Keto isomerase mutants and uses thereof

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to a ketol-isomerase mutant and application thereof. The ketol-isomerase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO. 2 by at least one of the following mutations: the 66th amino acid is mutated from N to Q; the 137th amino acid is mutated from I to L. After the ketol-isomerase shown in SEQ ID NO. 2 is subjected to the above-mentioned mutations, the ketol-isomerase mutant obtained is N66Q, I137L and N66Q / I137L, and the corresponding amino acid sequences are shown in SEQ ID NO. 10, SEQ ID NO. 12 and SEQ ID NO. 14, respectively. The ketol-isomerase mutant obtained has improved thermal stability in the range of 30 DEG C to 80 DEG C, prolonged half-life and improved enzyme activity, thereby further improving the conversion rate of muscle inositol and the yield of D-chiro-inositol.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a ketoisomerase mutant and its applications. Background Technology

[0002] D-chiro-inositol (DCI) is one of the nine isomers of inositol that is optically active. Recent studies have found that, in addition to its role in promoting hepatic lipid metabolism, D-chiro-inositol also possesses unique physiological functions such as insulin sensitization, blood sugar reduction, improvement of ovulation in patients with polycystic ovary syndrome, regulation of hormone balance, improvement of menstrual disorders, and antioxidant, anti-aging, and anti-inflammatory effects.

[0003] The combination of ketoisomerase and inositol dehydrogenase can convert the substrate muscle inositol into D-chiral inositol. However, the ketoisomerase in the existing technology has low enzyme activity and poor stability, which limits the overall conversion rate and is a key bottleneck restricting the production of D-chiral inositol.

[0004] Therefore, traditional techniques need to be improved. Summary of the Invention

[0005] The purpose of this invention is to provide a ketoisomerase mutant and its application, which can overcome the defects of low enzyme activity and poor stability of ketoisomerase, resulting in limited overall conversion rate.

[0006] In a first aspect, the present invention provides a ketoisomerase mutant, which is obtained by mutating the amino acid sequence shown in SEQ ID NO.2 through at least one of the following mutations: The 66th amino acid is mutated from N to Q; The 137th amino acid was mutated from I to L.

[0007] Compared with the prior art, the present invention mutates the ketoisomerase shown in SEQ ID NO.2 to obtain ketoisomerase mutants N66Q, I137L and N66Q / I137L, respectively, with corresponding amino acid sequences as shown in SEQ ID NO.10, SEQ ID NO.12 and SEQ ID NO.14, respectively. The ketoisomerase mutants obtained by the present invention exhibit improved thermostability, prolonged half-life and increased enzyme activity in the temperature range of 30℃ to 80℃. Therefore, when co-catalyzing the reaction of muscle inositol with inositol dehydrogenase to generate D-chiral inositol, the conversion rate of muscle inositol and the yield of D-chiral inositol are further improved.

[0008] Secondly, the present invention provides a biomaterial, comprising any one of the following: (1) A nucleic acid molecule encoding the above-mentioned ketoisomerase mutant; the sequence of which is shown in SEQ ID NO. 9, SEQ ID NO. 11 or SEQ ID NO. 13; (2) An expression vector containing the nucleic acid molecule described in (1); (3) A recombinant strain containing the nucleic acid molecule described in (1) or the expression vector described in (2).

[0009] Thirdly, the present invention provides a method for preparing a ketoisomerase mutant, which includes the following steps: The recombinant strain containing the encoding gene of the ketoisomerase mutant was seed cultured in LB medium until OD... 600 When the value reaches 0.8~1.0, the seed solution is obtained; The seed culture was inoculated into another LB medium at an inoculum volume of 1% to 5%, and cultured in shake flasks until OD reached. 600 The value reaches 0.6~0.8; cool down, add an inducer and induce culture for 10h~15h to obtain fermentation broth containing keto isomerase mutant; 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.

[0010] Compared with existing technologies, the present invention uses recombinant strains to prepare ketoisomerase mutants because the recombinant strains contain nucleic acid molecules that express the ketoisomerase mutants N66Q, I137L, or N66Q / I137L. Therefore, crude enzyme solution of ketoisomerase mutants can be obtained by culturing and fermenting the recombinant strains. This method improves production efficiency, is simple and easy to operate, and can quickly obtain crude enzyme solution, reducing cumbersome operations and time costs in the preparation process.

[0011] Furthermore, the seed culture temperature is 35℃~38℃, and the culture time is 8h~12h; the shake flask culture temperature is 35℃~38℃, and the rotation speed is 150rpm~200rpm; the cooling is to 25℃~30℃, the inducer is IPTG, and the final concentration of the inducer is 1mM~1.5mM.

[0012] Fourthly, the present invention provides uses of the above-mentioned ketoisomerase mutant or the above-mentioned biological material, including its use in the preparation of D-chiral inositol.

[0013] Compared with the prior art, the ketoisomerase mutant in this invention has improved stability, extended half-life, and increased enzyme activity. When co-catalyzing the reaction of muscle inositol with inositol dehydrogenase to generate D-chiral inositol, it can significantly improve the conversion rate of muscle inositol and the yield of D-chiral inositol.

[0014] Fifthly, the present invention provides a method for preparing D-chiral inositol, comprising: Add muscle inositol and NAD to the buffer solution + The crude enzyme solution of the ketoisomerase mutant and inositol dehydrogenase are reacted to produce a conversion solution containing D-chiral inositol.

