Process method for preparing D-chiro-inositol

Abstract

The invention discloses a process method for preparing D-chiro-inositol, and relates to the technical field of biology, IDH crude enzyme liquid, KMI crude enzyme liquid and NOX crude enzyme liquid are added into an inositol solution for conversion, after conversion is finished, inactivation treatment is performed, and the mixture passes through a ceramic membrane, an ultrafiltration membrane, cationic resin, decolorizing resin and anion resin to obtain a solution I; after the solution I is subjected to nanofiltration concentration, heat concentration continues to be conducted till the volume of the solution I is one third of the volume of a nanofiltration concentrated solution, then the temperature is reduced to 25 DEG C, suction filtration is conducted, a solution II and a solid II are obtained, and the solution II is a D-chiral inositol solution; continuously performing thermal concentration on the solution II until the solid content is 40-50% w / w, adding ethanol of which the volume is 1 time that of the solution II under the condition of 50 DEG C, performing heat preservation for 2 hours, then cooling to 25 DEG C at the speed of 5 DEG C / h, and performing suction filtration to respectively obtain a solution III and a solid III; the usage amount of the enzyme is greatly reduced, the production cost is reduced, and the production efficiency is improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a process for preparing D-chiral inositol. Background Technology

[0002] D-chiro-inositol (DCI) is an isomer of myo-inositol (MI), primarily found in nature in buckwheat and legumes. D-chiro-inositol has various physiological functions, including the treatment of type 2 diabetes, polycystic ovary syndrome, and inhibition of liver fibrosis; it can also be used as a raw material for health supplements and dietary supplements. Currently, several brands of D-chiro-inositol health supplements are available in the United States, indicating a broad market prospect.

[0003] Currently, D-chiral inositol is typically produced through plant extraction, chemical synthesis, or hydrolysis of kasugamycin. Plant-derived D-chiral inositol is generally extracted from plants such as buckwheat and carob. However, the low concentration of D-chiral inositol in these plants leads to high extraction costs. Chemical synthesis of D-chiral inositol is cumbersome, with difficult-to-separate byproducts and inevitable organic solvent residues, affecting product quality. Furthermore, the preparation process uses large amounts of organic solvents, which is environmentally unfriendly. Additionally, D-chiral inositol can be produced by hydrolyzing D-pineol or kasugamycin; however, the raw materials D-pineol or kasugamycin are expensive, making it uneconomical.

[0004] Biotransformation is a promising method for synthesizing D-chiral inositol due to its advantages of being mild, safe, and environmentally friendly. Chinese patent CN118853609A discloses engineered bacteria and their preparation methods for synthesizing D-chiral inositol using GT enzyme mutants and MA enzyme mutants. The method involves synthesizing D-chiral inositol via inositol dehydrogenase (IDH) and 2-ketoinositol isomerase (KMI). The specific synthetic route is as follows: .

[0005] Currently, the synthesis of D-chiral inositol suffers from the problem of high usage of inositol dehydrogenase and 2-ketoinositol isomerase, resulting in high production costs and low production efficiency. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a process for preparing D-chiral inositol that is low in production cost and high in production efficiency, in order to address the shortcomings of the existing technology.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0008] A process for preparing D-chiral inositol includes the following steps:

[0009] A: Inositol dehydrogenase, 2-ketoinositol isomerase and NADH oxidase were expressed separately. The resulting fermented cells were resuspended in dipotassium hydrogen phosphate, disodium hydrogen phosphate or sodium carbonate, and after cell disruption and centrifugation, crude IDH enzyme solution, crude KMI enzyme solution and crude NOX enzyme solution were obtained.

[0010] B: IDH crude enzyme solution, KMI crude enzyme solution and NOX crude enzyme solution were added to the inositol solution for conversion. After conversion, the solution was inactivated at 70℃~90℃ for 10 min and then passed through a ceramic membrane, ultrafiltration membrane, cation exchange resin, decolorizing resin and anion exchange resin to obtain solution I.

[0011] C: Solution I is concentrated by nanofiltration and then further concentrated by heat to one-third of the volume of nanofiltration concentrate. At this point, a large amount of solid precipitates out. Then, the temperature is lowered to 25°C and filtered to obtain solution II and solid II. Solution II is a D-chiral inositol solution.

