CN121022813A - A ketoisomerase mutant, its preparation method and application - Google Patents
A ketoisomerase mutant, its preparation method and applicationInfo
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Abstract
本发明公开一种酮基异构酶突变体及其制备方法与应用，涉及基因工程技术领域。酮基异构酶突变体由SEQ ID NO.2所示的氨基酸序列经过如下突变所得：第34位氨基酸由N突变为R；同时，第188位氨基酸由I突变为K。SEQ ID NO.2所示的氨基酸序列经过上述突变获得的酮基异构酶突变体的半衰期延长，酶活升高，从而进一步提高了D‑手性肌醇的产量。
This invention discloses a ketoisomerase mutant, its preparation method, and its applications, relating to the field of genetic engineering technology. The ketoisomerase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.2 as follows: amino acid position 34 is mutated from N to R; simultaneously, amino acid position 188 is mutated from I to K. The ketoisomerase mutant obtained by the above mutation of the amino acid sequence shown in SEQ ID NO.2 has a prolonged half-life and increased enzyme activity, thereby further improving the yield of D-chiral inositol.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a ketoisomerase mutant and a preparation method and application thereof.
Background
D-chiro-inositol (DCI) is one of the nine isomers of inositol that has optical activity. In recent years, research has found that D-chiro-inositol has not only the function of promoting liver lipid metabolism, but also the special physiological functions of resisting oxidation, aging and inflammation, such as insulin sensitization, reducing blood sugar, improving ovulation condition of polycystic ovary syndrome patients, regulating hormone balance, improving menstrual disorder.
The combination of the double enzymes of ketoisomerase and inositol dehydrogenase can convert substrate myo-inositol into D-chiro-inositol, but the ketoisomerase in the prior art has lower enzyme activity, shorter half-life and poorer heat resistance, so that the overall conversion rate is limited, and is a key bottleneck for limiting the yield of D-chiro-inositol.
Accordingly, the conventional technology is in need of improvement.
Disclosure of Invention
The invention aims to provide a ketoisomerase mutant, a preparation method and application thereof, which are used for overcoming the defects of low enzyme activity, short half-life period, poor heat resistance and limited overall conversion rate of the ketoisomerase.
In a first aspect, the invention provides a ketoisomerase mutant obtained from the amino acid sequence shown in SEQ ID NO.2 by mutation of:
The 34 th amino acid is mutated from N to R, and the 188 th amino acid is mutated from I to K.
Compared with the prior art, after the ketoisomerase shown in SEQ ID NO.2 is subjected to the mutation, the obtained ketoisomerase mutant has the advantages of improved thermal stability at 35 ℃ and 40 ℃, prolonged half-life and improved enzyme activity, so that the conversion rate of myo-inositol and the yield of D-chiral inositol are further improved.
Further, the amino acid sequence of the ketoisomerase mutant is shown in SEQ ID NO. 10. Wherein the ketoisomerase mutant is N34R/I188K.
In a second aspect, the invention provides a nucleic acid molecule encoding a ketoisomerase mutant N34R/I188K as described above.
Further, the nucleic acid molecule sequence for encoding the ketoisomerase mutant N34R/I188K is shown in SEQ ID NO. 9.
In a third aspect, the present invention provides an expression vector comprising a nucleic acid molecule as described above.
In a fourth aspect, the present invention provides a recombinant strain comprising said nucleic acid molecule or said expression vector.
In a fifth aspect, the invention provides a method for preparing a ketoisomerase mutant N34R/I188K, which is used for preparing the ketoisomerase mutant N34R/I188K, and comprises the following steps:
Seed culture is carried out on the recombinant strain in an LB culture medium to obtain seed liquid;
Inoculating the seed liquid into another LB culture medium according to the inoculum size of 1% -5% by volume, fermenting and culturing until the OD 600 value reaches 0.6-0.8, cooling, adding IPTG with the final concentration of 1 mM-1.5 mM, and performing induction culture until the OD 600 is 4-7, thus obtaining a fermentation liquid containing a ketoisomerase mutant N34R/I188K;
and centrifugally collecting thalli from the fermentation liquor, crushing cells after the thalli are re-suspended, and obtaining supernatant by centrifugation, namely the ketoisomerase mutant N34R/I188K crude enzyme solution.
