Inositol dehydrogenase mutant as well as preparation method and application thereof

Abstract

The invention discloses an inositol dehydrogenase mutant as well as a preparation method and application thereof, and belongs to the technical field of gene engineering. The inositol dehydrogenase mutant is obtained by mutating an amino acid sequence as shown in SEQ ID NO.1, and the mutation is that the 247th amino acid is mutated from K to R, or the 266th amino acid is mutated from E to D. The inositol dehydrogenase mutant disclosed by the invention has higher enzyme activity, and when the inositol dehydrogenase mutant and ketoisomerase are catalyzed to synthesize D-chiral inositol by taking myo-inositol and NADP < + > as substrates, the reaction is more inclined to a forward reaction, so that the yield of the D-chiral inositol and the conversion rate of the substrate myo-inositol are further improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, in particular to a myo-inositol dehydrogenase mutant, a preparation method and application thereof. BACKGROUND

[0002] D-chiro-inositol (DCI) is one of nine isomers of myo-inositol, and has physiological functions such as insulin-sensitizing effect, blood glucose-lowering effect, improvement of ovulation in patients with polycystic ovary syndrome, hormone balance regulation, improvement of menstrual disorders, and antioxidant, anti-aging and anti-inflammatory effects in addition to promoting liver lipid metabolism.

[0003] D-chiro-inositol can be extracted from plants such as buckwheat, but the content of D-chiro-inositol in plants is low, and the cost of separation and extraction is high. D-chiro-inositol can also be prepared by organic synthesis, but the steps are complicated, by-products are difficult to separate, and toxic substances may be left, affecting the quality of the product.

[0004] In addition, D-chiro-inositol can also be prepared by using myo-inositol dehydrogenase and ketol-isomerase with myo-inositol and NADP + as substrates, but the enzyme activity of wild-type myo-inositol dehydrogenase is low, and the reaction is a reversible reaction, which limits the yield and conversion rate of D-chiro-inositol. SUMMARY

[0005] Therefore, the present application aims to provide a myo-inositol dehydrogenase mutant, a preparation method and application thereof, to overcome the problem of low yield and conversion rate in the prior art when D-chiro-inositol is produced by using myo-inositol dehydrogenase and ketol-isomerase.

[0006] In a first aspect, the present application provides a myo-inositol dehydrogenase mutant, which is obtained by mutating the amino acid sequence shown in SEQ ID NO. 1, and the mutation is:

[0007] the 247th amino acid is mutated from K to R, or the 266th amino acid is mutated from E to D.

[0008] Compared with the existing wild-type myo-inositol dehydrogenase, the myo-inositol dehydrogenase mutant of the present application has higher enzyme activity after the above mutation, and when it is used as a catalyst for synthesizing D-chiro-inositol with myo-inositol and NADP + as substrates and ketol-isomerase, the reaction can be more inclined to the forward reaction, thereby further improving the yield of D-chiro-inositol and the conversion rate of the substrate myo-inositol.

[0009] Further, the amino acid sequence of the myo-inositol dehydrogenase mutant is shown in SEQ ID NO. 3 or SEQ ID NO. 5.

[0010] In a second aspect, the present application provides a coding gene having a nucleotide sequence coding the myo-inositol dehydrogenase mutant.

[0011] Optionally, the myo-inositol dehydrogenase mutant having the amino acid sequence shown in SEQ ID NO. 3 has a gene sequence shown in SEQ ID NO. 8.

[0012] The myo-inositol dehydrogenase mutant having the amino acid sequence shown in SEQ ID NO. 5 has a gene sequence shown in SEQ ID NO. 10.

[0013] In a third aspect, the present application provides an expression vector containing the coding gene.

[0014] In a fourth aspect, the present application provides a recombinant strain containing the coding gene or the expression vector.

