D-pinitol dehydrogenase mutant MtOEPb and application thereof in preparation of D-chiro-inositol

Abstract

The application discloses a D-pinitol dehydrogenase MtOEPb mutant and application thereof in preparation of D-chiro-inositol, and belongs to the technical field of genetic engineering. The amino acid sequence of the D-pinitol dehydrogenase MtOEPb mutant is shown as SEQ ID NO. 4. The wild-type D-pinitol dehydrogenase MtOEPb is subjected to mutation, the 119th amino acid in the amino acid sequence is mutated from H to Q, and the D-pinitol dehydrogenase MtOEPb mutant H119Q is obtained. The enzyme activity of the D-pinitol dehydrogenase MtOEPb mutant H119Q is significantly improved. When the D-pinitol dehydrogenase MtOEPb mutant H119Q is used together with D-mangiferoline dehydrogenase MtOEPa to catalyze a reaction of muscle inositol to generate D-chiro-inositol, the conversion rate of the reaction and the yield of D-chiro-inositol are further improved.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a D-pinel dehydrogenase MtOEPb 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 enzymatic methods typically utilize inositol dehydrogenase and ketoisomerase to produce D-chiral inositol. First, inositol dehydrogenase catalyzes the conversion of muscle inositol to glycosaminoglycans. Then, ketoisomerase catalyzes the conversion of glycosaminoglycans to 1-keto-chiral inositol. At this point, inositol dehydrogenase again catalyzes the conversion of 1-keto-chiral inositol to D-chiral inositol. Because these three steps are reversible and frequently affected by enzyme activity and half-life, the yield of D-chiral inositol is limited.

[0004] Studies have demonstrated that D-manganese dehydrogenase (MtOEPa) and D-pinel dehydrogenase (MtOEPb) from alfalfa can catalyze the reversible conversion between muscle inositol and D-chiral inositol by heterologous expression in Corynebacterium glutamicum. This has established a new pathway for the synthesis of D-chiral inositol, and the overall efficiency of this pathway depends on the activity levels of the two key enzymes mentioned above. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a D-pinel dehydrogenase MtOEPb mutant and its application in the preparation of D-chiral inositol, so as to further improve the enzyme activity of D-pinel dehydrogenase MtOEPb and the yield of D-chiral inositol.

[0006] In a first aspect, the present invention provides a D-pinel dehydrogenase MtOEPb mutant, the amino acid sequence of which is shown in SEQ ID NO.4.

[0007] Compared with the prior art, the present invention mutates wild-type D-pinel dehydrogenase MtOEPb by changing the 119th position of its amino acid sequence from H to Q, obtaining the D-pinel dehydrogenase MtOEPb mutant H119Q. The enzyme activity of the D-pinel dehydrogenase MtOEPb mutant H119Q is significantly improved. When it catalyzes the reaction of muscle inositol to produce D-chiral inositol together with D-monosoyl dehydrogenase MtOEPa, it further improves the conversion rate and the yield of D-chiral inositol.

[0008] Furthermore, the gene sequence of the D-pinel dehydrogenase MtOEPb mutant is shown in SEQ ID NO.3.

[0009] In a second aspect, the present invention provides a biomaterial comprising any one of the following: (A) An expression vector containing the gene sequence of a D-pinel dehydrogenase MtOEPb mutant; (B) A recombinant strain containing the gene sequence of a D-pinel dehydrogenase MtOEPb mutant or the expression vector described in (A).

[0010] Thirdly, the present invention provides a method for preparing a D-pinoxetine dehydrogenase MtOEPb mutant, which includes the following steps: Recombinant strains containing the gene sequence of the D-pinel dehydrogenase MtOEPb 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-pinel dehydrogenase MtOEPb 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-pinel dehydrogenase MtOEPb mutant.

[0011] Compared with existing technologies, the above preparation method can simply and quickly prepare crude enzyme solution with D-pinel dehydrogenase MtOEPb mutant enzyme activity.

[0012] Furthermore, the seed culture temperature is 35~40℃, the rotation speed is 120~250rpm, and the culture time is 8~14h.

