A ketol-isomerase mutant, its preparation method and application

Abstract

The application discloses a ketol-isomerase mutant, a preparation method and application thereof, and relates to the technical field of genetic engineering. The ketol-isomerase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO. 2 at the 34th amino acid from N to R and simultaneously mutating the 188th amino acid from I to K. The half-life of the ketol-isomerase mutant obtained by mutating the amino acid sequence shown in SEQ ID NO. 2 is prolonged, and the enzyme activity is increased, so that the yield of D-chiro-inositol is further improved.

Description

TECHNICAL FIELD

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

[0002] D-chiro-inositol (DCI) is one of nine isomers of inositol with optical activity. In recent years, people have found that D-chiro-inositol has special physiological functions such as insulin-sensitizing effect, blood glucose-lowering effect, improving ovulation of polycystic ovary syndrome patients, regulating hormone balance, improving menstrual disorders, and antioxidant, anti-aging and anti-inflammatory effects in addition to promoting liver lipid metabolism.

[0003] The double-enzyme combination of ketol-isomerase and inositol dehydrogenase can convert the substrate myo-inositol into D-chiro-inositol, but the enzyme activity of the ketol-isomerase in the prior art is low, the half-life is short, and the heat resistance is poor, which limits the overall conversion rate and is the key bottleneck limiting the production of D-chiro-inositol.

[0004] Therefore, the traditional technology needs to be improved. SUMMARY

[0005] The present application aims to provide a ketol-isomerase mutant and a preparation method and application thereof, which can overcome the defects of low enzyme activity, short half-life and poor heat resistance of the ketol-isomerase, thereby further improving the conversion rate of myo-inositol and the yield of D-chiro-inositol.

[0006] In a first aspect, the present application provides a ketol-isomerase mutant, which is obtained by mutating the amino acid sequence shown in SEQ ID NO. 2 as follows:

[0007] The 34th amino acid is mutated from N to R, and the 188th amino acid is mutated from I to K.

[0008] Compared with the prior art, after the above mutation of the ketol-isomerase shown in SEQ ID NO. 2, the thermal stability of the obtained ketol-isomerase mutant at 35℃ and 40℃ is improved, the half-life is prolonged, and the enzyme activity is improved, thereby further improving the conversion rate of myo-inositol and the yield of D-chiro-inositol.

[0009] Further, the amino acid sequence of the ketol-isomerase mutant is shown in SEQ ID NO. 10. The ketol-isomerase mutant is N34R / I188K.

[0010] In a second aspect, the present application provides a nucleic acid molecule encoding the ketol-isomerase mutant N34R / I188K described above.

[0011] Further, the nucleic acid molecule sequence encoding the ketol-isomerase mutant N34R / I188K is shown as SEQ ID NO. 9.

[0012] In a third aspect, the present application provides an expression vector containing the nucleic acid molecule mentioned above.

[0013] In a fourth aspect, the present application provides a recombinant strain containing the nucleic acid molecule or the expression vector.

[0014] In a fifth aspect, the present application provides a preparation method of the ketol-isomerase mutant N34R / I188K, for preparing the ketol-isomerase mutant N34R / I188K, comprising the following steps:

[0015] Seeding culture of the recombinant strain in LB medium to obtain a seed liquid;

[0016] The seed liquid is inoculated into another LB medium at a volume ratio of 1% to 5% to carry out fermentation culture until the OD value reaches 0.6 to 0.8, the temperature is lowered, and IPTG is added at a final concentration of 1 mM to 1.5 mM to induce culture until the OD value reaches 4 to 7. 600 600 The fermentation liquid containing the ketol-isomerase mutant N34R / I188K is obtained.

[0017] 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 ketol-isomerase mutant N34R / I188K.

[0018] Compared with the prior art, the present application uses a recombinant strain to prepare the ketol-isomerase mutant N34R / I188K, because the recombinant strain contains a nucleic acid molecule expressing the ketol-isomerase mutant N34R / I188K, and thus the crude enzyme liquid of the ketol-isomerase mutant N34R / I188K can be obtained by culturing and fermenting the recombinant strain, which improves the production efficiency, is simple and easy to operate, and can quickly obtain the crude enzyme liquid, reducing the cumbersome operation and time cost in the preparation process.

[0019] Further, the temperature of the seeding culture is 35℃ to 38℃, the rotation speed is 120r / min to 220r / min, and the culture time is 10h to 15h.