[0015] In a fifth aspect, the present application provides a preparation method of the myo-inositol dehydrogenase mutant, for preparing the myo-inositol dehydrogenase mutant, comprising the following steps:

[0016] The recombinant strain is cultured in the LB medium at 35-40°C and 120-220 r / min for 12-16 hours to obtain a seed liquid;

[0017] The seed liquid is inoculated into another LB medium at a volume ratio of 1-5% for fermentation culture, and the culture is performed at 35-40°C until the OD value reaches 0.6-0.8, and then the culture is cooled to 16-25°C, and IPTG with a final concentration of 0.1-0.5 mM is added for induction culture, and the induction culture is performed until the OD value reaches 6-10, to obtain a fermentation liquid containing the myo-inositol dehydrogenase mutant; 600 600

[0018] The fermentation liquid is centrifuged to collect the bacterial cells, the bacterial cells are resuspended and the cells are broken, and the supernatant obtained by centrifugation is the crude enzyme liquid of the myo-inositol dehydrogenase mutant.

[0019] Compared with the prior art, the myo-inositol dehydrogenase mutant crude enzyme liquid can be efficiently obtained by the above microbial fermentation technology, and the preparation efficiency of the myo-inositol dehydrogenase mutant is improved.

[0020] In a sixth aspect, the present application provides the use of the myo-inositol dehydrogenase mutant in the preparation of D-chiro-inositol.

[0021] Compared with the prior art, the myo-inositol dehydrogenase mutant has a one-way reaction characteristic, and can make the reaction more inclined to the forward reaction when synthesizing D-chiro-inositol, thereby further improving the conversion rate of the substrate myo-inositol and the yield of D-chiro-inositol. ​​

[0022] In a seventh aspect, the present application provides a method for preparing D-chiro-inositol, using myo-inositol, NADP + As the substrate, the D-chiro-inositol is synthesized using the myo-inositol dehydrogenase mutant and the ketol-isomerase described above.

[0023] Further, the concentrations of the components in the reaction system for catalytically synthesizing the D-chiro-inositol are as follows:

[0024] 10 g / L to 15 g / L of myo-inositol, 1 mM to 3 mM of NADP + , 3 mg / mL to 5 mg / mL of the ketol-isomerase, and 80 mM to 120 mM of the phosphate buffer.

[0025] The myo-inositol dehydrogenase mutant crude enzyme solution with a protein concentration of 4 mg / mL to 6 mg / mL in the reaction system described above should be understood as adding the myo-inositol dehydrogenase mutant crude enzyme solution so that the concentration of the myo-inositol dehydrogenase mutant in the reaction system is 4 mg / mL to 6 mg / mL.

[0026] Further, 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

[0027] In order to make the technical problems to be solved by the present application, the technical solutions and beneficial effects more clearly and clearly, the present application will be further described in detail below in combination with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0028] It should be understood that the raw materials used in the following examples are commercially available unless otherwise specified.

[0029] Example 1

[0030] Construction of recombinant plasmid pYB1S-IDH

[0031] After codon optimization of the nucleotide sequence of the wild-type myo-inositol dehydrogenase, sequence A (as shown in SEQ ID NO. 6) is obtained, which is artificially synthesized. Using sequence A as a template, F1 and R1 as primers, a target fragment IDH is amplified, which has the restriction enzyme cleavage sites of endonuclease Spe I and Sac I at both ends. The PCR amplification system is shown in Table 1, and the PCR amplification conditions are shown in Table 2.

[0032] Upstream primer F1: 5'-gagggtagatctggtactagtATGGCACTGACTGTAGGCGTTA-3' (SEQ ID NO. 11);

[0033] Downstream primer Rl: 5'-caccagctgcagaccgagctcTTAAATCGCGGCCGCTGG-3' (SEQ ID NO. 12).

[0034] Table 1

[0035] Ingredients Volume (μL) Sequence A 1 Upstream primer F1 (10 μmol / L) 1 Downstream primer R1 (10 μmol / L) 1 ddH2O 9.5 2 x Phanta Super High-fidelity Enzyme 12.5

[0036] Table 2

[0037] Step Temperature Time Cycle number Pre-denaturation 95℃ 1 min Denaturation 98℃ 10s 30 Annealing 53℃ 30s 30 Extension 72℃ 1 min 30 Final extension 72℃ 10 min Preservation 4℃ ∞

[0038] After the above PCR amplification, agarose gel recovery was performed to obtain the gene fragment IDH with higher purity.

