D-mycophenolic acid dehydrogenase MtOEPa mutant and application thereof in preparation of D-chiro-inositol

Abstract

The application discloses a D-mammary alcohol dehydrogenase MtOEPa mutant and application thereof in preparation of D-chiral inositol, and belongs to the technical field of genetic engineering. The D-mammary alcohol dehydrogenase MtOEPa mutant is any of the following: (1) a protein obtained by mutating the 88th amino acid of the amino acid sequence shown in SEQ ID NO. 1 into A; (2) a fusion protein obtained by connecting a terminal tag to the protein of (1). The wild-type D-mammary alcohol dehydrogenase MtOEPa derived from alfalfa is mutated to obtain the D-mammary alcohol dehydrogenase MtOEPa mutant G88A. The D-mammary alcohol dehydrogenase MtOEPa mutant G88A improves the enzyme activity on the substrate muscle inositol, and significantly improves the overall conversion rate and the yield of D-chiral inositol when catalyzing the muscle inositol reaction together with D-pine alcohol dehydrogenase MtOEPb to generate D-chiral inositol.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a D-manganese dehydrogenase MtOEPa mutant and its application in the preparation of D-chiral inositol. Background Technology

[0002] D-Chiro-inositol (DCI) is an important stereoisomer of inositol that has received much attention in the field of metabolic health in recent years. D-Chiro-inositol has a significant effect on improving polycystic ovary syndrome (PCOS) and insulin resistance.

[0003] Existing technologies for synthesizing D-chiral inositol typically involve constructing engineered strains of yeasts such as Saccharomyces cerevisiae and Corynebacterium glutamicum, enabling them to directly ferment and synthesize D-chiral inositol using inexpensive carbon sources like glucose. However, this method usually requires enhancing endogenous metabolic pathways, which may burden the strain's growth. Alternatively, D-chiral inositol can be extracted from plants (such as buckwheat) using physical, chemical, and enzymatic methods, but this is limited by plant resources and results in high extraction costs.

[0004] Studies have shown that D-monosoyl dehydrogenase (MtOEPa) and D-pinel dehydrogenase (MtOEPb) from alfalfa were heterologously expressed in Corynebacterium glutamicum, demonstrating that they can utilize substrate heterogeneity to catalyze the reversible conversion between muscle inositol and D-chiral inositol, establishing a new pathway for the synthesis of D-chiral inositol. However, this pathway is limited by the enzyme activity of D-monosoyl dehydrogenase (MtOEPa), and there is significant room for improvement in catalytic efficiency. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a D-manganese dehydrogenase MtOEPa mutant and its application in the preparation of D-chiral inositol, in order to overcome the problem that the enzyme activity of D-manganese dehydrogenase MtOEPa for the substrate muscle inositol is not high enough in the prior art.

[0006] In a first aspect, the present invention provides a D-monomentol dehydrogenase MtOEPa mutant, wherein the D-monomentol dehydrogenase MtOEPa mutant is any one of the following: (1): The protein obtained by mutating amino acid G to A at position 88 of the amino acid sequence shown in SEQ ID NO.1; (2): (1) The fusion protein obtained by attaching a tag to the end of the protein.

[0007] Compared with existing technologies, this invention mutates wild-type D-ononitol dehydrogenase MtOEPa (Medicago truncatula D-ononitol dehydrogenase) derived from alfalfa by changing the 88th amino acid from G to A, obtaining the D-ononitol dehydrogenase MtOEPa mutant G88A. This D-ononitol dehydrogenase MtOEPa mutant G88A increases the enzyme activity on the substrate muscle inositol. When catalyzing the reaction of muscle inositol to D-chiral inositol together with D-pinitol dehydrogenase MtOEPb (Medicago truncatula D-pinitol dehydrogenase), it significantly improves the overall conversion rate and the yield of D-chiral inositol.

[0008] In a second aspect, the present invention provides a biomaterial comprising any one of the following: (A) A nucleic acid molecule having a gene sequence encoding the above-described D-mangmenol dehydrogenase MtOEPa mutant; (B) An expression vector containing the nucleic acid molecule described in (A); (C) A recombinant strain containing the nucleic acid molecule described in (A) or the expression vector described in (B).