[0020] Further, the temperature of the fermentation culture is 35℃ to 38℃.

[0021] Further, the temperature is lowered to 16℃ to 20℃.

[0022] ​In a sixth aspect, the present application provides the use of the ketol-isomerase mutant N34R / I188K or the ketol-isomerase mutant N34R / I188K crude enzyme in the preparation of D-chiro-inositol.

[0023] Compared with the prior art, the ketol-isomerase mutant N34R / I188K or the ketol-isomerase mutant N34R / I188K crude enzyme in the present application has improved stability, prolonged half-life and increased enzyme activity, and can significantly improve the conversion rate of myo-inositol and the yield of D-chiro-inositol after mixing with myo-inositol.

[0024] In a seventh aspect, the present application provides a method for preparing D-chiro-inositol, which comprises adding myo-inositol, NADP + , the ketol-isomerase mutant N34R / I188K crude enzyme and myo-inositol dehydrogenase in a phosphate buffer to generate D-chiro-inositol.

[0025] Compared with the prior art, the ketol-isomerase mutant N34R / I188K crude enzyme is added to the reaction system in the present application, the half-life of the ketol-isomerase mutant N34R / I188K in the present application is significantly prolonged, the enzyme activity is higher, and the conversion rate of myo-inositol and the yield of D-chiro-inositol can be further improved.

[0026] Further, the concentrations of the components in the reaction system are as follows:

[0027] myo-inositol 8 mg / mL~12 mg / mL, NADP + 1 mM~3 mM, the ketol-isomerase mutant N34R / I188K crude enzyme 2 mg / mL~4 mg / mL, myo-inositol dehydrogenase 2 mg / mL~4 mg / mL, and phosphate buffer 80 mM~120 mM.

[0028] Further, the reaction temperature is 30℃~37℃, and the reaction pH value is 7.0~8.0. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Residual enzyme activity of wild-type ketol-isomerase crude enzyme and ketol-isomerase mutant N34R / I188K crude enzyme after incubation at 35℃ for 0h, 2h, 4h, 6h, 8h and 10h.

[0030] Figure 2 Residual enzyme activity of wild-type ketol-isomerase crude enzyme and ketol-isomerase mutant N34R / I188K crude enzyme after incubation at 40℃ for 0h, 2h, 4h, 6h, 8h and 10h. DETAILED DESCRIPTION

[0031] In order to make the technical problems to be solved by the present application, technical solutions and beneficial effects clearer, 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 not used to limit the present application.

[0032] It should be understood that the raw materials used in the following examples are commercially available unless otherwise specified.

[0033] Construction of recombinant plasmid pET28a-KMI in Example 1

[0034] After codon optimization of the nucleotide of wild-type ketol-isomerase, sequence 1 was obtained. Upstream primer F1 and downstream primer R1 were designed based on sequence 1 as a template, and PCR amplification was performed to obtain KMI target gene fragment with homologous arms of Hind Ⅲ and Xho Ⅰ enzyme cutting sites. The PCR amplification reaction system is shown in Table 1, and the PCR amplification reaction conditions are shown in Table 2.

[0035] The nucleotide sequence of sequence 1 is shown in SEQ ID NO. 1, and the amino acid sequence of the wild-type ketol-isomerase encoded by sequence 1 is shown in SEQ ID NO. 2.

[0036] F1: 5'-TCGAGCTCCGTCGACAAGCTTATGAAGACTACTCTGAACCACATGAC-3', SEQ ID NO. 3;

[0037] R1: 5'-GTGGTGGTGGTGGTGCTCGAGTTAAGCAGCACGTGCCTGC-3', SEQ ID NO. 4.

[0038] Table 1 PCR amplification reaction system

[0039]

[0040] Table 2 PCR amplification reaction conditions

[0041]

[0042] After the above-mentioned PCR amplification, the reaction product was recovered by agarose gel to obtain a KMI gene fragment with higher purity.

[0043] The expression vector pET28a was double digested using restriction enzymes Hind Ⅲ and Xho Ⅰ, and the double-digested product was recovered and purified to obtain a linearized vector pET28a with enzyme cutting sites. The enzyme cutting system is shown in Table 3.

