Method for producing D-glucuronic acid and process for producing glucuronolactone

Abstract

The present invention discloses a method for producing D-glucuronic acid by biotransformation and a method for producing glucuronolactone using D-glucuronic acid, and relates to the technical field of biological production. The method for producing D-glucuronic acid by biotransformation described above includes the steps of culturing a recombinant engineering bacterium to obtain a seed solution, inoculating the seed solution into a fermenter medium for fermentation culture, obtaining a fermentation broth through feed control and induction control, centrifuging or membrane filtering the fermentation broth to obtain wet bacterial cells, and adding the wet bacterial cells to a reaction solution for conversion to obtain D-glucuronic acid. Further, the method for producing glucuronolactone using the above-mentioned D-glucuronic acid includes further adding concentrated phosphoric acid to a D-glucuronic acid solution, performing an esterification reaction at a reaction temperature of 40°C to 80°C while stirring, and crystallizing to obtain a crude product of glucuronolactone. When the fermentation process of the present invention is adopted to produce D-glucuronic acid, the conversion rate of inositol can reach 95% or more, the content of the obtained D-glucuronic acid is greater than 76 g / L, and inositol oxidase can be repeatedly used. By adopting the production process of glucuronolactone of the present invention to produce glucuronolactone, the total time from the reaction to the completion of crystallization is less than 6 h, significantly reducing the reaction crystallization time, shortening the production cycle, and improving the production efficiency.

Description

[Technical Field]

[0001] This application claims priority to the Chinese patent filed with the China National Intellectual Property Office on February 28, 2023, with application number 202310177817.5, titled "Process for Producing Glucuronolactone," and to the Chinese patent filed with the China National Intellectual Property Office on March 22, 2023, with application number 202310283516.0, titled "Method for Producing D-Glucuronic Acid by Biofermentation," the contents of which are incorporated into this application by reference.

[0002] <Technical field> The present invention relates to the field of biological production technology, and more particularly to a method for producing D-glucuronic acid by biofermentation and a process for producing glucuronolactone using D-glucuronic acid. [Background technology]

[0005] Conventional methods for producing D-glucuronic acid mainly involve polysaccharide hydrolysis and chemical oxidation catalysts. Polysaccharide hydrolysis refers to the process of obtaining D-glucuronic acid by hydrolyzing uronic acid-containing polysaccharides. For example, D-glucuronic acid can be obtained from the water-soluble portion of hemicellulose in sunflower membranes, or by hydrolyzing cotton and cellulose with a base, extracting holocellulose with hot water, and oxidizing the cellulose with a chlorine aqueous solution. However, in polysaccharide hydrolysis, the glycosidic bonds linked to uronic acid are generally highly stable and difficult to hydrolyze. Therefore, it is necessary to use strong acids and strong bases in the hydrolysis process. Under these strong acid and base conditions, the D-glucuronic acid product is often decomposed, resulting in low oxidation selectivity, a large amount of by-products, and low product yield, making it impossible to meet production needs. Chemical oxidation refers to the production of D-glucuronic acid by oxidizing sugars and their derivatives with inorganic reagents. The most widespread application is the nitric acid oxidation method, which involves first oxidizing starch with concentrated nitric acid to obtain a crude starch oxidation solution, then heating and pressurizing the crude starch oxidation solution under acidic conditions to hydrolyze it, concentrating the resulting hydrolysate under reduced pressure, adding acetic acid for esterification, and finally freeze-crystallizing it to produce glucuronolactone. However, this method has drawbacks such as a relatively low overall yield (about 10%), high energy consumption, low selectivity, and serious environmental pollution, and therefore cannot meet production needs.

[0006] With increasing public awareness of microorganisms, research into producing D-glucuronic acid using biocatalysis is growing daily. Inositol oxidase is an enzyme that promotes the conversion of inositol to D-glucuronic acid. However, due to the low stability of this enzyme, incomplete conversion can occur during the conversion process, often leading to problems such as low product concentration and conversion rate when D-glucuronic acid is produced using inositol oxidase.

[0007] In addition, the main production method of glucuronolactone, which is a derivative of D-glucuronic acid, is to add starch to nitric acid with a content of about 80% (V / V) and oxidize it to obtain a starch oxidation solution. The starch oxidation solution is heated and pressurized under acidic conditions for hydrolysis to obtain a hydrolysis solution whose main component is D-glucuronic acid. When the hydrolysis solution is concentrated under reduced pressure until the Baumé degree reaches 44-49, a composite acid reagent composed of phosphoric acid and sulfuric acid is added to carry out an esterification reaction. The esterification temperature is 10-35°C, and it is left standing for 24 hours or more. After standing to complete the esterification, alcohol is added and stirred for crystallization. The temperature is decreased to 10-15°C in 0-24 hours, decreased to below 0°C in 24-48 hours, and decreased to below 8°C in 48-60 hours. In the above technical solution, the total time of cooling crystallization is generally 70-80 hours, the process steps are complicated, the production time is long, the efficiency is low, and the utilization rate of raw materials is low. Moreover, since two kinds of acids, sulfuric acid and phosphoric acid, are added, a large amount of heat is released by the mixing of the two kinds of acids, and there is a certain safety risk. On the other hand, the factors that cannot control the temperature of the reaction system increase, and it is necessary to strictly control the reaction temperature below 35°C during the reaction process. Therefore, the glucuronolactone crystal particles obtained by the reaction are fine and difficult to filter.

