Enzyme preparation for catalytic synthesis of D-chiro-inositol, preparation method of enzyme preparation and preparation method of D-chiro-inositol

Abstract

The invention discloses an enzyme preparation for catalytic synthesis of D-chiro-inositol, a preparation method of the enzyme preparation and a preparation method of the D-chiro-inositol, and belongs to the technical field of genetic engineering. The enzyme preparation has the enzyme activity of catalyzing the reaction of muscle inositol and NADP < + > to generate D-chiral inositol, and comprises inositol dehydrogenase and keto isomerase; and the amino acid sequence of the inositol dehydrogenase is as shown in SEQ ID NO.3. The enzyme activity of the inositol dehydrogenase with the amino acid sequence shown as SEQ ID NO.3 in the enzyme preparation is remarkably improved, so that when the enzyme preparation containing the inositol dehydrogenase and the keto isomerase catalyzes muscle inositol and NADP < + > to react to generate the D-chiral inositol, the yield of the D-chiral inositol can be further improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, in particular to an enzyme preparation for catalytic synthesis of D-chiro-inositol, a preparation method thereof and a preparation method of D-chiro-inositol. BACKGROUND

[0002] D-chiro-inositol is one of nine isomers of inositol with optical activity, which is an active factor in food crops such as buckwheat, soybean, rice bran and wheat, and exists in the form of a compound in plant seeds such as beans and buckwheat in nature, and has multiple effects such as regulating blood sugar, improving polycystic ovary syndrome and antioxidant.

[0003] D-chiro-inositol can be separated and purified from an aqueous solution of spring thunder, but the cost is high due to the influence of raw material price. D-chiro-inositol can also be obtained by taking inositol as a substrate and performing four-step reactions (protection, activation / derivation, isomerization / phenylboronyl introduction, and deprotection), but the operation time is long, the solvent consumption is large, and waste is also generated.

[0004] Meanwhile, using inositol dehydrogenase (IDH) and ketol isomerase (KMI), in the presence of NADP + , inositol can be converted into D-chiro-inositol, but the yield of D-chiro-inositol is not high due to the influence of the enzyme activity of inositol dehydrogenase and ketol isomerase. SUMMARY

[0005] Therefore, the present application aims to provide an enzyme preparation for catalytic synthesis of D-chiro-inositol, a preparation method thereof and a preparation method of D-chiro-inositol, to overcome the problem of low yield of D-chiro-inositol in the prior art due to the influence of enzyme activity.

[0006] In a first aspect, the present application provides an enzyme preparation for catalytic synthesis of D-chiro-inositol, which has enzyme activity of catalyzing inositol and NADP + to react to generate D-chiro-inositol, and the enzyme preparation comprises inositol dehydrogenase and ketol isomerase; the amino acid sequence of the inositol dehydrogenase is shown in SEQ ID NO. 3.

[0007] Compared with the prior art, the present application mutates wild-type inositol dehydrogenase to obtain an inositol dehydrogenase mutant with an amino acid sequence shown in SEQ ID NO. 3, and the enzyme activity of the mutant is improved, so that when the above-mentioned enzyme preparation containing inositol dehydrogenase and ketol isomerase is used to catalyze inositol and NADP + to react to generate D-chiro-inositol, the yield of D-chiro-inositol is further improved.

[0008] Further, the amino acid sequence of the ketol-isomerase is shown as SEQ ID NO. 7.

[0009] The technical solution above also mutates the wild-type ketol-isomerase, and obtains a ketol-isomerase mutant with an amino acid sequence shown as SEQ ID NO. 7, which, in combination with the myo-inositol dehydrogenase mutant with the amino acid sequence shown as SEQ ID NO. 3, can further improve the yield of D-chiro-inositol.

[0010] Further, the mass ratio of the myo-inositol dehydrogenase to the ketol-isomerase is (1-5) : (1-5).

[0011] Further, the enzyme activity modification method of the myo-inositol dehydrogenase is that the 201th amino acid in the amino acid sequence shown as SEQ ID NO. 1 is mutated from D to K.