[0009] Thirdly, the present invention provides a method for preparing a D-monospirol dehydrogenase MtOEPa mutant, which includes the following steps: Recombinant strains containing the gene encoding the D-manganese dehydrogenase MtOEPa mutant were seed cultured to obtain seed liquid; The seed culture was inoculated into the fermentation medium for fermentation culture until the OD reached... 600 The value was 0.6~0.8, the temperature was lowered, and an inducer was added for induction culture for 12~16h to obtain fermentation broth containing the D-monomentol dehydrogenase MtOEPa mutant; The fermentation broth was centrifuged to collect the cells. After the cells were resuspended, they were broken up and centrifuged again. The collected supernatant was the crude enzyme solution of the D-manganese dehydrogenase MtOEPa mutant.

[0010] Compared with the prior art, the present invention uses the above-mentioned technical solution to ferment and prepare the D-monomentol dehydrogenase MtOEPa mutant, which can simply and quickly prepare crude enzyme solution of D-monomentol dehydrogenase MtOEPa mutant with enzyme activity.

[0011] Furthermore, the seed culture temperature is 35~40℃, the rotation speed is 120~250rpm, and the culture time is 8~14h.

[0012] Furthermore, the fermentation temperature is 35~40℃ and the rotation speed is 120~250rpm.

[0013] Furthermore, the temperature will be lowered to 16-30℃.

[0014] Furthermore, the inducing agent includes IPTG, with a final concentration of 0.01~0.1mM.

[0015] Under the above conditions, the crude enzyme concentration in the prepared D-manganese dehydrogenase MtOEPa mutant was 2~6 mg / mL.

[0016] Fourthly, the present invention provides the application of the above-mentioned D-manganese dehydrogenase MtOEPa mutant or the above-mentioned biological material in the preparation of D-chiral inositol.

[0017] Fifthly, the present invention provides a method for preparing D-chiral inositol, which utilizes the above-mentioned D-manganese dehydrogenase MtOEPa mutant and D-pinel dehydrogenase MtOEPb to catalyze the reaction of the substrate muscle inositol to generate D-chiral inositol in the presence of a coenzyme factor.

[0018] Compared with the wild-type D-manganese dehydrogenase MtOEPa in the prior art, the D-manganese dehydrogenase MtOEPa of the present invention has higher enzyme activity, effectively improving the reaction efficiency in the preparation of D-chiral inositol, and further improving the conversion rate and the yield of D-chiral inositol.

[0019] Further, muscle inositol, coenzyme factor, crude enzyme solution of D-monomentol dehydrogenase MtOEPa mutant, and enzyme solution of D-pineol dehydrogenase MtOEPb were added to a 20-100mM buffer solution, so that the concentration of muscle inositol in the reaction system was 20-120 mg / mL, the concentration of crude enzyme solution of D-monomentol dehydrogenase MtOEPa mutant was 60-150 μL / mL, and the concentration of enzyme solution of D-pineol dehydrogenase MtOEPb was 60-150 μL / mL.

[0020] Furthermore, the buffer solution includes phosphate buffer, Tris-HCl buffer, or HEPES buffer.

[0021] Furthermore, coenzyme factors include NAD. ＋ and NADP ＋ In the reaction system, NAD ＋ The concentration is 0.5~2.0mM, NADP ＋ The concentration is 0.5~2.0mM.

[0022] Furthermore, the reaction temperature is 30~40℃, and the pH value of the reaction is 7.0~8.0. Detailed Implementation

[0023] 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.

[0024] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.

[0025] Example 1 Construction of recombinant plasmid pET21a-MtOEPA Wild-type D-ononitol dehydrogenase MtOEPa (Medicago truncatula D-ononitol dehydrogenase) derived from alfalfa was selected, and its amino acid sequence is shown in SEQ ID NO.1. Based on the codon preference of E. coli, the amino acid sequence of wild-type D-ononitol dehydrogenase MtOEPa was reverse-translated into DNA sequence to obtain the gene fragment MtOEPA of wild-type D-ononitol dehydrogenase MtOEPa, and its nucleotide sequence is shown in SEQ ID NO.2.

[0026] The MtOEPA gene fragment and the expression vector pET21a were double-digested using restriction endonucleases SalⅠ and XhoⅠ, respectively, at 37℃ for 20 min, yielding the digested MtOEPA gene fragment and the linearized vector pET21a, respectively. The double digestion systems are shown in Table 1.

[0027] Table 1 The enzyme-digested gene fragment MtOEPA and the linearized vector pET21a were ligated using T4 DNA ligase at a temperature of 37°C for 30 min. The ligation system is shown in Table 2.