[0015] Compared with the prior art, the present invention adds crude enzyme solution of ketoisomerase mutant to the reaction system. The half-life of the ketoisomerase mutant is significantly extended and the enzyme activity is higher, thereby further improving the conversion rate of muscle inositol and the yield of D-chiral inositol.

[0016] Furthermore, the concentrations of each component in the reaction system that generates the conversion solution containing D-chiral inositol are as follows: Muscle inositol 300mg / mL~500mg / mL, NAD + 1mM~3mM, crude ketoisomerase mutant enzyme 2mg / mL~4mg / mL, inositol dehydrogenase 2mg / mL~4mg / mL, buffer 80mM~120mM.

[0017] The buffer solutions mentioned above include phosphate buffer.

[0018] The reaction temperature is 30℃~80℃, the reaction time is 8h~16h, and the reaction pH is 7.0~8.0.

[0019] Furthermore, after obtaining the conversion solution containing D-chiral inositol, the method further includes extracting D-chiral inositol from the conversion solution, comprising the following steps: A precipitant is added to the conversion solution, and after heating, hot filtration is performed to collect filtrate 1 and filter cake. After cooling the filtrate 1, filter it to obtain filtrate 2; The filtrate 2 is filtered through an ultrafiltration membrane to obtain the ultrafiltration membrane filtrate; The cationic impurities, anionic impurities, and pigments in the ultrafiltration membrane filtrate are removed to obtain a treated solution. The treatment solution is concentrated to obtain concentrated solution 1 with a Baume degree of 50-80%. Ethanol is added to concentrated solution 1 at 45℃-55℃, and the temperature is lowered to 10℃-20℃ to precipitate muscle inositol. The solution is filtered to obtain filtrate 3. The filtrate 3 is concentrated to obtain concentrate 2 with a Baume degree of 40-80%. The concentrate is heated to 45℃-55℃, ethanol is added, and the concentrate is cooled to 25℃-35℃ to precipitate D-chiral inositol. The concentrate is filtered to obtain crude D-chiral inositol.

[0020] Compared with existing technologies, this invention first adds a precipitant to the conversion solution and then performs two filtrations to remove large molecular impurities such as bacterial proteins and cell debris. Then, it further removes residual colloidal impurities through ultrafiltration to obtain an ultrafiltration membrane filtrate. After removing cationic impurities, anionic impurities, and pigments from the ultrafiltration membrane filtrate, it is concentrated to obtain concentrate 1. Since the content of muscle inositol in concentrate 1 is higher than that of D-chiral inositol, and muscle inositol has lower solubility in ethanol-water solution, ethanol is added to concentrate 1 and the temperature is lowered to 10℃~20℃ to precipitate muscle inositol. The muscle inositol crystals are then removed by filtration to obtain filtrate 3. Filtrate 3 is then concentrated to obtain concentrate 2. After removing muscle inositol crystals in the above steps, the content of D-chiral inositol in concentrate 2 is significantly increased. Therefore, ethanol is added to concentrate 2 and the temperature is lowered to 25℃~35℃ for crystallization, at which point D-chiral inositol precipitates, yielding the target product, crude D-chiral inositol. This invention precipitates muscle inositol through a first alcohol precipitation crystallization, removes unreacted muscle inositol by filtration, and then obtains crude D-chiral inositol through a second alcohol precipitation crystallization. This effectively avoids the random loss of the target product during the extraction process and significantly improves the yield of crude D-chiral inositol.

[0021] Further, the precipitant includes calcium oxide; the temperature of the hot filtration is 65℃~75℃; the filtrate 1 is cooled to 10℃~20℃ before filtration; when the filtrate 2 is filtered through an ultrafiltration membrane, the pore size of the ultrafiltration membrane is 3000Da~3500Da; the amount of ethanol added to the concentrate 1 is 0.5~1.5 times the volume of the concentrate 1; the amount of ethanol added to the concentrate 2 is 0.5~1.5 times the volume of the concentrate 2.

[0022] After adding ethanol to concentrate 1 in the above steps, the temperature can be reduced to 10℃~20℃ at a rate of 4℃ / h~8℃ / h. After adding ethanol to concentrate 1 in the above steps, the temperature can be reduced to 25℃~35℃ at a rate of 4℃ / h~8℃ / h.

[0023] The above technical solution uses calcium oxide as a precipitant, which can efficiently precipitate acidic impurities and protein impurities. A two-step extraction method combining hot filtration at 65℃~75℃ and cold filtration at 10℃~20℃ is used to achieve the dual precipitation of soluble and insoluble impurities by utilizing the temperature difference, effectively improving the purity of filtrate 2.

[0024] Furthermore, after obtaining filtrate 1 and filter cake, before cooling filtrate 1, the process further includes rinsing the filter cake, and combining the rinsing liquid with filtrate 1 to obtain a combined liquid.

[0025] After obtaining the combined liquid, the combined liquid is further cooled to 10℃~20℃ and then filtered.

[0026] The above technical solution can further avoid the loss of the target product D-chiral inositol and improve the yield of crude D-chiral inositol by rinsing the filter cake and combining the rinsing liquid with filtrate 1.

[0027] Furthermore, the process of removing cationic impurities, anionic impurities, and pigments from the ultrafiltration membrane filtrate includes: The ultrafiltration membrane filtrate is passed through a cation exchange resin to remove cationic impurities, thereby obtaining a cation exchange resin effluent. The effluent from the cation exchange resin is passed through a macroporous adsorption resin to remove pigment impurities, thereby obtaining the macroporous adsorption resin effluent. The effluent from the macroporous adsorption resin is passed through anion exchange resin to remove anionic impurities, thus obtaining a treated solution.