Compared with the prior art, the method for preparing the ketoisomerase mutant N34R/I188K by using the recombinant strain has the advantages that the recombinant strain contains the nucleic acid molecule for expressing the ketoisomerase mutant N34R/I188K, so that the ketoisomerase mutant N34R/I188K crude enzyme liquid can be obtained by culturing and fermenting the recombinant strain, the production efficiency is improved, the operation is simple and easy, the crude enzyme liquid can be quickly obtained, and the complicated operation and time cost in the preparation process are reduced.
Further, the temperature of seed culture is 35-38 ℃, the rotating speed is 120-220 r/min, and the culture time is 10-15 h.
Further, the temperature of the fermentation culture is 35-38 ℃.
Further, the temperature is reduced to 16-20 ℃.
In a sixth aspect, the invention provides the use of said ketoisomerase mutant N34R/I188K or said ketoisomerase mutant N34R/I188K crude enzyme solution in the preparation of D-chiro-inositol.
Compared with the prior art, the ketoisomerase mutant N34R/I188K or the ketoisomerase mutant N34R/I188K crude enzyme solution has the advantages of improved stability, prolonged half-life period and improved enzyme activity, and can obviously improve the conversion rate of myo-inositol and the yield of D-chiral inositol after being mixed with myo-inositol.
In a seventh aspect, the invention provides a method for preparing D-chiro-inositol, wherein myo-inositol, NADP +, ketoisomerase mutant N34R/I188K crude enzyme solution and inositol dehydrogenase are added into phosphate buffer solution to react to generate D-chiro-inositol.
Compared with the prior art, the invention adds the crude enzyme solution of the ketoisomerase mutant N34R/I188K in the reaction system, wherein the half-life period of the ketoisomerase mutant N34R/I188K is obviously prolonged, the enzyme activity is higher, and the conversion rate of myo-inositol and the yield of D-chiral inositol can be further improved.
Further, the concentrations of the components in the reaction system are as follows:
myoinositol 8 mg/mL-12 mg/mL, NADP + mM-3 mM, ketoisomerase mutant N34R/I188K crude enzyme 2 mg/mL-4 mg/mL, inositol dehydrogenase 2 mg/mL-4 mg/mL, and phosphate buffer 80 mM-120 mM.
Further, the reaction temperature is 30-37 ℃, and the reaction pH value is 7.0-8.0.
Drawings
FIG. 1 shows the residual enzyme activities after incubation of the wild-type ketoisomerase crude enzyme and the ketoisomerase mutant N34R/I188K crude enzyme at 35℃for 0h, 2h, 4h, 6h, 8h, 10 h.
FIG. 2 shows the residual enzyme activities after incubation of the wild-type ketoisomerase crude enzyme and the ketoisomerase mutant N34R/I188K crude enzyme at 40℃for 0h, 2h, 4h, 6h, 8h, 10 h.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
It should be understood that the starting materials used in the examples below are commercially available unless otherwise specified.
EXAMPLE 1 construction of recombinant plasmid pET28a-KMI
The nucleotide of the wild ketoisomerase is subjected to codon optimization to obtain a sequence 1, an upstream primer F1 and a downstream primer R1 are designed by taking the sequence 1 as a template, and PCR amplification is carried out to obtain a KMI target gene fragment with HindIII and Xho I restriction enzyme sites homology arms. The PCR amplification reaction system is shown in Table 1, and the PCR amplification reaction conditions are shown in Table 2.
The nucleotide sequence of the sequence 1 is shown as SEQ ID NO.1, and the amino acid sequence of the encoded wild-type ketoisomerase is shown as SEQ ID NO. 2.
F1:5’-TCGAGCTCCGTCGACAAGCTTATGAAGACTACTCTGAACCACATGAC-3’,SEQ ID NO.3;
R1:5’-GTGGTGGTGGTGGTGCTCGAGTTAAGCAGCACGTGCCTGC-3’,SEQ ID NO.4。
TABLE 1PCR amplification reaction System
TABLE 2PCR amplification reaction conditions
And after the PCR amplification is finished, agarose gel recovery is carried out on the reaction product, so as to obtain KMI gene fragments with higher purity.
Double digestion is carried out on the expression vector pET28a by using restriction enzymes Hind III and Xho I, and a double digestion product is recovered and purified, so that a linearization vector pET28a with a digestion site is obtained, and the digestion system is shown in Table 3.