[0039] The expression vector pYBlS was double digested by restriction endonuclease Spe I and Sac I, and the linearized vector after enzyme digestion was connected with the IDH fragment obtained by the above PCR amplification using recombination enzyme Exnase II, and the IDH fragment was connected to pYBlS between Spe I / Sac I at 37°C for 30 min, to obtain the recombinant plasmid pYBlS-IDH.

[0040] The double digestion system is shown in Table 3, and the connection system is shown in Table 4.

[0041] Table 3

[0042] Ingredients Volume (μL) Expression vector pYB1S 12.5 10 x Buffer 2.5 Spe I 1 Sac I 1 ddH2O 8

[0043] Table 4

[0044] Ingredients Volume (μL) Linearized vector 4.5 Gene fragment IDH 1.5 5 x CE II Buffer 4 Exnase II 2 ddH2O 8

[0045] After the connection was completed, the chemical transformation method was used to transform the connection product into E. coli DH5a competent cells, and single bacteria were picked for plasmid extraction, and the extracted plasmid was subjected to DNA sequencing.

[0046] Example 2

[0047] Construction of mutant plasmid

[0048] The recombinant plasmid pYBlS-IDH verified by sequencing was used as a template to construct a mutant plasmid by reverse PCR, wherein the reverse PCR reaction system is shown in Table 5, and the reverse PCR reaction conditions are shown in Table 6.

[0049] 1. The recombinant plasmid pYBlS-IDH was used as a template, and the upstream primer F2 and the downstream primer R2 were used as primers to perform reverse PCR amplification to obtain the mutant plasmid pYBlS-IDH S159T .

[0050] 2. Using the recombinant plasmid pYB1S-IDH as a template, the upstream primer F3 and the downstream primer R3 as primers, reverse PCR amplification was performed to obtain the mutant plasmid pYB1S-IDH K247R .

[0051] 3. Using the recombinant plasmid pYB1S-IDH as a template, the upstream primer F4 and the downstream primer R4 as primers, reverse PCR amplification was performed to obtain the mutant plasmid pYB1S-IDH P257G .

[0052] 4. Using the recombinant plasmid pYB1S-IDH as a template, the upstream primer F5 and the downstream primer R5 as primers, reverse PCR amplification was performed to obtain the mutant plasmid pYB1S-IDH E266D .

[0053] The mutation sites and the primer sequences used in the above reverse PCR are shown in Table 7.

[0054] Table 5

[0055] Ingredients Volume (μL) sterile ddH2O 5 10 x buffer for PCR (10 x PCR buffer) 5 2 mM dNTPS (2 mM deoxyribonucleotide) 5 Upstream primer 1.5 Downstream primer 1.5 Template pYB1S-IDH 1 KDO-Plus (high-fidelity PCR enzyme) 1

[0056] Table 6

[0057] Temperature Time Cycle number 95℃ 1 min 95℃ 10s 30 54℃ 15s 30 72℃ 1 min 30 4℃ ∞

[0058] Table 7

[0059]

[0060] Elimination of templates: After the completion of each of the above reverse PCR reactions, 2 μL of restriction endonuclease Dpn I was added to the reaction liquid, the reaction liquid was gently blown with a pipette to mix the enzyme and the reaction liquid, and then the mixture was reacted at 37°C for 1 h to obtain four enzyme digestion liquids. The four enzyme digestion liquids were verified by agarose gel electrophoresis.

[0061] Self-circularization of the reverse PCR product: the reaction liquid of the above obtained enzyme digestion liquids was prepared according to Table 8, gently mixed, and reacted at 16°C for 1 h to obtain four mutant plasmids containing the inositol dehydrogenase mutant genes.

