A ketoisomerase mutant and a method for preparing D-chiral inositol

Abstract

This invention discloses a ketoisomerase mutant and a method for preparing D-chiral inositol, belonging to the field of genetic engineering technology. The ketoisomerase mutant is obtained by mutating the 73rd amino acid of the amino acid sequence shown in SEQ ID NO.3 from L to N. The ketoisomerase mutant L73N obtained by this invention further enhances enzyme activity. When catalyzing the reaction of muscle inositol to synthesize D-chiral inositol together with inositol dehydrogenase, it can further improve the conversion rate and the yield of D-chiral inositol.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a ketoisomerase mutant and a method for preparing D-chiral inositol. Background Technology

[0002] D-Chiral Inositol is the only one of the nine stereoisomers of inositol that exhibits optical activity. In nature, it mainly exists in its bound form in legumes, buckwheat, and some insects. In the pharmaceutical and health product fields, D-chiral inositol can be used as an adjunct to diabetes management and as a functional ingredient in products for the intervention of polycystic ovary syndrome. In the food industry, it can be added to beverages and dairy products as a nutritional fortifier, or processed into dietary supplements such as capsules and tablets. In the cosmetics field, D-chiral inositol, due to its antioxidant properties, is used in functional skincare products.

[0003] While the preparation of D-chiral inositol from glucose via microbial fermentation shows significant potential in terms of cost, it faces key challenges such as complex metabolic pathways and heavy metabolic burden on chassis cells. To reduce the complexity of initial research, a simplified biocatalytic system can be constructed: co-expressing the inositol dehydrogenase that catalyzes this core step with a ketoisomerase, using muscle inositol as a direct substrate to synthesize D-chiral inositol. However, this reaction is limited by the activity of the ketoisomerase, thus restricting the yield of D-chiral inositol. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a ketoisomerase mutant and a method for preparing D-chiral inositol.

[0005] In a first aspect, the present invention provides a ketoisomerase mutant, wherein the ketoisomerase mutant is obtained by mutating the 73rd amino acid of the amino acid sequence shown in SEQ ID NO.3 from L to N.

[0006] Compared with existing wild-type ketoisomerases, the ketoisomerase mutant L73N obtained by the present invention further improves enzyme activity. When catalyzing the reaction of muscle inositol to synthesize D-chiral inositol together with inositol dehydrogenase, it can further improve the conversion rate and the yield of D-chiral inositol.

[0007] Furthermore, the gene encoding the ketoisomerase mutant is shown in SEQ ID NO.6.

[0008] In a second aspect, the present invention provides a biomaterial, comprising any one of the following (1) to (2): (1) An expression vector containing the encoding gene of the above-mentioned ketoisomerase mutant; (2) A recombinant strain containing the coding gene of the above-mentioned ketoisomerase mutant or containing the expression vector described in (1).

[0009] Thirdly, the present invention provides the application of the above-mentioned ketoisomerase mutants or biological materials in the preparation of D-chiral inositol.

[0010] Fourthly, the present invention provides a method for preparing D-chiral inositol, which uses a crude enzyme solution containing both inositol dehydrogenase and ketoisomerase mutants to catalyze the reaction of muscle inositol to generate D-chiral inositol in the presence of a coenzyme factor.

[0011] Compared with the prior art, the crude enzyme solution of the present invention contains both inositol dehydrogenase and ketoisomerase mutants. The ketoisomerase mutant has higher enzyme activity, thus effectively improving the conversion rate and the yield of D-chiral inositol when catalyzing the reaction of muscle inositol to D-chiral inositol.

[0012] Furthermore, the method for preparing the crude enzyme solution containing both inositol dehydrogenase and ketoisomerase mutants includes: The inositol dehydrogenase gene and the ketoisomerase mutant gene were simultaneously transferred into the host strain to obtain a recombinant strain. The recombinant strain was subjected to seed culture to obtain seed solution; Inoculate the seed culture into the fermentation medium at an inoculation rate of 1%–5% by volume, and ferment until OD reaches 50%. 600 Once the OD reaches 0.6~0.8, add an inducing agent for induction culture until the OD reaches a certain level. 600 The fermentation broth containing both inositol dehydrogenase and ketoisomerase 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 ketoisomerase mutants.

[0013] Furthermore, the seed culture was carried out at 35℃~40℃ and 100r / min~250r / min for 8h~16h.