[0028] Table 2 After ligation, the ligation product was transformed into E. coli DH5α competent cells using chemical transformation. Single colonies were picked, plasmids were extracted, and sequencing was performed to obtain the correctly sequenced recombinant plasmid pET21a-MtOEPA.

[0029] Example 2 Construction of mutant plasmid pET21a-MtOEPA G88A Using the correctly sequenced recombinant plasmid pET21a-MtOEPA as a template, with F1 as the upstream primer and R1 as the downstream primer, a reverse PCR reaction was performed. The system of the reverse PCR reaction is shown in Table 3, and the reaction conditions are shown in Table 4.

[0030] F1: 5'-CCGAACATTATTgcgGAAGTGAAAGATCCGGAAAAACA-3' (SEQ ID NO. 5).

[0031] R1: 5'-TCcgcAATAATGTTCGGGCACGCCAGATGAAT-3' (SEQ ID NO. 6).

[0032] Table 3 Table 4 It should be understood that the pre-denaturation and incubation steps in the above-mentioned reverse PCR reaction do not participate in the cycling process, and the entire reverse PCR process is performed only once.

[0033] Template digestion: After the above reverse PCR reaction was completed, 2 μL of restriction endonuclease DpnⅠ was added to the reaction solution (50 μL) obtained after the above reverse PCR reaction, and the mixture was gently pipetted and aspirated. The mixture was then reacted at 37℃ for 1 h to obtain the enzyme digestion solution. After the reaction, the enzyme digestion solution was verified by agarose gel electrophoresis.

[0034] PCR product self-cyclization: Take 2 μL of the validated enzyme digestion solution, 1 μL of high-efficiency ligation reagent, 2 μL of T4 polynucleotide kinase, and 6 μL of ddH2O, mix gently, and cyclize at 16℃ for 1 hour to obtain the mutant plasmid pET21a-MtOEPA. G88A .

[0035] Mutant plasmid verification: The mutant plasmid pET21a-MtOEPA obtained after the above circularization was... G88A The cells were transformed into E. coli DH5α competent cells using chemical transformation. Single colonies on the plates were picked for plasmid extraction, and the extracted plasmids were sequenced for DNA.

[0036] Mutant plasmid pET21a-MtOEPA G88A The gene containing the D-monomentol dehydrogenase MtOEPa mutant G88A has the nucleotide sequence shown in SEQ ID NO.3 and the encoded amino acid sequence shown in SEQ ID NO.4.

[0037] Example 3 Preparation of crude enzyme solution Take 1 μL of the correctly sequenced mutant plasmid pET21a-MtOEPA G88A Add the mixture to E. coli BL21(DE3) competent cells. After mixing, place the reaction mixture on ice for 25 min, then heat shock it in a 42°C water bath for 60 s, followed by incubation on ice for 5 min. Transfer the mixture to 500 μL of LB liquid medium and incubate at 37°C with shaking for 1 h. Spread 100 μL of the bacterial culture onto LB agar plates containing 50 μg / mL ampicillin to screen for cells containing the mutant plasmid pET21a-MtOEPA. G88A The positive transformant yielded the mutant strain BL21-pET21a-MtOEPA. G88A .

[0038] The mutant strain BL21-pET21a-MtOEPA G88A Seed culture was carried out by inoculating the mutant strain into liquid LB medium (containing 50 μg / mL ampicillin) and culturing at 37℃ and 220 rpm for 8 h to obtain the seed culture.

[0039] The seed culture of the above mutant strain was inoculated into fresh LB liquid medium (containing 50 μg / mL ampicillin) at a 1% volume ratio for fermentation culture at 37°C and 220 rpm until OD500. 600 The value was 0.6, then the temperature was lowered to 28℃, and IPTG with a final concentration of 0.05mM was added for induction culture for 16h to obtain fermentation broth containing the D-monomentol dehydrogenase MtOEPa mutant G88A.

[0040] The fermentation broth was centrifuged at 4000 rpm for 15 min at 4℃ to collect the cells. The collected cells were resuspended in 20 mM phosphate buffer (pH 7.5). The cells were then disrupted using an ultrasonic cell disruptor at 450 W, with a 2-second break for 3 seconds, for a total of 30 min. After ultrasonic disruption, the cells were centrifuged at 12000 rpm at 4℃ to remove cell debris. The supernatant was collected to obtain the crude enzyme solution of the D-monomenthol dehydrogenase MtOEPa mutant G88A, with a crude enzyme concentration of 5 mg / mL.

