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

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 inositol dehydrogenase and 2-keto-inositol isomerase. The amino acid sequence of inositol dehydrogenase is shown in SEQ ID NO. 6, and the amino acid sequence of 2-keto-inositol isomerase is shown in SEQ ID NO. 8. Compared with the prior art, the enzyme activities of the inositol dehydrogenase and 2-keto-inositol isomerase with specific amino acid sequences of this invention are further improved. When catalyzing the reaction of muscle inositol to D-chiral inositol, both can further improve the conversion rate and the yield of D-chiral inositol. Furthermore, this inositol dehydrogenase has high tolerance to muscle inositol, and can still efficiently catalyze the reaction to produce D-chiral inositol even at high substrate concentrations, alleviating substrate inhibition.

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 isomer of inositol with important physiological activities, showing great potential for application in regulating blood sugar, improving insulin resistance, and treating polycystic ovary syndrome. Currently, the main production methods for D-chiro-inositol include chemical synthesis, natural extraction, and biosynthesis. Chemical synthesis typically uses inositol as a raw material, achieving configurational transformation through multiple chemical reactions; however, the steps are cumbersome, the reactions rely on large amounts of organic solvents, and are prone to environmental pollution. Natural extraction mainly involves separation from plant materials such as carob; however, limited by the source and content of the raw materials, the extraction efficiency is low, making it difficult to meet the needs of large-scale production.

[0003] Biosynthetic preparation of D-chiral inositol has attracted widespread attention due to its advantages such as mild conditions and environmental friendliness, and can be mainly divided into two categories: de novo synthesis and enzymatic synthesis. De novo synthesis relies on genetically engineered microorganisms (such as *E. coli* and yeast) to synthesize D-chiral inositol using inexpensive carbon sources such as glucose by constructing or enhancing endogenous metabolic pathways. However, the microbial endogenous metabolic network is complex, and the target product synthesis pathway often competes with cell growth for precursors and cofactors, resulting in low yields of the target product. Furthermore, multiple side reactions may occur in the pathway, making the separation and purification of the target product difficult and the process cost high.

[0004] Enzymatic catalysis utilizes the high specificity of specific enzymes to catalyze the production of D-chiral inositol from substrates such as inositol. It offers advantages such as simple reaction steps, high catalytic efficiency, and strong process controllability, making it suitable for large-scale preparation of high-purity D-chiral inositol. Currently, research and improvement of this enzyme catalysis method largely focuses on enhancing enzyme activity through protein engineering. However, achieving industrial-scale production requires not only highly active enzymes but, more importantly, the construction of a reaction system capable of efficiently catalyzing high concentrations of substrates at commonly used industrial temperatures. Existing technologies suffer from two main bottlenecks: first, insufficient attention is paid to the critical parameter of substrate concentration in the reaction system. Low substrate loading leads to low equipment utilization, weak reaction driving force, and low batch production intensity, severely restricting process economics; second, while thermostable enzymes are used to improve substrate solubility and reactions are conducted at high temperatures, excessively high operating temperatures do not meet the feasibility and energy consumption requirements of conventional industrial production. Therefore, it is urgent to establish an enzyme catalysis process adaptable to high substrate concentrations within a suitable industrial operating temperature range to achieve efficient, stable, and economical large-scale production. Summary of the Invention

[0005] Based on this, the present invention provides an enzyme preparation and a method for preparing D-chiral inositol. The enzyme preparation not only has higher enzyme activity, but also can efficiently catalyze the reaction to generate D-chiral inositol at high substrate concentrations.

[0006] In a first aspect, the present invention provides an enzyme preparation comprising inositol dehydrogenase and 2-keto-inositol isomerase, wherein the amino acid sequence of the inositol dehydrogenase is shown in SEQ ID NO.6 and the amino acid sequence of the 2-keto-inositol isomerase is shown in SEQ ID NO.8.

[0007] Compared with the prior art, the present invention mutates wild-type inositol dehydrogenase and wild-type 2-keto-inositol isomerase. The amino acid sequence of wild-type inositol dehydrogenase is mutated from G to A at position 234, from D to K at position 267, and from E to K at position 331, resulting in the inositol dehydrogenase mutant G234A / D267K / E331K with the amino acid sequence shown in SEQ ID NO.6. Similarly, the amino acid sequence of wild-type 2-keto-inositol isomerase is mutated from L to K at position 68 and from I to R at position 225, resulting in the 2-keto-inositol isomerase mutant L68K / I225R with the amino acid sequence shown in SEQ ID NO.8. After mutation, the enzyme activities of the inositol dehydrogenase mutants G234A / D267K / E331K and L68K / I225R, respectively, were further enhanced. These mutations improved the conversion rate and yield of D-chiral inositol in the catalytic reaction of muscle inositol to D-chiral inositol. Furthermore, the inositol dehydrogenase mutants G234A / D267K / E331K exhibit high tolerance to muscle inositol, allowing for efficient catalysis of D-chiral inositol production even at high substrate concentrations, thus mitigating substrate inhibition.

[0008] Furthermore, the gene sequence of inositol dehydrogenase is shown in SEQ ID NO.5; the gene sequence of 2-keto-inositol isomerase is shown in SEQ ID NO.7.

[0009] Furthermore, the mass ratio of inositol dehydrogenase to 2-keto-inositol isomerase is (0.5~30):(0.5~30).

[0010] In a second aspect, the present invention provides a method for preparing D-chiral inositol using the above-mentioned enzyme preparation, comprising the following steps:

[0011] Prepare the reaction system: Add crude enzyme solution of inositol dehydrogenase, crude enzyme solution of 2-keto-inositol isomerase, metal ions, and muscle inositol to the buffer solution.

[0012] The reaction system is subjected to a pH of 7.5-9.5 and a temperature of 30-50℃ to catalyze the reaction of muscle inositol to produce D-chiral inositol, resulting in a conversion solution containing D-chiral inositol.

[0013] Compared with existing technologies, this invention uses crude enzyme solutions of inositol dehydrogenase mutants G234A / D267K / E331K and 2-keto-inositol isomerase mutants L68K / I225R to catalyze the preparation of D-chiral inositol from muscle inositol. This alleviates the substrate inhibition problem at higher substrate concentrations under commonly used industrial temperatures, enabling the reaction system to generate more products within the same reaction time under higher substrate concentrations and conventional industrial temperatures, thereby further shortening the production cycle and improving overall efficiency and economic benefits.

[0014] Furthermore, this invention adds inositol dehydrogenase and 2-keto-inositol isomerase in the form of crude enzyme solution, so that the reaction system can utilize endogenous NAD(H) coenzyme without the need to add exogenous coenzyme, thus reducing raw material costs.

[0015] Furthermore, in the reaction system, the concentration of the buffer solution is 20~100mM, the amount of crude enzyme solution of inositol dehydrogenase added is 10%~90% of the total reaction volume, the amount of crude enzyme solution of 2-keto-inositol isomerase added is 10%~90% of the total reaction volume, the concentration of metal ions is 0.5~2mM, and the concentration of muscle inositol is 10~200g / L.

[0016] Furthermore, the method for preparing the crude enzyme solution of inositol dehydrogenase includes the following steps:

[0017] An expression vector containing the inositol dehydrogenase gene was constructed, and the expression vector was transformed into a host strain to obtain a recombinant strain containing the inositol dehydrogenase gene. The recombinant strain was fermented to express inositol dehydrogenase, and a fermentation broth containing inositol dehydrogenase was obtained.

[0018] Centrifuge the fermentation broth, collect the bacterial cells, resuspend the cells, and obtain the bacterial OD. 600 The bacterial cells in the resuspension were broken up, and the cell debris was removed by centrifugation to obtain the crude enzyme solution of inositol dehydrogenase.

[0019] Furthermore, the method for preparing the crude enzyme solution of 2-keto-inositol isomerase includes the following steps:

[0020] An expression vector containing the 2-keto-inositol isomerase gene was constructed, and the expression vector was transformed into a host strain to obtain a recombinant strain containing the 2-keto-inositol isomerase gene. The recombinant strain was fermented to express the 2-keto-inositol isomerase, and the fermentation broth of 2-keto-inositol isomerase was obtained.

[0021] Centrifuge the fermentation broth, collect the bacterial cells, resuspend the cells, and obtain the bacterial OD. 600 The bacterial cells in the resuspension were broken up, and the cell debris was removed by centrifugation to obtain a crude enzyme solution containing 2-keto-inositol isomerase.

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

[0023] Furthermore, the metal ions include manganese ions, cobalt ions, nickel ions, or zinc ions.

[0024] Furthermore, after obtaining the conversion solution containing D-chiral inositol, the method further includes extracting D-chiral inositol from the conversion solution, comprising the following steps:

[0025] The conversion solution was heated to 70-80℃ to inactivate the enzyme, and then cooled. The cooled conversion solution was filtered through a ceramic membrane with a pore size of 20-100nm to obtain the ceramic membrane permeate.