[0012] D: Solution II was further concentrated by heat until the solid content was 40-50% w / w. Ethanol was added at 50°C and kept at that temperature for 2 hours. Then the temperature was lowered to 25°C at a rate of 5°C / h and filtered to obtain solution III and solid III.

[0013] The specific synthetic route is as follows: .

[0014] Preferably, alcohol dehydrogenase (IDH) and 2-ketoinositol isomerase (KMI) are selected from the GT-P155H mutant and MA-P99R mutant in Chinese patent CN118853609A, respectively, and NADH oxidase (NOX) is selected from the NADH oxidase in Chinese patent CN120758469A.

[0015] Preferably, in step D, after cooling, ethanol is added according to the D-chiral inositol content in the liquid portion of the feed solution until the D-chiral inositol content in the liquid portion reaches more than 90%.

[0016] Preferably, solution III is thermally concentrated to a solid content of 40-50% w / w, ethanol is added at 50°C, the temperature is maintained for 2 hours, and the temperature is lowered to 25°C at a rate of 5°C / h. The solution is then filtered to obtain solution IV and solid IV, respectively.

[0017] Solid IV was dissolved in pure water at 80°C at a ratio of 100% w / v. After dissolution, 2% w / w activated carbon was added for decolorization. Ethanol of equal volume was added at 50°C and kept at this temperature for 2 hours. The temperature was then lowered to 25°C at a rate of 5°C / h. The mixture was filtered to obtain solution V and solid V. Solid V was dried to obtain D-chiral inositol.

[0018] Preferably, solid II is inositol, which is returned to the conversion system for the preparation of inositol solution;

[0019] Solid III is returned to solution I for further steps to recover D-chiral inositol;

[0020] After the alcohol is removed from solution IV, it is returned to solution II in step D;

[0021] Solution V is returned to solution III.

[0022] In step C, solution I is concentrated by nanofiltration to a solid content of 12% w / v and then further concentrated by heat.

[0023] The expression process of inositol dehydrogenase was as follows: a single colony was picked and cultured in a 4 mL LB tube (str 100 μg / mL) at 37℃ and 200 rpm for 10-12 h. The seed culture was then transferred to a shake flask containing 200 mL TB medium (str 100 μg / mL) at an inoculation rate of 1% v / v and cultured at 37℃ and 200 rpm until the bacterial culture reached OD. 600 =0.6-0.8, add 0.2% w / v L-arabinose, and induce expression of the target protein at 16℃ and 200rpm for 20h to obtain inositol dehydrogenase fermentation cells.

[0024] The expression process of 2-ketoinositol isomerase is as follows: A single colony is picked and cultured in a 4 mL LB tube (str 100 μg / mL) at 37℃ and 200 rpm for 10–12 h. The seed culture is then transferred to a shake flask containing 200 mL TB medium (str 100 μg / mL) at a 1% v / v inoculation rate and cultured at 37℃ and 200 rpm until the bacterial culture reaches OD500. 600 =0.6-0.8, add 0.2% w / v L-arabinose, and induce expression of the target protein at 16℃ and 200rpm for 20h to obtain 2-ketoinositol isomerase fermentation cells.

[0025] The expression process of NADH oxidase was as follows: a single colony was picked and cultured in a 4 mL LB tube (Kan 50 μg / mL) at 37℃ and 200 rpm for 10-12 h. The seed culture was then transferred to a shake flask containing 200 mL TB medium (Kan 50 μg / mL) at an inoculation rate of 1% v / v and cultured at 37℃ and 200 rpm until the bacterial culture reached OD. 600 =0.6-0.8, add IPTG to a final concentration of 0.2 mM, and induce the expression of the target protein at 16℃ and 200 rpm for 20 h to obtain NADH oxidase fermentation cells.

[0026] The LB medium (1L) consists of: 5g yeast extract, 10g peptone, and 10g sodium chloride.

[0027] The composition of TB medium (1L) is: 24g yeast extract, 12g peptone, 10g sodium chloride, 12.54g dipotassium hydrogen phosphate, 2.31g potassium dihydrogen phosphate, and 5mL glycerol. It is autoclaved at 121℃ for 20min.

[0028] The conversion system in step B is: inositol 50g / L, IDH 5 OD 600 KMI 5 OD 600 NOX 1 OD 600 In a 1mM NAD+ buffer solution, the mixture was converted at 37°C and 250 rpm for 8 hours.

