmid pETDuet-idh were ligated using T4 DNA ligase at 16°C for 2 hours. The ligation product was transformed into E. coli DH5α competent cells, and positive clones were screened on LB agar plates. Plasmids were extracted, and those with correct sequencing results were identified as mutant plasmids. The double enzyme digestion system is shown in Table 5, and the ligation system is shown in Table 6.

[0029] Table 5 Table 6 The above recombinant plasmid pETDuet-idh-iolI and mutant plasmid were used. Recombinant strains were obtained by chemical transformation into the expression host bacterium E. coli BL21(DE3). and mutant strains .

[0030] Among them, the recombinant strain BL21-pETDuet-idh-iolI contains the recombinant plasmid pETDuet-idh-iolI. Mutant strains Contains mutant plasmid .

[0031] Example 3 Preparation of crude enzyme solution The above recombinant strain BL21-pETDuet-idh-iolI and mutant strain Two seed cultures were obtained by culturing in LB medium at 37℃ and 120 r / min for 14 h. The two seed cultures were inoculated at a 2% (v / v) ratio into fresh LB liquid medium and cultured with shaking at 37°C and 120 rpm until... The concentration reached 0.7. IPTG was added to a final concentration of 0.5 mM for induction culture, and the culture was carried out at 25℃ and 200 r / min for 16 h. With a value of 3.0, fermentation broths containing both inositol dehydrogenase and wild-type 2-keto-inositol isomerase were obtained, as well as fermentation broths containing both inositol dehydrogenase and the 2-keto-inositol isomerase mutant D37W.

[0032] The two fermentation broths were centrifuged at 4°C and 8000 rpm for 10 minutes, and the cell pellets were collected separately. The cells were resuspended in pre-cooled 50 mM phosphate buffer (pH 7.5) to obtain two resuspensions. Both resuspensions were then sonicated on ice at a power of 300 W, with a 3-second sonication interval of 5 seconds, for a total duration of 15 minutes. The two sonicated bacterial suspensions were centrifuged at 4°C and 12000 rpm for 30 minutes, and the supernatants were collected separately to obtain crude enzyme solution 1, containing both inositol dehydrogenase and wild-type 2-keto-inositol isomerase, and crude enzyme solution 2, containing both inositol dehydrogenase and the 2-keto-inositol isomerase mutant D37W.

[0033] In crude enzyme solution 1, the concentration of crude inositol dehydrogenase was 3 mg / mL, and the concentration of crude wild-type 2-keto-inositol isomerase was 2 mg / mL. In crude enzyme solution 2, the concentration of crude inositol dehydrogenase was 3 mg / mL, and the concentration of crude 2-keto-inositol isomerase mutant D37W was 2 mg / mL.

[0034] Example 4 Preparation of D-chiral inositol Reaction system 1: Muscle inositol 30mM, NAD + A 100mM phosphate buffer solution (pH 7.5, 1.5mM manganese chloride, 1mM) was prepared, and crude enzyme solution 1 was added to bring the final concentration of total crude enzyme protein in the reaction system to 2.5 mg / mL. The reaction volume was 10 mL. The reaction system was incubated at 37°C for 90 min, followed by heating for 10 min to terminate the reaction. The concentration of D-chiral inositol in the reaction solution was determined using high-performance liquid chromatography (HPLC).

[0035] Reaction system 2: Muscle inositol 30mM, A 100mM phosphate buffer solution (pH 7.5) containing 1.5mM manganese chloride and 1mM crude enzyme solution 2 was added to bring the final concentration of total crude enzyme protein in the reaction system to 2.5 mg / mL. The reaction volume was 10 mL. The reaction system was incubated at 37°C for 90 min, followed by heating for 10 min to terminate the reaction. The concentration of D-chiral inositol in the reaction solution was determined using high-performance liquid chromatography (HPLC).

[0036] Methods for detecting D-chiral inositol: The chromatographic column was a 4.6 × 250 mm, 5 μm amino column; the mobile phase was acetonitrile: 50 mM ammonium acetate aqueous solution = 75: 25 (volume ratio); the column temperature was set at 30℃ and the flow rate was 1.0 mL / min.

[0037] Conversion rate = Amount of D-chiral inositol produced ÷ Amount of muscle inositol before reaction × 100%.

[0038] The results are shown in Table 7.

[0039] Table 7 From the above results, we can conclude that: Compared to the wild-type 2-keto-inositol isomerase, the 2-keto-inositol isomerase mutant D37W obtained by the present invention has higher enzyme activity, thereby further improving the conversion rate of the reaction and the yield of D-chiral inositol.

[0040] 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 37th amino acid of the amino acid sequence shown in SEQ ID NO.4 from aspartic acid to tryptophan.

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. 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.

6. A method for preparing D-chiral inositol, characterized in that, Using a crude enzyme solution containing both inositol dehydrogenase and 2-keto-inositol isomerase mutants, muscle inositol and NAD+ were catalyzed. + The reaction produces D-chiral inositol.

7. The preparation method according to claim 6, characterized in that, The method for preparing the crude enzyme solution containing both inositol dehydrogenase and 2-keto-inositol isomerase mutants includes the following steps: The inositol dehydrogenase gene and the 2-keto-inositol isomerase mutant gene were simultaneously transferred into the host strain to obtain recombinant strain 1. The recombinant strain 1 was cultured in LB medium at 35℃~40℃ and 120r / min~220r / min for 12h~16h to obtain seed culture; The seed culture was inoculated into another LB medium at an inoculation rate of 1% to 5% by volume, and cultured with shaking at 35℃ to 40℃ and 120 r / min to 220 r / min until OD. 600 Once the concentration reaches 0.6–0.8, add IPTG to a final concentration of 0.1–1.0 mM for induction culture, and incubate at 16–30℃ and 120–220 r / min until OD reaches 0.6–0.

8. 600 The fermentation broth containing both inositol dehydrogenase and 2-keto-inositol isomerase mutants was obtained by reaching a pH of 2.0~6.

0. Centrifuge the fermentation broth, collect the cells, resuspend them, break the cells, centrifuge again, and the resulting supernatant is the crude enzyme solution containing both inositol dehydrogenase and 2-keto-inositol isomerase mutants.

8. The preparation method according to claim 7, characterized in that, In the crude enzyme solution containing both inositol dehydrogenase and 2-keto-inositol isomerase mutants, the concentration ratio of inositol dehydrogenase to 2-keto-inositol isomerase mutant is (0.5 mg / mL~3 mg / mL): (0.5 mg / mL~3 mg / mL).

9. The preparation method according to claim 6, characterized in that, The preparation method for the reaction system that generates D-chiral inositol is as follows: Add muscle inositol and NAD to the reaction system + The reaction system contains crude enzyme solution containing both inositol dehydrogenase and 2-keto-inositol isomerase mutants, metal ions, and phosphate buffer, to achieve a muscle inositol concentration of 25-35 mM and NAD+ concentration. + The concentrations of the enzyme were 1-2 mM, the total crude enzyme protein concentration was 0.25-3 mg / mL, the metal ion concentration was 0.5-2 mM, and the phosphate buffer concentration was 50-150 mM.

10. The preparation method according to claim 9, characterized in that, The reaction temperature is 35℃~40℃, and the pH value is 7.0~8.0; and / or, The metal ions are derived from magnesium chloride, cobalt chloride, or manganese chloride.

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