[0026] The ceramic membrane permeate is filtered through an ultrafiltration membrane with a pore size of 2000~10000 Da to obtain the ultrafiltration membrane permeate;

[0027] The permeate from the ultrafiltration membrane is used to remove cationic impurities, anionic impurities, and pigments to obtain a treated solution with a conductivity of <300 μs / cm and a transmittance of >98%.

[0028] The treatment solution was concentrated to a solid content >15% by passing it through a nanofiltration membrane with a pore size of 150~200Da. The resulting nanofiltration membrane concentrate was then thermally concentrated at a temperature of 55~60℃ until muscle inositol crystals precipitated. After cooling to 20~25℃, the muscle inositol crystals were removed by filtration, and the first filtrate was collected.

[0029] The first filtrate is concentrated to 40% to 50% of its original volume, ethanol is added, and the mixture is heated, kept warm, and cooled in sequence to precipitate muscle inositol. The mixture is then filtered and the second filtrate is collected.

[0030] The second filtrate is distilled to remove alcohol, and the removed alcohol solution is concentrated to a solid content of 50% to 55%. The solution is heated and ethanol is added, then cooled and filtered. The resulting filter cake is crude D-chiral inositol.

[0031] Further, cationic impurities, anionic impurities, and pigments are removed from the ultrafiltration membrane permeate to obtain a treated solution with a conductivity <300 μS / cm and a transmittance >98%, including:

[0032] The permeate from the ultrafiltration membrane was treated with a cation exchange resin at a feed flow rate of 1-2 BV / h. The conductivity of the effluent from the cation exchange resin was controlled to be <4000 μs / cm and the pH to be <3.

[0033] The effluent from the cation exchange resin was treated with macroporous adsorption resin at a feed flow rate of 1-2 BV / h, and the transmittance of the effluent from the macroporous adsorption resin was controlled to be >98%.

[0034] The effluent from the macroporous adsorption resin was treated with anion exchange resin at a feed flow rate of 1-2 BV / h, and the conductivity of the effluent from the macroporous adsorption resin was controlled to be <300 μS / cm and the pH to be >8.

[0035] Further, after the first filtrate is concentrated, ethanol with a volume of 1 to 2 times that of the concentrated filtrate is added, and then the mixture is heated to 50 to 55°C, kept at that temperature for 1 to 2 hours, and then cooled to 20 to 25°C.

[0036] Furthermore, the distillation-dealcoholization process involves using a distillation column to dealcoholize the second filtrate, with the solid content of the dealcoholized solution being 12% to 18%.

[0037] Further, after the dealcoholized liquid is concentrated to a solid content of 50%~55%, it is heated to 45~50℃, and 0.5~1 times the volume of the concentrated liquid is added with ethanol, and then the temperature is lowered to 25~30℃.

[0038] Further, after obtaining crude D-chiral inositol, the process also includes adding water to the crude D-chiral inositol, with a mass ratio of crude D-chiral inositol to water of 1:(1~1.5), heating to 60~70℃ to dissolve the crude D-chiral inositol, and then performing a decolorization treatment to obtain a decolorized solution.

[0039] Add ethanol to the decolorizing solution, and after crystals precipitate, cool to 15-20℃ and filter to obtain D-chiral inositol.

[0040] Furthermore, the decolorization treatment of the solution of crude D-chiral inositol includes: decolorization with activated carbon, wherein the amount of activated carbon added is 1% to 3% of the crude D-chiral inositol mass, and the decolorization time is 20 to 40 minutes. Detailed Implementation

[0041] 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.

[0042] In a first aspect, embodiments of the present invention provide an enzyme preparation comprising inositol dehydrogenase and 2-keto-inositol isomerase, wherein the amino acid sequence of the inositol dehydrogenase is shown in SEQ ID NO.6 and the amino acid sequence of the 2-keto-inositol isomerase is shown in SEQ ID NO.8.

[0043] The activities of the aforementioned inositol dehydrogenase and 2-keto-inositol isomerase were further enhanced, resulting in increased conversion rates and D-chiral inositol yield in the catalytic reaction of muscle inositol to D-chiral inositol. Furthermore, the aforementioned inositol dehydrogenase exhibited high tolerance to the substrate muscle inositol, mitigating the inhibition of enzyme activity by high substrate concentrations and maintaining high catalytic efficiency even at high substrate concentrations.

[0044] Optionally, the gene sequence of the above-mentioned inositol dehydrogenase is shown in SEQ ID NO.5; the gene sequence of 2-keto-inositol isomerase is shown in SEQ ID NO.7.

[0045] Furthermore, the mass ratio of inositol dehydrogenase to 2-keto-inositol isomerase in the enzyme preparation is (0.5~30):(0.5~30), preferably (1~5):(1~5), and more preferably 1:1.

[0046] Secondly, embodiments of the present invention provide a method for preparing D-chiral inositol using the above-mentioned enzyme preparation, comprising the following steps:

[0047] Prepare the reaction system: Add crude enzyme solution of inositol dehydrogenase, crude enzyme solution of 2-keto-inositol isomerase, metal ions, and muscle inositol to the buffer solution.

[0048] The reaction system is subjected to a pH of 7.5-9.5 and a temperature of 30-50℃ to catalyze the reaction of muscle inositol to produce D-chiral inositol, resulting in a conversion solution containing D-chiral inositol.

[0049] For example, the pH value of the above reaction system can be 7.5, 8, 8.5, 9 or 9.5; the temperature can be 30°C, 35°C, 40°C, 45°C or 50°C, preferably 30~40°C.

[0050] In some embodiments, the method for preparing crude inositol dehydrogenase includes the following steps:

[0051] An expression vector containing the inositol dehydrogenase gene was constructed, and the expression vector was transformed into a host strain to obtain a recombinant strain containing the inositol dehydrogenase gene. The recombinant strain was fermented to express inositol dehydrogenase, and a fermentation broth containing inositol dehydrogenase was obtained.

[0052] Centrifuge the fermentation broth, collect the bacterial cells, resuspend the cells, and obtain the bacterial OD. 600 The bacterial cells in the resuspension were broken up to a concentration of 10-220. Cell debris was removed by centrifugation to obtain a crude enzyme solution of inositol dehydrogenase. For example, the bacterial OD value in the resuspension was... 600 The value can be 10, 40, 80, 120, 160, 180 or 220, preferably 160 to 220, and more preferably 180.

[0053] Furthermore, the amount of crude enzyme solution of inositol dehydrogenase added is 10% to 90% of the total reaction volume. For example, the amount added can be 10%, 20%, 30%, 40%, 50%, 70% or 90% of the total reaction volume, preferably 10% to 30%, and more preferably 20%.

[0054] In some embodiments, the preparation method of crude enzyme solution of 2-keto-inositol isomerase includes the following steps:

[0055] An expression vector containing the 2-keto-inositol isomerase gene was constructed, and the expression vector was transformed into a host strain to obtain a recombinant strain containing the 2-keto-inositol isomerase gene. The recombinant strain was fermented to express the 2-keto-inositol isomerase, and a fermentation broth containing the 2-keto-inositol isomerase was obtained.

[0056] Centrifuge the fermentation broth, collect the bacterial cells, resuspend the cells, and obtain the bacterial OD. 600 The bacterial cells in the resuspension were lysed to a concentration of 10–220. Cell debris was removed by centrifugation to obtain a crude enzyme solution of 2-keto-inositol isomerase. For example, the bacterial OD value in the resuspension was… 600 The value can be 10, 40, 80, 120, 160, 180 or 220, preferably 160 to 220, and more preferably 180.

[0057] Furthermore, the amount of crude enzyme solution of 2-keto-inositol isomerase added is 10% to 90% of the total reaction volume. For example, the amount added can be 10%, 20%, 30%, 40%, 50%, 70% or 90% of the total reaction volume, preferably 10% to 30%, and more preferably 20%.

[0058] In some embodiments, the buffer solution includes sodium carbonate buffer, phosphate buffer, or Tris-HCl buffer. The concentration of the buffer solution is 20-100 mM to ensure that the pH of the reaction system is 7.5-9.5.

[0059] In some embodiments, the metal ions include manganese ions, cobalt ions, nickel ions, or zinc ions; the concentration of the metal ions is 0.5~2 mM, for example, the concentration of the metal ions can be 0.5 mM, 1 mM, 1.5 mM, or 2 mM.

[0060] In some embodiments, the concentration of muscle inositol is 10-200 g / L. For example, the concentration of muscle inositol can be 10 g / L, 50 g / L, 100 g / L, 150 g / L or 200 g / L, preferably 100-200 g / L.

