An enzyme preparation and a method for preparing D-chiral inositol therein

Abstract

This invention discloses an enzyme preparation and a method for preparing D-chiral inositol, belonging to the field of genetic engineering technology. The enzyme preparation includes D-monosolenol dehydrogenase MtOEPa and D-pineol dehydrogenase MtOEPb; the amino acid sequence of D-monosolenol dehydrogenase MtOEPa is shown in SEQ ID NO.6, SEQ ID NO.8, or SEQ ID NO.10; the amino acid sequence of D-pineol dehydrogenase MtOEPb is shown in SEQ ID NO.3 or SEQ ID NO.12. This invention mutates wild-type D-monosolenol dehydrogenase MtOEPa and wild-type D-pineol dehydrogenase MtOEPb. The enzyme activities of the four mutants obtained through mutation are all significantly increased, which can significantly improve the conversion rate and yield of D-chiral inositol in the catalytic reaction of muscle inositol to D-chiral inositol.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to an enzyme preparation and a method for preparing D-chiral inositol. Background Technology

[0002] D-Chiro-inositol (DCI) is an important stereoisomer of inositol that has received much attention in the field of metabolic health in recent years. D-Chiro-inositol has a significant effect on improving polycystic ovary syndrome (PCOS) and insulin resistance.

[0003] The enzymatic route for producing D-chiral inositol generally involves using inositol dehydrogenase and ketoisomerase to catalyze the reaction of muscle inositol to D-chiral inositol. Inositol dehydrogenase first catalyzes the reaction of muscle inositol to 2-ketomusinotitanol, then ketoisomerase catalyzes the reaction of 2-ketomusinotitanol to 1-keto-D-chiral inositol. Finally, inositol dehydrogenase catalyzes the reduction of 1-keto-D-chiral inositol to the final product, D-chiral inositol. All three steps are reversible, leading to equilibrium constraints in the reaction system, a tendency for intermediate products to accumulate, and generally low yields and conversion efficiency of D-chiral inositol.

[0004] In addition, studies have demonstrated that D-monosodium dehydrogenase (MtOEPa) and D-pinel dehydrogenase (MtOEPb) from alfalfa can catalyze the reversible conversion between muscle inositol and D-chiral inositol by heterologous expression in Corynebacterium glutamicum, thus establishing a new pathway for the synthesis of D-chiral inositol. However, this pathway is limited by the enzyme activities of D-monosodium dehydrogenase (MtOEPa) and D-pinel dehydrogenase (MtOEPb), and there is significant room for improvement in the catalytic efficiency of this reaction. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an enzyme preparation and a method for preparing D-chiral inositol, to overcome the problem that the enzyme activity of D-manganese dehydrogenase MtOEPa and D-pinel dehydrogenase MtOEPb in the prior art is not high enough, resulting in limited D-chiral inositol production.

[0006] In a first aspect, the present invention provides an enzyme preparation comprising D-manganese dehydrogenase MtOEPa and D-pinel dehydrogenase MtOEPb.

[0007] The amino acid sequence of D-monomentol dehydrogenase MtOEPa is shown in SEQ ID NO.6, SEQ ID NO.8 or SEQ ID NO.10; the amino acid sequence of D-pineol dehydrogenase MtOEPb is shown in SEQ ID NO.3 or SEQ ID NO.12.

[0008] Compared with existing technologies, this invention mutates wild-type D-monomentol dehydrogenase MtOEPa to obtain D-monomentol dehydrogenase MtOEPa mutants I126K, L283Y, and I126K / L283Y, and mutates wild-type D-pineol dehydrogenase MtOEPb to obtain D-pineol dehydrogenase MtOEPb mutant G225A. The enzyme activities of the four mutants obtained through these mutations are all significantly increased, and they can significantly improve the conversion rate and yield of D-chiral inositol in the catalytic reaction of muscle inositol to D-chiral inositol.

[0009] Furthermore, the mass ratio of D-manganese dehydrogenase MtOEPa to D-pineol dehydrogenase MtOEPb in the enzyme preparation is (1~5):(1~5).

[0010] Optionally, the gene sequence of D-monomentol dehydrogenase MtOEPa is shown in SEQ ID NO.5, SEQ ID NO.7 or SEQ ID NO.9.

[0011] The gene sequence of D-pinel dehydrogenase MtOEPb is shown in SEQ ID NO.4 or SEQ ID NO.11.

[0012] Secondly, the present invention provides a method for preparing an enzyme preparation, comprising the following steps:

[0013] A recombinant expression vector containing both the D-manganese dehydrogenase MtOEPa and the D-pinel dehydrogenase MtOEPb encoding genes was constructed. The recombinant expression vector was then transformed into a host strain to obtain a recombinant strain.

[0014] The recombinant strain was fermented to induce the expression of D-manganese dehydrogenase MtOEPa and D-pinyl alcohol dehydrogenase MtOEPb, resulting in a fermentation broth containing both D-manganese dehydrogenase MtOEPa and D-pinyl alcohol dehydrogenase MtOEPb.