[0029] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0030] 1. Compared with the conversion process using inositol dehydrogenase (IDH) and 2-ketoinositol isomerase (KMI), the conversion system of this invention uses an IDH:KMI:NOX ratio of 5 OD. 600 5 OD 600 1 OD 600 The conversion rate reached the same level, approximately 22%, which is higher than that of the enzyme in the original synthetic route (IDH: KMI=20 OD). 600 20 OD 600 The usage was reduced by 72.5%. Under the condition that the existing fermentation tank conditions remain unchanged, the conversion rate of each batch of fermentation bacteria can be increased by 3 times, which greatly improves production efficiency.

[0031] 2. By inactivating, removing impurities and desalting the conversion solution, and then concentrating and crystallizing it multiple times, the proportion of D-chiral inositol in the solution is greatly increased, resulting in a significant improvement in the final yield and purity of D-chiral inositol.

[0032] 3. By improving the synthesis process and optimizing the extraction process, the same fermentation equipment increased the yield of D-chiral inositol, and the simplification of the extraction process improved production efficiency, laying a solid foundation for industrial production. Detailed Implementation

[0033] The present invention will be further illustrated below with reference to the embodiments. Example 1

[0034] Fermentation conditions:

[0035] LB medium (1L): 5g yeast extract, 10g peptone, 10g sodium chloride.

[0036] TB medium (1L): 24g yeast extract, 12g peptone, 10g sodium chloride, 12.54g dipotassium hydrogen phosphate, 2.31g potassium dihydrogen phosphate, 5mL glycerol, autoclaved at 121℃ for 20min.

[0037] The expression process of inositol dehydrogenase was as follows: Single colonies of inositol dehydrogenase (selected from the GT-P155H mutant in Chinese patent CN118853609A) were collected in 4 mL LB tubes (str 100 μg / mL) and cultured at 37℃ and 200 rpm for 10-12 h. The seed culture was then transferred to a shake flask containing 200 mL TB medium (str 100 μg / mL) at a 1% v / v inoculation rate and cultured at 37℃ and 200 rpm until the bacterial culture reached OD500. 600 =0.6-0.8, add 0.2% w / v L-arabinose, and induce expression of the target protein at 16℃ and 200rpm for 20h to obtain inositol dehydrogenase fermentation cells.

[0038] The expression process of 2-ketoinositol isomerase is as follows: A single colony of 2-ketoinositol isomerase (selected from the MA-P99R mutant in Chinese patent CN118853609A) is placed in a 4 mL LB tube (str 100 μg / mL) and cultured at 37℃ and 200 rpm for 10-12 h. The seed culture is then transferred to a shake flask containing 200 mL TB medium (str 100 μg / mL) at a 1% v / v inoculation rate and cultured at 37℃ and 200 rpm until the bacterial culture reaches OD500. 600 =0.6-0.8, add 0.2% w / v L-arabinose, and induce expression of the target protein at 16℃ and 200rpm for 20h to obtain 2-ketoinositol isomerase fermentation cells.

[0039] The expression process of NADH oxidase is as follows: A single colony of NADH oxidase (selected from the NADH oxidase in Chinese patent CN120758469A) is picked and placed in a 4 mL LB tube (Kan 50 μg / mL) and cultured at 37℃ and 200 rpm for 10-12 h. The seed culture is then transferred to a shake flask containing 200 mL TB medium (Kan 50 μg / mL) at a 1% v / v inoculation rate and cultured at 37℃ and 200 rpm until the bacterial culture reaches OD500. 600 =0.6-0.8, add IPTG to a final concentration of 0.2 mM, and induce the expression of the target protein at 16℃ and 200 rpm for 20 h to obtain NADH oxidase fermentation cells.

[0040] Preparation of crude enzyme solution:

[0041] Inositol dehydrogenase fermentation cells, 2-ketoinositol isomerase fermentation cells, and NADH oxidase fermentation cells were resuspended in 50 mM dipotassium hydrogen phosphate, disodium hydrogen phosphate, or sodium carbonate (pH 9.0), respectively. After cell wall disruption and centrifugation, crude enzyme solutions were obtained, namely IDH crude enzyme solution, KMI crude enzyme solution, and NOX crude enzyme solution.

