Enzyme formulation for catalytic synthesis of scyllo-inositol, and method for preparing scyllo-inositol

Abstract

The present invention belongs to the technical field of genetic engineering. Provided are an enzyme formulation for the catalytic synthesis of scyllo-inositol, and a method for preparing scyllo-inositol. The enzyme formulation has the enzymatic activity of catalyzing the reaction of myo-inositol to form scyllo-inositol, and comprises inositol dehydrogenase and scyllo-inositol dehydrogenase. After mutation, inositol dehydrogenase has a higher enzymatic activity, and scyllo-inositol dehydrogenase has a broader applicable pH range. Further provided is a method for preparing scyllo-inositol, which method comprises: (1) preparing a crude enzyme solution of inositol dehydrogenase and scyllo-inositol dehydrogenase, preparing a myo-inositol solution, and adding the myo-inositol solution to the crude enzyme solution; (2) adjusting a pH value to 7-10, controlling a temperature to 40-50°C, and performing a conversion reaction to obtain conversion solution A; and (3) subjecting conversion solution A to first cooling crystallization and filtration, and collecting a filtrate and a filter cake; adding water and a myo-inositol solid to the filtrate, and repeating step (2) and step (3) until no crystals precipitate; and subjecting the filter cake collected each time to first ethanol washing, second cooling crystallization, filtration, second ethanol washing and drying in sequence, to obtain scyllo-inositol. The method for preparing scyllo-inositol has good conversion performance, shortens the production cycle of scyllo-inositol, and reduces waste liquid generation and environmental pollution.

Description

An enzyme preparation for catalytic synthesis of squalinositol and a method for preparing squalinositol.

[0001] This application claims priority to Chinese Patent Application No. 202411684867.3, filed on November 22, 2024, and Chinese Patent Application No. 202510413089.2, filed on April 3, 2025, the disclosures of which are incorporated herein by reference in their entirety. Technical Field

[0002] This invention relates to the field of genetic engineering technology, and in particular to an enzyme preparation for catalytic synthesis of squalene and a method for preparing squalene. Background Technology

[0003] Squalinositol (SI) is an epimer of myoinositol (MI), found in plants, animals, and some bacteria, but its abundance in nature is far lower than that of myoinositol. It possesses a variety of biological activities, including therapeutic effects on Alzheimer's disease, antioxidant properties, metabolic regulation, and skin health maintenance. Furthermore, squalinositol is being widely used in clinical trials for behavioral and mental disorders, protein aggregation disorders, and anticancer drug combinations, and it still holds significant future development potential.

[0004] In traditional synthesis methods, squalene is mainly synthesized by chemical methods through complex steps using muscle inositol as raw material. The main drawbacks are as follows: (1) low inositol concentration and long production cycle; (2) long cell culture time, low utilization efficiency of inositol dehydrogenase and squalene dehydrogenase, and cannot be recycled; (3) large amounts of phosphoric acid, boric acid or borate are required in the production process, which is not environmentally friendly; (4) large amount of buffer salt is used in the conversion process.

[0005] The most likely industrial-scale biosynthesis of squalene is based on muscle inositol, utilizing inositol dehydrogenase (IDH) and squalene dehydrogenase (SID) with the aid of NAD+. + The NADH coenzyme cycle system converts muscle inositol into squalinositol. However, this method has the following drawbacks: First, the conversion rate of squalinositol is low, resulting in high costs; second, the reaction system after conversion contains not only squalinositol but also structurally similar muscle inositol and squalinositol monoketones, making squalinositol separation difficult; third, the preparation process requires the use of boric acid, which may have a certain impact on the environment. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide an enzyme preparation for the catalytic synthesis of squalinositol and a method for preparing squalinositol, thereby overcoming the problems of low inositol feed concentration, long production cycle, low utilization efficiency of inositol dehydrogenase and squalinositol dehydrogenase in the prior art, low conversion rate of squalinositol during preparation, difficult separation, and potential environmental impact from the use of boric acid.

[0007] In a first aspect, the present invention provides an enzyme preparation for catalyzing the synthesis of squalinositol, the enzyme preparation having enzymatic activity for catalyzing the reaction of muscle inositol to squalinositol, the enzyme preparation comprising inositol dehydrogenase and squalinositol dehydrogenase, the amino acid sequence of the inositol dehydrogenase being any of the following:

[0008] (1) The amino acid sequence shown in SEQ ID NO.3;

[0009] (2) The amino acid sequence shown in SEQ ID NO.4;

[0010] (3) The amino acid sequence shown in SEQ ID NO.5;

[0011] (4) The amino acid sequence shown in SEQ ID NO. 6;

[0012] The amino acid sequence of squalinositol dehydrogenase is shown in SEQ ID NO.7.

[0013] Compared with the prior art, the present invention mutates specific sites in wild-type inositol dehydrogenase (amino acid sequence as shown in SEQ ID NO.1) and wild-type squalinositol dehydrogenase (amino acid sequence as shown in SEQ ID NO.2), respectively. After mutation, the enzyme activity of inositol dehydrogenase is higher and the pH range applicable to squalinositol dehydrogenase is wider.

[0014] Furthermore, the enzyme activity ratio of inositol dehydrogenase to squalinositol dehydrogenase is (10-16):(10-16).

[0015] The above technical solution further limits the enzyme activity ratio of inositol dehydrogenase and squalinositol dehydrogenase. Within this enzyme activity ratio range, the two enzymes work together to further improve the conversion rate of squalinositol.

[0016] Furthermore, the methods for modifying the enzyme activity of inositol dehydrogenase include the following:

[0017] (1) The amino acid sequence shown in SEQ ID NO.3 is obtained by changing the N to Q of the 123rd amino acid in the amino acid sequence shown in SEQ ID NO.1;

[0018] (2) The amino acid sequence shown in SEQ ID NO.4 is obtained by mutating amino acid D to A at position 154 of the amino acid sequence shown in SEQ ID NO.1;

[0019] (3) The amino acid sequence shown in SEQ ID NO.5 is obtained by mutating amino acid F to P at position 277 of the amino acid sequence shown in SEQ ID NO.1;

[0020] (4) The amino acid sequence shown in SEQ ID NO.6 is obtained by changing the N to Q amino acid at position 123 of the amino acid sequence shown in SEQ ID NO.1, and changing the F to P amino acid at position 277.