TABLE 3 enzyme digestion system
The KMI gene fragment obtained by the PCR amplification is connected between the HindIII and Xho I cleavage sites of the linearization vector pET28a to obtain a recombinant plasmid pET28a-KMI, and the connection system is shown in Table 4. The reaction temperature was 37℃and the reaction time was 30 min.
Table 4 connection system
After the connection is completed, a chemical conversion method is adopted to convert the connection product into E.coli DH5 alpha competent cells, single colony is selected for plasmid extraction, and the extracted plasmid is subjected to DNA sequencing.
EXAMPLE 2 construction of mutant plasmids
Inverse PCR amplification was performed using the recombinant plasmid pET28a-KMI of example 1 as template and F-N34R and R-N34R as primers to obtain pET28a-KMI N34R.
The inverse PCR amplification reaction system is shown in Table 5, and the amplification reaction conditions are shown in Table 6.
TABLE 5 inverse PCR amplification reaction System
TABLE 6 inverse PCR amplification reaction conditions
Then inverse PCR amplification is carried out by taking pET28a-KMI N34R as a template and taking F-N34R/I188K and R-N34R/I188K as primers to obtain pET28a-KMI N34R/I188K. The inverse PCR amplification reaction system is shown in Table 7, and the amplification reaction conditions are shown in Table 6.
TABLE 7 inverse PCR amplification reaction System
Wherein:
F-N34R:5’-AGGTCCGTCGCGATATCGCTCGTCCGCTGTT-3’,SEQ ID NO.5;
R-N34R:5’-GATATCGCGACGGACCTCAACACCGATACA-3’,SEQ ID NO.6;
F-N34R/I188K:5’-GACGGGCAAAGTCCATATTTCCGCTGTTACGG-3’,SEQ ID NO.7;
R-N34R/I188K:5’-TATGGACTTTGCCCGTCTGTTCCGGGTA-3’,SEQ ID NO.8。
Template elimination, namely obtaining a reaction solution after the inverse PCR reaction is finished, sucking 2.5 mu L of restriction endonuclease DpnI into the reaction solution (25 mu L), carrying out gentle blowing and sucking uniformly, reacting at 37 ℃ for 1 h, and verifying the digestion solution by agarose gel electrophoresis.
And (3) cyclizing the inverse PCR product by using the obtained digestion solution, preparing a reaction solution according to the table 8, gently mixing, and reacting at 16 ℃ for 1h to obtain a mutant plasmid pET28a-KMI N34R/I188K containing the ketoisomerase mutant gene.
It will be appreciated that the template is eliminated each time the inverse PCR is completed, and that self-circularization of the inverse PCR product is performed.
TABLE 8 reaction solution
Mutant plasmid verification, namely converting the mutant plasmid obtained after cyclization into E. coliDH5 alpha competent cells by a chemical conversion method, picking a monoclonal colony on a flat plate for plasmid extraction, and carrying out DNA sequencing on the extracted plasmid. Wherein, the mutant plasmid pET28a-KMI N34R/I188K contains the gene of the ketoisomerase mutant N34R/I188K, the nucleotide sequence is shown as SEQ ID NO.9, and the encoded amino acid sequence is shown as SEQ ID NO. 10.
EXAMPLE 3 preparation of crude enzyme solution
Adding 1 mu L of mutant plasmid pET28a-KMI N34R/I188K which is sequenced correctly in the embodiment 2 into competent cells of escherichia coli BL21 (DE 3), placing the mixed reaction system on ice for 30min, performing heat shock for 60s in a 42 ℃ water bath, incubating on ice for 5min to obtain a recombinant strain, transferring the recombinant strain into 500 mu L of LB liquid culture medium, performing shaking recovery for 1h at 37 ℃, and taking 100 mu L of bacterial liquid coating plates, and screening positive transformants (namely mutant strains) containing the mutant plasmid pET28a-KMI N34R/I188K.
The mutant strain obtained by screening is subjected to seed culture in a liquid LB culture medium, and is cultured for 14 hours under the conditions of 37 ℃ and 120rpm, so as to obtain seed liquid of the mutant strain.
Inoculating the seed solution of the mutant strain into a new LB liquid culture medium according to the inoculation amount of 2% by volume ratio for fermentation culture, culturing at 37 ℃ until the OD 600 value is 0.7, then cooling to 18 ℃, adding IPTG with the final concentration of 1.0mM for induction culture until the OD 600 value reaches 5, and obtaining the fermentation liquor containing the ketoisomerase mutant N34R/I188K.