[0013] Furthermore, the fermentation temperature is 35~40℃ and the rotation speed is 120~250rpm.

[0014] Furthermore, the temperature will be lowered to 16-30℃.

[0015] Furthermore, the inducing agent includes IPTG, wherein the final concentration of IPTG is 0.01~0.1mM.

[0016] Under the above conditions, the concentration of crude enzyme in the crude enzyme solution of the D-pinel dehydrogenase MtOEPb mutant was 2~6 mg / mL.

[0017] Fourthly, the present invention provides the application of the above-mentioned D-pinel dehydrogenase MtOEPb mutant, or the above-mentioned biological material, in the preparation of D-chiral inositol.

[0018] Fifthly, the present invention provides a method for preparing D-chiral inositol, which utilizes D-manganese dehydrogenase MtOEPa and the above-mentioned D-pinene dehydrogenase MtOEPb mutant to catalyze the reaction of the substrate muscle inositol to generate D-chiral inositol in the presence of a coenzyme factor.

[0019] Compared with the prior art, the D-pinel dehydrogenase MtOEPb mutant obtained by mutation in this invention has higher enzyme activity, which effectively improves the conversion rate and the yield of D-chiral inositol when catalyzing the reaction of the substrate muscle inositol to produce D-chiral inositol.

[0020] Further, muscle inositol, coenzyme factor, D-monosodium dehydrogenase MtOEPa enzyme solution, and crude D-pineol dehydrogenase MtOEPb mutant enzyme solution were added to a 20-100 mM buffer solution, so that the concentration of muscle inositol in the reaction system was 70-125 mg / mL, the concentration of D-monosodium dehydrogenase MtOEPa enzyme solution was 80-150 μL / mL, and the concentration of crude D-pineol dehydrogenase MtOEPb mutant enzyme solution was 80-150 μL / mL.

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

[0022] 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.

[0023] Furthermore, the reaction temperature is 30~40℃, and the pH value of the reaction is 7.0~8.0. Detailed Implementation

[0024] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0025] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.

[0026] Example 1 Construction of recombinant plasmid pET21a-MtOEPB The wild-type D-pinitol dehydrogenase MtOEPb (Medicago truncatula D-pinitol dehydrogenase) derived from alfalfa was selected, and its amino acid sequence is shown in SEQ ID NO.1. Based on the codon bias of E. coli, the gene of wild-type D-pinitol dehydrogenase MtOEPb was optimized to obtain the optimized gene fragment MtOEPB, as shown in SEQ ID NO.2.

[0027] The gene fragment MtOEPB and the expression vector pET21a were double-digested using restriction endonucleases SalⅠ and NotⅠ, respectively, at 37℃ for 20 min each, yielding the digested gene fragment MtOEPB and the linearized expression vector pET21a. The double digestion system is shown in Table 1.

[0028] Table 1 Expression vector pET21a or gene fragment MtOEPB 25μL 10× buffer solution 5μL Restriction endonuclease SalⅠ 2μL Restriction endonuclease XhoⅠ 2μL <![CDATA[ddH2O]]> 16μL The enzyme-digested gene fragment MtOEPB and the linearized vector pET21a were ligated using T4 DNA ligase at 37°C for 30 min. The ligation system is shown in Table 2. Table 2 Linearized carrier pET21a 4.5μL The gene fragment MtOEPB after enzyme digestion 1.5μL T4 DNA Ligation Buffer 2μL T4 DNA ligase 1μL <![CDATA[ddH2O]]> 11μL 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-MtOEPB.

[0029] Example 2 Construction of mutant plasmid pET21a-MtOEPB H119Q Using the recombinant plasmid pET21a-MtOEPB with correct sequencing as a template, 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'-TACCTGATTcagTGGCCGGTGCGCCTGCGCCA-3' (SEQ ID NO. 5).