[0021] In the above technical solution, the amino acid sequences shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 are, in order, the amino acid sequences of the inositol dehydrogenase mutants N123Q, D154A, F277P, and N123Q / F277P.

[0022] Preferably, the gene sequences of the inositol dehydrogenase mutants N123Q, D154A, F277P and N123Q / F277P are shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13, respectively.

[0023] Furthermore, the methods for modifying the enzyme activity of squalinositol dehydrogenase include the following:

[0024] The amino acid sequence shown in SEQ ID NO.7 is obtained by mutating amino acid Q to Y at position 279 of the amino acid sequence shown in SEQ ID NO.2.

[0025] The amino acid sequence shown in SEQ ID NO.7 of the above technical solution is the amino acid sequence of the squalinositol dehydrogenase mutant Q279Y. Preferably, the gene sequence of the squalinositol dehydrogenase mutant Q279Y is shown in SEQ ID NO.14.

[0026] Furthermore, the preparation of inositol dehydrogenase includes the following methods:

[0027] 1) Provide the gene encoding the above-mentioned inositol dehydrogenase;

[0028] 2) Transform host cells using the above-mentioned genes;

[0029] 3) Obtain host cells that produce inositol dehydrogenase;

[0030] 4) Recover inositol dehydrogenase;

[0031] The preparation of squalene dehydrogenase includes the following methods:

[0032] 1) Provide the gene encoding the above-mentioned squalene dehydrogenase;

[0033] 2) Transform host cells using the above-mentioned genes;

[0034] 3) Obtain host cells that produce squalene dehydrogenase;

[0035] 4) Recover squalene dehydrogenase.

[0036] Specifically, the following methods can be used to prepare inositol dehydrogenase solution or squalinositol dehydrogenase solution:

[0037] Expression vectors containing the inositol dehydrogenase gene or the squalino dehydrogenase gene were transferred into host strains to obtain recombinant strains containing the inositol dehydrogenase gene or recombinant strains containing the squalino dehydrogenase gene.

[0038] The recombinant strains containing the inositol dehydrogenase gene or the recombinant strains containing the squalinositol dehydrogenase gene were respectively inoculated into fermentation medium for fermentation culture until the bacterial biomass reached OD. 600 The temperature was set at 70-90°C, then cooled to 25-30°C. L-arabinose was added to a final concentration of 1-2 g / L to begin induction culture until the biomass of each strain stopped increasing, and fermentation broth was obtained.

[0039] Centrifuge the two fermentation broths separately, collect the cells separately, resuspend the cells separately, homogenize and break the cells, centrifuge separately, and collect the supernatant separately to obtain the inositol dehydrogenase solution or squalinositol dehydrogenase solution.

[0040] During fermentation, the temperature is controlled at 35–40℃, the pH at 6.8–7.2, and the dissolved oxygen (DO) at 20%–30%.

[0041] Furthermore, in the preparation of squalene dehydrogenase, the fermentation medium contains PQQ and ammonium chloride; the concentration of PQQ in the fermentation medium is 0.015–0.025 mmol / L, and the concentration of ammonium chloride is 1–2 g / L.

[0042] In this invention, when fermenting a recombinant strain containing the squalene dehydrogenase gene, a specific concentration of PQQ and ammonium chloride is added to the fermentation medium. PQQ can increase the biomass of the recombinant strain, and ammonium chloride can further increase the expression level of squalene dehydrogenase in the recombinant strain.

[0043] Secondly, the present invention provides a method for preparing squalinositol, which utilizes the above-mentioned enzyme preparation to catalyze the reaction of muscle inositol to generate squalinositol.

[0044] Compared with existing technologies, using the enzyme preparation of the present invention, which includes specific inositol dehydrogenase and squalinositol dehydrogenase, to catalyze the reaction of muscle inositol to squalinositol can effectively improve the conversion rate of squalinositol.

[0045] Furthermore, the concentrations of each component in the reaction system that catalyzes the reaction of muscle inositol to produce squalene are as follows: muscle inositol 50-100 g / L, inositol dehydrogenase 10-16 U / mL, squalene dehydrogenase 10-16 U / mL, and dipotassium hydrogen phosphate 10-50 mM.

[0046] Furthermore, the reaction temperature of the reaction system is 35–60℃, and the pH value is 8–10.

[0047] Furthermore, the preparation method also includes: after the reaction is complete, maintaining the reaction system at a temperature of 115–130°C for 0.5–1 h to convert the intermediate product squalinositol monoketone into squalinositol, and then adding bacterial cells expressing inositol oxidase to make the concentration of the bacterial cells in the reaction system 10–50 OD. 600 The remaining muscle inositol in the reaction system was converted into glucuronic acid, and squalinositol and glucuronic acid were separated.

[0048] The above technical solution first converts the intermediate product squalinositol monoketone into squalinositol using high temperature, thus eliminating the intermediate product. Then, inositol oxidase is used to convert the remaining muscle inositol in the reaction system into glucuronic acid, resulting in a mixed solution of squalinositol and glucuronic acid. The squalinositol and glucuronic acid in this mixed solution are then separated to obtain high-purity squalinositol and glucuronic acid. Compared with existing technologies, the above method for preparing squalinositol converts squalinositol monoketone and muscle inositol, which have similar structures to squalinositol, into squalinositol and glucuronic acid, which is more conducive to the separation and extraction of squalinositol after the reaction. Furthermore, the above method eliminates the use of boric acid, making the preparation process more environmentally friendly.

[0049] Furthermore, the reaction temperature for converting the remaining muscle inositol in the reaction system into glucuronic acid is 35–40°C, and the pH value is 7.8–8.2.

[0050] Thirdly, this invention also provides a method for preparing squalene, comprising the following steps:

[0051] (1) Prepare raw materials: Prepare crude enzyme solutions of inositol dehydrogenase and squalinositol dehydrogenase, prepare muscle inositol solution, add muscle inositol solution to the crude enzyme solution, and control the temperature of the raw material system between 40-50℃.