[0028] The above technical solution treats the ultrafiltration membrane filtrate sequentially through cation exchange resin, macroporous adsorption resin, and anion exchange resin, thereby removing different types of impurities such as inorganic ions and residual pigments in a stepwise manner, and achieving the effect of enhancing the enrichment of the target product.

[0029] Further, after obtaining crude D-chiral inositol, the process further includes: adding water at 65°C to 75°C in an amount of 0.5 to 1.0 times the mass of the crude D-chiral inositol to dissolve the crude D-chiral inositol, obtaining a solution; decolorizing the solution with activated carbon to obtain a decolorized solution; adding ethanol at 0.5 to 1.5 times the volume of the decolorized solution; cooling to 25°C to 35°C; and filtering to obtain D-chiral inositol.

[0030] After adding ethanol to the decolorizing solution in the above steps, the temperature can be reduced to 25℃~35℃ at a rate of 4℃ / h~8℃ / h.

[0031] The above technical solution involves dissolving crude D-chiral inositol in water at 65℃~75℃, ensuring complete dissolution of the crude D-chiral inositol and reducing product loss during the dissolution process; activated carbon decolorization can efficiently remove pigment impurities and improve the appearance quality of the product; subsequent cooling to 25℃~35℃ can further precipitate D-chiral inositol, achieving deep purification. Attached Figure Description

[0032] Figure 1The residual enzyme activity of crude enzymes of ketoisomerase mutant N66Q / I137L, crude enzyme of ketoisomerase mutant N66Q, crude enzyme of ketoisomerase mutant I137L, and crude enzyme of wild-type ketoisomerase after incubation at 40°C for different times in Example 6.

[0033] Figure 2 The residual enzyme activity of crude enzymes of ketoisomerase mutant N66Q / I137L, crude enzyme of ketoisomerase mutant N66Q, crude enzyme of ketoisomerase mutant I137L, and crude enzyme of wild-type ketoisomerase after incubation at 60°C for different times in Example 6.

[0034] Figure 3 The residual enzyme activity of crude enzymes of ketoisomerase mutant N66Q / I137L, crude enzyme of ketoisomerase mutant N66Q, crude enzyme of ketoisomerase mutant I137L, and crude enzyme of wild-type ketoisomerase after incubation at 80°C for different times in Example 6. Detailed Implementation

[0035] 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.

[0036] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.

[0037] Example 1 Wild-type thermotolerant anaerobic bacteria *Pseudothermotoga thermarum* were screened from the database, and wild-type ketoisomerase was obtained from it. After codon optimization of the wild-type ketoisomerase nucleotides, sequence 1 was obtained. Using sequence 1 as a template, upstream primer F1 and downstream primer R1 were designed for PCR amplification, yielding the KMI target gene fragment with homologous arms. The PCR amplification reaction system is shown in Table 1, and the PCR amplification reaction conditions are shown in Table 2.

[0038] The nucleotide sequence of sequence 1 is shown in SEQ ID NO.1, and the amino acid sequence of the wild-type ketoisomerase it encodes is shown in SEQ ID NO.2.

[0039] F1:5'-tcgagctccgtcgacgagctcATGAAGACTACTCTGAACCACATGAC-3', SEQ ID NO.3; R1: 5'-gtggtggtggtggtgctcgagTTAAGCAGCACGTGCCTGC-3', SEQ ID NO. 4.

[0040] Table 1 PCR amplification reaction system Element Volume (μL) Amplified template sequence 1 1 Upstream primer F1 (10 μmol / L) 1 Downstream primer R1 (10 μmol / L) 1 <![CDATA[ddH2O]]> 9.5 2×Phanta Ultra-High Fidelity Enzyme 12.5 Table 2 PCR amplification reaction conditions step temperature time Cycle number Pre-variation 95℃ 1min / transsexual 98℃ 15s 35 annealing 57℃ 15s 35 extend 72℃ 45s 35 Final extension 72℃ 10min / Keep 4℃ ∞ / It should be understood that the pre-denaturation, final extension, and maintenance steps in the above PCR amplification reaction do not participate in the cycling process, and the entire PCR process is performed only once.

[0041] After the PCR amplification was completed, the reaction product was recovered by agarose gel extraction to obtain a KMI gene fragment with high purity.

[0042] The expression vector pET28a was double-digested with restriction endonucleases Sac I and Xho I. The double-digested products were recovered and purified to obtain the linearized vector pET28a. The digestion system is shown in Table 3.

[0043] Table 3 Enzyme digestion system Components Volume (μL) expression vector pET28a 12.5 10×Buffer 2.5 Sac Ⅰ 1 XhoⅠ 1 <![CDATA[ddH2O]]> 8 The KMI gene fragment obtained by PCR amplification was ligated between the Sac I and Xho I restriction sites of the linearized vector pET28a to obtain the recombinant plasmid pET28a-KMI. The ligation system is shown in Table 4. The ligation reaction temperature was 37℃ and the reaction time was 30 min.