[0015] The fermentation broth was centrifuged to collect the cells. After resuspending the cells, they were broken up, centrifuged again, and the supernatant was collected to obtain the crude enzyme solution containing the enzyme preparation.

[0016] The mass ratio of D-manganese dehydrogenase MtOEPa to D-pineol dehydrogenase MtOEPb in the crude enzyme solution was (1~5):(1~5).

[0017] Compared with existing technologies, this invention constructs an engineered bacterium capable of co-expressing D-manganese dehydrogenase MtOEPa and D-pinel dehydrogenase MtOEPb, integrating the expression of the two enzymes into the same strain, thus avoiding the operational complexity and cost associated with separate expression.

[0018] Thirdly, the present invention provides the application of the above-mentioned enzyme preparation in the preparation of D-chiral inositol.

[0019] Fourthly, the present invention provides a method for preparing D-chiral inositol, wherein the above-mentioned enzyme preparation is used to catalyze the reaction of the substrate muscle inositol to generate D-chiral inositol in the presence of a coenzyme factor.

[0020] Compared with the prior art, the enzyme preparations of the present invention contain D-manganese dehydrogenase MtOEPa and D-pineol dehydrogenase MtOEPb with specific amino acid sequences, which have further improved enzyme activity, thereby further improving the conversion rate of the reaction and the yield of D-chiral inositol.

[0021] Further, muscle inositol, coenzyme factor, and crude enzyme solution containing the above-mentioned enzyme preparation are added to a 20-100mM buffer solution, so that the concentration of muscle inositol in the reaction system is 5-120g / L and the total concentration of crude enzyme protein is 0.5-100mg / mL.

[0022] Furthermore, the buffer solution includes HEPE buffer, Tris-HCl buffer, or phosphate buffer.

[0023] Furthermore, coenzyme factors include NAD. ＋ NADP ＋ .

[0024] Furthermore, in the reaction system, NAD ＋ The concentration is 0.5~2.0mM, NADP ＋ The concentration is 0.5~2.0mM.

[0025] Furthermore, the reaction temperature is 30~40℃, and the pH value of the reaction is 7.0~8.0. Detailed Implementation

[0026] 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.

[0027] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.

[0028] Example 1

[0029] Construction of recombinant plasmid pETDuet-MtOEPA-MtOEPB

[0030] The amino acid sequence of wild-type D-ononitol dehydrogenase MtOEPa (Medicago truncatula D-ononitol dehydrogenase) derived from alfalfa (as shown in SEQ ID NO.1) was reverse-translated into a gene sequence and optimized according to the codon preference of Escherichia coli to obtain the optimized gene sequence of wild-type D-ononitol dehydrogenase MtOEPa, as shown in SEQ ID NO.2.

[0031] The amino acid sequence of wild-type D-pinitol dehydrogenase MtOEPb (Medicago truncatula D-pinitol dehydrogenase) derived from alfalfa (as shown in SEQ ID NO.3) was reverse-translated into a gene sequence and optimized according to the codon preference of Escherichia coli to obtain the optimized gene sequence of wild-type D-pinitol dehydrogenase MtOEPb, as shown in SEQ ID NO.4.

[0032] Using the optimized wild-type D-mangmenol dehydrogenase MtOEPa gene sequence as a template, PCR amplification was performed with F1 as the upstream primer and R1 as the downstream primer to obtain the target gene MtOEPA with homologous arms.

[0033] F1:5'-ccacagccaggatccgaattcATGAGCAAAACCGTGTGCG-3' (SEQ ID NO. 13).

[0034] R1:5'-gcattatgcggccgcaagcttCTACACCAGGCCGCGGCT-3' (SEQ ID NO. 14).

[0035] Using the optimized wild-type D-pinel dehydrogenase MtOEPb gene sequence as a template, PCR amplification was performed with F2 as the upstream primer and R2 as the downstream primer to obtain the target gene MtOEPB with homologous arms.

[0036] F2: 5'-taagaaggagatatacatatgATGGCGGGCAACAAAATTC-3' (SEQ ID NO. 15).

[0037] R2: 5'-tttaccagactcgagggtaccCTACACATCGCCATCCCACAG-3' (SEQ ID NO. 16).

[0038] The reaction systems for the two PCR amplification reactions are shown in Table 1, and the reaction procedures are shown in Table 2.

[0039] Table 1

[0040]

[0041] Table 2

[0042]

[0043] It should be understood that the pre-denaturation at 98°C, the final extension at 72°C, and the storage at 4°C in the two PCR amplification reactions mentioned above are not included in the cycle, and the entire PCR process is performed only once.

[0044] After the two PCR amplification reactions were completed, agarose gel extraction was performed to obtain the target gene MtOEPA with homologous arms and the target gene MtOEPB with homologous arms with high purity, respectively.

[0045] The expression vector pETDuet-1 was double-digested with restriction endonucleases EcoRⅠ and HindⅢ at 37℃ for 20 min to obtain the linearized vector pETDuet-1. The double digestion system is shown in Table 3.