[0052] (2) Conversion reaction: Adjust the pH value to 7-10, control the temperature to 40-50℃, carry out the conversion reaction, and obtain conversion liquid A. Perform the first cooling crystallization and filtration on the conversion liquid A, and collect the filtrate and filter cake respectively.

[0053] (3) Add water and muscle inositol solid to the filtrate, repeat steps (2) and (3) until no crystals precipitate, and wash the filter cake collected each time with ethanol for the first time, cool and crystallize for the second time, filter, wash with ethanol for the second time, and dry to obtain squalene product.

[0054] Compared with existing technologies, firstly, this invention, after reacting the crude enzyme solution and muscle inositol solution to obtain the conversion solution, directly cools and crystallizes the conversion solution, replacing the commonly used method of extracting squalene from the conversion solution with boric acid. This avoids the use of acids such as boric acid, simplifies the reaction steps, improves production safety, and yields squalene products with a purity of up to 99.8%. Secondly, water and muscle inositol solids are added to the filtrate after cooling, crystallization, and filtration of the conversion solution. Preferably, this ensures that the concentration of muscle inositol during the cyclic conversion process is consistent with the concentration of the muscle inositol solution in the raw material system, allowing for cyclic conversion. This enables the recycling of inositol dehydrogenase and squalene dehydrogenase in the filtrate, reducing the amount of crude enzyme solution used and ensuring good conversion performance. The preparation method of this invention significantly shortens the squalene production cycle, significantly reduces production costs, and reduces waste liquid generation and environmental pollution.

[0055] Further, in step (1), the preparation method of the crude enzyme solution is as follows: bacterial cells containing inositol dehydrogenase and bacterial cells containing squalinositol dehydrogenase are resuspended separately, the two resuspensions are mixed, the mixed resuspension is homogenized to break the cell wall, centrifuged, and the supernatant is taken as the crude enzyme solution; the OD of the mixed resuspension is... 600 The OD values ​​are 150-200 for the resuspension of bacterial cells containing inositol dehydrogenase and the resuspension of bacterial cells containing squalene dehydrogenase. 600 The ratio is 1:(1-3).

[0056] Furthermore, the concentration of the muscle inositol solution is 160-200 g / L.

[0057] Compared with the prior art, the above technical solution limits the concentration of the muscle inositol solution to 160-200 g / L. Muscle inositol has low solubility in water, with a solubility of about 140 g / L at room temperature (25°C) and 170-310 g / L at 40-50°C. The muscle inositol solution used in this invention is a solution with a concentration of 160-200 g / L prepared at 40-50°C. Using a higher concentration of muscle inositol solution, combined with a cyclic conversion process, can reduce the amount of enzyme solution used and greatly improve the conversion rate of subsequent conversion reactions, resulting in a higher yield of squalinositol.

[0058] Further, in step (1), the crude enzyme solution accounts for 10-20% of the volume of the muscle inositol solution.

[0059] Furthermore, in step (2), the conversion reaction takes 8-18 hours.

[0060] Furthermore, in step (2), the cooling rate of the cooling crystallization is 3-5℃ / h, and crystallization stops when the temperature drops to 5-15℃.

[0061] Compared with the existing technology, the above technical solution limits the cooling rate of the conversion solution and the cooling crystallization temperature. At the above cooling rate and cooling crystallization temperature, the high concentration of squalene in the conversion solution can be precipitated more efficiently. If the cooling rate is too fast or the cooling temperature is too low, crystals cannot be fully precipitated and will affect the crystal form of the product. If the cooling rate is too slow or the cooling temperature is too high, the purpose of crystal precipitation cannot be achieved and energy is wasted.

[0062] Furthermore, in step (2), the filtration method is to perform filtration by suction filtration using a suction filtration flask.

[0063] Furthermore, in step (3), the purity of the added muscle inositol solid is above 99 wt%; after adding water and muscle inositol solid, the total volume of the circulating solution is the same as the total volume of the raw material system in step (1), and the concentration of muscle inositol in the circulating solution is the same as the concentration of the muscle inositol solution in step (1).

[0064] Further, in step (3), during washing, the volume fraction of ethanol is 95 wt%, and the ratio of the amount of ethanol added to the mass of the filter cake is (0.5~1):1.

[0065] Furthermore, in step (3), the cooling rate of the cooling crystallization is 3-5℃ / h, and the crystallization stops when the temperature drops to 5-15℃.

[0066] Compared with the prior art, the above technical solution limits the cooling temperature and cooling rate when purifying the obtained squalene crystallization. Its beneficial effects are the same as those of cooling crystallization in step (2), and will not be repeated here. Detailed Implementation

[0067] 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.

[0068] Example 1

[0069] Mutant modification was performed on wild-type squalene dehydrogenase (SID, named PT) and wild-type inositol dehydrogenase (IDH, named GT). Alpha-folding site prediction was used, and saturation mutation screening was performed on the predicted key sites. The PT mutant Q279Y and the GT mutants N123Q, D154A, and F277P were obtained. The GT mutants were then combined with other mutants to obtain GT mutants N123Q / D154A, D154A / F277P, N123Q / F277P, and N123Q / D154A / F277P.

[0070] The plasmid containing the above-mentioned GT mutant gene or PT mutant gene is constructed using the following method:

[0071] The wild-type inositol dehydrogenase gene (SEQ ID NO.8) and the wild-type squalinositol dehydrogenase gene (SEQ ID NO.9) were cloned into the vector pYB1s using the restriction endonucleases BamHI / SpeI, respectively, to obtain plasmids pYB1s-GT and pYB1s-PT.

[0072] N123Q mutant plasmid: Using N123QF and N123QR as primers and pYB1s-GT as template, a mutant plasmid containing the mutant N123Q gene was obtained by PCR amplification, which is plasmid 1.

[0073] D154A mutant plasmid: Using D154AF and D154AR as primers and pYB1s-GT as a template, a mutant plasmid containing the mutant D154A gene was obtained by PCR amplification, which is plasmid 2.