[0061] Furthermore, after obtaining the conversion solution containing D-chiral inositol, this embodiment of the invention also provides a method for extracting D-chiral inositol from the above-mentioned conversion solution containing D-chiral inositol, comprising the following steps:

[0062] S1. Heat the conversion solution to 70~80℃ to inactivate the enzyme, then cool it down. After cooling, filter the conversion solution through a ceramic membrane with a pore size of 20~100nm to obtain the ceramic membrane permeate.

[0063] In the above steps, the conversion solution is heated to 70-80°C and then inactivated at 70-80°C for 20-40 minutes to cause the enzyme protein to flocculate. The temperature is then lowered to 30-40°C. For example, the temperature can be raised to 70°C, 75°C, or 80°C; and lowered to 30°C, 35°C, or 40°C.

[0064] In the above steps, the pore size of the ceramic membrane is 20~100nm during ceramic membrane filtration to remove flocculated enzyme proteins and obtain ceramic membrane permeate. For example, the pore size of the ceramic membrane can be 20nm, 50nm, 80nm or 100nm.

[0065] S2. The ceramic membrane permeate is filtered through an ultrafiltration membrane with a pore size of 2000~10000 Da to obtain the ultrafiltration membrane permeate.

[0066] The above steps remove unflocculated large molecular weight proteins and other organic impurities with molecular weights greater than 2000~10000 Da by ultrafiltration membrane filtration. For example, the pore size of the ultrafiltration membrane can be 2000 Da, 5000 Da, 8000 Da or 10000 Da.

[0067] S3. Remove cationic impurities, anionic impurities, and pigments from the ultrafiltration membrane permeate to obtain a treated solution with a conductivity <300 μs / cm and a transmittance >98%, including the following steps:

[0068] S31. The permeate from the ultrafiltration membrane is treated with a cation exchange resin to remove cationic impurities. The feed flow rate is 1~2 BV / h, and the conductivity of the effluent from the cation exchange resin is controlled to be <4000 μS / cm, and the pH is <3. For example, the feed flow rate can be 1 BV / h, 1.5 BV / h, or 2 BV / h.

[0069] In the above steps, before feeding, the cation exchange resin can be regenerated using 4%~5% hydrochloric acid by mass, and then washed with deionized water until neutral. For example, the cation exchange resin can be D001, D113, or SQD-80.

[0070] S32. The effluent from the cation exchange resin is treated with a macroporous adsorption resin to remove pigments and other organic impurities. The feed flow rate is 1~2 BV / h, and the transmittance of the effluent from the macroporous adsorption resin is controlled to be >98%. For example, the feed flow rate can be 1 BV / h, 1.5 BV / h, or 2 BV / h.

[0071] The macroporous adsorption resin can be selected from LX-60, LS-305 or LS-406.

[0072] S33. The effluent from the macroporous adsorption resin is treated with anion exchange resin to remove anionic impurities. The feed flow rate is 1~2 BV / h, and the conductivity of the effluent from the macroporous adsorption resin is controlled to be <300 μS / cm, and the pH is >8. For example, the feed flow rate can be 1 BV / h, 1.5 BV / h, or 2 BV / h.

[0073] In the above steps, before feeding, the anion exchange resin can be regenerated using sodium hydroxide with a mass concentration of 4%~5%, and then washed with deionized water until neutral. For example, the anion exchange resin can be selected as D-201, D-301, or D-311.

[0074] S4. After concentrating the treatment solution through a nanofiltration membrane with a pore size of 150~200Da to a solid content >15%, the obtained nanofiltration membrane concentrate is thermally concentrated at a temperature of 55~60℃ until muscle inositol crystals precipitate. After cooling to 20~25℃, the muscle inositol crystals are removed by filtration, and the first filtrate is collected.

[0075] Using the above steps, the treated solution is concentrated using a nanofiltration membrane with a pore size of 150~200 Da. During the concentration process, salt ions in the treated solution are further removed to obtain a nanofiltration membrane concentrate with a solid content >15%. For example, the pore size of the nanofiltration membrane can be 150 Da, 170 Da, or 200 Da.

[0076] The obtained nanofiltration membrane concentrate is thermally concentrated at a temperature of 55~60℃. Muscle inositol has a lower solubility in water than D-chiral inositol, and the proportion of muscle inositol in the nanofiltration membrane concentrate is higher. Therefore, when the nanofiltration membrane concentrate is thermally concentrated at the above-mentioned temperature of 55℃~60℃, the solid content of the obtained thermal concentrate can be selected to be 30~35%. At this time, muscle inositol crystals will precipitate first. After cooling to 20~25℃, the muscle inositol crystals are removed by filtration, and the first filtrate is collected. The mass content of D-chiral inositol in the first filtrate is 25~30%.

[0077] S5. Concentrate the first filtrate to 40%~50% of its original volume, add ethanol, and then heat, keep warm, and cool down in sequence to precipitate muscle inositol. Filter and collect the second filtrate.

[0078] In the above steps, the first filtrate is concentrated to 40%~50% of its original volume, then ethanol of 1~2 times the volume of the concentrated filtrate is added, and then heated to 50~55℃ and kept at that temperature for 1~2 hours. Then the temperature is lowered to 20~25℃ to precipitate muscle inositol. The filtrate is filtered and the second filtrate is collected. The mass ratio of D-chiral inositol to muscle inositol in the second filtrate is (80%~85%): (15%~20%).

[0079] After concentrating the first filtrate and adding ethanol, the ethanol is quickly and evenly mixed with the concentrated first filtrate through heating and heat preservation. This further reduces the solubility of muscle inositol in the ethanol-water solution, making it easier to precipitate. D-chiral inositol has a higher solubility in the ethanol-water solution than muscle inositol. Therefore, by controlling the amount of ethanol added, muscle inositol can be precipitated a second time.

[0080] When concentrating the first filtrate, single-effect concentration can be performed. Under conditions of temperature of 50~70℃ and vacuum degree <-0.09MPa, the first filtrate can be concentrated to 40%~50% of its original volume.

[0081] For example, the first filtrate can be concentrated to 40%, 45%, or 50% of its original volume; the amount of ethanol added can be 1, 1.5, or 2 times the volume of the concentrated liquid; the heating temperature can be 50°C, 52°C, or 55°C; the holding time can be 1 h, 1.5 h, or 2 h; and the cooling temperature can be 20°C, 22°C, or 25°C.

[0082] S6. Distill the second filtrate to remove alcohol, and concentrate the alcohol to a solid content of 50%~55%. Heat and add ethanol, cool, and filter. The resulting filter cake is crude D-chiral inositol.

[0083] In the above steps, a distillation column can be used to remove alcohol from the second filtrate to obtain a de-alcoholized liquid with a solid content of 12% to 18%.

[0084] When concentrating the dealcoholized liquid, single-effect concentration can be performed. Under conditions of temperature of 50~70℃ and vacuum degree <-0.09MPa, the dealcoholized liquid can be concentrated to a solid content of 50%~55%.

[0085] The above steps involve concentrating the dealcoholized liquid to a solid content of 50%–55%, heating it to 45–50°C, adding 0.5–1 times the volume of the concentrated liquid in ethanol, stirring thoroughly, then cooling to 25–30°C and filtering. The resulting filter cake is the crude D-chiral inositol product. For example, the dealcoholized liquid can be concentrated to a solid content of 50%, 52%, or 55%; heated to 45°C, 48°C, or 50°C; 0.5, 0.8, or 1 times the volume of the concentrated liquid in ethanol can be added; and the temperature can be lowered to 25°C, 28°C, or 30°C.

[0086] By separating muscle inositol from the treatment solution, the proportion of D-chiral inositol in the second filtrate can be significantly increased. After distillation and deethanolination of the second filtrate and concentration of the deethanolination solution, the concentration of D-chiral inositol in the deethanolination concentrate is further increased. Ethanol is then added back to the deethanolination concentrate, and the mixture is heated to 45-50°C to allow the ethanol and deethanolination concentrate to mix rapidly and evenly, thereby further reducing the solubility of D-chiral inositol. D-chiral inositol can then be precipitated by cooling. After filtration, crude D-chiral inositol is obtained.

[0087] The crude D-chiral inositol product contains >95% D-chiral inositol by mass. The filtrate obtained from filtration can be returned to the first filtrate in step S5 for repeated crystallization.

[0088] Furthermore, after obtaining crude D-chiral inositol, embodiments of the present invention also provide a method for purifying the crude D-chiral inositol, comprising:

[0089] Water was added to crude D-chiral inositol at a mass ratio of 1:(1~1.5). The mixture was heated to 60~70℃ to dissolve the crude D-chiral inositol, followed by decolorization to obtain a decolorized solution. Ethanol was added to the decolorized solution, and after crystals precipitated, the solution was cooled to 15~20℃ and filtered to obtain D-chiral inositol.