[0046] Table 3

[0047]

[0048] The linearized vector pETDuet-1 was ligated with the target gene MtOEPA obtained by PCR amplification using the recombinase Exnase II at a temperature of 37°C for 30 min to obtain the intermediate plasmid pETDuet-MtOEPA. The ligation system is shown in Table 4.

[0049] Table 4

[0050]

[0051] After ligation, the intermediate plasmid pETDuet-MtOEPA was transformed into E. coli DH5α competent cells using chemical transformation. Single colonies were picked, plasmids were extracted, and samples were sent for sequencing to obtain the correctly sequenced intermediate plasmid pETDuet-MtOEPA.

[0052] The correctly sequenced intermediate plasmid pETDuet-MtOEPA was double-digested using restriction endonucleases NdeⅠ and KpnⅠ at 37℃ for 20 min to obtain the linearized plasmid pETDuet-MtOEPA. The double digestion system is shown in Table 5.

[0053] Table 5

[0054]

[0055] The linearized plasmid pETDuet-MtOEPA was ligated with the target gene MtOEPB obtained by the above PCR amplification using recombinase Exnase II at a temperature of 37℃ for 30 min to obtain the recombinant plasmid pETDuet-MtOEPA-MtOEPB. The ligation system is shown in Table 6.

[0056] Table 6

[0057]

[0058] After ligation, the ligation product recombinant plasmid pETDuet-MtOEPA-MtOEPB was transformed into E. coli DH5α competent cells using chemical transformation. Single colonies were picked, plasmids were extracted, and sent for sequencing to obtain the correctly sequenced recombinant plasmid pETDuet-MtOEPA-MtOEPB.

[0059] Example 2

[0060] Constructing mutant plasmids

[0061] Using the correctly sequenced recombinant plasmid pETDuet-MtOEPA-MtOEPB as a template, reverse PCR amplification was performed with F3 as the upstream primer and R3 as the downstream primer. After removing the template from the reverse PCR product, self-circularization was performed to obtain the mutant plasmid pETDuet-MtOEPA. I126K -MtOEPB.

[0062] F3: AGCAGCaaaAGCGCGATTATTCCGAGCCCGAG (SEQ ID NO. 17).

[0063] R3: ATCGCGCTtttGCTGCTGGTCGCCACCACGCG (SEQ ID NO. 18).

[0064] Using the correctly sequenced recombinant plasmid pETDuet-MtOEPA-MtOEPB as a template, reverse PCR amplification was performed with F4 as the upstream primer and R4 as the downstream primer. After removing the template from the reverse PCR product, self-circularization was performed to obtain the mutant plasmid pETDuet-MtOEPA. L283Y -MtOEPB.

[0065] F4: CCTGtatCGCGCGAAAAACGCGAGCAAAAAAC (SEQ ID NO. 19).

[0066] R4:TTTTCGCGCGataCAGGCCCGGCTGGGTATCG (SEQ ID NO. 20).

[0067] With the mutant plasmid pETDuet-MtOEPA I126K Using MtOEPB as a template, reverse PCR amplification was performed with F4 as the upstream primer and R4 as the downstream primer. After template removal, the reverse PCR product was self-circulated to obtain the mutant plasmid pETDuet-MtOEPA. I126K / L283Y -MtOEPB.

[0068] With the mutant plasmid pETDuet-MtOEPA I126K / L283Y Using MtOEPB as a template, reverse PCR amplification was performed with F5 as the upstream primer and R5 as the downstream primer. After template removal, the reverse PCR product was self-circulated to obtain the mutant plasmid pETDuet-MtOEPA. I126K / L283Y -MtOEPB G225A .

[0069] F5: TAAAGTGACCTGGgccAGCGGCGCGGTGGTGGAA (SEQ ID NO. 21).

[0070] R5: TggcCCAGGTCACTTTATACGCGCCCAGCGCG (SEQ ID NO. 22).

[0071] The systems for each of the above reverse PCR amplification reactions are shown in Table 7, and the reaction procedures are shown in Table 8.

[0072] Table 7

[0073]

[0074] Table 8

[0075]

[0076] It should be understood that the pre-denaturation at 94°C and the storage at 4°C are not included in the cycle for each of the above reverse PCR amplification reactions, and each reverse PCR process is performed only once.

[0077] Template elimination: After the above reverse PCR reactions were completed, 2 μL of restriction endonuclease DpnⅠ was added to each reaction solution (50 μL), and the mixture was gently blown and aspirated. The mixture was then reacted at 37℃ for 1 h to obtain the enzyme digestion solution. The enzyme digestion solution was verified by agarose gel electrophoresis.