[0074] F277P mutant plasmid: Using F277PF and F277PR as primers and pYB1s-GT as a template, a mutant plasmid containing the mutant F277P gene was obtained by PCR amplification, which is plasmid 3.

[0075] N123Q / D154A mutant plasmid: Using N123Q / D154AF and N123Q / D154AR as primers and plasmid 1 as a template, the mutant plasmid containing the mutant N123Q / D154A gene was obtained by PCR amplification, which is plasmid 4.

[0076] D154A / F277P mutant plasmid: Using D154A / F277PF and D154A / F277PR as primers and plasmid 2 as a template, the mutant plasmid containing the mutant D154A / F277P gene was obtained by PCR amplification, which is plasmid 5.

[0077] N123Q / F277P mutant plasmid: Using D154A / F277PF and D154A / F277PR as primers and plasmid 1 as a template, the mutant plasmid containing the mutant N123Q / F277P gene was obtained by PCR amplification, which is plasmid 6.

[0078] N123Q / D154A / F277P mutant plasmid: Using D154A / F277PF and D154A / F277PR as primers and plasmid 4 as a template, the mutant plasmid containing the mutant N123Q / D154A / F277P gene was obtained by PCR amplification, which is plasmid 7.

[0079] Q279Y mutant plasmid: Using Q279YF and Q279YR as primers and pYB1s-PT as a template, a mutant plasmid containing the mutant Q279Y gene was obtained by PCR amplification, which is plasmid 8.

[0080] The PCR system described above is shown in Table 1, and the primers used for PCR are shown in Table 2.

[0081] Table 1 PCR system (10 μL )

[0082] PCR reaction program: Pre-denaturation: denaturation at 98℃ for 3 min; Amplification cycle: denaturation at 98℃ for 10 s, annealing at 55℃ for 30 s, extension at 72℃ for 4.5 min, 32 cycles; Finish-up: extension at 72℃ for 10 min.

[0083] Table 2 Primers

[0084] After the PCR reaction was completed, the template was digested using the restriction endonuclease DpnI. The digestion reaction system was: 1 μL of 10× buffer, 8 μL of PCR product, and 1 μL of DpnI. The digestion reaction was carried out at 37°C for 1 h.

[0085] After digestion, 10 μL of the digest was added to *E. coli* DH5α competent cells, incubated on ice for 30 min, heat-shocked at 42°C for 90 s, and then placed on ice for 5 min. The mixture was then added to 600 μL of LB liquid medium and spread onto LB agar plates (containing 50 μg / mL streptomycin sulfate) until the bacterial culture was completely absorbed. The plates were inverted and incubated at 37°C for 14 h. The plasmid was then extracted and sent to BGI Genomics for sequencing verification.

[0086] Example 2

[0087] Preparation of expression strains

[0088] Take 1 μL of plasmids 1-8 from Example 1 and add them to E. coli BW25113 competent cells respectively. Incubate on ice for 30 min, heat shock at 42°C for 90 s, and then stand on ice for 5 min. Add each plasmid to 8 600 μL LB liquid medium and spread them onto 8 LB agar plates (containing 50 μg / mL streptomycin sulfate) until the bacterial culture is completely absorbed. Invert the plates and incubate at 37°C for 14 h to obtain 8 colonies. The successfully transformed strains were named BW25113::pYB1s-GT-N123Q, BW25113::pYB1s-GT-D154A, BW25113::pYB1s-GT-F277P, BW25113::pYB1s-GT-N123Q / D154A, BW25113::pYB1s-GT-D154A / F277P, BW25113::pYB1s-GT-N123Q / F277P, BW25113::pYB1s-GT-N123Q / D154A / F277P, and BW25113::pYB1s-PT-Q279Y, and were stored at -80℃ for later use.

[0089] Example 3

[0090] Preparation of crude enzyme solution

[0091] The eight strains successfully transformed in Example 2 were inoculated into the fermentation medium in eight fermenters at a 5% (v / v) inoculum. The temperature was controlled at 37°C, the pH was controlled at 7.0 with 50% (v / v) ammonia, the tank pressure was 0.05 MPa, the initial rotation speed was 300 rpm, the dissolved oxygen was 20%, and the dissolved oxygen (DO) was controlled between 20% and 30%. When the base sugar in the fermentation medium was depleted, feed medium was added to replenish the culture. The amount of feed was adjusted according to the dissolved oxygen, and the DO was always controlled between 20% and 30%. The fermentation was continued until the bacterial biomass reached the OD value. 600 After the value reached 70, the temperature was lowered to 30℃ and L-arabinose with a final concentration of 2g / L was added to start induction culture. After the biomass of the cells stopped increasing, the culture was transferred to a tank to obtain 8 kinds of fermentation broth.

[0092] The eight fermentation broths obtained above were centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were collected separately. The bacterial cells were resuspended in 20 mM dipotassium hydrogen phosphate buffer to make the bacterial cell concentration in the resuspended solution 200 OD. 600The obtained 8 resuspensions were homogenized and the cells were disrupted under a pressure of 80 MPa. Then, they were centrifuged at 10,000 rpm and 4 °C for 10 min, and the precipitates were discarded to obtain crude enzyme solutions containing inositol dehydrogenase mutants N123Q, D154A, F277P, N123Q / D154A, D154A / F277P, N123Q / F277P, and N123Q / D154A / F277P, and crude enzyme solution containing squalene dehydrogenase mutant Q279Y.

[0093] The fermentation medium in the fermenter for preparing inositol dehydrogenase (GT) enzyme solution was: citric acid 2 g / L, potassium dihydrogen phosphate 14 g / L, dipotassium hydrogen phosphate 4.5 g / L, ammonium sulfate 4 g / L, glucose 20 g / L, magnesium sulfate 0.6 g / L, yeast powder 1 g / L, defoamer 0.1 g / L, and 100× trace element-10 g / L.

[0094] The supplemental culture medium consisted of: 600 g / L glucose, 2 g / L magnesium sulfate, 10 g / L yeast extract, and 100 × trace element 2 at 10 mL / L.