[0044] Table 3 Enzyme cutting system

[0045]

[0046] The KMI gene fragment amplified by PCR was ligated into the linearized vector pET28a between the Hind III and Xho I enzyme sites to obtain the recombinant plasmid pET28a-KMI. The ligation system is shown in Table 4. The reaction temperature was 37°C, and the reaction time was 30 min.

[0047] Table 4 Ligation system

[0048]

[0049] After the ligation was completed, the ligation product was transformed into E. coli DH5a competent cells by chemical transformation, a single colony was picked for plasmid extraction, and the extracted plasmid was subjected to DNA sequencing.

[0050] Example 2 Construction of mutant plasmid

[0051] The recombinant plasmid pET28a-KMI of Example 1 was used as a template, and F-N34R and R-N34R were used as primers to perform reverse PCR amplification to obtain pET28a-KMI N34R .

[0052] The reverse PCR amplification reaction system is shown in Table 5, and the amplification reaction conditions are shown in Table 6.

[0053] Table 5 Reverse PCR amplification reaction system

[0054]

[0055] Table 6 Reverse PCR amplification reaction conditions

[0056]

[0057] Then, pET28a-KMI N34R was used as a template, and F-N34R / I188K and R-N34R / I188K were used as primers to perform reverse PCR amplification to obtain pET28a-KMI N34R / I188K . The reverse PCR amplification reaction system is shown in Table 7, and the amplification reaction conditions are shown in Table 6.

[0058] Table 7 Reverse PCR amplification reaction system

[0059]

[0060] Among them:

[0061] F-N34R: 5'-AGGTCCGTCGCGATATCGCTCGTCCGCTGTT-3', SEQ ID NO. 5;

[0062] R-N34R: 5'-GATATCGCGACGGACCTCAACACCGATACA-3', SEQ ID NO. 6;

[0063] F-N34R / I188K: 5'-GACGGGCAAAGTCCATATTTCCGCTGTTACGG-3', SEQ ID NO. 7;

[0064] R-N34R / I188K: 5'-TATGGACTTTGCCCGTCTGTTCCGGGTA-3', SEQ ID NO. 8.

[0065] Elimination of template: after the above reverse PCR reaction is completed, 2.5 μL of restriction endonuclease Dpn I is taken and added to the reaction solution (25 μL) for gentle blowing and mixing, and the enzyme digestion solution is verified by agarose gel electrophoresis.

[0066] Self-circularization of reverse PCR product: using the above obtained enzyme digestion solution, the reaction solution is prepared according to Table 8, gently mixed, and placed at 16°C for 1 h to obtain the mutant plasmid pET28a-KMI N34R / I188K containing the ketol-isomerase mutant gene.

[0067] It should be understood that the template is eliminated and the self-circularization of the reverse PCR product is performed after each reverse PCR is completed.

[0068] Table 8 Reaction solution

[0069]

[0070] Mutant plasmid verification: the mutant plasmid obtained after the above circularization is transformed into E. coli DH5α competent cells by chemical transformation method, a single colony on the plate is picked for plasmid extraction, and the extracted plasmid is subjected to DNA sequencing. Among them, the mutant plasmid pET28a-KMI N34R / I188K containing the ketol-isomerase mutant N34R / I188K has a nucleotide sequence as shown in SEQ ID NO. 9, and an encoded amino acid sequence as shown in SEQ ID NO. 10.

[0071] Example 3 Preparation of crude enzyme solution

[0072] Take 1 μL of the above mutant plasmid pET28a-KMI N34R / I188KThe mixed reaction system was placed on ice for 30 min, and then heat shocked at 42°C for 60 s, followed by incubation on ice for 5 min to obtain the recombinant strain. Then the recombinant strain was transferred into 500 μL of LB liquid medium and shaken at 37°C for 1 h to recover, and 100 μL of the bacterial liquid was plated to screen positive transformants (i.e. mutant strains) containing the mutant plasmid pET28a-KMI N34R / I188K .

[0073] The mutant strain obtained by screening was seed cultured in liquid LB medium at 37°C and 120 rpm for 14 h to obtain a seed liquid of the mutant strain.

[0074] The seed liquid of the mutant strain was inoculated into new LB liquid medium at a volume ratio of 2% for fermentation culture at 37°C until the OD 600 value was 0.7, and then the temperature was lowered to 18°C, and 1.0 mM of IPTG was added for induction culture until the OD 600 value reached 5 to obtain a fermentation liquid containing the ketol-isomerase mutant N34R / I188K.