[0014] Furthermore, the fermentation temperature was 35℃~40℃, and the rotation speed was 100r / min~250r / min.

[0015] Furthermore, the induction culture temperature is 16℃~30℃, and the rotation speed is 100r / min~250r / min.

[0016] Furthermore, in the crude enzyme solution containing both inositol dehydrogenase and ketoisomerase mutants, the mass ratio of inositol dehydrogenase to ketoisomerase mutants is (0.5~3):(0.5~3).

[0017] Furthermore, the preparation method for the reaction system to generate D-chiral inositol is as follows: Add muscle inositol, coenzyme factor, crude enzyme solution containing both inositol dehydrogenase and ketoisomerase mutants, and buffer solution to the reaction system to make the concentration of muscle inositol 20-50 mM, coenzyme factor 1-2 mM, total protein of crude enzyme containing inositol dehydrogenase and ketoisomerase mutants 1-6 mg / mL, and buffer solution 50-150 mM.

[0018] Furthermore, the reaction temperature is 35℃~40℃, and the pH value is 7.0~8.0.

[0019] Furthermore, coenzyme factors include NAD. + or NADP ＋ .

[0020] It should be understood that appropriate coenzyme factors can be selected based on the different sources of inositol dehydrogenase.

[0021] Furthermore, the buffer solution includes phosphate buffer, Tris-HCl buffer, or HEPES buffer. Detailed Implementation

[0022] 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.

[0023] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.

[0024] Example 1 Inositol dehydrogenase derived from *Bacillus thermophilus* was selected, and its amino acid sequence is shown in SEQ ID NO.1. The amino acid sequence of the above inositol dehydrogenase was reverse-translated into a DNA sequence, and optimized according to the codon bias of *E. coli*, resulting in the optimized gene sequence idh (as shown in SEQ ID NO.2). The optimized gene sequence idh was artificially synthesized, with an EcoRI restriction site introduced at the 5' end and a SalRI restriction site introduced at the 3' end.

[0025] A wild-type ketoisomerase derived from *Bacillus atrophus* was selected, and its amino acid sequence is shown in SEQ ID NO.3. The amino acid sequence of the wild-type ketoisomerase was reverse-translated into a DNA sequence, and optimized according to the codon bias of *E. coli*, resulting in the optimized gene sequence iolI (as shown in SEQ ID NO.4). The optimized gene sequence iolI was artificially synthesized, with a BglⅡ restriction site introduced at the 5' end and an XhoⅠ restriction site introduced at the 3' end.

[0026] The amino acid sequence of the above-mentioned wild-type ketoisomerase was mutated by changing the 73rd amino acid from L (leucine) to N (asparagine) in the amino acid sequence shown in SEQ ID NO.3, resulting in the ketoisomerase mutant L73N, whose amino acid sequence is shown in SEQ ID NO.5. The codon encoding the 73rd amino acid L (leucine) in the above-mentioned wild-type ketoisomerase gene sequence iolI was replaced with the codon encoding N (asparagine), resulting in the gene sequence iolI of the ketoisomerase mutant L73N. L73N Its nucleotide sequence is shown in SEQ ID NO.6. The artificially synthesized gene sequence iolI L73N During synthesis, a BglⅡ restriction site is introduced at the 5' end of the gene and an XhoⅠ restriction site is introduced at the 3' end.

[0027] Example 2 Preparation of recombinant strains and mutant strains The artificially synthesized gene sequence idh and expression vector pETDuet-1 from Example 1 were double-digested using restriction endonucleases EcoRI and SalRI, respectively, at 37°C for 20 min, to obtain the digested gene sequence idh and linearized vector pETDuet-1. The double digestion system is shown in Table 1.

[0028] Table 1 The enzyme-digested gene sequence idh and the linearized vector pETDuet-1 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 plates. The plasmid was extracted and sequenced to obtain the intermediate plasmid pETDuet-idh. The ligation system is shown in Table 2.

[0029] Table 2 Components Volume (μL) Linearized carrier pETDuet-1 4.5 Gene sequence idh after enzyme digestion 1.5 T4 DNA ligase buffer (10×) 2 T4 DNA ligase 1 <![CDATA[ddH2O]]> 11 The artificially synthesized gene sequence iolI and the intermediate plasmid pETDuet-idh from Example 1 were double-digested using restriction endonucleases BglⅡ and XhoⅠ, respectively, at 37℃ for 20 min, to obtain the digested gene sequence iolI and the linearized plasmid pETDuet-idh. The double digestion system is shown in Table 3.