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

[0032] Table 3 pET21a-MtOEPB 2μL 10 pmol / μL F1 2μL 10 pmol / μL R1 2μL 2mM deoxynucleoside triphosphates (dNTPs) 5μL KOD-Plus DNA polymerase 2μL 10×PCR buffer 5μL <![CDATA[Sterile ddH2O]]> 32μL Table 4 Pre-variation 94℃ 2min / transsexual 98℃ 10s 10 annealing 55℃ 30s 10 extend 68℃ 30s 10 Insulation 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-circulation: 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 circulate at 16℃ for 1 hour to obtain the mutant plasmid pET21a-MtOEPB. H119Q .

[0035] Mutant plasmid verification: The mutant plasmid pET21a-MtOEPB obtained after circularization was... H119Q 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-MtOEPB H119Q The gene sequence containing the D-pinel dehydrogenase MtOEPb mutant is shown in SEQ ID NO.3, and the amino acid sequence of the D-pinel dehydrogenase MtOEPb mutant H119Q encoded by this gene sequence is shown in SEQ ID NO.4.

[0037] Example 3 Preparation of crude enzyme solution Take 1 μL of the correctly sequenced mutant plasmid pET21a-MtOEPB H119Q Add the mixture to E. coli BL21(DE3) competent cells. After mixing, the reaction system is placed on ice for 25 min, then heat-shocked in a 42°C water bath for 60 s, followed by incubation on ice for 5 min. The mixture is then transferred to 500 μL of LB liquid medium and incubated at 37°C with shaking for 1 h. 100 μL of the bacterial culture is then plated on LB agar plates containing 50 μg / mL ampicillin to screen for the mutant plasmid pET21a-MtOEPB. H119Q The positive transformant was obtained, that is, the mutant strain BL21-pET21a-MtOEPB. H119Q .

[0038] The mutant strain BL21-pET21a-MtOEPB H119QSeed 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 0.7, 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-pinel dehydrogenase MtOEPb mutant H119Q.

[0040] The fermentation broth was centrifuged at 4000 rpm for 15 min at 4°C, and the cells were collected. 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°C to remove cell debris, and the supernatant was collected to obtain the crude enzyme solution of the D-pinel dehydrogenase MtOEPb mutant H119Q, with a crude enzyme concentration of 5 mg / mL.

[0041] Take 1 μL of the correctly sequenced recombinant plasmid pET21a-MtOEPB and prepare the crude enzyme solution of wild-type D-pineol dehydrogenase MtOEPb 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 Configure the reaction system: Add muscle inositol and NAD to the reaction system. ＋ NADP ＋ The reaction mixture consisted of D-monomentol dehydrogenase MtOEPa (Medicago truncatula D-ononitol dehydrogenase) enzyme solution and wild-type D-pineol dehydrogenase MtOEPb crude enzyme solution, which were then adjusted to a reaction volume of 10 mL using 20 mM phosphate buffer (pH 7.5). The concentration of muscle inositol in the reaction system was 20 mM, and NAD+... ＋ The concentration was 2 mM NADP ＋ The concentrations of the enzymes were 2 mM, the concentration of D-manganese dehydrogenase MtOEPa was 100 μL / mL, and the concentration of wild-type D-pineol dehydrogenase MtOEPb crude enzyme solution was 100 μL / mL. The prepared system was mixed well and reacted at 37°C for 60 min. The reaction was then immediately terminated by heating in a boiling water bath.

[0043] Add muscle inositol and NAD to the reaction system. ＋ NADP ＋ D-Momenhydrinate dehydrogenase MtOEPa enzyme solution and D-pinel dehydrogenase MtOEPb mutant H119Q crude enzyme solution were added to a reaction volume of 10 mL using 20 mM phosphate buffer (pH 7.5). The concentration of muscle inositol in the reaction system was 20 mM, and NAD+ was... ＋ The concentration was 2 mM NADP ＋ The concentrations of the enzymes were 2 mM, 100 μL / mL for D-manganese dehydrogenase MtOEPa, and 100 μL / mL for crude D-pineol dehydrogenase MtOEPb mutant H119Q. The prepared system was mixed and reacted at 37°C for 60 min. The reaction was then immediately terminated by heating in a boiling water bath.