[0012] Further, the enzyme activity modification method of the ketol-isomerase is that the 206th amino acid in the amino acid sequence shown as SEQ ID NO. 5 is mutated from D to K.

[0013] In the second aspect, the application provides a preparation method of an enzyme preparation, for preparing the enzyme preparation above, comprising the following steps: transforming the coding gene of the myo-inositol dehydrogenase into a host strain to obtain a recombinant strain 1; transforming the coding gene of the ketol-isomerase into a host strain to obtain a recombinant strain 2; respectively fermenting and culturing the recombinant strain 1 and the recombinant strain 2 to respectively obtain a fermentation broth containing the myo-inositol dehydrogenase and a fermentation broth containing the ketol-isomerase; respectively centrifuging the fermentation broth containing the myo-inositol dehydrogenase and the fermentation broth containing the ketol-isomerase, respectively collecting the bacterial cells, respectively resuspending and crushing the cells, and respectively centrifuging to obtain a crude enzyme solution of the myo-inositol dehydrogenase and a crude enzyme solution of the ketol-isomerase.

[0014] Compared with the prior art, the application obtains the crude enzyme solution of the myo-inositol dehydrogenase and the crude enzyme solution of the ketol-isomerase with enzyme activity by the simple, convenient and fast microbial fermentation method.

[0015] Further, the fermentation and culture is high-density fermentation. The steps of the high-density fermentation of the recombinant strain 1 include: inoculating the recombinant strain 1 into LB liquid culture medium, and culturing at 37℃ until OD 600 is 2.0-2.5 to obtain a seed liquid; The seed liquid is inoculated into a fermenter 1 containing base medium 1, and cultured under the conditions of tank pressure 0.035-0.045 MPa, initial rotating speed 115-125 rpm, temperature 35-40 DEG C, and pH value 7.0-8.0, until OD 600 is 28-32 DEG C, and the culture is continued until OD 600 is 28-32 DEG C, and the culture is continued until OD 3 is 28-32 DEG C, and the culture is continued until OD 3 is 28-32 DEG C, and the culture is continued until OD 3 is 28-32 DEG C, and the culture is continued until OD 600 stops, and the fermentation is ended; and / or; The steps of high-density fermentation of the recombinant strain 2 include: The recombinant strain 2 is inoculated into LB liquid medium, and cultured at 37 DEG C until OD 600 is 28-32 DEG C, and the culture is continued until OD The seed liquid is inoculated into a fermenter 2 containing base medium 2, and cultured under the conditions of tank pressure 0.040-0.050 MPa, initial rotating speed 75-85 rpm, temperature 35-40 DEG C, and pH value 7.0-8.0, until DO rises, and then the temperature is lowered to 28-32 DEG C, and the culture is continued until OD 600 is 28-32 DEG C, and the culture is continued until OD 3 is 28-32 DEG C, and the culture is continued until OD 3 is 28-32 DEG C, and the culture is continued until OD 3 is 28-32 DEG C, and the culture is continued until OD 600 stops, and the fermentation is ended.

[0016] The above technical solution further limits the specific steps of high-density fermentation of the recombinant strain 1 and the recombinant strain 2, and the high-density fermentation greatly increases the total expression amount of the target protein enzyme, is suitable for large-scale preparation of enzyme preparation, and reduces the unit cost.

[0017] In a third aspect, the application provides a preparation method of D-chiro-inositol, which utilizes the enzyme preparation described above to catalyze myo-inositol and NADP+ The reaction generates D-chiro-inositol.

[0018] Compared with the prior art, the enzyme preparation of the present application contains inositol dehydrogenase with the amino acid sequence shown as SEQ ID NO. 3 and ketone isomerase with the amino acid sequence shown as SEQ ID NO. 7, and the enzyme activity is higher, thereby effectively increasing the reaction yield of D-chiro-inositol.

[0019] Further, in the reaction for generating D-chiro-inositol, the reaction system comprises the following components: phosphate buffer 80-120 mM, myo-inositol 25-35 mM, NADP + 1-2 mM, inositol dehydrogenase crude enzyme 1-5 mg / mL, ketone isomerase crude enzyme 1-5 mg / mL.