[0044] Table 4 Connection System Components Volume (μL) Linearized vector pET28a 4.5 KMI gene fragment 1.5 5×CE II Buffer 4 Exnase II 2 <![CDATA[ddH2O]]> 8 After the above ligation was completed, the ligation product recombinant plasmid pET28a-KMI was transformed into E. coli DH5α competent cells using chemical transformation. Single colonies were picked for plasmid extraction, and the extracted plasmids were sequenced for DNA.

[0045] Example 2: Construction of the mutant plasmid pET28a-KMI N66Q Using the recombinant plasmid pET28a-KMI from Example 1 as a template, PCR amplification was performed using F-N66Q and R-N66Q primers to obtain the mutant plasmid pET28a-KMI. N66Q .

[0046] The PCR amplification reaction system described above is shown in Table 5, and the amplification reaction conditions are shown in Table 6.

[0047] Table 5 PCR amplification reaction system Element Volume (μL) Amplification of template pET28a-KMI (2 ng / μl) 3 Upstream primer F-N66Q (9.9 ng / μl) 21 Downstream primer R-N66Q (9.9 ng / μl) 21 2×PrimeStar max 45 Table 6 PCR amplification reaction conditions step temperature time Cycle number Pre-variation 95℃ 5 min / transsexual 95℃ 30 s 35 annealing 60℃ 30 s 35 extend 72℃ 2 min 35 Complete extension 72℃ 14 min / save 4℃ 10 min / It should be understood that the pre-denaturation, thorough extension, and preservation steps in the above PCR amplification process do not participate in the cycling, and the entire PCR process is performed only once.

[0048] in: F-N66Q: 5'-TACGATTCAAAGCGTGGAACAGTTCACCTTTT-3', SEQ ID NO.5; R-N66Q: 5'-CCACGCTTTGAATCGTAGATGGTAAAATCGAGTAATCT-3', SEQ ID NO. 6.

[0049] Example 3: Construction of the mutant plasmid pET28a-KMI I137L Using the recombinant plasmid pET28a-KMI from Example 1 as a template, PCR amplification was performed using F-I137L and R-I137L primers to obtain the mutant plasmid pET28a-KMI. I137L .

[0050] The PCR amplification reaction system described above is shown in Table 7, and the amplification reaction conditions are shown in Table 8.

[0051] Table 7 PCR amplification reaction system Element Volume (μL) Amplification of template pET28a-KMI (2 ng / μl) 3 Upstream primer F-I137L (9.9 ng / μl) 21 Downstream primer R-I137L (9.9 ng / μl) 21 2×PrimeStar max 45 Table 8 PCR amplification reaction conditions step temperature time Cycle number Pre-variation 95℃ 5 min / transsexual 95℃ 30 s 35 annealing 60℃ 30 s 35 extend 72℃ 2 min 35 Complete extension 72℃ 14 min / save 4℃ 10 min / It should be understood that the pre-denaturation, thorough extension, and preservation steps in the above PCR amplification process do not participate in the cycling, and the entire PCR process is performed only once.

[0052] in: F-I137L: 5'-TGTTAAAACTCGGTTTCGAATTCTTGGGGTTCG-3', SEQ ID NO.7; R-I137L: 5'-CGAAACCGAGTTTAACATTGTATAATTTGGCAAGATCC-3', SEQ ID NO. 8.

[0053] Example 4: Construction of the mutant plasmid pET28a-KMI N66Q / I137L pET28a-KMI prepared in Example 2 N66Q Using F-I137L (SEQ ID NO.7) and R-I137L (SEQ ID NO.8) as primers, PCR amplification was performed to obtain the mutant plasmid pET28a-KMI. N66Q / I137LThe PCR amplification reaction system is shown in Table 9, and the amplification reaction conditions are shown in Table 10.

[0054] Table 9 PCR amplification reaction system Element Volume (μL) <![CDATA[Amplification template pET28a-KMI N66Q > 3 Upstream primer F-I137L (9.9 ng / μl) 21 Downstream primer R-I137L (9.9 ng / μl) 21 2×PrimeStar max 45 Table 10 PCR Amplification Reaction Conditions step temperature time Cycle number Pre-variation 95℃ 5 min / transsexual 95℃ 30 s 35 annealing 60℃ 30 s 35 extend 72℃ 2 min 35 Complete extension 72℃ 14 min / save 4℃ 10 min / It should be understood that the pre-denaturation, thorough extension, and preservation steps in the above PCR amplification process do not participate in the cycling, and the entire PCR process is performed only once.

[0055] The mutant plasmid pET28a-KMI obtained in Example 2 above was used. N66Q The mutant plasmid pET28a-KMI obtained in Example 3 I137L and the mutant plasmid pET28a-KMI obtained in Example 4. N66Q / I137L 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.

[0056] Among them, the mutant plasmid pET28a-KMI N66Q The gene containing the ketoisomerase mutant N66Q has the nucleotide sequence shown in SEQ ID NO.9 and the encoded amino acid sequence shown in SEQ ID NO.10.

[0057] Mutant plasmid pET28a-KMI I137L The gene containing the ketoisomerase mutant I137L has the nucleotide sequence shown in SEQ ID NO.11 and the encoded amino acid sequence shown in SEQ ID NO.12.

[0058] Mutant plasmid pET28a-KMI N66Q / I137L The gene containing the ketoisomerase mutant N66Q / I137L has the nucleotide sequence shown in SEQ ID NO.13 and the encoded amino acid sequence shown in SEQ ID NO.14.