[0044] Enzyme activity is defined as the amount of enzyme required to generate 1 μmol of product D-chiral inositol within 1 minute under the above reaction system.

[0045] Methods for detecting D-chiral 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.

[0046] The relative enzyme activity of the D-pinel dehydrogenase MtOEPb mutant H119Q was calculated with the wild-type D-pinel dehydrogenase MtOEPb activity as 100%, and the results are shown in Table 5.

[0047] Table 5

[0048] The results above show that the present invention mutates wild-type D-pinel dehydrogenase MtOEPb by changing the 119th position of its amino acid sequence from H to Q, resulting in the D-pinel dehydrogenase MtOEPb mutant H119Q, which has significantly increased enzyme activity.

[0049] 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 +The reaction system was prepared using D-monosodium dehydrogenase MtOEPa enzyme solution and wild-type D-pinel dehydrogenase MtOEPb crude enzyme solution to achieve a muscle inositol concentration of 100 mg / mL and NAD+ concentration of 100 mg / mL. + The concentration was 2 mM, NADP + The concentration of the enzyme was 2 mM, the concentration of D-manganese dehydrogenase MtOEPa was 150 μL / mL, and the concentration of wild-type D-pineol dehydrogenase MtOEPb crude enzyme solution was 150 μL / mL.

[0050] Add muscle inositol and NAD to a 20mM phosphate buffer solution with a pH of 7.5. + NADP + The D-monomentol dehydrogenase MtOEPa enzyme solution and the crude D-pinelol dehydrogenase MtOEPb mutant H119Q 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 D-manganese dehydrogenase MtOEPa was 2 mM, the concentration of D-manganese dehydrogenase MtOEPb mutant H119Q crude enzyme solution was 150 μL / mL, and the concentration of D-pineol dehydrogenase MtOEPb mutant H119Q crude enzyme solution was 150 μL / mL.

[0051] 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.

[0052] Conversion rate: (D-chiral inositol content after reaction ÷ initial muscle inositol content in the reaction system) × 100%

[0053] 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.

[0054] Table 6

[0055] The results above show that, compared with wild-type D-pinel dehydrogenase MtOEPb, the MtOEPb mutant H119Q of the present invention has increased enzyme activity, thereby further improving 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-pinel dehydrogenase MtOEPb mutant, characterized in that, The amino acid sequence of the D-pinel dehydrogenase MtOEPb mutant is shown in SEQ ID NO.

4.

2. The D-pinel dehydrogenase MtOEPb mutant according to claim 1, characterized in that, The gene sequence of the D-pinel dehydrogenase MtOEPb mutant is shown in SEQ ID NO.

3.

3. A biomaterial, characterized in that, Includes any one of the following: (A) An expression vector containing the gene sequence of a D-pinel dehydrogenase MtOEPb mutant; (B) A recombinant strain containing the gene sequence of a D-pinel dehydrogenase MtOEPb mutant or the expression vector described in (A).

4. A method for preparing a D-pinel dehydrogenase MtOEPb mutant, characterized in that, The preparation of the D-pinel dehydrogenase MtOEPb mutant according to claim 1 includes the following steps: Recombinant strains containing the gene sequence of the D-pinel dehydrogenase MtOEPb 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-pinel dehydrogenase MtOEPb 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-pinel dehydrogenase MtOEPb mutant.

5. The preparation method according to claim 4, 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.

6. The use of the D-pinel dehydrogenase MtOEPb mutant according to claim 1 or 2, or the biomaterial according to claim 3, in the preparation of D-chiral inositol.

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

8. The preparation method according to claim 7, characterized in that, Add muscle inositol, coenzyme factor, D-monosodium dehydrogenase MtOEPa enzyme solution, and crude D-pineol dehydrogenase MtOEPb mutant enzyme solution to a 20-100 mM buffer solution, so that the concentration of muscle inositol in the reaction system is 70-125 mg / mL, the concentration of D-monosodium dehydrogenase MtOEPa enzyme solution is 80-150 μL / mL, and the concentration of crude D-pineol dehydrogenase MtOEPb mutant enzyme solution is 80-150 μL / mL.

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