[0062] Table 8

[0063] Ingredients Volume (μL) Enzyme digestion solution 2 Ligation high (high-efficiency ligation reagent) 5 T4 Polynucleotide Kinase 1 sterile ddH2O 7

[0064] Mutant plasmid verification: the four reaction products obtained after the above cyclization were respectively transformed into four groups of E. coli DH5α competent cells by chemical transformation method. The specific method was as follows: the above reaction products were added into E. coli DH5α competent cells, the mixed reaction system was placed on ice for 30 min, then was heat shocked at 42℃ water bath for 45 s, and then was incubated on ice for 2 min, was transferred into 500 μL of LB liquid medium, and was shaken at 37℃ for 1 h for recovery, and 200 μL of bacterial liquid was plated. Single colony on the plate was picked for plasmid extraction, and the extracted plasmid was subjected to DNA sequencing.

[0065] Mutant plasmid pYB1S-IDH S159T containing the gene of myo-inositol dehydrogenase mutant S159T, and the sequence of the gene is shown as SEQ ID NO. 7.

[0066] Mutant plasmid pYB1S-IDH K247R containing the gene of myo-inositol dehydrogenase mutant K247R, and the sequence of the gene is shown as SEQ ID NO. 8.

[0067] Mutant plasmid pYB1S-IDH P257G containing the gene of myo-inositol dehydrogenase mutant P257G, and the sequence of the gene is shown as SEQ ID NO. 9.

[0068] Mutant plasmid pYB1S-IDH E266D containing the gene of myo-inositol dehydrogenase mutant E266D, and the sequence of the gene is shown as SEQ ID NO. 10.

[0069] Example 3

[0070] 1 μL of each of the recombinant plasmid pYB1S-IDH verified successfully in Example 1, the mutant plasmid pYB1S-IDH S159T , the mutant plasmid pYB1S-IDH K247R , the mutant plasmid pYB1S-IDH P257G , and the mutant plasmid pYB1S-IDH E266D were respectively transformed into E. coli DH5α competent cells by chemical transformation method, and positive transformants were screened to obtain recombinant strain 1 containing the recombinant plasmid pYB1S-IDH, recombinant strain 2 containing the mutant plasmid pYB1S-IDH S159T , recombinant strain 3 containing the mutant plasmid pYB1S-IDH K247R , recombinant strain 4 containing the mutant plasmid pYB1S-IDH P257G , and recombinant strain 5 containing the mutant plasmid pYB1S-IDH E266D .

[0071] The above recombinant strains 1-5 were respectively subjected to seed culture in liquid LB medium, and were respectively cultured at 37℃, 120 rpm for 14 h to obtain seed liquid of the recombinant strains 1-5. The seed liquid of the recombinant strains 1-5 was respectively inoculated into another 5 LB mediums at a volume ratio of 2% for fermentation culture, and was cultured at 37℃ until the OD 600 value was 0.7, and then was cooled to 18℃, and was respectively induced by adding IPTG at a final concentration of 0.3 mM to culture, and was induced to culture until the OD 600 value was 10 to obtain fermentation liquid 1 containing wild-type inositol dehydrogenase, fermentation liquid 2 containing inositol dehydrogenase mutant S159T, fermentation liquid 3 containing inositol dehydrogenase mutant K247R, fermentation liquid 4 containing inositol dehydrogenase mutant P257G, and fermentation liquid 5 containing inositol dehydrogenase mutant E266D, respectively.

[0072] The above fermentation liquids 1-5 were respectively centrifuged at 4000 r / min for 15 min at 4℃ to collect the bacterial bodies. The obtained 5 bacterial bodies were respectively resuspended using 100 mM phosphate buffer with a pH value of 7.5, and were respectively broken after resuspension. The cells were broken by ultrasonic crushing at a power of 400 W, and were stopped for 2 s for every 3 s of ultrasonic crushing for a total of 25 min. After ultrasonic crushing, the bacterial bodies were respectively centrifuged at 4000 r / min for 15 min at 4℃ to discard the precipitates, and 5 supernatants were respectively obtained, which were crude enzyme liquid 1 of wild-type inositol dehydrogenase, crude enzyme liquid 2 of inositol dehydrogenase mutant S159T, crude enzyme liquid 3 of inositol dehydrogenase mutant K247R, crude enzyme liquid 4 of inositol dehydrogenase mutant P257G, and crude enzyme liquid 5 of inositol dehydrogenase mutant E266D, respectively.