[0095] The fermentation medium in the fermenter for preparing squalene dehydrogenase (PT) enzyme solution was as follows: citric acid 2 g / L, potassium dihydrogen phosphate 14 g / L, dipotassium hydrogen phosphate 4.5 g / L, ammonium sulfate 4 g / L, glucose 20 g / L, magnesium sulfate 0.6 g / L, yeast powder 1 g / L, defoamer 0.1 g / L, PQQ 0.02 mmol / L, ammonium chloride 1.5 g / L, and 100× trace element-10 g / L.

[0096] The supplemental culture medium consisted of: 600 g / L glucose, 2 g / L magnesium sulfate, 10 g / L yeast extract, and 100 × trace element 2 at 10 mL / L.

[0097] The concentrations of each component in the above 100× Trace Element I are: CoCl2 6H2O 25mg / L, MnCl2 4H2O 150mg / L, CuCl2 2H2O 15mg / L, H3BO3 30mg / L, Na2MoO4 2H2O 25mg / L, ZnSO4 7H2O 130mg / L, and ferric citrate 1g / L.

[0098] The concentrations of each component in the above 100× Trace Element II are: CoCl2 6H2O 40mg / L, MnCl2 4H2O 24mg / L, CuCl2 2H2O 25mg / L, H3BO3 50mg / L, Na2MoO4 2H2O 40mg / L, ZnSO4 7H2O 160mg / L, and ferric citrate 0.4g / L.

[0099] The inventors discovered that when preparing squalinositol dehydrogenase (PT) enzyme solution, the presence of a small amount of PQQ in the fermentation medium can increase the biomass of strain BW25113::pYB1s-PT-Q279Y by 12.8%, and the presence of a certain amount of PQQ and ammonium chloride in the fermentation medium can double the expression level of squalinositol dehydrogenase.

[0100] Example 4

[0101] Determination of enzyme activity

[0102] GT enzyme activity assay: The concentrations of each component in the reaction system are as follows: muscle inositol 20mM, NAD(P) + The reaction mixture consisted of 1 mM GT enzyme crude solution (14 U / mL), wild-type PT enzyme crude solution (14 U / mL), and 50 mM PBS buffer. The reaction system was incubated at pH 8.0 and 37°C for 8 h to obtain the conversion solution. The conversion rate of squalene was determined by high-performance liquid chromatography (HPLC). The results are shown in Table 3.

[0103] Table 3

[0104] As shown in Table 3, compared with wild-type inositol dehydrogenase, the enzyme activities of inositol dehydrogenase mutants N123Q, D154A, F277P, and N123Q / F277P are increased, thereby improving the conversion rate of squalene in the reaction system.

[0105] PT enzyme activity assay: The concentrations of each component in the reaction system are as follows: muscle inositol 20mM, NAD(P) + 1 mM squalene, 14 U / mL crude wild-type GT enzyme solution, 14 U / mL crude PT enzyme solution, and 50 mM PBS buffer were used. The above reaction systems were reacted at different pH values ​​and 37℃ for 8 h to obtain the conversion solution. The conversion rate of squalene was detected by high-performance liquid chromatography (HPLC). The results are shown in Table 4.

[0106] Table 4

[0107] As shown in Table 4, compared with wild-type squalene dehydrogenase, the squalene dehydrogenase mutant Q279Y has a wider applicable pH range while ensuring that the conversion rate of squalene is not reduced.

[0108] Example 5

[0109] Preparation of squalene

[0110] The concentrations of each component in the conversion system were as follows: muscle inositol 75 g / L, inositol dehydrogenase enzyme solution 12 U / mL, squalinositol dehydrogenase enzyme solution 14 U / mL, dipotassium hydrogen phosphate 30 mM. The pH was adjusted with sodium hydroxide to 8. The system was heated to 40°C and stirred to begin the conversion. The conversion ended when the squalinositol conversion rate reached its maximum. The reaction system was then heated to 120°C and maintained for 40 min to inactivate the enzymes and convert the intermediate product squalinositol monoketone to squalinositol. Finally, bacterial cells expressing inositol oxidase were added to achieve a concentration of 30 Od in the reaction system. 600 The remaining muscle inositol in the reaction system was catalyzed to glucuronic acid, and the conversion was carried out at pH 8 and temperature 37 for 12 hours to obtain the conversion solution.

[0111] The conversion rate of squalene was determined by high performance liquid chromatography (HPLC). The results are shown in Table 5.

[0112] Table 5

[0113] As shown in Table 5, compared with wild-type inositol dehydrogenase and wild-type squalinositol dehydrogenase, the conversion rate of squalinositol in the reaction system was improved by using inositol dehydrogenase mutants N123Q, D154A, F277P or N123Q / F277P, and squalinositol dehydrogenase mutant Q279Y.

[0114] Example 6

[0115] Squalin extract

[0116] The conversion solution obtained in Example 5 was filtered through a ceramic membrane with a pore size of 50 nm to remove bacterial cells. The resulting filtrate was then passed through a 5000 Da ultrafiltration membrane at a pressure of 4 MPa and a flow rate of 8 L / h to remove proteins. The supernatant was collected and passed through a cation exchange resin at a flow rate of 1 BV / h to remove cations. The effluent was then passed through an anion exchange resin at a flow rate of 1 BV / h, yielding a squalene solution. The squalene solution was then concentrated by nanofiltration through a 150 Da nanofiltration membrane at a pressure of 3 MPa until the solid content of the solution was 13%. This was followed by thermal concentration at 65°C until the solid content was 30%. 0.5 times the volume of ethanol was added, and the solution was cooled to 20°C at a rate of 5°C / h to crystallize, yielding squalene. The anion exchange resin was eluted with sodium hydroxide at a flow rate of 1 BV / h, yielding a sodium glucuronide solution, which can be used to prepare glucuronolactone.