[0059] Example 5: Preparation of crude enzyme solution Take 5 μL of the mutant plasmid pET28a-KMI from the above examples that was correctly sequenced. N66Q Mutant plasmid pET28a-KMI I137L Mutant plasmid pET28a-KMI N66Q / I137LAdd the mixture to E. coli BL21(DE3) competent cells, mix separately, and incubate the mixture on ice for 30 min, then heat shock at 42°C for 90 s, followed by incubation on ice for 2 min to obtain recombinant strains. Then, transfer each recombinant strain to 500 μL of LB broth and incubate at 37°C with shaking for 1.5 h. Spread 100 μL of the bacterial culture onto plates and screen for strains containing the mutant plasmid pET28a-KMI. N66Q The mutant strain KMI-N66Q, containing the mutant plasmid pET28a-KMI I137L The mutant strain KMI-137L, containing the mutant plasmid pET28a-KMI N66Q / I137L The mutant strain KMI-N66Q / 137L.

[0060] The three mutant strains obtained from screening were seed cultured in liquid LB medium and cultured at 37℃ and 200 rpm until OD. 600 The concentration was 0.8, and seed cultures of mutant strains KMI-N66Q, KMI-137L, and KMI-N66Q / 137L were obtained respectively.

[0061] The seed cultures of the three mutant strains were transferred to new shake flasks containing LB liquid medium at an inoculum volume of 1% and cultured until the bacterial cell concentration reached OD500. 600 When the concentration reached 0.7, the temperature was then lowered to 30°C, and IPTG with a final concentration of 1.0 mM was added for induction culture for 12 h. Fermentation broth containing ketoisomerase mutant N66Q, fermentation broth containing ketoisomerase mutant I137L, and fermentation broth containing ketoisomerase mutants N66Q / I137L were obtained.

[0062] The fermentation broths containing the ketoisomerase mutant N66Q, the ketoisomerase mutant I137L, and the ketoisomerase mutants N66Q / I137L were centrifuged at 4°C and 4000 rpm for 15 min, respectively. The cells were collected separately and resuspended in 50 mM dipotassium hydrogen phosphate buffer (pH 8.5). The resuspended cells were then homogenized twice at 700 bar. After centrifugation at 4000 rpm for 15 min, the supernatants were collected separately to obtain crude enzyme solutions of ketoisomerase mutants N66Q, I137L, and N66Q / I137L, respectively.

[0063] The crude enzyme concentration in the crude enzyme solution of ketoisomerase mutant N66Q was 6 mg / mL, the crude enzyme concentration in the crude enzyme solution of ketoisomerase mutant I137L was 4 mg / mL, and the crude enzyme concentration in the crude enzyme solution of ketoisomerase mutant N66Q / I137L was 5 mg / mL.

[0064] Take 5 μL of the correctly sequenced recombinant plasmid pET28a-KMI from Example 1 and prepare a crude enzyme solution of wild-type ketoisomerase with a concentration of 5 mg / mL using the same method described above.

[0065] Example 6: Detection of enzyme activity and half-life Add muscle inositol and NAD to a 100mM phosphate buffer solution with a pH of 7.5. + Wild-type ketoisomerase crude enzyme solution and inositol dehydrogenase enzyme solution were used to prepare 10 mL of reaction system one. The concentrations of each component in reaction system one were: muscle inositol 30 mM, NAD + 1.5mM, crude wild-type ketoisomerase 3mg / mL, inositol dehydrogenase 3mg / mL.

[0066] Add muscle inositol and NAD to a 100mM phosphate buffer solution with a pH of 7.5. + A reaction system II was prepared by mixing crude enzyme solution of ketoisomerase mutant N66Q and enzyme solution of inositol dehydrogenase, resulting in a 10 mL volume. The concentrations of each component in reaction system II were: muscle inositol 30 mM, NAD... + 1.5 mM, crude enzyme of ketoisomerase mutant N66Q 3 mg / mL, and inositol dehydrogenase 3 mg / mL.

[0067] Add muscle inositol and NAD to a 100mM phosphate buffer solution with a pH of 7.5. + The crude enzyme solution of ketoisomerase mutant I137L and the enzyme solution of inositol dehydrogenase were used to prepare a 10 mL reaction system three. The concentrations of each component in reaction system three were: muscle inositol 30 mM, NAD3 10 mM, and 10 mL of crude enzyme solution of ketoisomerase mutant I137L. + 1.5 mM, crude enzyme I137L (ketoisomerase mutant) 3 mg / mL, and inositol dehydrogenase 3 mg / mL.

[0068] Add muscle inositol and NAD to a 100mM phosphate buffer solution with a pH of 7.5. + A 10 mL reaction system (reaction system four) was prepared by combining crude enzyme solution of ketoisomerase mutant N66Q / I137L and inositol dehydrogenase solution. The concentrations of each component in reaction system four were: muscle inositol 30 mM, NAD... + 1.5 mM, crude enzyme of ketoisomerase mutant N66Q / I137L 3 mg / mL, and inositol dehydrogenase 3 mg / mL.

[0069] The four reaction systems were reacted at 40℃ for 5 min, and the enzyme activity was measured.

[0070] Enzyme activity is defined as the amount of enzyme required to consume 1 μmol of muscle inositol within 1 minute under the above reaction system and conditions.

[0071] Enzyme activity calculation method: Enzyme activity (U) = (ΔA×V×103) / (6220×1).

[0072] Where ΔA refers to the change in absorbance at 340 nm over 1 min; V is the total volume of the reaction system (mL); and 6220 refers to the molar extinction coefficient of NADH (L·mol⁻¹). −1 cm −1 ); l is the optical path distance, i.e., the optical path of the cuvette (cm).