[0020] Further, the reaction temperature is 30-40°C, and the reaction pH value is 7.0-8.0. DETAILED DESCRIPTION

[0021] In order to make the technical problems to be solved by the present application, the technical solutions and beneficial effects more clear and explicit, 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 are not used to limit the present application.

[0022] It should be understood that the raw materials used in the following examples are all commercially available unless otherwise specified.

[0023] Example 1 Construction of recombinant plasmid pET28a-IDH The coding gene of wild-type inositol dehydrogenase (IDH) was codon-optimized, and the IDH gene fragment was artificially synthesized. The IDH gene fragment was used as a template, and upstream primer F1 and upstream primer R1 were designed to perform PCR amplification reaction, thereby obtaining the IDH target gene fragment with homologous arms. The system of PCR amplification reaction is shown in Table 1, and the reaction conditions are shown in Table 2.

[0024] The amino acid sequence of the wild-type inositol dehydrogenase is shown as SEQ ID NO. 1, and the optimized coding gene sequence is shown as SEQ ID NO. 2.

[0025] F1: 5'-cagcaaatgggtcgcggatccATGGCACTGACTGTAGGCGTTA-3' (SEQ ID NO. 9); R1: 5'-ctcgagtgcggccgcaagcttTTAAATCGCGGCCGCTGG-3' (SEQ ID NO. 10).

[0026] Table 1 Table 2 It should be understood that the three steps of pre-denaturation, final extension and preservation do not involve cycling, and the entire PCR process is performed only once.

[0027] After the above PCR amplification reaction, the reaction product is recovered by agarose gel to obtain a higher purity IDH target gene fragment.

[0028] The expression vector pET28a is double-digested using restriction endonuclease BamH I and Hind III to obtain linearized expression vector pET28a, and the enzyme digestion system is shown in Table 3.

[0029] Table 3 After enzyme digestion, the double-digested product is recovered using a gel recovery kit.

[0030] The target gene fragment recovered by the above PCR amplification reaction is connected between the enzyme digestion sites BamH I and Hind III of the linearized expression vector pET28a to obtain recombinant plasmid pET28a-IDH, and the connection system is shown in Table 4. After mixing the connection system, the reaction is carried out at 37°C for 30 min.

[0031] Table 4 After the connection is completed, the connection product is transformed into E. coli DH5α competent cells by chemical transformation method, and single colonies are picked to extract plasmids for sequencing.

[0032] Example 2 Construction of mutant plasmid pET28a-IDH D201K The plasmid ET28a-IDH successfully verified by sequencing in Example 1 is used as a template, F2 and R2 are used as primers, and a mutant plasmid is constructed by reverse PCR. The reverse PCR reaction system is shown in Table 5, and the reaction conditions are shown in Table 6.

[0033] F2: 5'-CTGGTGaaaCCGGCAATCGGTGAAGCTGGCGA-3' (SEQ ID NO. 11); R2: 5'-ATTGCCGGtttCACCAGCACAGCACCGTACGC-3' (SEQ ID NO. 12).

[0034] Table 5 Table 6 It should be understood that the above two steps of 94°C pre-denaturation and 4°C preservation do not involve cycling, and the entire reverse PCR process is only performed once.

[0035] Template elimination: after the above reverse PCR reaction is completed, 2 μL of restriction endonuclease Dpn I is added to the reaction solution, the reaction solution is gently blown with a pipette to mix the enzyme and the reaction solution, and then the mixture is reacted at 37°C for 1 h to obtain a digestion solution. The digestion solution is verified using agarose gel electrophoresis.

[0036] PCR product self-circularization: the reaction solution of the circularization system is prepared according to Table 7, gently mixed, and then reacted at 16°C for 1 h to obtain the mutant plasmid pET28a-IDH D201K .