[0090] For example, the mass ratio of crude D-chiral inositol to water can be 1:1, 1:1.2, or 1:1.5; and the temperature can be raised to 60°C, 65°C, or 70°C.

[0091] The above steps for decolorizing the solution of crude D-chiral inositol include: decolorizing with activated charcoal at an amount of 1%–3% of the crude D-chiral inositol mass for 20–40 minutes. After decolorization, the activated charcoal is removed to obtain the decolorized solution. For example, the amount of activated charcoal added can be 1%, 2%, or 3% of the crude D-chiral inositol mass; the decolorization time can be 20, 30, or 40 minutes.

[0092] When adding ethanol to the decolorizing solution in the above steps, the ethanol should be added slowly. Stop adding ethanol after crystals precipitate in the system, cool to 15-20°C, filter, and obtain a filter cake. Wash the filter cake with ethanol and dry it to obtain the D-chiral inositol product, wherein the mass content of D-chiral inositol is >99%. For example, the temperature can be lowered to 15°C, 18°C, or 20°C.

[0093] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.

[0094] Example 1

[0095] Construct recombinant plasmids pYB1s-BsiolG and pYB1s-BaiolI

[0096] Based on the codon bias of Escherichia coli, codon optimization was performed on the wild-type inositol dehydrogenase gene and the wild-type 2-keto-inositol isomerase gene, respectively.

[0097] The nucleotide sequence of the optimized wild-type inositol dehydrogenase gene is shown in SEQ ID NO.1, and the amino acid sequence of the wild-type inositol dehydrogenase it encodes is shown in SEQ ID NO.2.

[0098] The nucleotide sequence of the optimized wild-type 2-keto-inositol isomerase gene is shown in SEQ ID NO.3, and the amino acid sequence of the wild-type 2-keto-inositol isomerase it encodes is shown in SEQ ID NO.4.

[0099] Using the optimized wild-type inositol dehydrogenase gene as a template, PCR amplification was performed with F1 as the upstream primer and R1 as the downstream primer to obtain the target gene BsiolG with homologous arms.

[0100] F1: 5'-gagggtagatctggtactagtATGAGTCTGCGCATCGGCG-3' (SEQ ID NO. 9).

[0101] R1: 5'-caccagctgcagaccgagctcTTAGTTCTGCACGGTCGTAAAGC-3' (SEQ ID NO. 10).

[0102] Using the optimized wild-type 2-keto-inositol isomerase gene as a template, PCR amplification was performed with F2 as the upstream primer and R2 as the downstream primer to obtain the target gene BaiolI with homologous arms.

[0103] F2: 5'-gagggtagatctggtactagtATGAAACTGTGCTTTAACGAAGCA-3' (SEQ ID NO. 11).

[0104] R2: 5'-caccagctgcagaccgagctcTCAGGCCTCTTTCATGAAATACTTAC-3' (SEQ ID NO. 12).

[0105] Both PCR amplification reactions were performed using the reaction systems shown in Table 1 and according to the reaction procedures shown in Table 2.

[0106] Table 1

[0107]

[0108] Table 2

[0109]

[0110] It should be understood that the above-mentioned pre-denaturation, thorough extension, and maintenance steps are not involved in the cycle, and the entire PCR process is performed only once.

[0111] After the PCR amplification was completed, the reaction products were recovered by agarose gel extraction to obtain the target gene BsiolG and the target gene BaiolI with high purity.

[0112] The expression vector pYB1s was double-digested with restriction endonucleases SpeⅠ and SacⅠ at 37℃ for 20 min. After digestion, the double-digested products were recovered and purified to obtain the linearized vector pYB1s. The digestion system is shown in Table 3.

[0113] Table 3

[0114]

[0115] The target genes BsiolG and BaiolI, obtained by PCR amplification, were ligated into the linearized vector pYB1s to obtain recombinant plasmids pYB1s-BsiolG and pYB1s-BaiolI, respectively. The ligation system is shown in Table 4, and ligation was performed at 37℃ for 30 min.

[0116] Table 4

[0117]

[0118] After the above ligation was completed, the ligation products, recombinant plasmids pYB1s-BsiolG and pYB1s-BaiolI, were transformed into E. coli DH5α competent cells by chemical transformation. Single colonies were picked from each cell for plasmid extraction, and the extracted plasmids were sequenced for DNA.

[0119] Example 2

[0120] Construction of mutant plasmid pYB1s-BsiolG G234A / D267K / E331K

[0121] Using the correctly sequenced recombinant plasmid pYB1s-BsiolG from Example 1 above as a template, and with G234A-F as upstream primers and G234A-R as downstream primers, a reverse PCR amplification reaction was performed. After removing the template from the reaction product, the reverse PCR product was self-circularized to obtain the mutant plasmid pYB1s-BsiolG. G234A .

[0122] G234A-F: CTGCAAATACgccTATGATATTCAGTGTGAAATTGTAGGGG (SEQ ID NO. 13).

[0123] G234A-R: CATAggcGTATTTGCAGTTCACATAAATCTCTGC (SEQ ID NO. 14).

[0124] Using the above mutant plasmid pYB1s-BsiolG G234A Using D267K-F as the upstream primer and D267K-R as the downstream primer, a reverse PCR amplification reaction was performed. After removing the template from the reaction product, the reverse PCR product was self-circularized to obtain the mutant plasmid pYB1s-BsiolG. G234A / D267K .

[0125] D267K-F: GCCGTTTCTCTACGaaaATCCTCATGGATTGGCAGCG (SEQ ID NO. 15).

[0126] D267K-R: tttCGTAGAGAAACGGCCCTCTTTGCGTAATG (SEQ ID NO. 16).

[0127] Using the above mutant plasmid pYB1s-BsiolG G234A / D267K Using E331K-F as the upstream primer and E331K-R as the downstream primer, a reverse PCR amplification reaction was performed. After removing the template from the reaction product, the reverse PCR product was self-circularized to obtain the mutant plasmid pYB1s-BsiolG. G234A / D267K / E331K .

[0128] E331K-F: GCCGTTTCTCTACGaaaATCCTCATGGATTGGCAGCG (SEQ ID NO. 17).

[0129] E331K-R: CCGGCTTtttCTTGAGTTCGACTTTTTCCTTCTGG (SEQ ID NO. 18).

[0130] The reverse PCR amplification reaction systems described above are shown in Table 5, and the amplification reaction conditions are shown in Table 6.

[0131] Table 5

[0132]

[0133] Table 6

[0134]

[0135] It should be understood that during the above reverse PCR amplification, the pre-denaturation at 94℃ and storage at 4℃ do not participate in the cycling, and the entire reverse PCR process is performed only once.

[0136] After each of the above reverse PCR amplification reactions was completed, the template was removed from the reaction products using the following methods:

[0137] Add 2 μL of restriction endonuclease DpnⅠ to the reaction product (50 μL), mix gently by pipetting, and react at 37 °C for 1 h to obtain the enzyme digestion solution. The enzyme digestion solution was verified by agarose gel electrophoresis.

[0138] After template removal, the reverse PCR products were self-circularized using the following methods:

[0139] Using the enzyme digestion solution verified above, prepare the reaction solution according to Table 7, mix gently, and react at 16℃ for 1 hour.

[0140] Table 7

[0141]

[0142] Mutant plasmid verification: The above mutant plasmid pYB1s-BsiolG was used. G234A / D267K / E331K The plasmid was transformed into *E. coli* DH5α competent cells using chemical transformation. Single colonies were picked from the plates for plasmid extraction, and the extracted plasmids were then sequenced. The mutant plasmid pYB1s-BsiolG was used. G234A / D267K / E331K It contains the gene of the inositol dehydrogenase mutant G234A / D267K / E331K, the nucleotide sequence of which is shown in SEQ ID NO.5 and the encoded amino acid sequence is shown in SEQ ID NO.6.

[0143] Example 3

[0144] Construction of mutant plasmid pYB1s-BaiolI L68K / I225R

[0145] Using the correctly sequenced recombinant plasmid pYB1s-BaiolI from Example 1 above as a template, and with L68K-F as the upstream primer and L68K-R as the downstream primer, a reverse PCR amplification reaction was performed. After removing the template from the reaction product, the reverse PCR product was self-circularized to obtain the mutant plasmid pYB1s-BaiolI. L68K .

[0146] L68K-F: GAACGCCaaaGTCTTCTTCAACAATCGTGACGAA (SEQ ID NO. 19).

[0147] L68K-R: AGAAGACtttGGCGTTCAGGGCCAGCGGCTTA (SEQ ID NO. 20).