[0117] Example 7

[0118] A method for preparing squalene includes the following steps:

[0119] (1) Prepare the raw materials:

[0120] Prepare 2 L of a 160 g / L muscle inositol solution at 40 °C with thorough stirring. Add the muscle inositol solution to the crude enzyme solutions of inositol dehydrogenase and squalinositol dehydrogenase, maintaining the overall temperature at 40 °C. The crude enzyme solution is prepared as follows: resuspend the bacterial cells containing inositol dehydrogenase and squalinositol dehydrogenase separately in PBS (pH 7.0, 50 mM). Mix the two resuspensions and homogenize the mixture three times at 40 MPa and 16 °C. Centrifuge and collect the supernatant as the crude enzyme solution. The OD of the mixed resuspension is... 600 The OD values ​​were 150 for a resuspension of bacterial cells containing inositol dehydrogenase and a resuspension of bacterial cells containing squalene dehydrogenase. 600 The ratio is 1:2, the amount of crude enzyme solution added accounts for 20% of the volume of muscle inositol solution, and the total volume of the raw material system is 2.4L.

[0121] (2) Transformation reaction:

[0122] The pH of the system was adjusted to 7.5 using sodium hydroxide, the conversion temperature was 40℃, the conversion time was 8h, and 2.2L of conversion solution was obtained by carrying out the conversion reaction.

[0123] After the first conversion, the conversion solution was cooled to 5℃ at a rate of 5℃ / h, stirred for 1h, and then filtered. The filtrate and filter cake were collected separately. The solid wet product in the filter cake weighed 95.8g.

[0124] At room temperature, 95 wt% ethanol was added to the filter cake for the first wash. The mass ratio of 95 wt% ethanol to filter cake was 0.5:1. The mixture was cooled to 5°C at a rate of 5°C / h, and then filtered to obtain a solid crude product. The solid crude product was then washed a second time with 95 wt% ethanol, filtered again to obtain a filter cake, and dried to obtain 93.4 g of white granular squalene product with a squalene content of 99.5%.

[0125] (3) Add muscle inositol solid and water to the collected filtrate, stir until the solid is completely dissolved, the solution volume is 2.4L, the concentration of muscle inositol in the solution is 160g / L, repeat steps (2) and (3) for four cycles, and obtain the solid dry weight of white granular squalinositol product as follows: 94.1g, 92.6g, 90.5g, 90.6g, and squalinositol content as follows: 99.8%, 99.2%, 99.1%, 99.7%.

[0126] Example 8

[0127] A method for preparing squalene includes the following steps:

[0128] (1) Prepare the raw materials:

[0129] Prepare 3 L of a 180 g / L muscle inositol solution at 42 °C with thorough stirring. Add the muscle inositol solution to the crude enzyme solutions of inositol dehydrogenase and squalinositol dehydrogenase, maintaining the overall temperature at 42 °C. The crude enzyme solution is prepared as follows: resuspend the bacterial cells containing inositol dehydrogenase and squalinositol dehydrogenase separately in PBS (pH 7.0, 50 mM). Mix the two resuspensions and homogenize the mixture three times at 120 MPa and 4 °C. Centrifuge and collect the supernatant as the crude enzyme solution. The OD of the mixed resuspension... 600 The OD values ​​were 200 for a resuspension of bacterial cells containing inositol dehydrogenase and a resuspension of bacterial cells containing squalene dehydrogenase. 600 The ratio is 1:1, the amount of crude enzyme solution added accounts for 10% of the volume of muscle inositol solution, and the total volume of the raw material system is 3.3L.

[0130] (2) Transformation reaction:

[0131] The pH of the system was adjusted to 7.5 using sodium hydroxide, the conversion temperature was 42℃, the conversion time was 10h, and 3.1L of conversion solution was obtained by the conversion reaction.

[0132] After the first conversion, the conversion solution was cooled to 10℃ at a rate of 3℃ / h, stirred for 1.5h, and then filtered. The filtrate and filter cake were collected separately. The solid wet product in the filter cake weighed 106.8g.

[0133] At room temperature, 95 wt% ethanol was added to the filter cake for the first wash. The mass ratio of 95 wt% ethanol to filter cake was 0.5:1. The temperature was lowered to 10°C at a rate of 3°C / h, and the mixture was filtered to obtain a solid crude product. The solid crude product was then washed a second time with 95 wt% ethanol, filtered again to obtain a filter cake, and dried to obtain 104.3 g of white granular squalene product with a squalene content of 99.2%.

[0134] (3) Add muscle inositol solid and water to the collected filtrate, stir until the solid is completely dissolved, the solution volume is 3.3L, the concentration of muscle inositol in the solution is 180g / L, repeat steps (2) and (3) five times, and obtain the solid dry weight of white granular squalene product as follows: 108.2g, 101.4g, 109.5g, 104.7g, 99.6g, and squalene content as follows: 99.1%, 99.2%, 99.1%, 99.3%, 99.0%.

[0135] Example 9

[0136] A method for preparing squalene includes the following steps:

[0137] (1) Prepare the raw materials:

[0138] Prepare 2.5 L of a 200 g / L muscle inositol solution at 47 °C with thorough stirring. Add the muscle inositol solution to the crude enzyme solutions of inositol dehydrogenase and squalinositol dehydrogenase, maintaining the overall temperature at 47 °C. The crude enzyme solution is prepared as follows: resuspend the bacterial cells containing inositol dehydrogenase and squalinositol dehydrogenase separately in PBS (pH 7.0, 50 mM). Mix the two resuspensions and homogenize the mixture three times at 80 MPa and 10 °C. Centrifuge and collect the supernatant as the crude enzyme solution. The OD of the mixed resuspension is... 600 The OD values ​​were 180 for a resuspension of bacterial cells containing inositol dehydrogenase and a resuspension of bacterial cells containing squalene dehydrogenase. 600 The ratio is 1:3, the amount of crude enzyme solution added accounts for 10% of the volume of muscle inositol solution, and the total volume of the raw material system is 2.75L.

[0139] (2) Transformation reaction:

[0140] The pH of the system was adjusted to 8.0 using sodium hydroxide, the conversion temperature was 47℃, the conversion time was 12h, and 2.6L of conversion solution was obtained by carrying out the conversion reaction.

[0141] After the first conversion, the conversion solution was cooled to 15℃ at a rate of 5℃ / h, stirred for 1.5h, and then filtered. The filtrate and filter cake were collected separately. The solid wet product in the filter cake weighed 125.4g.