[0073] Using the wild-type ketoisomerase activity as 100%, the relative enzyme activities of the ketoisomerase mutants N66Q / I137L, N66Q, and I137L were calculated according to the enzyme activity calculation method described above. The results are shown in Table 11.

[0074] Table 11 Enzyme activity assay results Ketoisomerase relative enzyme activity Crude wild-type ketoisomerase 100% Ketoisomerase mutant N66Q / I137L crude enzyme 300% crude enzyme of ketoisomerase mutant N66Q 500% Ketoisomerase mutant I137L crude enzyme 200% The results above show that, compared with wild-type ketoisomerase, the crude enzyme activities of the ketoisomerase mutants N66Q / I137L, N66Q, and I137L are all significantly increased.

[0075] The crude enzyme solutions of the above-mentioned ketoisomerase mutants N66Q / I137L, N66Q, and I137L, as well as the crude enzyme solution of wild-type ketoisomerase, were incubated at 40℃, 60℃, and 80℃ for 0h, 2h, 4h, 8h, 12h, and 24h, respectively. Then, the crude enzyme solutions after incubation at different temperatures and times were prepared according to reaction systems one through four described above. With enzyme activity at 0h incubation as 100%, the residual enzyme activity of the crude enzymes of ketoisomerase mutants N66Q / I137L, N66Q, and I137L, as well as wild-type ketoisomerase, was determined according to the enzyme activity calculation method described above after incubation at different times. The results are shown in […]. Figures 1-3 The half-life at 40℃, 60℃, and 80℃ was obtained based on the residual enzyme activity. The results are shown in Table 12.

[0076] Table 12 Half-life of wild-type keto isomerases and their mutants 40℃ 60℃ 80℃ Wild-type ketoisomerase 11.64 h 9.84 h 6.6 h mutant N66Q / I137L 16.8 h 12 h 10.66 h mutant N66Q 21.6 h 17.33 h 12 h mutant I137L 12 h 11.55 h 10.04 h Depend on Figure 1 , Figure 2 , Figure 3As shown in Table 12, the thermostability of the ketoisomerase mutants N66Q / I137L, N66Q, and I137L is further improved compared with the wild-type ketoisomerase at 40℃, 60℃, and 80℃.

[0077] Example 7: Preparation of D-chiral inositol Add muscle inositol and NAD to a phosphate buffer solution with a pH of 8.0 and a concentration of 80 mM. + Wild-type ketoisomerase crude enzyme solution and inositol dehydrogenase enzyme solution were used to prepare a 30L reaction system five. The concentrations of each component in reaction system five were: muscle inositol 400mg / mL, NAD... + 1 mM, crude wild-type ketoisomerase 3 mg / mL, inositol dehydrogenase 3 mg / mL.

[0078] Add muscle inositol and NAD to a phosphate buffer solution with a pH of 8.0 and a concentration of 80 mM. + The crude enzyme solution of ketoisomerase mutant N66Q and the enzyme solution of inositol dehydrogenase were used to prepare a 30L reaction system six. The concentrations of each component in reaction system six were: muscle inositol 400mg / mL, NAD400mg / mL, and NAD400mg / mL, respectively. + 1 mM, crude enzyme of ketoisomerase mutant N66Q 3 mg / mL, and inositol dehydrogenase 3 mg / mL.

[0079] Add muscle inositol and NAD to a phosphate buffer solution with a pH of 8.0 and a concentration of 80 mM. + A 30L reaction system (reaction system seven) was prepared by mixing crude enzyme solution of ketoisomerase mutant I137L and inositol dehydrogenase solution. The concentrations of each component in reaction system seven were: muscle inositol 400mg / mL, NAD+, and so on. + 1 mM, crude enzyme of ketoisomerase mutant I137L 3 mg / mL, and inositol dehydrogenase 3 mg / mL.

[0080] Add muscle inositol and NAD to a phosphate buffer solution with a pH of 8.0 and a concentration of 80 mM. + A 30L reaction system was prepared by mixing crude enzyme solution of ketoisomerase mutant N66Q / I137L and inositol dehydrogenase solution. The concentrations of each component in reaction system eight were: muscle inositol 400mg / mL, NAD... + 1 mM, crude enzyme of ketoisomerase mutant N66Q / I137L 3 mg / mL, and inositol dehydrogenase 3 mg / mL.

[0081] After converting reaction systems five to eight at 70℃ for 16 h, they were inactivated to obtain reaction solutions 1 to 4 containing crude D-chiral inositol. The D-chiral inositol content in reaction solutions 1 to 4 containing crude D-chiral inositol was detected by high performance liquid chromatography, and the conversion rate of the substrate muscle inositol was calculated. The results are shown in Table 13.

[0082] High performance liquid chromatography: Column: 4.6×250 mm amino column with a particle size of 5 μm; mobile phase: acetonitrile and 50 mM ammonium acetate aqueous solution in a volume ratio of 75:25; column temperature: 30℃; flow rate: 1.0 mL / min; detector: differential refractive index detector.

[0083] The conversion rate is calculated as follows: (D-chiral inositol content generated ÷ initial muscle inositol content) × 100%.