[0037] Table 7 Mutant plasmid verification: the above reaction product is transformed into competent cells by a chemical transformation method, and the specific method is as follows: the above reaction product is added to competent cells, the mixed reaction system is placed on ice for 30 min, then heat shocked at 42°C for 45 s, then incubated on ice for 2 min, transferred to 500 μL of LB liquid medium, and shaken at 37°C for 1 h to recover, and 200 μL of bacterial solution is plated. A single colony is picked from the plate and inoculated in liquid LB to culture to an OD 600 value of 5.0, and the plasmid is extracted and sent to a detection agency for sequencing.

[0038] The mutant plasmid contains a coding gene of the inositol dehydrogenase mutant D201K, the amino acid sequence of the inositol dehydrogenase mutant D201K is shown in SEQ ID NO. 3, and the sequence of the inositol dehydrogenase mutant D201K coding gene is shown in SEQ ID NO. 4.

[0039] Compared with the amino acid sequence of the wild-type inositol dehydrogenase, the amino acid sequence of the inositol dehydrogenase mutant D201K is mutated from D (aspartic acid) to K (lysine) at the 201st amino acid.

[0040] Example 3 1 μL of the mutant plasmid with correct sequencing in Example 2 is taken The mixed reaction system is placed on ice for 30 min, and then heat shocked at 42℃ for 60 s, and then incubated on ice for 5 min. It is transferred into 500 μL of LB liquid medium and shaken at 37℃ for 1 h. 100 μL of the bacterial solution is plated to obtain positive transformants (i.e. mutant strains) containing the mutant plasmid.

[0041] The mutant strains are subjected to high-density fermentation, and the specific steps are as follows: The mutant strains are inoculated into LB liquid medium and cultured at 37℃ until the OD 600 reaches 2.2 to obtain a seed solution.

[0042] The seed solution is inoculated into a fermenter containing base medium 1 at a volume ratio of 2%, the tank pressure of the fermenter is 0.04 MPa, the initial rotation speed is 120 rpm, and the culture is carried out at 37℃ and a pH of 7.5 until the OD 600 reaches 30. The temperature is lowered to 30℃, and the culture is continued until the OD 600 reaches 70. IPTG is added at a final concentration of 2 g / L for induction culture. The induction culture temperature is 30℃, the initial air flow is 300 m 3 / h, when the rotation speed reaches 200 rpm, the air flow is adjusted to 350 m 3 / h, when the rotation speed reaches 400 rpm, the air flow is adjusted to 400 m 3 / h, and when the dissolved oxygen rebounds, the feed medium is added to keep the DO value always >20%, until the OD 600 does not increase for 3 h in succession, the fermentation is ended.

[0043] The composition of the above-mentioned base medium 1 is: citric acid monohydrate 2 g / L, potassium dihydrogen phosphate 14 g / L, potassium phosphate dibasic trihydrate 4.5 g / L, ammonium sulfate 4 g / L, glucose 20 g / L, magnesium sulfate heptahydrate 1 g / L, yeast powder 1 g / L, and trace element solution 2 mL / L.

[0044] The composition of the trace element solution is: MnSO4·7H2O 0.5 g / L, FeSO4·7H2O 10.0 g / L, CaCl2 2.0 g / L, (NH4)Mo7O 24 0.1 g / L, CuSO4·5H2O 3.0 g / L, NiCl2·7H2O 0.02 g / L, and ZnSO4·7H2O 5.25 g / L, dissolved in sterile water.

[0045] The composition of the above-mentioned feed medium is: glucose 600 g / L, magnesium sulfate 2 g / L, and yeast powder 10 g / L. ​

[0046] After high density fermentation, the fermentation broth containing the myo-inositol dehydrogenase mutant D201K was obtained, and the bacterial cells were collected by centrifugation of the fermentation broth using a high-speed refrigerated centrifuge at 8000 rpm for 15 min at 4°C. After centrifugation, the collected bacterial cells were resuspended with phosphate buffer, and the resuspension OD 600 value was 80, and then the cells were broken using a high-pressure homogenizer at 750 bar for 15 min. The cell fragments were removed by centrifugation again using a high-speed refrigerated centrifuge at 12000 r for 15 min at 4°C, and the obtained supernatant was the crude enzyme solution of the myo-inositol dehydrogenase mutant D201K, and the concentration of the crude enzyme in the crude enzyme solution was 10 mg / mL.