[0042] Transformation system:

[0043] Inositol 50g / L, IDH 5 OD 600 KMI 5 OD 600 NOX 1 OD 600 The product was converted at 37°C and 250 rpm for 8 hours in 1 mM NAD+ and 50 mM buffer salt to obtain a conversion solution containing 11.2 g of product and 38.8 g of substrate, with a conversion rate of 22.4%. Example 2

[0044] An extraction process for D-chiral inositol includes the following steps:

[0045] The conversion solution from Example 1 was inactivated at 70-90°C for 10 minutes and then passed through a ceramic membrane, an ultrafiltration membrane, a cation exchange resin, a decolorizing resin, and an anion exchange resin. The resulting conversion solution after impurity removal and desalting was Solution I.

[0046] Solution I was concentrated by nanofiltration to a solid content of 12% w / w, and then further concentrated by heat to one-third of its volume. At this point, a large amount of solid precipitated out. The solution was then cooled to 25°C and filtered to obtain solution II and solid II. Solution II is a D-chiral inositol solution, and solid II is inositol. Both can be added to the conversion system.

[0047] Solution II is further concentrated by heat until the solid content is 40-50% w / w. While stirring, one volume of ethanol is slowly added at 50°C, and this condition is maintained for approximately 2 hours. The solution is then slowly cooled to 25°C at a rate of 5°C / h. A sample is taken and filtered to determine the D-chiral inositol content. If the D-chiral inositol content reaches 90% or more, this crystallization step can be terminated. If the content is less than 90%, ethanol can be added in small amounts multiple times until the content reaches 90% or more. The solution is then filtered to obtain solution III and solid III. Solution III is used for subsequent steps, while solid III can be added to solution I for further D-chiral inositol recovery.

[0048] Solution III was further concentrated by heat until the solid content reached 40-50% w / w. Then, 1 volume of ethanol was slowly added while stirring at 50°C, and this condition was maintained for about 2 hours. The solution was then slowly cooled to 25°C at a rate of 5°C / h. The solution was filtered to obtain solution IV and solid IV. Solid IV was used for subsequent steps, while solution IV, after being de-ethanolified, was combined with solution II for further steps.

[0049] Solid IV was dissolved in pure water at 80°C at a ratio of 100% w / v. After dissolution, 2% w / w activated carbon was added for decolorization. Ethanol (1 volume) was slowly added while stirring at 50°C and maintained at this temperature for about 2 hours. The temperature was then slowly lowered to 25°C at a rate of 5°C / h. The mixture was filtered to obtain solution V and solid V. Solid V, after drying, became D-chiral inositol. Solution V and solution III were combined for further steps. Comparative Example 1

[0050] The preparation of inositol dehydrogenase fermentation cells and 2-ketoinositol isomerase fermentation cells was exactly the same as in Example 1, except that NOX and OD were omitted from the transformation system. 600 The rest was exactly the same as in Example 1, except that the amount of product in the conversion solution was 5.1 g, the amount of substrate was 44.9 g, and the conversion rate was 10.2%. Comparative Example 2

[0051] The preparation of inositol dehydrogenase fermentation cells and 2-ketoinositol isomerase fermentation cells was exactly the same as in Example 1, except that the transformation system was modified to: inositol 50 g / L, IDH 20 OD 600 KMI 20 OD 600 The product was converted at 1 mM NAD+ and 50 mM buffer salt at 37°C and 250 rpm for 8 h. The amount of product in the conversion solution was 10.9 g, the amount of substrate was 38.5 g, and the conversion rate was 21.8%.

[0052] In Example 1, the conversion rate of D-chiral inositol was increased by 120% compared to Comparative Example 1 and by 2.8% compared to Comparative Example 2. The total amount of enzyme used in Example 1 was reduced by 72.5% compared to Comparative Example 2.