[0148] Using the above mutant plasmid pYB1s-BaiolI L68K Using I225R-F as the upstream primer and I225R-R as the downstream primer, a reverse PCR amplification reaction was performed. After removing the template from the reaction product, the reverse PCR product was self-circularized to obtain the mutant plasmid pYB1s-BaiolI. L68K / I225R .

[0149] I225R-F: GAGCAcgcGATCTGGATGCCCACCTGAGCGCT (SEQ ID NO. 21).

[0150] I225R-R: ATCCAGATCgcgTGCTCCCTGACCCCGGCCAAA (SEQ ID NO. 22).

[0151] The two reverse PCR amplification reaction systems are shown in Table 5, and the amplification reaction conditions are shown in Table 8.

[0152] Table 8

[0153]

[0154] It should be understood that during the above reverse PCR amplification, the pre-denaturation at 94℃ and storage at 4℃ do not participate in the cycling, and the entire reverse PCR process is performed only once.

[0155] After each of the above reverse PCR amplification reactions is completed, the template is removed from the reaction product according to the method in Example 2 above, and the reverse PCR product is self-circularized.

[0156] Mutant plasmid verification: The above mutant plasmid pYB1s-BaiolI was used. L68K / I225R The plasmid was transformed into E. coli DH5α competent cells using chemical transformation. Single colonies on the plates were picked for plasmid extraction, and the extracted plasmids were sequenced for DNA. The mutant plasmid pYB1s-BaiolI was used. L68K / I225R The gene contains the 2-keto-inositol isomerase mutant L68K / I225R, the nucleotide sequence of which is shown in SEQ ID NO.7 and the encoded amino acid sequence is shown in SEQ ID NO.8.

[0157] Example 4

[0158] Preparation of crude enzyme solution

[0159] Take 1 μL of the above recombinant plasmid pYB1s-BsiolG, 1 μL of the recombinant plasmid pYB1s-BaiolI, and 1 μL of the mutant plasmid pYB1s-BsiolG respectively. G234A / D267K / E331K And 1 μL of mutant plasmid pYB1s-BaiolI L68K / I225R The culture medium was added separately to E. coli BW25113 competent cells and gently mixed. The mixture was then transferred to pre-chilled 1 mm electroporation cuvettes. The cuvettes were placed in an electroporator and subjected to single electroporation at 1.8 kV, 200 Ω resistance, and 25 μF capacitance. After electroporation, 450 μL of pre-chilled LB medium was added to each cuvette and gently mixed by pipetting. The bacterial culture was then transferred to sterile centrifuge tubes and incubated at 37°C and 200 rpm with shaking for 1 hour. After resuscitation and culture, 100 μL of bacterial suspension was spread onto LB agar plates containing 50 μg / ml streptomycin sulfate and incubated upside down at 37°C for 12 hours to obtain recombinant strains BW25113-pYB1s-BsiolG containing the recombinant plasmid pYB1s-BsiolG, BW25113-pYB1s-BaiolI containing the recombinant plasmid pYB1s-BaiolI, and BW25113-pYB1s-BaiolI containing the mutant plasmid pYB1s-BsiolG. G234A / D267K / E331K The mutant strain BW25113-pYB1s-BsiolG G234A / D267K / E331K and containing the mutant plasmid pYB1s-BaiolI L68K / I225R The mutant strain BW25113-pYB1s-BaiolI L68K / I225R .

[0160] The two recombinant strains and two mutant strains were inoculated into liquid LB medium (containing 50 μg / ml streptomycin sulfate) for seed culture and cultured at 37℃ and 180 rpm for 12 h to obtain seed cultures of recombinant strain BW25113-pYB1s-BsiolG, recombinant strain BW25113-pYB1s-BaiolI, and mutant strain BW25113-pYB1s-BsiolG, respectively. G234A / D267K / E331K Seed culture and mutant strain BW25113-pYB1s-BaiolI L68K / I225R Seed liquid.

[0161] The four seed cultures were inoculated into fresh LB liquid medium (containing 50 μg / ml streptomycin sulfate) at a 2% volume ratio for fermentation and cultured at 37°C until OD reached. 600The value was 0.6, then the temperature was lowered to 20℃, and L-arabinose was added to a final concentration of 2 g / L for induction culture for 16 h. Fermentation broths of recombinant strain BW25113-pYB1s-BsiolG, recombinant strain BW25113-pYB1s-BaiolI, and mutant strain BW25113-pYB1s-BsiolG were obtained respectively. G234A / D267K / E331K The fermentation broth, and the mutant strain BW25113-pYB1s-BaiolI L68K / I225R The fermentation broth.

[0162] The four fermentation broths were centrifuged at 4℃ and 4000 r / min for 20 min, respectively, and the bacterial cells were collected. The collected bacterial cells were resuspended in a 50 mM sodium carbonate buffer solution with a pH of 7.5. The OD of the resuspended solutions was measured. 600 The concentration was 40. Then, an ultrasonic cell disruptor was used to disrupt the bacterial cells. The ultrasonic power was 450W, with a 3-second disruption followed by a 2-second pause, for a total of 30 minutes. After ultrasonic disruption, the cells were centrifuged at 4℃ and 12000r / min to remove cell debris. The supernatants were collected to obtain crude enzyme solutions of wild-type inositol dehydrogenase, inositol dehydrogenase mutant G234A / D267K / E331K, wild-type 2-keto-inositol isomerase, and 2-keto-inositol isomerase mutant L68K / I225R.

[0163] Example 5

[0164] Detecting the transformation efficiency of mutants

[0165] Reaction system 1: Add 20 mM muscle inositol, 1.0 mM manganese chloride, the crude enzyme solution of wild-type inositol dehydrogenase prepared in Example 4, and the crude enzyme solution of wild-type 2-keto-inositol isomerase prepared in Example 4 to 50 mM sodium carbonate buffer, so that the final concentration of wild-type inositol dehydrogenase is 3.5 mg / mL and the final concentration of wild-type 2-keto-inositol isomerase is 2.5 mg / mL, to prepare a 10 mL reaction system. Adjust the pH of the reaction system to 8.5.

[0166] Reaction system 2: Add 20 mM muscle inositol, 1.0 mM manganese chloride, crude enzyme solution of inositol dehydrogenase mutant G234A / D267K / E331K prepared in Example 4, and crude enzyme solution of wild-type 2-keto-inositol isomerase prepared in Example 4 to 50 mM sodium carbonate buffer, so that the final concentration of inositol dehydrogenase mutant G234A / D267K / E331K is 3.5 mg / mL and the final concentration of wild-type 2-keto-inositol isomerase is 2.5 mg / mL, to prepare a 10 mL reaction system, and adjust the pH of the reaction system to 8.5.

[0167] Reaction system 3: Add 20 mM muscle inositol, 1.0 mM manganese chloride, the crude enzyme solution of wild-type inositol dehydrogenase prepared in Example 4, and the crude enzyme solution of 2-keto-inositol isomerase mutant L68K / I225R prepared in Example 4 to 50 mM sodium carbonate buffer to prepare a 10 mL reaction system. The final concentration of wild-type inositol dehydrogenase is 3.5 mg / mL, and the final concentration of 2-keto-inositol isomerase mutant L68K / I225R is 2.5 mg / mL. Adjust the pH of the reaction system to 8.5.

[0168] Reaction system 4: Add 20 mM muscle inositol, 1.0 mM manganese chloride, crude enzyme solution of inositol dehydrogenase mutant G234A / D267K / E331K prepared in Example 4, and crude enzyme solution of 2-keto-inositol isomerase mutant L68K / I225R prepared in Example 4 to 50 mM sodium carbonate buffer to prepare a 10 mL reaction system. The final concentration of inositol dehydrogenase mutant G234A / D267K / E331K is 3.5 mg / mL, and the final concentration of 2-keto-inositol isomerase mutant L68K / I225R is 2.5 mg / mL. Adjust the pH of the reaction system to 8.5.

[0169] The above reaction systems were reacted at 35℃ for 6 hours, and then the reaction was terminated by boiling for 10 minutes. The content of D-chiral inositol in the reaction solution was detected by high performance liquid chromatography, and the conversion rate was calculated. The results are shown in Table 9.

[0170] Conversion rate = D-chiral inositol content generated ÷ initial inositol content × 100%.

[0171] High performance liquid chromatography detection method:

[0172] The chromatographic column was a 4.6 × 250 mm, 5 μm amino column; the mobile phase was acetonitrile: 50 mM ammonium acetate aqueous solution = 75: 25 (volume ratio); the column temperature was set at 30℃ and the flow rate was 1.0 mL / min.

[0173] Table 9

[0174]

[0175] The results above show that, compared with wild-type inositol dehydrogenase and wild-type 2-keto-inositol isomerase, the inositol dehydrogenase mutants G234A / D267K / E331K and 2-keto-inositol isomerase mutants L68K / I225R of the present invention can further improve the conversion rate and the yield of D-chiral inositol.