[0078] Self-circularization of reverse PCR products: Take 2 μL of each of the enzyme digestion solutions obtained above, add 1 μL of high-efficiency ligation reagent, 1 μL of T4 polynucleotide kinase, and 6 μL of ddH2O respectively, mix gently, and incubate at 16℃ for 1 h to obtain the mutant plasmid pETDuet-MtOEPA. I126K -MtOEPB, mutant plasmid pETDuet-MtOEPA L283Y -MtOEPB, mutant plasmid pETDuet-MtOEPA I126K / L283Y -MtOEPB, and the mutant plasmid pETDuet-MtOEPA I126K / L283Y -MtOEPB G225A .

[0079] Mutant plasmid verification: Each mutant plasmid obtained after circularization was transformed into E. coli DH5α competent cells by chemical transformation. Single colonies on the plates were picked for plasmid extraction, and the extracted plasmids were sequenced for DNA.

[0080] Mutant plasmid pETDuet-MtOEPA I126K MtOEPB contains both the encoding gene for the D-monomentol dehydrogenase MtOEPa mutant I126K (shown in SEQ ID NO.5) and the encoding gene for the wild-type D-pinelol dehydrogenase MtOEPb. The amino acid sequence of the D-monomentol dehydrogenase MtOEPa mutant I126K is shown in SEQ ID NO.6.

[0081] Mutant plasmid pETDuet-MtOEPA L283Y MtOEPB contains both the encoding gene for the D-monomentol dehydrogenase MtOEPa mutant L283Y (shown in SEQ ID NO.7) and the encoding gene for the wild-type D-pinelol dehydrogenase MtOEPb. The amino acid sequence of the D-monomentol dehydrogenase MtOEPa mutant L283Y is shown in SEQ ID NO.8.

[0082] Mutant plasmid pETDuet-MtOEPA I126K / L283Y MtOEPB contains both the encoding gene for the D-monomentol dehydrogenase MtOEPa mutant I126K / L283Y (shown in SEQ ID NO. 9) and the encoding gene for the wild-type D-pinelol dehydrogenase MtOEPb. The amino acid sequence of the D-monomentol dehydrogenase MtOEPa mutant I126K / L283Y is shown in SEQ ID NO. 10.

[0083] Mutant plasmid pETDuet-MtOEPA I126K / L283Y -MtOEPB G225AIt contains the encoding genes for both the D-mannosyl dehydrogenase MtOEPa mutant I126K / L283Y and the D-pinoxetine dehydrogenase MtOEPb mutant G225A (as shown in SEQ ID NO. 11). The amino acid sequence of the D-pinoxetine dehydrogenase MtOEPb mutant G225A is shown in SEQ ID NO. 12.

[0084] Example 3

[0085] Preparation of crude enzyme solution

[0086] Take 2 μL of the mutant plasmid pETDuet-MtOEPA I126K -MtOEPB, 2μL mutant plasmid pETDuet-MtOEPA L283Y -MtOEPB, 2μL mutant plasmid pETDuet-MtOEPA I126K / L283Y -MtOEPB, and 2 μL of the mutant plasmid pETDuet-MtOEPA I126K / L283Y -MtOEPB G225A The recombinant strains were added to *E. coli* BL21(DE3) competent cells. The mixtures were incubated on ice for 30 min, followed by heat shock at 42°C for 60 s, and then incubated on ice for 5 min. The resulting recombinant strains were then transferred to 500 μL of LB broth and incubated at 37°C with shaking for 1 h. 100 μL of each bacterial culture was then plated onto LB agar plates containing 50 μg / mL ampicillin and incubated upside down at 37°C for 12 h. Mutant strains BL21-MtOEPA were obtained through screening. I126K -MtOEPB, mutant strain BL21-MtOEPA L283Y -MtOEPB, mutant strain BL21-MtOEPA I126K / L283Y -MtOEPB, and the mutant strain BL21-MtOEPA I126K / L283Y -MtOEPB G225A .

[0087] The four mutant strains were inoculated into liquid LB medium (containing 50 μg / mL ampicillin) for seed culture and cultured at 37℃ and 220 rpm for 12 h to obtain mutant strain BL21-MtOEPA. I126K Seed culture of MtOEPB, mutant strain BL21-MtOEPA L283Y Seed culture of MtOEPB, mutant strain BL21-MtOEPA I126K / L283Y Seed culture of MtOEPB and mutant strain BL21-MtOEPA I126K / L283Y -MtOEPB G225A Seed liquid.

[0088] The above four seed solutions were inoculated into fresh LB liquid medium (containing 50 μg / mL ampicillin) at a 2% (v / v) inoculum and cultured at 37°C until OD500. 600 The value was 0.7. IPTG was added to a final concentration of 0.05 mM and the mixture was induced and cultured at 20℃ for 16 h. Fermentation broths containing D-manganese dehydrogenase MtOEPa mutant I126K and wild-type D-pineol dehydrogenase MtOEPb, D-manganese dehydrogenase MtOEPa mutant L283Y and wild-type D-pineol dehydrogenase MtOEPb, D-manganese dehydrogenase MtOEPa mutant I126K / L283Y and wild-type D-pineol dehydrogenase MtOEPb, and D-pineol dehydrogenase MtOEPb mutant G225A were obtained.