[0084] Table 13 D-chiral inositol content and muscle inositol conversion rate Ketoisomerase D-chiroinositol content (mg / mL) Conversion rate (%) Crude wild-type ketoisomerase 43.2 10.8 Ketoisomerase mutant N66Q / I137L crude enzyme 47.2 11.8 crude enzyme of ketoisomerase mutant N66Q 50.4 12.6 Ketoisomerase mutant I137L crude enzyme 45.2 11.3 The results above show that the thermostability of wild-type ketoisomerase is improved after mutation at the above specific sites, which in turn increases the content and conversion rate of D-chiral inositol.

[0085] Example 8: Extraction of D-chiral inositol from the reaction solution Take 30 L each of reaction solutions 2 to 4 containing crude D-chiral inositol, and extract the D-chiral inositol from them using the following methods: 750 g of calcium oxide was added to the reaction solution, heated to 70°C, and then hot-filtered under a vacuum of -0.095 MPa. Filtrate 1 and filter cake were collected. The filter cake was rinsed with 10 L of hot water, and the rinsing solution was combined with filtrate 1 to obtain 40 L of combined solution.

[0086] The combined liquid was cooled to 15°C and then filtered under a vacuum of -0.095 MPa to obtain filtrate 2. Filtrate 2 was filtered through an ultrafiltration membrane with a pore size of 3000 Da, and the ultrafiltration membrane filtrate was collected. The ultrafiltration membrane was washed with water, and the water was combined with the ultrafiltration membrane filtrate to obtain 53 L of ultrafiltration membrane filtrate.

[0087] The ultrafiltration membrane filtrate was passed through 12L of cation exchange resin D67 to remove cationic impurities. The feed flow rate was 1 BV / h to obtain the cation exchange resin effluent. After the feed was completed, the resin was washed with 12L of water, and the wash liquid was collected and combined with the cation exchange resin effluent.

[0088] The combined cation exchange resin effluent was passed through 3L of macroporous adsorption resin 109D to remove pigment impurities. The feed flow rate was 3BV / h to obtain macroporous adsorption resin effluent. After the feed was completed, the resin was washed with 3L of water, and the wash liquid was collected and combined with the macroporous adsorption resin effluent.

[0089] The combined macroporous adsorption resin effluent was passed through 12L of anion exchange resin LS4539 to remove anion impurities. The feed flow rate was 1 BV / h to obtain anion exchange resin effluent. After the feed was completed, the resin was washed with 12L of water. The washed liquid was collected and combined with the anion exchange resin effluent to obtain a total of 80L of decolorization and desalination solution.

[0090] 80L of decolorizing and desalting solution was vacuum concentrated and dehydrated at 70℃ and -0.095MPa to obtain 6.5L of concentrate 1, at which time the Baumé degree of concentrate 1 was 55%.

[0091] The concentrate was heated to 50°C, and ethanol of equal volume to the concentrate was added. Then the temperature was lowered to 15°C at a rate of 5°C / h to allow muscle inositol to crystallize out. The solution was then filtered under a vacuum of -0.095 MPa, and the filtrate 3 was collected.

[0092] Filtrate 3 was concentrated under vacuum at 70℃ and -0.095MPa to a Baume degree of 45%, yielding concentrate 2, which had a volume of 1.2 L. The concentrate was then heated to 50℃ and an equal volume of ethanol was added. The temperature was then lowered to 30℃ at a rate of 5℃ / h to allow D-chiral inositol to crystallize out. The crystals were then filtered under vacuum at -0.095MPa to obtain crude D-chiral inositol.

[0093] The above-mentioned reaction solution 2 containing crude D-chiral inositol (catalyzed by crude enzyme of ketoisomerase mutant N66Q) was extracted using the above method to obtain crude D-chiral inositol 1 with a mass of 653.4 g. The content of crude D-chiral inositol after drying was detected by liquid chromatography and found to be 91.4%. The above-mentioned reaction solution 3 containing crude D-chiral inositol (catalyzed by crude enzyme of ketoisomerase mutant I137L) was extracted using the above method to obtain crude D-chiral inositol 2 with a mass of 603 g. The content of crude D-chiral inositol after drying was detected by liquid chromatography and found to be 90.6%. The above-mentioned reaction solution 4 containing crude D-chiral inositol (catalyzed by crude enzyme of ketoisomerase mutant N66Q / I137L) was extracted using the above method to obtain crude D-chiral inositol 3 with a mass of 630.7 g. The content of crude D-chiral inositol after drying was detected by liquid chromatography and found to be 90.3%.

[0094] After obtaining crude D-chiral inositol 1 to crude D-chiral inositol 3, the following steps were taken to purify crude D-chiral inositol 1 to crude D-chiral inositol 3: The crude D-chiral inositol 1 to 3 were completely dissolved in 650 mL, 600 mL, and 630 mL of pure water at 70 °C, respectively, to obtain solution 1 to solution 3. 12 g of activated carbon was added to solution 1 to solution 3 for decolorization. After decolorization, the activated carbon was removed to obtain decolorized solution 1 to decolorized solution 3.

[0095] Add ethanol equal to the volume of the decolorizing solution to each of the decolorizing solutions 1 to 3, and cool to 30°C at a rate of 5°C / h to allow D-chiral inositol to crystallize out. Filter under vacuum of -0.095 MPa to obtain D-chiral inositol 1 to D-chiral inositol 3.

[0096] The mass of D-chiral inositol 1 was 424g, and the content of the dried D-chiral inositol product 1 obtained by liquid chromatography was 99.5%.