[0073] The amino acid sequence of the wild-type inositol dehydrogenase is shown in SEQ ID NO. 1;

[0074] The amino acid sequence of the inositol dehydrogenase mutant S159T is shown in SEQ ID NO. 2;

[0075] The amino acid sequence of the inositol dehydrogenase mutant K247R is shown in SEQ ID NO. 3;

[0076] The amino acid sequence of the inositol dehydrogenase mutant P257G is shown in SEQ ID NO. 4;

[0077] The amino acid sequence of the inositol dehydrogenase mutant E266D is shown in SEQ ID NO. 5.

[0078] Example 4

[0079] Determination of enzyme activity of inositol dehydrogenase and mutants thereof

[0080] Reaction system (5 mL) for measuring enzyme activity: 30 mM myo-inositol, 1.5 mM NADP + , 100 mM phosphate buffer with pH value of 7.5, and the above-mentioned crude enzyme solution was added respectively to make the concentration of enzyme protein in the reaction system 5 mg / mL.

[0081] The above-mentioned reaction system was reacted at 35℃ for 5 min to obtain a reaction solution.

[0082] The above-mentioned reaction was carried out in the presence of NADP + , and the myo-inositol dehydrogenase in the crude enzyme solution could reversibly catalyze the conversion of myo-inositol into scyllitol, and NADP + was converted into NADPH.

[0083] Definition of enzyme activity: the amount of enzyme required for consuming 1 μmol myo-inositol within 1 min under the above-mentioned reaction system and conditions.

[0084] Method for calculating enzyme activity: enzyme activity (U) = (ΔA x V x 103) / (6220 x 1).

[0085] Wherein, ΔA refers to the change of absorbance value at 340 nm within 1 min; V is the total volume of the reaction system (mL); 6220 refers to the molar extinction coefficient of NADPH (L·mol -1 cm -1 ); and l refers to the optical path distance, i.e. the light path of the cuvette (cm).

[0086] The results are shown in Table 9.

[0087] Table 9

[0088] Enzyme Relative enzyme activity Wild-type inositol dehydrogenase 100％ Inositol dehydrogenase mutant S159T 43％ Inositol dehydrogenase mutant K247R 357％ Inositol dehydrogenase mutant P257G 135％ Inositol dehydrogenase mutant E266D 187％

[0089] From the above results, it can be seen that the enzyme activity of the myo-inositol dehydrogenase mutant K247R, the myo-inositol dehydrogenase mutant P257G and the myo-inositol dehydrogenase mutant E266D is further improved compared with the wild-type myo-inositol dehydrogenase.

[0090] Example 5

[0091] Preparation of D-chiral inositol

[0092] A reaction system of 5 mL was configured, and the concentration of each component in the reaction system was as follows:

[0093] 10 g / L myo-inositol, 2 mM NADP + , 4 mg / mL ketoisomerase, 100 mM phosphate buffer with pH value of 7.5, and the crude enzyme solution 1 was added to make the concentration of enzyme protein in the reaction system 5 mg / mL.

[0094] 10 g / L myo-inositol, 2 mM NADP + 4 mg / mL ketol-isomerase, 100 mM phosphate buffer, pH 7.5, and crude enzyme solution 3 was added to make the concentration of enzyme protein in the reaction system 5 mg / mL.

[0095] 10 g / L myo-inositol, 2 mM NADP + 4 mg / mL ketol-isomerase, 100 mM phosphate buffer, pH 7.5, and crude enzyme solution 4 was added to make the concentration of enzyme protein in the reaction system 5 mg / mL.

[0096] 10 g / L myo-inositol, 2 mM NADP + 4 mg / mL ketol-isomerase, 100 mM phosphate buffer, pH 7.5, and crude enzyme solution 5 was added to make the concentration of enzyme protein in the reaction system 5 mg / mL.