[0142] At room temperature, 95 wt% ethanol was added to the filter cake for the first wash. The ratio of 95 wt% ethanol to filter cake mass was 1:1. The temperature was lowered to 15°C at a rate of 5°C / h, and the mixture was filtered to obtain a solid crude product. The solid crude product was then washed a second time with 95 wt% ethanol, filtered again to obtain a filter cake, and dried to obtain 120.9 g of white granular squalene product with a squalene content of 99.3%.

[0143] (3) Add muscle inositol solid and water to the collected filtrate, stir until the solid is completely dissolved, the solution volume is 2.75L, the concentration of muscle inositol in the solution is 200g / L, repeat steps (2) and (3) for six cycles, and obtain the solid dry weights of the white granular squalene product as follows: 126.3g, 122.4g, 118.5g, 119.9g, 117.4g, 115.6g, and the squalene contents as follows: 99.0%, 99.2%, 99.1%, 99.2%, 99.1%, 99.1%, 99.5%.

[0144] Example 10

[0145] A method for preparing squalene includes the following steps:

[0146] (1) Prepare the raw materials:

[0147] Prepare 2 L of a 160 g / L muscle inositol solution at 50 °C with thorough stirring. Add the muscle inositol solution to the crude enzyme solutions of inositol dehydrogenase and squalinositol dehydrogenase, maintaining the overall temperature at 50 °C. The crude enzyme solution is prepared as follows: resuspend the bacterial cells containing inositol dehydrogenase and squalinositol dehydrogenase separately in PBS (pH 7.0, 50 mM). Mix the two resuspensions and homogenize the mixture three times at 60 MPa and 8 °C. Centrifuge and collect the supernatant as the crude enzyme solution. The OD of the mixed resuspension... 600 The OD values ​​were 180 for a resuspension of bacterial cells containing inositol dehydrogenase and a resuspension of bacterial cells containing squalene dehydrogenase. 600 The ratio is 1:2, the amount of crude enzyme solution added accounts for 15% of the volume of muscle inositol solution, and the total volume of the raw material system is 2.3L.

[0148] (2) Transformation reaction:

[0149] The pH of the system was adjusted to 10 using sodium hydroxide, the conversion temperature was 50℃, the conversion time was 18h, and the conversion reaction was carried out to obtain 2.1L of conversion solution;

[0150] After the first conversion, the conversion solution was cooled to 12℃ at a rate of 4℃ / h, stirred for 1.3h, and then filtered. The filtrate and filter cake were collected separately. The solid wet product in the filter cake weighed 95.2g.

[0151] At room temperature, 95 wt% ethanol was added to the filter cake for the first wash. The mass ratio of 95 wt% ethanol to filter cake was 0.8:1. The temperature was lowered to 12°C at a rate of 4°C / h, and the mixture was filtered to obtain a solid crude product. The solid crude product was then washed a second time with 95 wt% ethanol, filtered again to obtain a filter cake, and dried to obtain 90.1 g of white granular squalene product with a squalene content of 99.2%.

[0152] (3) Add muscle inositol solid and water to the collected filtrate, stir until the solid is completely dissolved, the solution volume is 2.3L, the concentration of muscle inositol in the solution is 160g / L, repeat steps (2) and (3) for six cycles, and obtain the solid dry weights of the white granular squalene product as follows: 89.5g, 91.3g, 90.5g, 91.1g, 92.4g, and 91.9g, and the squalene contents as follows: 99.5%, 99.1%, 99.1%, 99.3%, 99.7%, and 99.7%.

[0153] Comparative Example 1

[0154] A method for preparing squalene includes the following steps:

[0155] (1) Prepare the raw materials:

[0156] Prepare 2 L of a 50 g / L muscle inositol solution at 40 °C with thorough stirring. Add the muscle inositol solution to the crude enzyme solutions of inositol dehydrogenase and squalinositol dehydrogenase, maintaining the overall temperature at 40 °C. The crude enzyme solution is prepared as follows: resuspend the bacterial cells containing inositol dehydrogenase and squalinositol dehydrogenase separately in PBS (pH 7.0, 50 mM). Mix the two resuspensions and homogenize the mixture three times at 40 MPa and 16 °C. Centrifuge and collect the supernatant as the crude enzyme solution. The OD of the mixed resuspension... 600 The OD values ​​were 150 for a resuspension of bacterial cells containing inositol dehydrogenase and a resuspension of bacterial cells containing squalene dehydrogenase. 600 The ratio is 1:2, the amount of crude enzyme solution added accounts for 20% of the volume of muscle inositol solution, and the total volume of the raw material system is 2.4L.

[0157] (2) Transformation reaction:

[0158] The pH of the system was adjusted to 7.5 using sodium oxide, the conversion temperature was 40℃, the conversion time was 8h, and 2.2L of conversion solution was obtained by carrying out the conversion reaction.

[0159] After the conversion was completed, the conversion solution was cooled to 5℃ at a rate of 5℃ / h, and no crystals were precipitated.

[0160] As can be seen from Examples 7 to 10 and Comparative Example 1 above, the squalene content in the squalene product prepared by the preparation method of the present invention is above 99.1%, and can reach up to 99.8%. Using the preparation method of the present invention, the product obtained has high purity, a short production cycle, and is suitable for large-scale production. The utilization efficiency of inositol dehydrogenase and squalene dehydrogenase is high, replacing the use of large amounts of phosphate, boric acid, or borate in existing technologies, and is environmentally friendly.

[0161] 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 method for preparing squalene, characterized in that, Using enzyme preparations to catalyze the reaction of muscle inositol to produce squalinositol; The enzyme preparation has enzymatic activity that catalyzes the reaction of muscle inositol to produce squalinositol. The enzyme preparation includes inositol dehydrogenase and squalinositol dehydrogenase, and the amino acid sequence of the inositol dehydrogenase is shown in any of the following examples: (1) The amino acid sequence shown in SEQ ID NO.3; (2) The amino acid sequence shown in SEQ ID NO.4; (3) The amino acid sequence shown in SEQ ID NO.5; (4) The amino acid sequence shown in SEQ ID NO. 6; The amino acid sequence of the squalinositol dehydrogenase is shown in SEQ ID NO.