[0089] The four fermentation broths were centrifuged at 4℃ and 4000 rpm for 15 min, and the bacterial cells were collected. The collected bacterial cells were resuspended in 50 mM phosphate buffer (pH 7.5). Then, the bacterial cells were disrupted using an ultrasonic cell disruptor at 450 W, with a 2-second disruption followed by a 3-second pause, for a total of 30 min. After ultrasonic disruption, the cells were centrifuged at 4℃ and 12000 rpm to remove cell debris, and the supernatant was collected. These yielded crude enzyme solutions containing both the D-mannone dehydrogenase MtOEPa mutant I126K and wild-type D-pineol dehydrogenase MtOEPb, and solutions containing D-mannone dehydrogenase MtOEPb. Crude enzyme solutions containing the D-pineol dehydrogenase MtOEPa mutant L283Y and wild-type D-pineol dehydrogenase MtOEPb; crude enzyme solutions containing both the D-pineol dehydrogenase MtOEPa mutant I126K / L283Y and wild-type D-pineol dehydrogenase MtOEPb; and crude enzyme solutions containing both the D-pineol dehydrogenase MtOEPa mutant I126K / L283Y and the D-pineol dehydrogenase MtOEPb mutant G225A.

[0090] The recombinant plasmid pETDuet-MtOEPA-MtOEPB with correct sequencing from Example 1 above was transformed into Escherichia coli BL21(DE3) competent cells using the same method as described above to obtain the recombinant strain BL21-MtOEPA-MtOEPB. Crude enzyme solution containing both wild-type D-manganese dehydrogenase MtOEPa and wild-type D-pineol dehydrogenase MtOEPb was prepared using the same method as described above.

[0091] Example 4

[0092] Preparation of reaction system 1: Add muscle inositol and NAD to a 50mM phosphate buffer solution with a pH of 7.5. ＋ NADP + Example 3 prepared a crude enzyme solution containing both wild-type D-manganese dehydrogenase MtOEPa and wild-type D-pineol dehydrogenase MtOEPb, and prepared a 10 mL reaction system. The concentrations of each component in the reaction system were: muscle inositol 30 mM, NAD... ＋ 0.5mM, NADP + 0.5mM, total crude enzyme protein concentration 1.5mg / mL.

[0093] Preparation of reaction system 2: Add muscle inositol and NAD to a 50mM phosphate buffer solution with a pH of 7.5. ＋ NADP + Example 3 prepared a crude enzyme solution containing both the D-mannosyl alcohol dehydrogenase MtOEPa mutant I126K and the wild-type D-pineol dehydrogenase MtOEPb. A 10 mL reaction system was prepared, with the following concentrations of components: muscle inositol 30 mM, NAD+... ＋ 0.5mM, NADP + 0.5mM, total crude enzyme protein concentration 1.5mg / mL.

[0094] Preparation of reaction system 3: Add muscle inositol and NAD to a 50mM phosphate buffer solution with a pH of 7.5. ＋ NADP + Example 3 prepared a crude enzyme solution containing both the D-manganese dehydrogenase MtOEPa mutant L283Y and the wild-type D-pinene dehydrogenase MtOEPb. A 10 mL reaction system was prepared, with the following concentrations: muscle inositol 30 mM, NAD+... ＋ 0.5mM, NADP + 0.5mM, total crude enzyme protein concentration 1.5mg / mL.

[0095] Preparation of reaction system 4: Add muscle inositol and NAD to a 50mM phosphate buffer solution with a pH of 7.5. ＋ NADP + Example 3 prepared a crude enzyme solution containing both the D-mannosyl alcohol dehydrogenase MtOEPa mutant I126K / L283Y and the wild-type D-pineol dehydrogenase MtOEPb. A 10 mL reaction system was prepared, with the following concentrations: muscle inositol 30 mM, NAD+... ＋ 0.5mM, NADP + 0.5mM, total crude enzyme protein concentration 1.5mg / mL.

[0096] Preparation of reaction system 5: Add muscle inositol and NAD to a 50mM phosphate buffer solution with a pH of 7.5. ＋ NADP + Example 3 prepared a crude enzyme solution containing both D-mannosyl alcohol dehydrogenase MtOEPa mutant I126K / L283Y and D-pinel alcohol dehydrogenase MtOEPb mutant G225A. A 10 mL reaction system was prepared, with the following concentrations: muscle inositol 30 mM, NAD+... ＋ 0.5mM, NADP + 0.5mM, total crude enzyme protein concentration 1.5mg / mL.

[0097] Each of the above reaction systems was reacted at 37℃ for 20 min. The content of D-chiral inositol in each reaction solution was detected by high performance liquid chromatography after the reaction, and the conversion rate was calculated. The results are shown in Table 9.

[0098] Conversion rate: D-chiral inositol content ÷ initial muscle inositol content × 100%.

[0099] High-performance liquid chromatography (HPLC) detection methods:

[0100] The chromatographic column is a 4.6 × 250 mm, 5 μm amino column;

[0101] Mobile phase: Acetonitrile: 50 mM ammonium acetate aqueous solution = 75: 25 (volume ratio);

[0102] The column temperature was set to 30℃ and the flow rate to 1.0 mL / min.