[0047] 1 μL of the plasmid ET28a-IDH successfully sequenced in Example 1 was added to the E. coli BL21 (DE3) competent cells, and the crude enzyme solution of the wild-type myo-inositol dehydrogenase with a concentration of 10 mg / mL was prepared in the same manner as in Example 3.

[0048] Example 4 Detection of enzyme activity Reaction system one: myo-inositol 30 mM, NADP + 1.5 mM, wild-type myo-inositol dehydrogenase crude enzyme 3 mg / mL, phosphate buffer with a pH value of 7.5 100 mM, and the total volume of the reaction system was 10 mL.

[0049] Reaction system two: myo-inositol 30 mM, NADP + 1.5 mM, myo-inositol dehydrogenase mutant D201K crude enzyme 3 mg / mL, phosphate buffer with a pH value of 7.5 100 mM, and the total volume of the reaction system was 10 mL.

[0050] The above two reaction systems were respectively reacted at 35°C for 1 min to obtain reaction solutions. The absorbance of the two reaction solutions was detected respectively, and the enzyme activity was calculated.

[0051] In the above reaction, when NADP + was present, the myo-inositol dehydrogenase crude enzyme solution could catalyze the substrate myo-inositol to be converted into scyllite, and NADP + was converted into NADPH.

[0052] Definition of enzyme activity: the amount of enzyme required to consume 1 μmol of myo-inositol in 1 min under the above reaction system and conditions.

[0053] Method for calculating enzyme activity: enzyme activity (U) = (ΔA x V x 10 3 ) / (6220 x 1).

[0054] ΔA refers to the change in absorbance value at 340 nm within 1 min; V refers to the total volume of the reaction system (mL); 6220 refers to the molar extinction coefficient of NADPH ); and l refers to the optical path distance, i.e. the light path of the cuvette (cm).

[0055] The relative enzyme activity of the myo-inositol dehydrogenase mutant D201K was calculated based on the enzyme activity of the wild-type myo-inositol dehydrogenase as 100%, and the results are shown in Table 8.

[0056] Table 8 From the above results, it can be seen that the enzyme activity of the myo-inositol dehydrogenase mutant D201K of the application is significantly improved compared with the wild-type myo-inositol dehydrogenase.

[0057] Example 5 Construction of recombinant plasmid pET28a-KMI and mutant plasmid pET28a-KMI D206K The coding gene of the wild-type ketoisomerase (KMI) was codon-optimized and artificially synthesized to obtain a KMI gene fragment. The KMI gene fragment was used as a template, and upstream primers F3 and upstream primers R3 were designed to perform PCR amplification reaction to obtain a KMI target gene fragment with homologous arms. The PCR amplification reaction system is shown in Table 9, and the reaction conditions are shown in Table 10.

[0058] The amino acid sequence of the wild-type myo-inositol dehydrogenase is shown in SEQ ID NO. 5, and the optimized coding gene sequence is shown in SEQ ID NO. 6.

[0059] F3: 5'-cagcaaatgggtcgcggatccATGAAGACTACTCTGAACCACATGACC-3' (SEQ ID NO. 13); R3: 5'-ctcgagtgcggccgcaagcttTTAAGCAGCACGTGCCTGC-3' (SEQ ID NO. 14).

[0060] Table 9 Table 10 It should be understood that the three steps of pre-denaturation, final extension and preservation do not involve cycling, and the entire PCR process is performed only once.

[0061] After the above PCR amplification reaction is completed, the reaction product is recovered by agarose gel to obtain a KMI target gene fragment with high purity. ​

[0062] According to the method in Embodiment 1 above, the KMI target gene fragment amplified by PCR was cloned into the expression vector pET28a to obtain the recombinant plasmid pET28a-KMI, which was sequenced.

[0063] The successfully sequenced recombinant plasmid pET28a-KMI was used as a template, and F4 and R4 were used as primers to construct a mutant plasmid by reverse PCR. The reverse PCR reaction system is shown in Table 11, and the reaction conditions are shown in Table 6.