The fermentation broth containing the ketoisomerase mutant N34R/I188K was centrifuged at 4℃and 4000R/min for 15min, the cells were collected, and the collected cells were resuspended in phosphate buffer having a pH of 7.5. Then respectively crushing the bacterial cells by using an ultrasonic cell crusher, stopping ultrasonic power for 2s every 3s of crushing for 30min, centrifuging at 4 ℃ and 12000R/min after ultrasonic crushing to remove cell fragments, and collecting supernatant to obtain crude enzyme liquid of the ketoisomerase mutant N34R/I188K, wherein the concentration of crude enzyme of the ketoisomerase mutant N34R/I188K in the crude enzyme liquid is 4mg/mL.
1. Mu.L of recombinant plasmid pET28a-KMI of example 1, which was sequenced correctly, was taken out, and a crude enzyme solution of wild-type ketoisomerase, in which the concentration of the crude enzyme of wild-type ketoisomerase was 4mg/mL, was prepared in the same manner as above.
Example 4 detection of enzyme Activity and half-life
The myo-inositol, NADP +, crude enzyme solution of wild ketoisomerase and enzyme solution of inositol dehydrogenase are added into phosphate buffer solution with pH value of 7.5 and 100mM, and 10mL of reaction system I is prepared, wherein the concentration of each component in the reaction system I is 30mM myo-inositol, 1.5mM NADP +, 3mg/mL crude enzyme of wild ketoisomerase and 3mg/mL inositol dehydrogenase.
Meanwhile, myoinositol, NADP +, ketoisomerase mutant N34R/I188K crude enzyme solution and inositol dehydrogenase enzyme solution are added into phosphate buffer solution with the pH value of 7.5 and 100mM, and 10mL of reaction system II is prepared, wherein the concentration of each component in the reaction system II is that myoinositol 30mM, NADP + 1.5.5 mM, ketoisomerase mutant N34R/I188K crude enzyme 3mg/mL and inositol dehydrogenase 3mg/mL.
The two reaction systems are respectively reacted for 5min at 35 ℃ to determine the enzyme activity.
The enzyme activity is defined as the amount of enzyme required to consume 1. Mu. Mol myo-inositol within 1min under the above reaction system and conditions.
The relative enzyme activities of the ketoisomerase mutants N34R/I188K were calculated using the enzyme activities of the wild-type ketoisomerase as 100%, and the results are shown in Table 9.
TABLE 9 results of enzyme activity measurements
As is clear from the above results, the enzyme activity of the ketoisomerase mutant N34R/I188K crude enzyme was significantly improved as compared with the wild-type ketoisomerase crude enzyme.
And respectively incubating the crude enzyme solution of the ketoisomerase mutant N34R/I188K and the crude enzyme solution of the wild ketoisomerase at 35 ℃ and 40 ℃ for 0h, 2h, 4h, 6h, 8h and 10h, preparing a reaction system according to the first reaction system and the second reaction system after incubation at different temperatures and different time, taking the enzyme activity of the incubated crude enzyme solution of the ketoisomerase mutant N34R/I188K as 100%, and respectively measuring the residual enzyme activities of the crude enzyme of the ketoisomerase mutant N34R/I188K and the crude enzyme of the wild ketoisomerase at the incubation for different times according to the enzyme activity calculation method to obtain half lives at 35 ℃ and 40 ℃. The results are shown in FIGS. 1 and 2.
As can be seen from FIGS. 1 and 2, the crude enzyme of the ketoisomerase mutant N34R/I188K has improved thermostability at 35 ℃ and 40 ℃ compared with the crude enzyme of the wild-type ketoisomerase, and the half-life at 35 ℃ is prolonged from 3h to 6h, and the half-life at 40 ℃ is improved from 2h to 3.7h.
EXAMPLE 5 preparation of D-chiro-inositol
The myo-inositol, NADP +, crude enzyme solution of wild ketoisomerase and enzyme solution of inositol dehydrogenase are added into phosphate buffer solution with the pH value of 7.5 and 100mM, and 10mL of reaction system III is prepared, wherein the concentration of each component in the reaction system III is that myo-inositol 10mg/mL, NADP + mM, crude enzyme of wild ketoisomerase 3mg/mL and inositol dehydrogenase 3mg/mL.