[0041] Take 1 μL of the correctly sequenced recombinant plasmid pET21a-MtOEPA and prepare the crude enzyme solution of wild-type D-manganese dehydrogenase MtOEPa using the same method as described above. The concentration of crude enzyme in the crude enzyme solution is 5 mg / mL.

[0042] Example 4 Enzyme activity detection The enzyme activities of wild-type D-monomentol dehydrogenase MtOEPa and the D-monomentol dehydrogenase mutant G88A were determined using muscle inositol as a substrate: Add muscle inositol and NAD to the reaction system ＋ The crude enzyme solution of wild-type D-monosodium dehydrogenase MtOEPa was added to a reaction volume of 10 mL with 20 mM phosphate buffer (pH 7.5). The concentration of muscle inositol in the reaction system was 20 mM, and NAD+ was... ＋ The concentration of the enzyme was 2 mM and the concentration of the crude wild-type D-manganese dehydrogenase MtOEPa was 80 μL / mL. The prepared system was mixed well and reacted at 37°C for 5 min. The reaction was then immediately terminated by heating in a boiling water bath.

[0043] Add muscle inositol and NAD to the reaction system ＋ The crude enzyme solution of D-monosodium dehydrogenase MtOEPa mutant G88A was added, and the reaction volume was brought up to 10 mL with 20 mM phosphate buffer (pH 7.5). The concentration of muscle inositol in the reaction system was 20 mM, and NAD+... ＋ The concentration of the enzyme was 2 mM, and the concentration of the crude enzyme solution of D-monomentol dehydrogenase MtOEPa mutant G88A was 80 μL / mL. The prepared system was mixed well, and after reacting at 37℃ for 5 min, it was immediately taken out and heated in a boiling water bath to terminate the reaction.

[0044] The enzyme activity unit (U) is defined as the amount of enzyme required to consume 1 μmol of the substrate muscle inositol within 1 minute under the above reaction system and analytical conditions.

[0045] The enzyme activity is calculated as follows: Enzyme activity (U) = (ΔA × V × 10³) / 6220 × 1. ΔA refers to the change in absorbance at 340 nm over 5 minutes; V is the total volume of the reaction system (mL); 6220 refers to the molar extinction coefficient of NADH (…). ); l is the optical path distance, i.e., the optical path of the cuvette (cm).

[0046] Using the activity of wild-type D-monomentol dehydrogenase MtOEPa as 100%, the relative enzyme activity of the D-monomentol dehydrogenase MtOEPa mutant G88A was calculated, and the results are shown in Table 5.

[0047] Methods for detecting muscle inositol: High performance liquid chromatography: 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.

[0048] Table 5

[0049] As can be seen from the above results, the present invention mutates the amino acid sequence of wild-type D-monomentol dehydrogenase MtOEPa by changing the 88th amino acid from G to A, resulting in the D-monomentol dehydrogenase MtOEPa mutant G88A. This D-monomentol dehydrogenase MtOEPa mutant G88A significantly increases the enzyme activity of the substrate muscle inositol.

[0050] Example 5 Preparation of D-chiral inositol Add muscle inositol and NAD to a 20mM phosphate buffer solution with a pH of 7.5. + NADP + Wild-type D-mannosyl dehydrogenase MtOEPa crude enzyme solution and D-pinel dehydrogenase MtOEPb enzyme solution were used to make the concentration of muscle inositol in the reaction system 100 mg / mL, NAD+ + The concentration was 2 mM, NADP + The concentration of the enzyme was 2 mM, the concentration of the crude enzyme solution of wild-type D-manganese dehydrogenase MtOEPa was 140 μL / mL, and the concentration of the enzyme solution of D-pineol dehydrogenase MtOEPb was 140 μL / mL.

[0051] Add muscle inositol and NAD to a 20mM phosphate buffer solution with a pH of 7.5. + NADP + The crude enzyme solution of D-monosodium dehydrogenase MtOEPa mutant G88A and the enzyme solution of D-pineol dehydrogenase MtOEPb were used to make the concentration of muscle inositol in the reaction system 100 mg / mL, and NAD+ + The concentration was 2 mM, NADP + The concentration of the enzyme was 2 mM, the concentration of the crude enzyme solution of D-manganese dehydrogenase MtOEPa mutant G88A was 140 μL / mL, and the concentration of the enzyme solution of D-pineol dehydrogenase MtOEPb was 140 μL / mL.