[0097] The mass of D-chiral inositol 2 was 408g, and the content of the dried D-chiral inositol product 1 was 99.3% as determined by liquid chromatography.

[0098] The mass of D-chiral inositol 3 was 416g, and the content of the dried D-chiral inositol product 1 was 99.4% as determined by liquid chromatography.

[0099] 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 mutant ketol-isomerase characterized in that, The ketoisomerase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.2 through at least one of the following mutations: The 66th amino acid is mutated from N to Q; The 137th amino acid was mutated from I to L.

2. A biomaterial, characterized by, Including any of the following: (1) A nucleic acid molecule encoding the ketoisomerase mutant of claim 1; (2) An expression vector containing the nucleic acid molecule described in (1); (3) A recombinant strain containing the nucleic acid molecule described in (1) or the expression vector described in (2).

3. A method for producing a mutant ketol-isomerase according to claim 1, characterized by, Includes the following steps: The recombinant strain containing the gene encoding the ketol-isomerase mutant is seed-cultured in LB medium until the OD 600 value reaches 0.8-1.0, to obtain a seed liquid; The seed liquid is inoculated into another LB medium at an inoculation amount of 1% to 5% by volume, and is cultured in a shake flask to an OD 600 value of 0.6 to 0.8 is reached; the temperature is lowered, and an inducer is added for induction culture for 10 to 15 hours to obtain a fermentation liquid containing the ketol-isomerase mutant. 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.

4. The method for preparing the ketoisomerase mutant according to claim 3, characterized in that, The seed culture temperature is 35℃~38℃, and the culture time is 8h~12h; and / or, The temperature for the shake flask culture is 35℃~38℃, and the rotation speed is 150rpm~200rpm; and / or, The cooling is described as reducing the temperature to 25°C~30°C; and / or, The inducer is IPTG, and the final concentration of the inducer is 1 mM to 1.5 mM.

5. Use of the ketol-isomerase mutant according to claim 1 or of the biological material according to claim 2, characterized in that, The uses include its application in the preparation of D-chiral inositol.

6. A method for the preparation of D-chiro-inositol, characterized in that, include: In the buffer was added myo-inositol, NAD + , ketol-isomerase mutant crude enzyme solution, inositol dehydrogenase, and a reaction solution containing D-chiral inositol was produced.

7. The production method according to claim 6, characterized by, The concentrations of each component in the reaction system that generates the conversion solution containing D-chiral inositol are as follows: Muscle inositol 300 mg / mL to 500 mg / mL, NAD + 1 mM to 3 mM, ketol-isomerase mutant crude enzyme 2 mg / mL to 4 mg / mL, inositol dehydrogenase 2 mg / mL to 4 mg / mL, buffer 80 mM to 120 mM; and / or, The buffer solution includes phosphate buffer; and / or, The reaction temperature is 30℃~80℃, the reaction time is 8h~16h, and the reaction pH is 7.0~8.

0.

8. The preparation method according to claim 6, characterized in that, After obtaining the conversion solution containing D-chiral inositol, the method further includes extracting D-chiral inositol from the conversion solution, comprising the following steps: A precipitant was added to the conversion liquid, and after heating, hot filtration was performed to collect filtrate 1 and filter cake. After cooling the filtrate 1, it is filtered to obtain filtrate 2. The filtrate 2 is filtered through an ultrafiltration membrane to obtain the ultrafiltration membrane filtrate; The cationic impurities, anionic impurities, and pigments in the ultrafiltration membrane filtrate are removed to obtain a treated solution. The treatment solution is concentrated to obtain concentrated solution 1 with a Baume degree of 50-80%. Ethanol is added to concentrated solution 1 at 45℃-55℃, and the temperature is lowered to 10℃-20℃ to precipitate muscle inositol. The solution is filtered to obtain filtrate 3. The filtrate 3 is concentrated to obtain concentrate 2 with a Baume degree of 40-80%. The concentrate is heated to 45℃-55℃, ethanol is added, and the concentrate is cooled to 25℃-35℃ to precipitate D-chiral inositol. The concentrate is filtered to obtain crude D-chiral inositol.

9. The preparation method according to claim 8, characterized in that, The precipitant includes: calcium oxide; and / or, The temperature of the hot filtration is 65℃~75℃; and / or, The filtrate 1 is cooled to 10℃~20℃ and then filtered by vacuum filtration; and / or, When the filtrate 2 is filtered through an ultrafiltration membrane, the pore size of the ultrafiltration membrane is 3000 Da~3500 Da; and / or, The amount of ethanol added to concentrate 1 is 0.5 to 1.5 times the volume of concentrate 1; and / or, The amount of ethanol added to the concentrate 2 is 0.5 to 1.5 times the volume of the concentrate 2.

10. The preparation method according to claim 8, characterized in that, After obtaining filtrate 1 and filter cake, before cooling filtrate 1, the process further includes rinsing the filter cake and combining the rinsing liquid with filtrate 1 to obtain a combined liquid.

11. The preparation method according to claim 8, characterized in that, After obtaining crude D-chiral inositol, the process also includes: Add 0.5 to 1.0 times the mass of crude D-chiral inositol to the crude D-chiral inositol at 65℃ to 75℃ to dissolve the crude D-chiral inositol and obtain a solution. Decolorize the solution with activated carbon to obtain a decolorized solution. Add 0.5 to 1.5 times the volume of the decolorized solution of ethanol, cool to 25℃ to 35℃, and filter to obtain D-chiral inositol.

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