[0064] F4: 5'-GATGGAGaaaGAGCATCGTGTACTGGTCGACG-3' (SEQ ID NO. 15); R4: 5'-GATGCTCtttCTCCATCTCGTGAACAGCCAGC-3' (SEQ ID NO. 16).

[0065] Table 11

[0066] Template elimination: After the reverse PCR reaction, 2 μL of restriction endonuclease Dpn I was added to the reaction solution, and the reaction solution was gently blown with a pipette to mix the enzyme and the reaction solution. Then, the mixture was incubated at 37°C for 1 h to obtain a digestion solution. The digestion solution was verified by agarose gel electrophoresis.

[0067] PCR product self-circularization: The digestion product after enzyme digestion was prepared into a reaction solution for circularization according to Table 7, gently mixed, and incubated at 16°C for 1 h to obtain the mutant plasmid pET28a-KMI. D206K .

[0068] Mutant plasmid verification: The reaction product was transformed into E. coli DH5α competent cells by chemical transformation, and the plasmid was extracted and sequenced.

[0069] The mutant plasmid pET28a-KMI D206K contains a gene encoding the ketol-isomerase mutant D206K. The amino acid sequence of the ketol-isomerase mutant D206K is shown in SEQ ID NO. 7, and the sequence of the gene encoding the ketol-isomerase mutant D206K is shown in SEQ ID NO. 8.

[0070] Compared with the amino acid sequence of the wild-type ketol-isomerase, the amino acid sequence of the ketol-isomerase mutant D206K has a D (aspartic acid) mutation to K (lysine) at position 206.

[0071] Embodiment 6 Take 1 μL of the mutant plasmid pET28a-KMI sequenced correctly in Example 5 D206K The mixed reaction system was placed on ice for 30 min and then heat shocked at 42°C for 60 s, and then incubated on ice for 5 min. It 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 solution was plated. Positive transformants (i.e. mutant strains) containing the mutant plasmid pET28a-KMI were obtained. D206K

[0072] The mutant strain BL21-pET28a-KMI D206K was subjected to high-density fermentation, and the specific steps were as follows: The mutant strain BL21-pET28a-KMI D206K was inoculated into LB liquid medium and cultured at 37°C until the OD 600 was 2.2, to obtain a seed solution.

[0073] The seed solution was inoculated into a fermenter containing base medium 2 at a volume ratio of 2%, the tank pressure of the fermenter was 0.045 MPa, the initial rotation speed was 80 rpm, and the culture was carried out at 37°C and pH 7.5 until the dissolved oxygen rebounded. The temperature was reduced to 30°C, and the culture was continued until the OD 600 was 70, and IPTG was added at a final concentration of 2 g / L for induction culture. The temperature of the induction culture was reduced to 25°C, the initial air flow was 300 m 3 / h, when the rotation speed reached 200 rpm, the air flow was adjusted to 350 m 3 / h, when the rotation speed reached 400 rpm, the air flow was adjusted to 400 m 3 / h, and when the dissolved oxygen rebounded, the feed medium was added to keep the DO value always >20%, until the OD 600 did not increase for 3 h in succession, the fermentation was ended.

[0074] The composition of the above base medium 2 was: citric acid monohydrate 2 g / L, potassium dihydrogen phosphate 14 g / L, potassium phosphate dibasic trihydrate 4.5 g / L, ammonium sulfate 4 g / L, glucose 20 g / L, magnesium sulfate heptahydrate 0.6 g / L, yeast powder 1 g / L, and trace element solution 2 mL / L.

[0075] The composition of the trace element solution was: MnSO4·7H2O 0.5 g / L, FeSO4·7H2O 10.0 g / L, CaCl2 2.0 g / L, (NH4)Mo7O 24 0.1 g / L, CuSO4·5H2O 3.0 g / L, NiCl2·7H2O 0.02 g / L, and ZnSO4·7H2O 5.25 g / L, dissolved in sterile water. ​

[0076] The composition of the above-mentioned feed medium is: glucose 600 g / L, magnesium sulfate 2 g / L, yeast powder 10 g / L.