[0176] Example 6

[0177] High-density fermentation for the preparation of crude enzyme solution

[0178] Seed cultures of recombinant strain BW25113-pYB1s-BsiolG, recombinant strain BW25113-pYB1s-BaiolI, and mutant strain BW25113-pYB1s-BsiolG from Example 4 were taken respectively. G234A / D267K / E331K Seed culture and mutant strain BW25113-pYB1s-BaiolI L68K / I225R The seed culture was inoculated into 5L fermenters at a 5% (v / v) inoculum and cultured at 0.04 MPa, 300 rpm, 37°C, and pH 7.0 until OD500. 600 Cool to 35°C, then reduce to 30°C and continue culturing until OD (October Expiratory Time) reaches 35°C. 600 Induction culture was performed at 45°C. The induction culture conditions were as follows: L-arabinose was added to a final concentration of 2 g / L; the initial airflow rate was 4 L / min; when the rotation speed reached 400 rpm, the airflow rate was adjusted to 4.2 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 DO value at 20-40% until OD... 600 Fermentation was stopped when the temperature reached 120°C. Fermentation broths of recombinant strain BW25113-pYB1s-BsiolG, recombinant strain BW25113-pYB1s-BaiolI, and mutant strain BW25113-pYB1s-BsiolG were obtained, respectively. G234A / D267K / E331K The fermentation broth, and the mutant strain BW25113-pYB1s-BaiolI L68K / I225R The fermentation broth.

[0179] The four fermentation broths obtained above were centrifuged at 4℃ and 4000 r / min for 20 min, and the bacterial cells were collected. The collected bacterial cells were resuspended in a 50 mM sodium carbonate buffer solution with a pH of 7.5. The OD of the resuspended solution was measured. 600The concentration was 180. Then, a high-pressure homogenizer was used to disrupt the bacterial cells at 450W for 30 minutes. After ultrasonic disruption, the cells were centrifuged at 4℃ and 12000r / min to remove cell debris. The supernatants were collected to obtain crude enzyme solutions of wild-type inositol dehydrogenase, inositol dehydrogenase mutants G234A / D267K / E331K, wild-type 2-keto-inositol isomerase, and 2-keto-inositol isomerase mutants L68K / I225R.

[0180] In this embodiment, the culture medium in the fermenter consists of: 8 g / L yeast extract, 2.5 g / L ammonium sulfate, 12 g / L peptone, 0.5 g / L magnesium sulfate heptahydrate, 12 g / L glucose, 3 g / L sodium chloride, 2.1 g / L citric acid monohydrate, and 4 g / L potassium phosphate.

[0181] The fed culture medium consisted of 500 g / L glucose, 2 g / L magnesium sulfate heptahydrate, and 2 g / L yeast extract.

[0182] Example 7

[0183] Detecting substrate tolerance of inositol dehydrogenase

[0184] Add the crude enzyme solution of wild-type inositol dehydrogenase prepared in Example 6 to 50 mM sodium carbonate buffer, the amount of which is 20% of the total reaction volume; add the crude enzyme solution of wild-type 2-keto-inositol isomerase prepared in Example 6, the amount of which is 40% of the total reaction volume; add manganese chloride to a final concentration of 1 mM; add the substrate muscle inositol; adjust the reaction pH to 8.5; and set the reaction temperature to 35°C.

[0185] Three reaction systems were prepared according to the above method, and the concentrations of the substrate muscle inositol in the three reaction systems were set to 100 g / L, 150 g / L, and 200 g / L, respectively. Samples were taken from each reaction system at 0 min, 30 min, 60 min, and 90 min, respectively, and the concentration of D-chiral inositol in each reaction solution was detected by HPLC to obtain the initial rate of D-chiral inositol formation.

[0186] The crude enzyme solution of the inositol dehydrogenase mutant G234A / D267K / E331K prepared in Example 6 was added to 50 mM sodium carbonate buffer, with the amount added being 20% ​​of the total reaction volume; the crude enzyme solution of wild-type 2-keto-inositol isomerase prepared in Example 6 was added, with the amount added being 40% of the total reaction volume; manganese chloride was added to a final concentration of 1 mM; the substrate muscle inositol was added; the reaction pH was adjusted to 8.5; and the reaction temperature was 35°C.

[0187] Three reaction systems were prepared according to the above method, and the concentrations of the substrate muscle inositol in the three reaction systems were set to 100 g / L, 150 g / L, and 200 g / L, respectively. Samples were taken from each reaction system at 0 min, 30 min, 60 min, and 90 min, respectively, and the concentration of D-chiral inositol in each reaction solution was detected by HPLC to obtain the initial rate of D-chiral inositol formation.

[0188] The results are shown in Table 10.

[0189] HPLC detection method: The chromatographic column was a 4.6 × 250 mm, 5 μm amino column; the mobile phase was acetonitrile: 50 mM ammonium acetate aqueous solution = 75: 25 (volume ratio); the column temperature was set at 30℃ and the flow rate was 1.0 mL / min.

[0190] Table 10

[0191]

[0192] The results above show that in the presence of excessive wild-type 2-keto-inositol isomerase, wild-type inositol dehydrogenase reaches the highest rate of D-chiral inositol production (0.13 g / L / min) when the substrate concentration of myositol is 150 g / L. However, when the substrate concentration of myositol is further increased to 200 g / L, the rate of D-chiral inositol production decreases to 0.06 g / L / min, indicating that substrate inhibition exists. When the substrate concentration is too high, it will inhibit the catalytic efficiency of wild-type inositol isomerase.

[0193] Compared to wild-type inositol dehydrogenase, the inositol dehydrogenase mutant G234A / D267K / E331K of the present invention maintains a high rate of D-chiral inositol production (0.17 g / L / min) even when the concentration of the substrate muscle inositol reaches 200 g / L. This indicates that the inositol dehydrogenase mutant G234A / D267K / E331K of the present invention can alleviate substrate inhibition and can efficiently catalyze the reaction to produce D-chiral inositol at high substrate concentrations.

[0194] Example 8

[0195] Preparation of D-chiral inositol by a dual-enzyme cascade

[0196] System 1: Add the crude enzyme solution of wild-type inositol dehydrogenase prepared in Example 6 to 50mM sodium carbonate buffer, the amount of which is 20% of the total reaction volume. Add the crude enzyme solution of wild-type 2-keto-inositol isomerase prepared in Example 6, the amount of which is 20% of the total reaction volume. Add manganese chloride to a final concentration of 1mM. Add muscle inositol to a final concentration of 200g / L. Adjust the pH of the reaction to 8.5. After reacting at 35℃ for 8h, the concentration of D-chiral inositol is detected by HPLC to obtain the conversion rate and space-time yield of D-chiral inositol.

[0197] System 2: The crude enzyme solution of the inositol dehydrogenase mutant G234A / D267K / E331K prepared in Example 6 was added to 50mM sodium carbonate buffer, with the amount added being 20% ​​of the total reaction volume. The crude enzyme solution of wild-type 2-keto-inositol isomerase prepared in Example 6 was also added, with the amount added being 20% ​​of the total reaction volume. Manganese chloride was added to a final concentration of 1mM, and muscle inositol was added to a final concentration of 200g / L. The pH of the reaction was adjusted to 8.5, and after reacting at 35℃ for 8h, the concentration of D-chiral inositol was detected by HPLC to obtain the D-chiral inositol conversion rate and space-time yield.

[0198] System 3: The crude enzyme solution of the inositol dehydrogenase mutant G234A / D267K / E331K prepared in Example 6 was added to 50 mM sodium carbonate buffer, at a volume of 20% of the total reaction volume. The crude enzyme solution of the 2-keto-inositol isomerase mutant L68K / I225R prepared in Example 6 was also added, at a volume of 20% of the total reaction volume. Manganese chloride was added to a final concentration of 1 mM, and muscle inositol was added to a final concentration of 200 g / L. The pH of the reaction was adjusted to 8.5. After reacting at 35°C for 8 h, the concentration of D-chiral inositol was detected using HPLC to obtain the D-chiral inositol conversion rate and space-time yield.

[0199] HPLC detection: The chromatographic column was a 4.6 × 250 mm, 5 μm amino column; the mobile phase was acetonitrile: 50 mM ammonium acetate aqueous solution = 75: 25 (volume ratio); the column temperature was set at 30℃ and the flow rate was 1.0 mL / min.

[0200] The results are shown in Table 11.

[0201] Table 11

[0202]

[0203] The results above show that, compared to wild-type inositol dehydrogenase, the inositol dehydrogenase mutant G234A / D267K / E331K of the present invention can alleviate substrate inhibition and effectively improve the conversion rate and space-time yield of D-chiral inositol under conditions of high substrate concentration and conventional industrial temperature. Furthermore, when the inositol dehydrogenase mutant G234A / D267K / E331K is used in combination with the 2-keto-inositol isomerase mutant L68K / I225R, the conversion rate and space-time yield of D-chiral inositol are further improved.