[0075] The fermentation liquid containing the ketol-isomerase mutant N34R / I188K was centrifuged at 4°C and 4000 r / min for 15 min to collect the bacterial cells, and the collected bacterial cells were resuspended with phosphate buffer having a pH value of 7.5. Then the bacterial cells were broken by using an ultrasonic cell crusher, the ultrasonic power was 450 W, and each breaking was for 3 s and stopping for 2 s, and the total ultrasonic time was 30 min. After the ultrasonic breaking, the cell debris was removed by centrifugation at 4°C and 12000 r / min, and the supernatant was collected to obtain a crude enzyme liquid of the ketol-isomerase mutant N34R / I188K, and the concentration of the crude enzyme of the ketol-isomerase mutant N34R / I188K in the crude enzyme liquid was 4 mg / mL.

[0076] 1 μL of the recombinant plasmid pET28a-KMI correctly sequenced in Example 1 was taken to prepare a crude enzyme liquid of the wild-type ketol-isomerase by the same method as above, and the concentration of the crude enzyme of the wild-type ketol-isomerase in the crude enzyme liquid was 4 mg / mL.

[0077] Example 4: Detection of enzyme activity and half-life

[0078] In a phosphate buffer having a pH value of 7.5 and a concentration of 100 mM, muscle inositol, NADP + , a crude enzyme liquid of the wild-type ketol-isomerase, and an inositol dehydrogenase enzyme liquid were added to prepare 10 mL of a reaction system one, and the concentration of each component in the reaction system one was as follows: muscle inositol 30 mM, NADP +1.5mM, wild type ketol-isomerase crude enzyme 3mg / mL, myo-inositol dehydrogenase 3mg / mL.

[0079] Meanwhile, in 100mM phosphate buffer solution with pH value of 7.5, myo-inositol, NADP + , ketol-isomerase mutant N34R / I188K crude enzyme solution, myo-inositol dehydrogenase enzyme solution, 10mL of reaction system two was configured, and the concentration of each component in the reaction system two was: myo-inositol 30mM, NADP + 1.5mM, ketol-isomerase mutant N34R / I188K crude enzyme 3mg / mL, myo-inositol dehydrogenase 3mg / mL.

[0080] The above two reaction systems were respectively reacted at 35℃ for 5min, and the enzyme activity was measured.

[0081] Enzyme activity definition: the amount of enzyme required to consume 1μmol of myo-inositol within 1min under the above reaction system and conditions.

[0082] The relative enzyme activity of the ketol-isomerase mutant N34R / I188K was calculated with the enzyme activity of the wild type ketol-isomerase as 100%, and the results are shown in Table 9.

[0083] Table 9 Enzyme activity determination results

[0084]

[0085] From the above results, it can be seen that the enzyme activity of the ketol-isomerase mutant N34R / I188K crude enzyme is significantly improved compared with the wild type ketol-isomerase crude enzyme.

[0086] The above ketol-isomerase mutant N34R / I188K crude enzyme solution and wild type ketol-isomerase crude enzyme solution were respectively incubated at 35℃ and 40℃ for 0h, 2h, 4h, 6h, 8h and 10h, then the crude enzyme solutions incubated at different temperatures and for different times were configured into reaction systems according to the above reaction system one and reaction system two, and the residual enzyme activity of the ketol-isomerase mutant N34R / I188K crude enzyme and the wild type ketol-isomerase crude enzyme after incubation for different times was measured according to the above enzyme activity calculation method, with the enzyme activity of incubation for 0h as 100%, to obtain the half-life at 35℃ and 40℃. The results are shown in Figure 1 and Figure 2 .

[0087] From Figure 1 and Figure 2 , it can be seen that the thermal stability of the ketol-isomerase mutant N34R / I188K crude enzyme is improved compared with the wild type ketol-isomerase crude enzyme at 35℃ and 40℃, the half-life at 35℃ is prolonged from 3h to 6h, and the half-life at 40℃ is improved from 2h to 3.7h.

[0088] Example 5 Preparation of D-chiro-inositol

[0089] In a 100 mM phosphate buffer solution with pH value of 7.5, add myo-inositol, NADP + , wild-type ketol-isomerase crude enzyme, myo-inositol dehydrogenase enzyme solution, to configure 10 mL of reaction system three, the concentration of each component in the reaction system three is: myo-inositol 10 mg / mL, NADP + 2 mM, wild-type ketol-isomerase crude enzyme 3 mg / mL, myo-inositol dehydrogenase 3 mg / mL.