[0077] After the above-mentioned high-density fermentation is completed, a fermentation liquor containing the ketoisomerase mutant D206K is obtained, and the bacterial cells are collected by centrifuging the fermentation liquor using a high-speed refrigerated centrifuge at 4°C and 8000 rpm for 15 min. After centrifugation, the collected bacterial cells are resuspended with a phosphate buffer, and the resuspension OD 600 value is 80, and then the cells are broken using a high-pressure homogenizer at 750 bar for 15 min. The cell fragments are removed by centrifuging the broken cells again using a high-speed refrigerated centrifuge at 4°C and 12000 r for 15 min, and the obtained supernatant is the crude enzyme liquor of the ketoisomerase mutant D206K, and the concentration of the crude enzyme in the crude enzyme liquor is 10 mg / mL.

[0078] 1 μL of the recombinant plasmid pET28a-KMI correctly sequenced in Example 5 is added to the E. coli BL21 (DE3) competent cells, and the wild-type ketoisomerase crude enzyme liquor with a concentration of 10 mg / mL is prepared in the same manner as in Example 6.

[0079] Example 7 Preparation of D-chiro-inositol The reaction system for preparing D-chiro-inositol is configured as follows: Reaction system 1: myo-inositol 30 mM, NADP + 1.5 mM, wild-type inositol dehydrogenase crude enzyme 2 mg / mL, wild-type ketoisomerase crude enzyme 2 mg / mL, phosphate buffer with a pH value of 7.5 100 mM, and the total volume of the reaction system is 10 mL.

[0080] Reaction system 2: myo-inositol 30 mM, NADP + 1.5 mM, inositol dehydrogenase mutant D201K crude enzyme 2 mg / mL, wild-type ketoisomerase crude enzyme 2 mg / mL, phosphate buffer with a pH value of 7.5 100 mM, and the total volume of the reaction system is 10 mL.

[0081] Reaction system 3: myo-inositol 30 mM, NADP + 1.5 mM, inositol dehydrogenase mutant D201K crude enzyme 2 mg / mL, ketoisomerase mutant D206K crude enzyme 2 mg / mL, phosphate buffer with a pH value of 7.5 100 mM, and the total volume of the reaction system is 10 mL.

[0082] Each of the above-mentioned reaction systems is reacted at 35°C for 30 min, and reaction liquors are obtained, respectively.

[0083] The concentration of D-chiral inositol in each reaction solution was determined by high performance liquid chromatography, and the conversion rate was calculated. The results are shown in Table 12.

[0084] Detection method: The chromatographic column is a 4.6 × 250 mm, 5 μm amino column; Mobile phase: Acetonitrile: 50 mM ammonium acetate aqueous solution = 75: 25 (volume ratio); The column temperature was set to 30℃ and the flow rate to 1.0 mL / min.

[0085] Conversion rate = Amount of D-chiral inositol generated / Amount of initial muscle inositol in the reaction.

[0086] Table 12 From the above results, we can conclude that: The inositol dehydrogenase mutant D201K and ketoisomerase mutant D206K of the present invention have higher enzyme activity than their wild type, which can improve the yield of D-chiral inositol and the conversion rate of muscle inositol in the preparation of D-chiral inositol.

[0087] 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. An enzyme preparation for use in the catalytic synthesis of D-chiro- inositol, characterized in that, The enzyme preparation has catalytic myo-inositol and NADP + reacting to produce D-chiro-inositol, the enzyme preparation comprising myo-inositol dehydrogenase and ketol-isomerase; The amino acid sequence of the myo-inositol dehydrogenase is shown as SEQ ID NO.

3.

2. The enzyme preparation of claim 1, characterized in that, The amino acid sequence of the ketol-isomerase is shown as SEQ ID NO.

7.

3. The enzyme preparation according to claim 1 or 2, characterized in that, The mass ratio of the myo-inositol dehydrogenase to the ketol-isomerase is (1-5) : (1-5).

4. The enzyme formulation of claim 1, wherein, The enzyme activity modification method of the myo-inositol dehydrogenase is to mutate the 201st amino acid of the amino acid sequence shown as SEQ ID NO. 1 from D to K.