[0030] Table 3 The enzyme-digested gene sequence iolI and the linearized 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 plates. The plasmid was extracted and sequenced to obtain the recombinant plasmid pETDuet-idh-iolI. The ligation system is shown in Table 4.

[0031] Table 4 Components Volume (μL) Linearized plasmid pETDuet-idh 4.5 Gene sequence after enzyme digestion: iolI 1.5 T4 DNA ligase buffer (10×) 2 T4 DNA ligase 1 <![CDATA[ddH2O]]> 11 The artificially synthesized gene sequence iolI from Example 1 was processed using restriction endonucleases BglⅡ and XhoⅠ. L73N The intermediate plasmid pETDuet-idh was double-digested with enzymes at 37°C for 20 min to obtain the digested gene sequence iolI. L73N And linearized plasmid pETDuet-idh. The double digestion system is shown in Table 5.

[0032] Table 5 Components Volume (μL) <![CDATA[Gene sequence iolI L73N or intermediate plasmid pETDuet-idh]]> 25 10×Buffer 5 Restriction endonuclease BglII 2 Restriction endonuclease XhoⅠ 2 <![CDATA[ddH2O]]> 16 The above-mentioned enzyme-digested gene sequence iolI L73N The linearized plasmid pETDuet-idh was 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. The plasmid was extracted and sequenced to obtain the mutant plasmid pETDuet-idh-iolI. L73N The connection system is shown in Table 6.

[0033] Table 6 Components Volume (μL) Linearized plasmid pETDuet-idh 4.5 <![CDATA[The gene sequence iolI after enzymatic digestion L73N > 1.5 T4 DNA ligase buffer (10×) 2 T4 DNA ligase 1 <![CDATA[ddH2O]]> 11 The above recombinant plasmid pETDuet-idh-iolI and mutant plasmid pETDuet-idh-iolI were used. L73N The recombinant strain BL21-pETDuet-idh-iolI and the mutant strain BL21-pETDuet-idh-iolI were obtained by chemical transformation into the host strain E. coli BL21(DE3). L73N .

[0034] Example 3 Preparation of crude enzyme solution The above recombinant strain BL21-pETDuet-idh-iolI and mutant strain BL21-pETDuet-idh-iolI were used. L73NThe culture was inoculated into two LB liquid media (containing 50 μg / mL ampicillin) and cultured at 37℃ and 120 r / min for 14 h, respectively, to obtain seed culture of recombinant strain BL21-pETDuet-idh-iolI and mutant strain BL21-pETDuet-idh-iolI, respectively. L73N Seed liquid.

[0035] Seed culture of the above recombinant strain BL21-pETDuet-idh-iolI and mutant strain BL21-pETDuet-idh-iolI were compared. L73N The seed culture was inoculated into two separate LB liquid media (containing 50 μg / mL ampicillin) at a 2% (v / v) inoculation ratio, and cultured at 37°C and 120 rpm with shaking until OD. 600 Once the concentration reached 0.7, IPTG was added to a final concentration of 0.5 mM for induction culture, and the culture was induced to OD at 25℃ and 200 r / min. 600 With a value of 3.0, fermentation broths containing both inositol dehydrogenase and wild-type ketoisomerase were obtained, as well as fermentation broths containing both inositol dehydrogenase and the ketoisomerase mutant L73N.

[0036] 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-chilled 50 mM phosphate buffer (pH 7.5), and the cells were 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. After sonication, the cells were centrifuged at 4°C and 12000 rpm for 30 minutes, and the supernatants were collected to obtain crude enzyme solution 1, containing both inositol dehydrogenase and wild-type ketoisomerase, and crude enzyme solution 2, containing both inositol dehydrogenase and the ketoisomerase mutant L73N.

[0037] Example 4 Preparation of D-chiral inositol Reaction System 1: Add muscle inositol and NAD to the reaction system. + A crude enzyme solution containing both inositol dehydrogenase and wild-type ketoisomerase, along with phosphate buffer at pH 7.5, was used to prepare a 10 mL reaction system. The concentration of muscle inositol in the reaction system was 30 mM, and NAD+ was also present. + The concentration of the enzyme was 1.5 mM, the concentration of crude enzyme total protein of inositol dehydrogenase and wild-type ketoisomerase was 2.5 mg / mL, and the concentration of phosphate buffer was 100 mM.