The myo-inositol, NADP +, ketoisomerase mutant N34R/I188K crude enzyme solution and inositol dehydrogenase enzyme solution are added into phosphate buffer solution with the pH value of 7.5 and 100mM, and a reaction system IV with the concentration of 10mg/mL myo-inositol, NADP + mM, ketoisomerase mutant N34R/I188K crude enzyme 3mg/mL and inositol dehydrogenase 3mg/mL is prepared.
And (3) respectively reacting the third reaction system and the fourth reaction system for 1h at 35 ℃ to respectively obtain reaction solutions, respectively detecting the D-chiro-inositol content in the reaction solutions by using a high performance liquid chromatography, and calculating the conversion rate of the myo-inositol of the substrate.
The detection conditions were a chromatographic column with a specification of 4.6X1250 mm, an amino column with a specification of 5 μm, a mobile phase (volume ratio of acetonitrile to 50 mM aqueous ammonium acetate solution: 75:25), a column temperature of 30℃and a flow rate of 1.0 mL/min were set. The detection was performed by a differential refraction detector, and the detection results are shown in table 10.
Where conversion = D-chiro-inositol content produced/initial myo-inositol content.
TABLE 10D chiral inositol content and myo-inositol conversion
From the above results, it was found that the crude enzyme of ketoisomerase mutant N34R/I188K of the present invention can further improve the conversion rate of myo-inositol of the substrate and thus the yield of D-chiro-inositol, compared with the crude enzyme of wild-type ketoisomerase.
The foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102740709A (en) * | 2009-12-09 | 2012-10-17 | 雀巢产品技术援助有限公司 | Hydrolyzed whole grain composition |
| CN105229152A (en) * | 2012-09-26 | 2016-01-06 | (株)韩国迪外天然健康 | Method for producing D-chiro-inositol using microorganisms |
| US20200407766A1 (en) * | 2016-06-20 | 2020-12-31 | Pharmazell Gmbh | Coupled, Self-Sufficient Biotransformation of Chenodeoxcholic Acid to Ursodeoxycholic Acid and Novel Enzyme Mutants Applicable in said Process |
| CN117088757A (en) * | 2023-08-21 | 2023-11-21 | 诸城市浩天药业有限公司 | A kind of preparation method of D-chiral inositol |
| CN120624387A (en) * | 2025-08-13 | 2025-09-12 | 诸城市浩天药业有限公司 | An enzyme preparation for catalytic synthesis of D-chiro-inositol and a preparation method of D-chiro-inositol |
-
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- 2025-10-31 CN CN202511582479.9A patent/CN121022813B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102740709A (en) * | 2009-12-09 | 2012-10-17 | 雀巢产品技术援助有限公司 | Hydrolyzed whole grain composition |
| CN105229152A (en) * | 2012-09-26 | 2016-01-06 | (株)韩国迪外天然健康 | Method for producing D-chiro-inositol using microorganisms |
| US20200407766A1 (en) * | 2016-06-20 | 2020-12-31 | Pharmazell Gmbh | Coupled, Self-Sufficient Biotransformation of Chenodeoxcholic Acid to Ursodeoxycholic Acid and Novel Enzyme Mutants Applicable in said Process |
| CN117088757A (en) * | 2023-08-21 | 2023-11-21 | 诸城市浩天药业有限公司 | A kind of preparation method of D-chiral inositol |
| CN120624387A (en) * | 2025-08-13 | 2025-09-12 | 诸城市浩天药业有限公司 | An enzyme preparation for catalytic synthesis of D-chiro-inositol and a preparation method of D-chiro-inositol |
Non-Patent Citations (3)
| Title |
|---|
| FRY, J.等: ""TIM barrel protein [Marinovum algicola]，NCBI Reference Sequence: WP_283638873.1"", 《NCBI GENPEPT》, 16 March 2025 (2025-03-16), pages 1 * |
| JAMES B. THODEN等: ""The molecular architecture of QdtA, a sugar 3, 4-ketoisomerase from Thermoanaerobacterium thermosaccharolyticum"", 《PROTEIN SCIENCE》, vol. 23, 10 March 2014 (2014-03-10), pages 683 - 692 * |
| 杨丹等: ""棉花查尔酮异构酶基因家族的鉴定及表达分析"", 《华中农业大学学报》, vol. 42, no. 5, 30 September 2023 (2023-09-30), pages 94 - 104 * |
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