[0204] Example 9

[0205] After the reaction of System 3 in Example 8 above was completed, a conversion solution was obtained. D-chiral inositol was extracted from this conversion solution, and the specific steps are as follows:

[0206] The conversion solution was heated to 75°C for enzyme inactivation for 30 minutes, and then cooled to 35°C. The cooled conversion solution was then filtered through a ceramic membrane with a pore size of 50 nm to obtain the ceramic membrane permeate.

[0207] The ceramic membrane permeate is then filtered through an ultrafiltration membrane with a pore size of 5000 Da to obtain the ultrafiltration membrane permeate.

[0208] The permeate from the ultrafiltration membrane was passed through cation exchange resin D001 at a feed flow rate of 1.5 BV / h, and the conductivity of the effluent was controlled to be <4000 μs / cm and the pH to be <3, to obtain the cation exchange resin effluent.

[0209] The effluent from the cation exchange resin was passed through macroporous adsorption resin LX-60 at a feed flow rate of 1.5 BV / h, and the transmittance of the effluent was controlled to be >98% to obtain the macroporous adsorption resin effluent.

[0210] The effluent from the macroporous adsorption resin was passed through anion exchange resin D-201 at a feed flow rate of 1.5 BV / h, and the conductivity of the effluent was controlled to be <300 μS / cm and the pH to be >8 to obtain the treated solution.

[0211] The treated solution was concentrated using a nanofiltration membrane with a pore size of 170 Da until the solid content was >15%. The resulting nanofiltration concentrate was then thermally concentrated at 58°C until muscle inositol crystals precipitated, yielding a thermally concentrated solution. The thermally concentrated solution was cooled to 22°C and filtered to remove the muscle inositol crystals using a 500-mesh filtration filter, collecting the first filtrate.

[0212] The first filtrate was concentrated to 45% of its original volume under single-effect conditions of 60℃ and vacuum degree < -0.09MPa. Then, ethanol with a volume of 1.5 times that of the concentrated filtrate was added, and the mixture was heated to 52℃ and kept at that temperature for 1.5 hours. The mixture was then cooled to 22℃ to precipitate muscle inositol. The mixture was then filtered through a 500-mesh filtration filter to obtain the second filtrate.

[0213] The second filtrate was de-alcoholized using a distillation column to obtain a de-alcoholized liquid with a solid content of 15%. The de-alcoholized liquid was then concentrated in a single-effect manner at a temperature of 60℃ and a vacuum degree of <-0.09MPa until the solid content reached 52%. The solution was then heated to 48℃, and 0.8 times the volume of the concentrated liquid was added with ethanol. The mixture was stirred evenly, then cooled to 28℃ and filtered. The filter was filtered through a 500-mesh pore size, and the resulting filter cake was the crude D-chiral inositol product.

[0214] Add water at 1.2 times the mass of crude D-chiral inositol to the crude D-chiral inositol, heat to 65°C to dissolve the crude D-chiral inositol, and then add activated carbon at 2% of the mass of crude D-chiral inositol for decolorization. Decolorize for 30 min, remove the activated carbon, and obtain the decolorized solution.

[0215] Ethanol was slowly added to the decolorizing solution. Once crystals precipitated, the addition was stopped, and the solution was cooled to 18°C. The solution was then filtered through a 500-mesh sieve to obtain a filter cake. The filter cake was washed with ethanol and dried to obtain the D-chiral inositol product, which had a D-chiral inositol content of 99.6%.

[0216] Example 10

[0217] After the reaction of System 3 in Example 8 above was completed, a conversion solution was obtained. D-chiral inositol was extracted from this conversion solution, and the specific steps are as follows:

[0218] The conversion solution was heated to 70°C for enzyme inactivation for 20 minutes, and then cooled to 30°C. The cooled conversion solution was then filtered through a ceramic membrane with a pore size of 20 nm to obtain the ceramic membrane permeate.

[0219] The ceramic membrane permeate is then filtered through an ultrafiltration membrane with a pore size of 2000 Da to obtain the ultrafiltration membrane permeate.

[0220] The permeate from the ultrafiltration membrane was passed through cation exchange resin D113 at a feed flow rate of 1 BV / h, and the conductivity of the effluent was controlled to be <4000 μs / cm and the pH to be <3, to obtain the cation exchange resin effluent.

[0221] The effluent from the cation exchange resin was passed through macroporous adsorption resin LS-305 at a feed flow rate of 1 BV / h, and the transmittance of the effluent was controlled to be >98% to obtain the macroporous adsorption resin effluent.

[0222] The effluent from the macroporous adsorption resin was passed through anion exchange resin D-301 at a feed flow rate of 1 BV / h, and the conductivity of the effluent was controlled to be <300 μs / cm and the pH to be >8 to obtain the treated solution.

[0223] The treated solution was concentrated using a nanofiltration membrane with a pore size of 150 Da until the solid content was >15%. The resulting nanofiltration membrane concentrate was then thermally concentrated at 55°C until muscle inositol crystals precipitated, yielding a thermally concentrated solution. The thermally concentrated solution was cooled to 20°C and filtered to remove the muscle inositol crystals using a 500-mesh filtration filter, collecting the first filtrate.

[0224] The first filtrate was concentrated to 40% of its original volume under a single-effect concentration at 60°C and a vacuum of <-0.09MPa. Ethanol with a volume equal to 1 volume of the concentrate was added, and the solution was then heated to 50°C and kept at that temperature for 1 hour. The solution was then cooled to 20°C to precipitate muscle inositol. The solution was filtered through a 500-mesh filtration filter to obtain the second filtrate.

[0225] The second filtrate was de-alcoholized using a distillation column to obtain a de-alcoholized liquid with a solid content of 12%. The de-alcoholized liquid was then concentrated in a single-effect manner at a temperature of 60℃ and a vacuum degree of <-0.09MPa until the solid content reached 50%. The solution was then heated to 45℃, and 0.5 times the volume of the concentrated liquid was added with ethanol. The mixture was stirred evenly, then cooled to 25℃ and filtered. The filter was filtered through a 500-mesh pore size, and the resulting filter cake was the crude D-chiral inositol product.

[0226] Add water equal to the mass of crude D-chiral inositol to the crude product, heat to 60°C to dissolve the crude D-chiral inositol, and then add activated carbon at 1% of the mass of crude D-chiral inositol for decolorization. Decolorize for 40 min, remove the activated carbon, and obtain the decolorized solution.

[0227] Ethanol was slowly added to the decolorizing solution. Once crystals precipitated, the addition was stopped, and the solution was cooled to 15°C. The solution was then filtered through a 500-mesh sieve to obtain a filter cake. The filter cake was washed with ethanol and dried to obtain the D-chiral inositol product, which had a D-chiral inositol content of 99.5%.

[0228] Example 11

[0229] After the reaction of System 3 in Example 8 above was completed, a conversion solution was obtained. D-chiral inositol was extracted from this conversion solution, and the specific steps are as follows:

[0230] The conversion solution was heated to 80°C for enzyme inactivation for 40 minutes, and then cooled to 40°C. The cooled conversion solution was then filtered through a ceramic membrane with a pore size of 100 nm to obtain the ceramic membrane permeate.

[0231] The ceramic membrane permeate is then filtered through an ultrafiltration membrane with a pore size of 10,000 Da to obtain the ultrafiltration membrane permeate.

[0232] The permeate from the ultrafiltration membrane was passed through cation exchange resin SQD-80 at a feed flow rate of 2 BV / h, and the conductivity of the effluent was controlled to be <4000 μs / cm and the pH to be <3, to obtain the cation exchange resin effluent.

[0233] The effluent from the cation exchange resin was passed through macroporous adsorption resin LS-406 at a feed flow rate of 2 BV / h, and the transmittance of the effluent was controlled to be >98% to obtain the macroporous adsorption resin effluent.

[0234] The effluent from the macroporous adsorption resin was passed through anion exchange resin D-311 at a feed flow rate of 2 BV / h, and the conductivity of the effluent was controlled to be <300 μs / cm and the pH to be >8 to obtain the treated solution.

[0235] The treated solution was concentrated using a nanofiltration membrane with a pore size of 200 Da until the solid content was >15%. The resulting nanofiltration membrane concentrate was then thermally concentrated at 60°C until muscle inositol crystals precipitated, yielding a thermally concentrated solution. The thermally concentrated solution was cooled to 25°C and filtered to remove the muscle inositol crystals using a 500-mesh filtration filter, collecting the first filtrate.