[0038] Reaction System 2: Add muscle inositol and NAD to the reaction system.+ 1. A crude enzyme solution containing both inositol dehydrogenase and the ketoisomerase mutant L73N, and a phosphate buffer solution with a pH of 7.5 were used to prepare a 10 mL reaction system. The concentration of muscle inositol in the reaction system was 30 mM, and NAD+ was... + The concentration of the enzyme was 1.5 mM, the concentration of crude enzyme total protein of inositol dehydrogenase and ketoisomerase mutant L73N was 2.5 mg / mL, and the concentration of phosphate buffer was 100 mM.

[0039] The two reaction systems were reacted at 37℃ for 90 min, and then heated for 10 min to terminate the reaction. The concentration of D-chiral inositol in the reaction solution after the reaction was completed was detected by high performance liquid chromatography, and the conversion rate was calculated. The results are shown in Table 7.

[0040] 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.

[0041] Conversion rate = Amount of D-chiral inositol produced ÷ Amount of muscle inositol added before the reaction × 100%.

[0042] Table 7 reaction system D-chiroinositol concentration (mM) Conversion rate Reaction system 1 4.65 15.5% Reaction system 2 5.86 19.5% From the above results, we can conclude that: Compared to the wild-type ketoisomerase, the ketoisomerase mutant L73N obtained by the present invention has higher enzyme activity, thereby further improving the conversion rate and the yield of D-chiral inositol.

[0043] 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 ketoisomerase mutant, characterized in that, The ketoisomerase mutant is obtained by mutating the 73rd amino acid of the amino acid sequence shown in SEQ ID NO.3 from L to N.

2. The ketoisomerase mutant according to claim 1, characterized in that, The gene encoding the ketoisomerase mutant is shown in SEQ ID NO.

6.

3. A biomaterial, characterized in that, Including any one of the following (1) to (2): (1) An expression vector containing the gene encoding the ketoisomerase mutant of claim 1; (2) A recombinant strain containing the coding gene of the ketoisomerase mutant of claim 1, or containing the expression vector of (1).

4. The use of the ketoisomerase mutant of claim 1 or 2 or the biomaterial of claim 3 in the preparation of D-chiral inositol.

5. A method for preparing D-chiral inositol, characterized in that, Using a crude enzyme solution containing both inositol dehydrogenase and ketoisomerase mutants, in the presence of coenzyme factors, the reaction of muscle inositol is catalyzed to produce D-chiral inositol.

6. The preparation method according to claim 5, characterized in that, The method for preparing the crude enzyme solution containing both inositol dehydrogenase and ketoisomerase mutants includes: The inositol dehydrogenase gene and the ketoisomerase mutant gene were simultaneously transferred into the host strain to obtain a recombinant strain. The recombinant strain was subjected to seed culture to obtain seed solution; The seed culture was inoculated into the fermentation medium at an inoculation rate of 1% to 5% by volume, and fermentation was carried out until the OD reached the specified level. 600 Once the OD reaches 0.6~0.8, add an inducing agent for induction culture until the OD reaches a certain level. 600 The fermentation broth containing both inositol dehydrogenase and ketoisomerase 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 ketoisomerase mutants.

7. The preparation method according to claim 6, characterized in that, The seed culture is performed by culturing at 35℃~40℃ and 100r / min~250r / min for 8h~16h; and / or, The fermentation culture temperature is 35℃~40℃, and the rotation speed is 100r / min~250r / min; and / or, The induction culture temperature is 16℃~30℃, and the rotation speed is 100r / min~250r / min.

8. The preparation method according to claim 6, characterized in that, In the crude enzyme solution containing both inositol dehydrogenase and ketoisomerase mutants, the mass ratio of inositol dehydrogenase to ketoisomerase mutants is (0.5~3):(0.5~3).

9. The preparation method according to claim 5, characterized in that, The preparation method for the reaction system that generates D-chiral inositol is as follows: Add muscle inositol, coenzyme factor, crude enzyme solution containing both inositol dehydrogenase and ketoisomerase mutants, and buffer solution to the reaction system to make the concentration of muscle inositol 20-50 mM, coenzyme factor 1-2 mM, total protein of crude enzyme containing inositol dehydrogenase and ketoisomerase mutants 1-6 mg / mL, and buffer solution 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 coenzyme factor includes NAD. + or NADP ＋ ; and / or, The buffer solution includes phosphate buffer, Tris-HCl buffer, or HEPES buffer.

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