[0103] Table 9

[0104]

[0105] The results above show that the D-monosoline dehydrogenase MtOEPa mutant I126K, D-monosoline dehydrogenase MtOEPa mutant L283Y, and D-monosoline dehydrogenase MtOEPa mutant I126K / L283Y in this invention all further improved the conversion rate and the yield of D-chiral inositol by increasing enzyme activity. Furthermore, the combined use of D-monosoline dehydrogenase MtOEPa mutant I126K / L283Y and D-pinel dehydrogenase MtOEPb mutant G225A resulted in the highest conversion rate and the highest yield of D-chiral inositol.

[0106] Example 5

[0107] High-density fermentation for the preparation of crude enzyme solution

[0108] The mutant strain BL21-MtOEPA from Example 3 I126K-MtOEPB, mutant strain BL21-MtOEPA L283Y -MtOEPB, mutant strain BL21-MtOEPA I126K / L283Y -MtOEPB, and the mutant strain BL21-MtOEPA I126K / L283Y -MtOEPB G225A The samples were inoculated into liquid culture medium for seed culture, with LB liquid medium containing 50 μg / mL ampicillin as a suitable seed medium, and cultured at 37°C until OD500. 600 Up to version 2.0, mutant strain BL21-MtOEPA was obtained. I126K Seed culture of MtOEPB, mutant strain BL21-MtOEPA L283Y Seed culture of MtOEPB, mutant strain BL21-MtOEPA I126K / L283Y Seed culture of MtOEPB and mutant strain BL21-MtOEPA I126K / L283Y -MtOEPB G225A Seed liquid.

[0109] The four seed cultures were inoculated into fermenters containing culture medium at a volume ratio of 5%, and cultured at a pressure of 0.045 MPa, an initial rotation speed of 350 rpm, a temperature of 37°C, and a pH of 7.0 until OD. 600 Cool to 30°C and continue culturing until OD reaches 30°C. 600 Induction culture was performed separately at a concentration of 40 μM. The induction culture conditions were as follows: IPTG was added to a final concentration of 0.05 mM; the initial airflow rate was 4.0 L / min; when the rotation speed reached 450 rpm, the airflow rate was adjusted to 5 L / min; when the rotation speed reached 600 rpm, the airflow rate was adjusted to 5.5 L / min; when dissolved oxygen rebounded, supplemental culture medium was added to maintain the dissolved oxygen (DO) value between 20% and 40%, until the OD value was reached. 600 Fermentation was stopped when the temperature reached 120°C, yielding fermentation broths containing both the D-monocarboxyl dehydrogenase MtOEPa mutant I126K and wild-type D-pinyl dehydrogenase MtOEPb, the D-monocarboxyl dehydrogenase MtOEPa mutant L283Y and wild-type D-pinyl dehydrogenase MtOEPb, the D-monocarboxyl dehydrogenase MtOEPa mutant I126K / L283Y and wild-type D-pinyl dehydrogenase MtOEPb, and the D-monocarboxyl dehydrogenase MtOEPa mutant I126K / L283Y and D-pinyl dehydrogenase MtOEPb mutant G225A.

[0110] The four fermentation broths were centrifuged separately using a high-speed refrigerated centrifuge at 4°C and 8000 rpm for 15 min. The supernatant was discarded, and the bacterial cells were collected. The collected bacterial cells were resuspended in 50 mM phosphate buffer (pH 7.5). The OD of the resuspended cells was measured. 600 The cell value was 80. Cells were then homogenized at 750 bar for 30 minutes using a high-pressure homogenizer. After cell disruption, the cells were centrifuged again at 4°C and 12000 rpm for 60 minutes to remove cell debris. The supernatants were collected to obtain crude enzyme solutions containing both the D-manganese dehydrogenase MtOEPa mutant I126K and wild-type D-pinene dehydrogenase MtOEPb, and crude enzyme solutions containing both D-manganese dehydrogenase MtOEPa mutant I126K and wild-type D-pinene dehydrogenase MtOEPb. Crude enzyme solutions containing D-pineol dehydrogenase MtOEPa mutant L283Y and wild-type D-pineol dehydrogenase MtOEPb; crude enzyme solutions containing both D-pineol dehydrogenase MtOEPa mutant I126K / L283Y and wild-type D-pineol dehydrogenase MtOEPb; and crude enzyme solutions containing both D-pineol dehydrogenase MtOEPa mutant I126K / L283Y and D-pineol dehydrogenase MtOEPb mutant G225A.

[0111] Take the recombinant strain BL21-MtOEPA-MtOEPB from Example 3 and prepare a crude enzyme solution containing both wild-type D-manganese dehydrogenase MtOEPa and wild-type D-pineol dehydrogenase MtOEPb using the same method described above.