[0236] The first filtrate was concentrated to 50% of its original volume under a single-effect concentration at 60°C and a vacuum of <-0.09MPa. Ethanol with a volume twice that of the concentrate was added, and the solution was then heated to 55°C and kept at that temperature for 2 hours. The solution was then cooled to 25°C to precipitate muscle inositol. The solution was then filtered through a 500-mesh filtration filter to obtain the second filtrate.

[0237] The second filtrate was de-alcoholized using a distillation column to obtain a de-alcoholized liquid with a solid content of 18%. The de-alcoholized liquid was then concentrated in a single-effect manner at a temperature of 60°C and a vacuum degree of <-0.09MPa until the solid content reached 55%. The solution was then heated to 50°C, and ethanol with a volume equal to that of the concentrate was added. The mixture was stirred until homogeneous, then cooled to 30°C and filtered through a 500-mesh filtration filter. The resulting filter cake was the crude D-chiral inositol product.

[0238] Add water at 1.5 times the mass of crude D-chiral inositol to the crude D-chiral inositol, heat to 70°C to dissolve the crude D-chiral inositol, and then add activated carbon for decolorization. The amount of activated carbon added is 3% of the mass of crude D-chiral inositol. Decolorize for 20 minutes, remove the activated carbon, and obtain the decolorized solution.

[0239] Ethanol was slowly added to the decolorizing solution. Once crystals precipitated, the addition was stopped, and the solution was cooled to 20°C. The solution was then filtered through a 500-mesh sieve to obtain a filter cake. The filter cake was washed with ethanol and dried to obtain the D-chiral inositol product, which had a D-chiral inositol content of 99.5%.

[0240] 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 comprising inositol dehydrogenase and 2-keto-inositol isomerase, characterized in that, The amino acid sequence of the inositol dehydrogenase is shown in SEQ ID NO.6, and the amino acid sequence of the 2-keto-inositol isomerase is shown in SEQ ID NO.

8.

2. The enzyme preparation according to claim 1, characterized in that, The gene sequence of the inositol dehydrogenase is shown in SEQ ID NO. 5; the gene sequence of the 2-keto-inositol isomerase is shown in SEQ ID NO.

7.

3. The enzyme preparation according to claim 1, characterized in that, The mass ratio of the inositol dehydrogenase to the 2-keto-inositol isomerase is (0.5~30):(0.5~30).

4. A method for preparing D-chiral inositol using the enzyme preparation of claim 1, characterized in that, Includes the following steps: Prepare the reaction system: Add crude enzyme solution of inositol dehydrogenase, crude enzyme solution of 2-keto-inositol isomerase, metal ions and muscle inositol to the buffer solution. The reaction system catalyzes the reaction of muscle inositol to generate D-chiral inositol under conditions of pH 7.5~9.5 and temperature 30~50℃, resulting in a conversion solution containing D-chiral inositol.

5. The method according to claim 4, characterized in that, In the reaction system, the concentration of the buffer solution is 20-100 mM, the amount of crude enzyme solution of inositol dehydrogenase added is 10%-90% of the total reaction volume, the amount of crude enzyme solution of 2-keto-inositol isomerase added is 10%-90% of the total reaction volume, the concentration of the metal ion is 0.5-2 mM, and the concentration of muscle inositol is 10-200 g / L.

6. The method according to claim 4, characterized in that, The method for preparing the crude enzyme solution of inositol dehydrogenase includes the following steps: An expression vector containing the inositol dehydrogenase gene was constructed, and the expression vector was transformed into a host strain to obtain a recombinant strain containing the inositol dehydrogenase gene. The recombinant strain was fermented to express the inositol dehydrogenase, and a fermentation broth containing inositol dehydrogenase was obtained. centrifuging the fermentation broth, collecting the bacterial cells, resuspending the bacterial cells to obtain a bacterial cell suspension having an OD of 10-220 600 crushing the bacterial cells in the resuspension, centrifuging to remove cell debris, and obtaining a crude enzyme solution of the myo-inositol dehydrogenase.

7. The method according to claim 4, characterized in that, The method for preparing the crude enzyme solution of the 2-keto-inositol isomerase includes the following steps: An expression vector containing the 2-keto-inositol isomerase gene was constructed, and the expression vector was transformed into a host strain to obtain a recombinant strain containing the 2-keto-inositol isomerase gene. The recombinant strain was fermented to express the 2-keto-inositol isomerase, and a fermentation broth containing the 2-keto-inositol isomerase was obtained. Centrifuge the fermentation broth, collect the bacterial cells, resuspend the bacterial cells, and obtain the bacterial OD. 600 The bacterial cells in the resuspension were broken up, and the cell debris was removed by centrifugation to obtain the crude enzyme solution of the 2-keto-inositol isomerase.

8. The method according to claim 4, characterized in that, The buffer solution includes sodium carbonate buffer, phosphate buffer, or Tris-HCl buffer; the metal ions include manganese ions, cobalt ions, nickel ions, or zinc ions.

9. The method according to claim 4, characterized in that, After obtaining the conversion solution containing D-chiral inositol, the method further includes extracting D-chiral inositol from the conversion solution, comprising the following steps: The conversion solution is heated to 70-80°C for enzyme inactivation, then cooled, and the cooled conversion solution is filtered through a ceramic membrane with a pore size of 20-100 nm to obtain ceramic membrane permeate. The ceramic membrane permeate is filtered through an ultrafiltration membrane with a pore size of 2000~10000 Da to obtain an ultrafiltration membrane permeate. The cationic impurities, anionic impurities, and pigments in the permeate of the ultrafiltration membrane are removed to obtain a treated solution with a conductivity of <300 μs / cm and a transmittance of >98%. The treatment solution is concentrated to a solid content >15% by passing it through a nanofiltration membrane with a pore size of 150~200Da. The resulting nanofiltration membrane concentrate is then thermally concentrated at a temperature of 55~60°C until muscle inositol crystals precipitate. After cooling to 20~25°C, the muscle inositol crystals are removed by filtration, and the first filtrate is collected. The first filtrate is concentrated to 40% to 50% of its original volume, ethanol is added, and the mixture is heated, kept warm, and cooled in sequence to precipitate muscle inositol. The mixture is then filtered and the second filtrate is collected. The second filtrate is distilled to remove alcohol, and the removed alcohol solution is concentrated to a solid content of 50% to 55%. The solution is heated and ethanol is added, then cooled and filtered. The resulting filter cake is crude D-chiral inositol.

10. The method according to claim 9, characterized in that, The process removes cationic impurities, anionic impurities, and pigments from the ultrafiltration membrane permeate to obtain a treated solution with a conductivity <300 μS / cm and a transmittance >98%, comprising: The permeate from the ultrafiltration membrane is treated with a cation exchange resin at a feed flow rate of 1-2 BV / h, and the conductivity of the effluent from the cation exchange resin is controlled to be <4000 μs / cm and the pH to be <3. The effluent from the cation exchange resin was treated with macroporous adsorption resin at a feed flow rate of 1-2 BV / h, and the transmittance of the effluent from the macroporous adsorption resin was controlled to be >98%. The effluent from the macroporous adsorption resin is treated with anion exchange resin at a feed flow rate of 1-2 BV / h, and the conductivity of the effluent from the macroporous adsorption resin is controlled to be <300 μs / cm and the pH to be >8.

11. The method according to claim 9, characterized in that, After the first filtrate is concentrated, ethanol with a volume of 1 to 2 times that of the concentrated filtrate is added, and then the mixture is heated to 50 to 55°C, kept at that temperature for 1 to 2 hours, and then cooled to 20 to 25°C.

12. The method according to claim 9, characterized in that, The distillation and de-alcoholization process involves using a distillation column to de-alcoholize the second filtrate, and the solid content of the de-alcoholized solution is 12% to 18%.

13. The method according to claim 9, characterized in that, After the dealcoholized liquid is concentrated to a solid content of 50% to 55%, it is heated to 45 to 50°C, and 0.5 to 1 times the volume of the concentrated liquid is added with ethanol. Then the temperature is lowered to 25 to 30°C.

14. The method according to claim 8, characterized in that, After obtaining crude D-chiral inositol, the process further includes adding water to the crude D-chiral inositol, wherein the mass ratio of crude D-chiral inositol to water is 1:(1~1.5), heating to 60~70℃, dissolving the crude D-chiral inositol, and then performing a decolorization treatment to obtain a decolorized solution. Ethanol was added to the decolorizing solution. After crystals precipitated, the solution was cooled to 15-20°C and filtered to obtain D-chiral inositol.

15. The method according to claim 14, characterized in that, The decolorization treatment of the solution of crude D-chiral inositol includes: decolorization with activated carbon, wherein the amount of activated carbon added is 1% to 3% of the crude D-chiral inositol, and the decolorization time is 20 to 40 minutes.

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