[0097] The above reaction systems were respectively reacted at 35°C for 5 min, and reaction solutions were respectively obtained. The concentration of D-chiro-inositol in the reaction solution was detected by high performance liquid chromatography, and the conversion rate of substrate myo-inositol was calculated. The results are shown in Table 10.

[0098] Table 10

[0099] Crude enzyme solution Yield of D-chiral inositol Conversion rate (%) Wild-type inositol dehydrogenase crude enzyme solution 1 1.044 g / L 10.44 Inositol dehydrogenase mutant K247R crude enzyme solution 3 2.436 g / L 24.36 Inositol dehydrogenase mutant P257G crude enzyme solution 4 0.98 g / L 9.8 Inositol dehydrogenase mutant E266D crude enzyme solution 5 1.724 g / L 17.24

[0100] From the above results, compared with the wild-type inositol dehydrogenase, the inositol dehydrogenase mutant K247R and the inositol dehydrogenase mutant E266D not only have higher enzyme activity, but also have a one-way reaction characteristic, which can make the reaction more inclined to the forward reaction, thereby further improving the yield of D-chiro-inositol and the conversion rate of substrate myo-inositol.

[0101] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can modify the technical solutions described in the foregoing embodiments or make equivalent replacements to some technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A mutant of myo-inositol dehydrogenase, characterized in that, The myo-inositol dehydrogenase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO. 1, and the mutation is that: the amino acid at position 247 is mutated from K to R, or the amino acid at position 266 is mutated from E to D.

2. The inositol dehydrogenase mutant according to claim 1, characterized in that, The amino acid sequence of the myo-inositol dehydrogenase mutant is shown in SEQ ID NO. 3 or SEQ ID NO.

5.

3. A gene encoding a gene, characterized in that, A nucleotide sequence encoding the myo-inositol dehydrogenase mutant of claim 1 or 2.

4. An expression vector, characterized by, The expression vector contains the coding gene of claim 3.

5. A recombinant bacterial strain, characterized in that, The recombinant strain contains the coding gene of claim 3 or the expression vector of claim 4.

6. A method for producing the myo-inositol dehydrogenase mutant according to claim 1 or 2, characterized by, The method comprises the following steps: The recombinant strain of claim 5 is cultured in LB medium at 35-40°C and 120-220 r / min for 12-16 hours to obtain a seed liquid; The seed liquid is inoculated into another LB medium for fermentation culture at an inoculation amount of 1% to 5% by volume, and cultured at 35°C to 40°C until the OD 600 value reaches 0.6 to 0.8, and then the temperature is lowered to 16°C to 25°C, and IPTG is added at a final concentration of 0.1 mM to 0.5 mM for induction culture, and the induction culture is performed until the OD 600 value reaches 6 to 10, and a fermentation liquid containing the myo-inositol dehydrogenase mutant is obtained. The bacterial cells are collected by centrifugation, resuspended, and then the cells are broken, and the supernatant obtained by centrifugation is the crude enzyme solution of the myo-inositol dehydrogenase mutant.

7. Use of the myo-inositol dehydrogenase mutant of claim 1 or 2 in the preparation of D-chiro-inositol.

8. A process for the preparation of D-chiro-inositol, characterized in that, with muscle inositol, NADP + using the inositol dehydrogenase mutant according to claim 1 or 2 and the ketol isomerase to synthesize D-chiral inositol.

9. The production method according to claim 8, characterized by, The concentrations of the components in the reaction system for catalytically synthesizing D-chiro-inositol are as follows: 10g / L to 15g / L myo-inositol, 1mM to 3mM NADP + myo-inositol dehydrogenase mutant crude enzyme solution with a protein concentration of 4mg / mL to 6mg / mL, 3mg / mL to 5mg / mL ketol-isomerase, 80mM to 120mM phosphate buffer.

10. The method of claim 9, wherein, The reaction temperature of the reaction system is 30-37°C, and the pH value is 7.0-8.0.

Images