[0112] The culture medium in the above fermenter consists of: 2 g / L citric acid monohydrate, 14 g / L potassium dihydrogen phosphate, 4.5 g / L dipotassium hydrogen phosphate trihydrate, 6 g / L ammonium sulfate, 10 g / L glucose, 1 g / L magnesium sulfate heptahydrate, 1 g / L yeast extract, and 1 mL / L trace element solution.

[0113] The trace element solution consists of: 1M concentrated hydrochloric acid, 20g / L ferric chloride hexahydrate, 2g / L zinc chloride, 2g / L cobalt dichloride hexahydrate, and 2g / L copper chloride dihydrate.

[0114] The composition of the above-mentioned feed culture medium is: glucose 600g / L, magnesium sulfate 2g / L, and yeast extract 10g / L.

[0115] Example 6

[0116] Preparation of D-chiral inositol

[0117] Preparation of reaction system one: Add muscle inositol and NAD to a 50mM phosphate buffer solution with a pH of 7.5. ＋ NADP +Example 5 prepared a crude enzyme solution containing both wild-type D-manganese dehydrogenase MtOEPa and wild-type D-pineol dehydrogenase MtOEPb. The concentrations of each component in the reaction system were: muscle inositol 120 g / L, NAD... ＋ 1.5mM, NADP + 1.5mM, crude enzyme protein total concentration 80mg / mL.

[0118] Preparation of reaction system two: Add muscle inositol and NAD to a 50mM phosphate buffer solution with a pH of 7.5. ＋ NADP + Example 5 prepared a crude enzyme solution containing both the D-manganese dehydrogenase MtOEPa mutant I126K and the wild-type D-pinene dehydrogenase MtOEPb. The concentrations of each component in the reaction system were: muscle inositol 120 g / L, NAD+, and so on. ＋ 1.5mM, NADP + 1.5mM, crude enzyme protein total concentration 80mg / mL.

[0119] Preparation of reaction system three: Add muscle inositol and NAD to a 50mM phosphate buffer solution with a pH of 7.5. ＋ NADP + Example 5 prepared a crude enzyme solution containing both the D-manganese dehydrogenase MtOEPa mutant L283Y and the wild-type D-pinene dehydrogenase MtOEPb. The concentrations of each component in the reaction system were: muscle inositol 120 g / L, NAD+, and so on. ＋ 1.5mM, NADP + 1.5mM, crude enzyme protein total concentration 80mg / mL.

[0120] Preparation of reaction system four: Add muscle inositol and NAD to a 50mM phosphate buffer solution with a pH of 7.5. ＋ NADP + Example 5 prepared a crude enzyme solution containing both the D-mannosyl alcohol dehydrogenase MtOEPa mutant I126K / L283Y and the wild-type D-pineol dehydrogenase MtOEPb. The concentrations of each component in the reaction system were: muscle inositol 120 g / L, NAD+, and so on. ＋ 1.5mM, NADP + 1.5mM, crude enzyme protein total concentration 80mg / mL.

[0121] Preparation of reaction system five: Add muscle inositol and NAD to a 50mM phosphate buffer solution with a pH of 7.5. ＋ NADP +Example 5 prepared a crude enzyme solution containing both D-mannosyl alcohol dehydrogenase MtOEPa mutant I126K / L283Y and D-pinel alcohol dehydrogenase MtOEPb mutant G225A. The concentrations of each component in the reaction system were: muscle inositol 120 g / L, NAD... ＋ 1.5mM, NADP + 1.5mM, crude enzyme protein total concentration 80mg / mL.

[0122] Each of the above reaction systems was reacted at 35℃ for 8 hours. The content of D-chiral inositol in each reaction solution was detected by high performance liquid chromatography (HPLC) after the reaction, and the conversion rate was calculated. The results are shown in Table 10.

[0123] Conversion rate: D-chiral inositol content ÷ initial muscle inositol content × 100%.

[0124] High-performance liquid chromatography (HPLC) detection methods:

[0125] The chromatographic column is a 4.6 × 250 mm, 5 μm amino column;

[0126] Mobile phase: Acetonitrile: 50 mM ammonium acetate aqueous solution = 75: 25 (volume ratio);

[0127] The column temperature was set to 30℃ and the flow rate to 1.0 mL / min.

[0128] Table 10

[0129]

[0130] The results above show that the D-mannone dehydrogenase MtOEPa and wild-type D-pineol dehydrogenase MtOEPb mutants obtained by the present invention, namely, the D-mannone dehydrogenase MtOEPa mutant I126K, the D-mannone dehydrogenase MtOEPa mutant L283Y, the D-mannone dehydrogenase MtOEPa mutant I126K / L283Y, and the D-pineol dehydrogenase MtOEPb mutant G225A, can still achieve high conversion rates and increase the yield of D-chiral inositol even at high substrate concentrations.

[0131] Example 7

[0132] After the reaction in Example 6 was completed, five conversion solutions containing D-chiral inositol were obtained. D-chiral inositol could be extracted from each conversion solution using the following method, the specific steps of which are as follows:

[0133] The conversion solution was heated at 95°C for 10 min to inactivate it, and then filtered using an ultrafiltration membrane with a pore size of 10000 Da to remove proteins and other biomacromolecules from the conversion solution, thus obtaining the ultrafiltration membrane permeate.