[0090] In a 100 mM phosphate buffer solution with pH value of 7.5, add myo-inositol, NADP + , ketol-isomerase mutant N34R / I188K crude enzyme solution, myo-inositol dehydrogenase enzyme solution, to configure 10 mL of reaction system four, the concentration of each component in the reaction system four is: myo-inositol 10 mg / mL, NADP + 2 mM, ketol-isomerase mutant N34R / I188K crude enzyme 3 mg / mL, myo-inositol dehydrogenase 3 mg / mL.

[0091] The above reaction system three and reaction system four are respectively reacted at 35°C for 1 h, and reaction solutions are respectively obtained, and the content of D-chiro-inositol in the reaction solution is detected by high performance liquid chromatography, and the conversion rate of the substrate myo-inositol is calculated.

[0092] The detection condition is that the chromatographic column is 4.6*250 mm in size, the amino column is 5 μm, the mobile phase is a mixture of acetonitrile and 50 mM ammonium acetate aqueous solution in a volume ratio of 75:25, the column temperature is set to 30°C, and the flow rate is 1.0 mL / min. The detection is performed by using a differential refractometer detector, and the detection result is shown in Table 10.

[0093] The conversion rate = the content of generated D-chiro-inositol / the initial content of myo-inositol.

[0094] Table 10 D-chiro-inositol content and conversion rate of myo-inositol

[0095]

[0096] From the above results, it can be seen that, compared with the wild-type ketol-isomerase crude enzyme, the ketol-isomerase mutant N34R / I188K crude enzyme in the application can further improve the conversion rate of the substrate myo-inositol, thereby further improving the yield of D-chiro-inositol.

[0097] The above merely describes the preferred embodiments of the present application and is not used to limit the present application, and although the present application is described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments, or equivalently replace some technical features thereof. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A ketoisomerase mutant, characterized in that, The ketoisomerase mutant was obtained by mutagenesis of the amino acid sequence shown in SEQ ID NO.2 as follows: The 34th amino acid is mutated from N to R; at the same time, the 188th amino acid is mutated from I to K. The amino acid sequence of the ketoisomerase mutant is shown in SEQ ID NO.

10.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the ketoisomerase 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. A method for preparing a ketoisomerase mutant, used to prepare the ketoisomerase mutant of claim 1, characterized in that, Includes the following steps: The recombinant strain described in claim 4 was seed cultured in LB medium to obtain a seed solution; The seed culture was inoculated into another LB medium at an inoculum volume of 1% to 5%, and fermented until OD reached. 600 Once the pH reaches 0.6-0.8, cool the temperature and add IPTG to a final concentration of 1-1.5 mM for induction culture until OD reaches 0.

6. 600 The ratio was 4-7, and a fermentation broth containing a ketoisomerase mutant was obtained. The fermentation broth was centrifuged to collect the bacterial cells. After the bacterial cells were resuspended, the cells were broken up. The supernatant obtained by centrifugation was the crude enzyme solution of the ketoisomerase mutant.

6. The preparation method according to claim 5, characterized in that, The seed culture temperature is 35℃~38℃, the rotation speed is 120r / min~220r / min, and the culture time is 10h~15h; and / or, The fermentation culture temperature is 35℃~38℃; and / or, The cooling refers to reducing the temperature to 16℃~20℃.

7. The application of the ketoisomerase mutant of claim 1, or the crude enzyme solution of the ketoisomerase mutant prepared by the preparation method of claim 5, in the preparation of D-chiral inositol.

8. A method for preparing D-chiral inositol, characterized in that, Add muscle inositol and NADP to phosphate buffer. + The crude enzyme solution of the ketoisomerase mutant prepared by the preparation method described in claim 5, and inositol dehydrogenase, react to generate D-chiral inositol.

9. The preparation method according to claim 8, characterized in that, The concentrations of each component in the reaction system are as follows: Muscle inositol 8mg / mL~12mg / mL, NADP + 1 mM~3 mM, crude ketoisomerase mutant 2 mg / mL~4 mg / mL, inositol dehydrogenase 2 mg / mL~4 mg / mL, phosphate buffer 80 mM~120 mM; and / or, The reaction temperature is 30℃~37℃, and the reaction pH is 7.0~8.0.

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