7.

2. The preparation method according to claim 1, characterized in that, The enzyme activity ratio of the inositol dehydrogenase and squalinositol dehydrogenase is (10-16):(10-16).

3. The preparation method according to claim 1, characterized in that, The method for modifying the enzyme activity of inositol dehydrogenase includes the following methods: (1) The amino acid sequence shown in SEQ ID NO.3 is obtained by changing the N to Q of the 123rd amino acid in the amino acid sequence shown in SEQ ID NO.1; (2) The amino acid sequence shown in SEQ ID NO.4 is obtained by mutating amino acid D to A at position 154 of the amino acid sequence shown in SEQ ID NO.1; (3) The amino acid sequence shown in SEQ ID NO.5 is obtained by mutating amino acid F to P at position 277 of the amino acid sequence shown in SEQ ID NO.1; (4) The amino acid sequence shown in SEQ ID NO.6 is obtained by changing the N to Q amino acid at position 123 of the amino acid sequence shown in SEQ ID NO.1, and changing the F to P amino acid at position 277.

4. The preparation method according to claim 1, characterized in that, The method for modifying the enzyme activity of squalinositol dehydrogenase includes the following methods: The amino acid sequence shown in SEQ ID NO.7 is obtained by mutating amino acid Q to Y at position 279 of the amino acid sequence shown in SEQ ID NO.

2.

5. The preparation method according to claim 1, characterized in that, The preparation of the inositol dehydrogenase includes the following method: 1) Provide a gene encoding the inositol dehydrogenase according to any one of claims 1 to 4; 2) Transform host cells using the aforementioned gene; 3) Obtain host cells that produce the inositol dehydrogenase; 4) Recover the inositol dehydrogenase; The preparation of the squalinositol dehydrogenase includes the following method: 1) Provide a gene encoding the squalene dehydrogenase according to any one of claims 1 to 4; 2) Transform host cells using the aforementioned gene; 3) Obtain a host cell that produces the squalene dehydrogenase; 4) Recover the squalinositol dehydrogenase.

6. The preparation method according to claim 1, characterized in that, The concentrations of each component in the reaction system for catalyzing the reaction of muscle inositol to produce squalinositol are as follows: muscle inositol 50-100 g / L, inositol dehydrogenase 10-16 U / mL, squalinositol dehydrogenase 10-16 U / mL, and dipotassium hydrogen phosphate 10-50 mM.

7. The preparation method according to claim 1, characterized in that, The reaction system has a reaction temperature of 35–60°C and a pH value of 8–10.

8. The preparation method according to claim 1, characterized in that, The preparation method further includes: after the reaction is completed, maintaining the reaction system at a temperature of 115-130°C for 0.5-1 hour to convert the intermediate product squalinositol monoketone into squalinositol, and then adding bacterial cells expressing inositol oxidase to make the concentration of the bacterial cells in the reaction system 10-50 OD. 600 The remaining muscle inositol in the reaction system was converted into glucuronic acid, and squalinositol and glucuronic acid were separated.

9. The preparation method according to claim 8, characterized in that, The reaction temperature for converting the remaining muscle inositol in the reaction system into glucuronic acid is 35–40°C, and the pH value is 7.8–8.

2.

10. A method for preparing squalene, characterized in that, Includes the following steps: (1) Prepare raw materials: Prepare crude enzyme solutions of inositol dehydrogenase and squalinositol dehydrogenase, prepare muscle inositol solution, add muscle inositol solution to crude enzyme solution, and control the temperature of raw material system between 40-50℃. (2) Conversion reaction: Adjust the pH value to 7-10, control the temperature to 40-50℃, carry out the conversion reaction, and obtain conversion liquid A. Perform the first cooling crystallization and filtration on the conversion liquid A, and collect the filtrate and filter cake respectively. (3) Add water and muscle inositol solid to the filtrate, repeat steps (2) and (3) until no crystals precipitate, and wash the filter cake collected each time with ethanol for the first time, cool and crystallize for the second time, filter, wash with ethanol for the second time, and dry to obtain squalene product.

11. The preparation method according to claim 10, characterized in that, In step (1), the preparation method of the crude enzyme solution is as follows: resuspend the bacterial cells containing inositol dehydrogenase and the bacterial cells containing squalinositol dehydrogenase respectively, mix the two resuspended solutions, homogenize and break the cell wall of the mixed resuspended solution, centrifuge, and take the supernatant as the crude enzyme solution. OD of the mixed resuspension 600 The OD values ​​are 150-200 for the resuspension of bacterial cells containing inositol dehydrogenase and the resuspension of bacterial cells containing squalene dehydrogenase. 600 The ratio is 1:(1-3).

12. The preparation method according to claim 10, characterized in that, In step (1), the concentration of the muscle inositol solution is 160-200 g / L.

13. The preparation method according to claim 10, characterized in that, In step (1), the crude enzyme solution accounts for 10-20% of the volume of the muscle inositol solution.

14. The preparation method according to claim 10, characterized in that, In step (2), the conversion reaction takes 8-18 hours.

15. The preparation method according to claim 10, characterized in that, In step (2), the cooling rate of the cooling crystallization is 3-5℃ / h, and the crystallization stops when the temperature drops to 5-15℃.

16. The preparation method according to claim 10, characterized in that, In step (2), the filtration method is to perform filtration by vacuum filtration using a vacuum filtration flask.

17. The preparation method according to claim 10, characterized in that, In step (3), the purity of the added muscle inositol solid is above 99 wt%; after adding water and muscle inositol solid, the total volume of the circulating solution is the same as the total volume of the raw material system in step (1), and the concentration of muscle inositol in the circulating solution is the same as the concentration of muscle inositol solution in step (1).

18. The preparation method according to claim 10, characterized in that, In step (3), during washing, the volume fraction of ethanol is 95%, and the ratio of the amount of ethanol added to the mass of the filter cake is (0.5~1):

1.

19. The preparation method according to claim 10, characterized in that, In step (3), the cooling rate of the cooling crystallization is 3-5℃ / h, and the crystallization stops when the temperature drops to 5-15℃.

Images