5. The enzyme preparation of claim 2, wherein The enzyme activity modification method of the ketol-isomerase is to mutate the 206th amino acid of the amino acid sequence shown as SEQ ID NO. 5 from D to K.

6. A method for producing an enzyme preparation according to any one of claims 1 to 5, characterized in that The method comprises the following steps: a coding gene of the myo-inositol dehydrogenase is transformed into a host strain to obtain a recombinant strain 1; a coding gene of the ketol-isomerase is transformed into a host strain to obtain a recombinant strain 2; the recombinant strain 1 and the recombinant strain 2 are respectively subjected to fermentation culture to respectively obtain a fermentation liquid containing the myo-inositol dehydrogenase and a fermentation liquid containing the ketol-isomerase; the fermentation liquid containing the myo-inositol dehydrogenase and the fermentation liquid containing the ketol-isomerase are respectively subjected to centrifugation, and the bacterial bodies are respectively collected, resuspended, and then broken and centrifuged to respectively obtain a crude enzyme liquid of the myo-inositol dehydrogenase and a crude enzyme liquid of the ketol-isomerase.

7. The production method according to claim 6, wherein The fermentation culture is high-density fermentation. The step of high-density fermentation of the recombinant strain 1 comprises: The recombinant strain 1 was inoculated into LB liquid medium and cultured at 37°C to OD 600 2.0~2.5, to obtain seed liquid; The seed liquid is inoculated into a fermenter 1 containing base medium 1, and cultured under the conditions of tank pressure 0.035-0.045 MPa, initial rotation speed 115-125 rpm, temperature 35-40℃, pH value 7.0-8.0, to OD 600 28-32, and the temperature is lowered to 28-32℃, and the culture is continued to OD 600 68-72, and induction culture is carried out by adding IPTG at a final concentration of 1.8-2.2 g / L, the temperature of the induction culture is 28-32℃, the initial air flow is 300 m 3 / h, when the rotation speed reaches 180-220 rpm, the air flow is adjusted to 330-370 m 3 / h, when the rotation speed reaches 380-420 rpm, the air flow is adjusted to 380-420 m 3 / h, and when the dissolved oxygen rebounds, the feed medium is added to keep the DO value always >20%, until OD 600 No longer increases, and the fermentation is ended; and / or, The step of high-density fermentation of the recombinant strain 2 comprises: The recombinant strain 2 was inoculated into LB liquid medium and cultured at 37°C to OD 600 2.0~2.5, to obtain seed liquid; The seed liquid is inoculated into the fermenter 2 containing base medium 2, and cultured under the conditions of tank pressure 0.040-0.050 MPa, initial rotating speed 75-85 rpm, temperature 35-40℃, pH value 7.0-8.0, until the dissolved oxygen rebounds, then the temperature is lowered to 28-32℃, and the culture is continued until OD 600 68-72, and induced culture is carried out by adding IPTG with final concentration of 1.8-2.2 g / L, the temperature of the induced culture is 23-27℃, the initial air flow is 280-320 m 3 / h, when the rotating speed reaches 180-220 rpm, the air flow is adjusted to 330-370 m 3 / h, when the rotating speed reaches 380-420 rpm, the air flow is adjusted to 380-420 m 3 / h, the feeding medium is added when the dissolved oxygen rebounds, so that the DO value is always >20%, until OD 600 No longer growth ends the fermentation.

8. A process for the preparation of D-chiro-inositol, characterized in that, The enzyme preparation according to any one of claims 1 to 7 is used to catalyze the reaction of muscle inositol and NADP + to produce D-chiro-inositol.

9. The production method according to claim 8, characterized by, When the reaction generates D-chiral inositol, the reaction system comprises the following components: Phosphate buffer 80-120 mM, myo-inositol 25-35 mM, NADP + 1-2 mM, crude inositol dehydrogenase 1-5 mg / mL, crude ketol-isomerase 1-5 mg / mL.

10. The production method according to claim 8 or 9, characterized by, The reaction temperature is 30-40℃, and the reaction pH value is 7.0-8.0.

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