[0052] The two reaction systems were reacted at 35℃ for 6 hours to obtain reaction solutions. The content and conversion rate of D-chiral inositol in each reaction solution were measured, and the results are shown in Table 6.

[0053] Conversion rate: D-chiral inositol content after reaction ÷ initial muscle inositol content in the reaction system × 100%.

[0054] Detection method: High performance liquid chromatography (HPLC) was used. 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.

[0055] Table 6 The results above show that, compared with wild-type D-mannose dehydrogenase MtOEPa, the D-mannose dehydrogenase MtOEPa mutant G88A of the present invention further improves the conversion rate and the yield of D-chiral inositol.

[0056] 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 D-monomentol dehydrogenase MtOEPa mutant, characterized in that, The D-monomentol dehydrogenase MtOEPa mutant is any one of the following: (1): The protein obtained by mutating amino acid G to A at position 88 of the amino acid sequence shown in SEQ ID NO.1; (2): (1) The fusion protein obtained by attaching a tag to the end of the protein.

2. A biomaterial, characterized in that, Includes any one of the following: (A) A nucleic acid molecule having a gene sequence encoding the D-mangmenol dehydrogenase MtOEPa mutant of claim 1; (B) An expression vector containing the nucleic acid molecule described in (A); (C) A recombinant strain containing the nucleic acid molecule described in (A) or the expression vector described in (B).

3. A method for preparing a D-mannoselide dehydrogenase MtOEPa mutant, characterized in that, The preparation of the D-monomentol dehydrogenase MtOEPa mutant according to claim 1 includes the following steps: Recombinant strains containing the gene encoding the D-manganese dehydrogenase MtOEPa mutant were seed cultured to obtain seed liquid; The seed culture was inoculated into a fermentation medium for fermentation culture until the OD value reached... 600 The value was 0.6~0.8, the temperature was lowered, and an inducer was added for induction culture for 12~16h to obtain fermentation broth containing the D-monomentol dehydrogenase MtOEPa mutant; The fermentation broth was centrifuged to collect the bacterial cells. After the bacterial cells were resuspended, they were broken up and centrifuged again. The collected supernatant was the crude enzyme solution of the D-monomentol dehydrogenase MtOEPa mutant.

4. The preparation method according to claim 3, characterized in that, The seed culture temperature is 35-40℃, the rotation speed is 120-250 rpm, and the culture time is 8-14 hours; and / or, The fermentation culture is carried out at a temperature of 35-40℃ and a rotation speed of 120-250 rpm; and / or, The cooling is to reduce the temperature to 16~30℃; and / or, The inducer includes IPTG, and the final concentration of IPTG is 0.01~0.1mM.

5. The application of the D-manganese dehydrogenase MtOEPa mutant of claim 1 or the biomaterial of claim 2 in the preparation of D-chiral inositol.

6. A method for preparing D-chiral inositol, characterized in that, Using the D-manganese dehydrogenase MtOEPa mutant and D-pinel dehydrogenase MtOEPb as described in claim 1, in the presence of coenzyme factors, the substrate muscle inositol is catalyzed to produce D-chiral inositol.

7. The preparation method according to claim 6, characterized in that, Add muscle inositol, coenzyme factor, crude enzyme solution of D-manganese dehydrogenase MtOEPa mutant, and enzyme solution of D-pineol dehydrogenase MtOEPb to a 20-100 mM buffer solution, so that the concentration of muscle inositol in the reaction system is 20-120 mg / mL, the concentration of crude enzyme solution of D-manganese dehydrogenase MtOEPa mutant is 60-150 μL / mL, and the concentration of enzyme solution of D-pineol dehydrogenase MtOEPb is 60-150 μL / mL.

8. The preparation method according to claim 7, characterized in that, The buffer solution includes phosphate buffer, Tris-HCl buffer, or HEPES buffer.

9. The preparation method according to claim 7, characterized in that, The coenzyme factor includes NAD. ＋ and NADP ＋ In the reaction system, the NAD ＋ The concentration of NADP is 0.5~2.0 mM. ＋ The concentration is 0.5~2.0mM.

10. The preparation method according to claim 7, characterized in that, The reaction temperature is 30~40℃, and the pH value of the reaction is 7.0~8.0.

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