[0134] The permeate from the ultrafiltration membrane was concentrated and desalted through a nanofiltration membrane with a pore size of 200 Da until the solid content of the nanofiltration membrane concentrate was 15%. The nanofiltration membrane concentrate was then filtered under vacuum conditions of ≤-0.09 MPa to remove solid impurities, resulting in a first filtrate containing 50% D-chiral inositol by mass.

[0135] The first filtrate was heated to 45°C, and 70% of the volume of the first filtrate was added with ethanol. The mixture was kept at this temperature for 1 hour and then cooled to 25°C to crystallize the muscle inositol. The muscle inositol crystals were removed by filtration under a vacuum of ≤-0.09 MPa to obtain a second filtrate with a D-chiral inositol content of 80%.

[0136] The second filtrate was de-alcoholized using a rotary evaporator to obtain a de-alcoholized solution with an alcohol content of less than 5%. The de-alcoholized solution was then concentrated using a rotary evaporator to a solid content of 50%. The solution was heated to 50°C, and 70% of the volume of the de-alcoholized solution of ethanol was added. The solution was then slowly cooled to 30°C at a rate of 5°C / h to allow the D-chiral inositol to crystallize. The solution was then filtered under vacuum conditions of ≤-0.09 MPa, and the filter cake was collected to obtain solid D-chiral inositol with a mass content >90%.

[0137] 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, characterized in that, Including D-monomentol dehydrogenase MtOEPa and D-pinelol dehydrogenase MtOEPb; The amino acid sequence of the D-mannosyl dehydrogenase MtOEPa is shown in SEQ ID NO. 6, SEQ ID NO. 8 or SEQ ID NO. 10, and the amino acid sequence of the D-pinel dehydrogenase MtOEPb is shown in SEQ ID NO. 3; or, The amino acid sequence of the D-mannosyl dehydrogenase MtOEPa is shown in SEQ ID NO.10, and the amino acid sequence of the D-pinel dehydrogenase MtOEPb is shown in SEQ ID NO.

12.

2. The enzyme preparation according to claim 1, characterized in that, The mass ratio of the D-manganese dehydrogenase MtOEPa to the D-pineol dehydrogenase MtOEPb is (1~5):(1~5).

3. The enzyme preparation according to claim 1, characterized in that, The gene sequence of the D-mannosyl dehydrogenase MtOEPa is shown in SEQ ID NO.5, SEQ ID NO.7 or SEQ ID NO.9, and the gene sequence of the D-pinel dehydrogenase MtOEPb is shown in SEQ ID NO.4; or, The gene sequence of the D-mannosyl dehydrogenase MtOEPa is shown in SEQ ID NO.9, and the gene sequence of the D-pinel dehydrogenase MtOEPb is shown in SEQ ID NO.

11.

4. A method for preparing an enzyme preparation, used to prepare the enzyme preparation of claim 1, characterized in that, Includes the following steps: A recombinant expression vector containing both the D-manganese dehydrogenase MtOEPa encoding gene and the D-pinyl alcohol dehydrogenase MtOEPb encoding gene was constructed, and the recombinant expression vector was transformed into a host strain to obtain a recombinant strain. The recombinant strain was fermented to induce the expression of D-manganese dehydrogenase MtOEPa and D-pinyl alcohol dehydrogenase MtOEPb, resulting in a fermentation broth containing both D-manganese dehydrogenase MtOEPa and D-pinyl alcohol dehydrogenase MtOEPb. The fermentation broth is centrifuged to collect the cells, which are then resuspended and broken up. The cells are centrifuged again, and the supernatant is collected to obtain a crude enzyme solution containing the enzyme preparation.

5. The use of the enzyme preparation according to claim 1 in the preparation of D-chiral inositol.

6. A method for preparing D-chiral inositol, characterized in that, Using the enzyme preparation of claim 1, in the presence of a coenzyme factor, the substrate muscle inositol is catalyzed to react and generate D-chiral inositol.

7. The preparation method according to claim 6, characterized in that, Add muscle inositol, coenzyme factor, and crude enzyme solution containing enzyme preparation to a 20-100mM buffer solution to make the concentration of muscle inositol in the reaction system 5-120g / L and the total concentration of crude enzyme protein 0.5-100mg / mL.

8. The preparation method according to claim 7, characterized in that, The buffer solution includes HEPE buffer, Tris-HCl buffer, or phosphate buffer.

9. The preparation method according to claim 6, characterized in that, The coenzyme factor includes NAD. ＋ NADP ＋ ; In the reaction system, the NAD ＋ The concentration of NADP is 0.5~2.0 mM. ＋ The concentration is 0.5~2.0mM.

10. The preparation method according to claim 6, characterized in that, The reaction temperature is 30~40℃, and the pH value of the reaction is 7.0~8.0.

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