[0053] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A process for preparing D-chiral inositol, characterized in that... Includes the following steps: A: Inositol dehydrogenase, 2-ketoinositol isomerase and NADH oxidase were expressed separately. The resulting fermented cells were resuspended in dipotassium hydrogen phosphate, disodium hydrogen phosphate or sodium carbonate, and after cell disruption and centrifugation, crude IDH enzyme solution, crude KMI enzyme solution and crude NOX enzyme solution were obtained. B: IDH crude enzyme solution, KMI crude enzyme solution and NOX crude enzyme solution are added to the inositol solution for conversion. After conversion, the solution is inactivated and then passed through a ceramic membrane, ultrafiltration membrane, cation exchange resin, decolorizing resin and anion exchange resin to obtain solution I. C: Solution I was concentrated by nanofiltration and then further concentrated by heat to one-third of the volume of the nanofiltration concentrate. It was then cooled to 25°C and filtered to obtain solution II and solid II. Solution II is a D-chiral inositol solution. D: Solution II was further concentrated by heat until the solid content was 40-50% w / w. Ethanol was added at 50°C and kept at that temperature for 2 hours. Then the temperature was lowered to 25°C at a rate of 5°C / h and filtered to obtain solution III and solid III.

2. The process for preparing D-chiral inositol as described in claim 1, characterized in that: After cooling in step D, ethanol is added according to the D-chiral inositol content in the liquid portion of the feed solution until the D-chiral inositol content in the liquid portion reaches more than 90%.

3. The process for preparing D-chiral inositol as described in claim 1, characterized in that: Solution III was thermally concentrated to a solid content of 40-50% w / w, and 1 volume of ethanol was added at 50°C. The mixture was kept at this temperature for 2 hours and then cooled to 25°C at a rate of 5°C / h. The mixture was then filtered to obtain solution IV and solid IV, respectively. Solid IV was dissolved in pure water at 80°C at a ratio of 100% w / v. After dissolution, 2% w / w activated carbon was added for decolorization. Ethanol of equal volume was added at 50°C and kept at this temperature for 2 hours. The temperature was then lowered to 25°C at a rate of 5°C / h. The mixture was filtered to obtain solution V and solid V. Solid V was dried to obtain D-chiral inositol.

4. The process for preparing D-chiral inositol as described in claim 3, characterized in that: Solid II is inositol, which is returned to the conversion system for the preparation of inositol solution; Solid III is returned to solution I for further steps to recover D-chiral inositol; After the alcohol is removed from solution IV, it is returned to solution II in step D; Solution V is returned to solution III.

5. The process for preparing D-chiral inositol as described in claim 1, characterized in that: In step C, solution I is concentrated by nanofiltration to a solid content of 12% w / v and then further concentrated by heat.

6. The process for preparing D-chiral inositol as described in claim 1, characterized in that: The expression process of inositol dehydrogenase was as follows: a single colony was picked and cultured in an LB tube at 37°C and 200 rpm for 10-12 h. The seed culture was then transferred to a shake flask containing TB medium at an inoculation rate of 1% v / v and cultured at 37°C and 200 rpm until the bacterial culture reached OD. 600 =0.6-0.8, add 0.2% w / v L-arabinose, and induce expression of the target protein at 16℃ and 200rpm for 20h to obtain inositol dehydrogenase fermentation cells.

7. The process for preparing D-chiral inositol as described in claim 1, characterized in that: The expression process of 2-ketoinositol isomerase is as follows: Single colonies are picked and cultured in LB tubes at 37°C and 200 rpm for 10-12 h. The seed culture is then transferred to a shake flask containing TB medium at an inoculation rate of 1% v / v and cultured at 37°C and 200 rpm until the bacterial culture reaches OD. 600 =0.6-0.8, add 0.2% w / v L-arabinose, and induce expression of the target protein at 16℃ and 200rpm for 20h to obtain 2-ketoinositol isomerase fermentation cells.

8. The process for preparing D-chiral inositol as described in claim 1, characterized in that: The expression process of NADH oxidase is as follows: single colonies are picked and cultured in LB tubes at 37℃ and 200 rpm for 10-12 h. The seed culture is then transferred to a shake flask containing TB medium at an inoculation rate of 1% v / v and cultured at 37℃ and 200 rpm until the bacterial culture reaches OD. 600 =0.6-0.8, add IPTG to a final concentration of 0.2 mM, and induce the expression of the target protein at 16℃ and 200 rpm for 20 h to obtain NADH oxidase fermentation cells.

9. The process for preparing D-chiral inositol as described in claim 1, characterized in that, The conversion system in step B is: inositol 50g / L, IDH 5 OD 600 KMI 5 OD 600 NOX 1 OD 600 In a 1mM and 50mM buffer of NAD+, the conversion was carried out at 37°C and 250rpm for 8 hours.