Summary of the Invention

Problems to be Solved by the Invention

[0008] In view of this, the object of the present invention is to cause the phenomenon that the conversion is incomplete during the conversion process due to the low stability of inositol oxidase in this field, and finally cause the problems that the concentration and conversion rate of the product when producing D-glucuronic acid by inositol oxidase are low, and the production process of glucuronolactone in the prior art is complicated, the production time is long, the efficiency is low, and the crystal particles of glucuronolactone are fine and difficult to filter. The purpose is to provide a method for producing D-glucuronic acid and a method for producing glucuronolactone using D-glucuronic acid to overcome these problems.

Means for Solving the Problems

[0009] To achieve the above object, in a first aspect, the present invention provides the following technical solution. The present invention provides a method for producing D-glucuronic acid, (1) culturing a recombinant engineering bacterium to obtain a seed solution; (2) inoculating the seed solution into a fermenter medium and performing fermentation culture to obtain a fermentation broth; (3) centrifuging or membrane filtering the fermentation broth and collecting to obtain wet bacterial cells; (4) adding the wet bacterial cells to a reaction solution for conversion to obtain D-glucuronic acid, where step (2) includes performing feed control and induction control after inoculating the seed solution into the fermenter medium and culturing.

[0010] The method for producing D-glucuronic acid by bioconversion provided by the present invention includes culturing a recombinant engineering bacterium to obtain a seed solution, inoculating the seed solution into a fermenter medium and performing fermentation culture to obtain a fermentation broth through feed control and induction control, centrifuging or membrane filtering the fermentation broth to obtain wet bacterial cells, and adding the wet bacterial cells to a reaction solution for conversion to obtain D-glucuronic acid. By controlling the parameters of the fermentation process and the conversion process, especially by controlling the components of the medium (seed tank medium and fermenter medium) and adopting feed control in the fermentation process, the excellent expression of the engineering bacterium is promoted, highly active, highly stable and high-content inositol oxidase in the wet bacterial cells is obtained, the conversion rate of inositol in the conversion process is further promoted, and the concentration of D-glucuronic acid in the product is improved. When the method for producing D-glucuronic acid of the present invention is adopted, the conversion rate is improved, the inositol conversion rate can reach 95% or more, the content of the obtained D-glucuronic acid is 76 g / L, the difficulty of extraction is reduced, and at the same time, inositol oxidase can be reused repeatedly to reduce the production cost.

[0011] In a second aspect, the present invention provides a production process of glucuronolactone, The process includes adding concentrated phosphoric acid to a D-glucuronic acid solution, stirring, and carrying out an esterification reaction at a reaction temperature of 40°C to 80°C to crystallize and obtain the crude product glucuronolactone.

[0012] Compared to conventional techniques, the glucuronolactone production process of the present invention uses concentrated phosphoric acid instead of the mixed acid of phosphoric acid and sulfuric acid used in conventional techniques, and controls the concentration of phosphoric acid and the reaction temperature to produce glucuronolactone by esterifying it with a D-glucuronic acid solution while stirring. When glucuronolactone is produced using the technology of the present invention, the total time from reaction to completion of crystallization is less than 6 hours, significantly reducing the reaction crystallization time, shortening the production cycle, and improving production efficiency. Furthermore, in the esterification reaction system of the present invention, only concentrated phosphoric acid is used, the temperature of the reaction system can be controlled, and the resulting glucuronolactone has advantages such as large crystal particles, ease of filtration, and high product yield. [Brief explanation of the drawing]

[0013] [Figure 1] This is a liquid chromatography diagram of the glucuronolactone product produced in Example 4 of the present invention. [Modes for carrying out the invention]

[0014] To further clarify the technical problems, technical solutions, and beneficial effects that this invention aims to solve, the invention will be described in more detail below in combination with specific examples. It should be understood that the specific examples described herein are merely for interpretation purposes and do not limit the invention.

[0015] The present invention provides a method for producing D-glucuronic acid. (1) A step of culturing recombinant engineered bacteria to obtain seed solution, (2) The step of inoculating the seed liquid into a fermentation vessel culture medium and performing fermentation culture to obtain a fermented liquid, (3) The fermentation liquid is centrifuged or filtered through a membrane and collected to obtain wet microbial cells, (4) The step of adding the wet bacterial cells to the reaction solution and converting them to obtain D-glucuronic acid.

[0016] The present invention relates to culturing recombinant engineered bacteria to obtain seed solution. In the present invention, the cultivation of the recombinant engineered bacteria comprises two steps: primary seed culture and secondary seed culture. The primary seed culture involves inoculating the recombinant engineered bacteria into LB medium and culturing them for 5-6 hours under conditions of 200-240 rpm and 34°C-40°C. The recombinant engineered bacteria in the present invention are purchased from an external source, the Institute of Microbiology, Chinese Academy of Sciences, and the bacteria have already been patented and approved, with application number CN201710790108.9. It should be understood that in the present invention, the substance that essentially catalytically converts inositol to D-glucuronic acid is not the recombinant engineered bacteria themselves, but rather inositol oxidase produced by the expression of the functional protein of the engineered bacteria. In the present invention, there are no special requirements for LB medium; LB medium is a known medium in the art, and any commercially available LB medium can be selected in this invention. The present invention does not require any special container / container for primary seed culture, and any corresponding container or container capable of achieving shaking culture can be used, preferably using a shaking bottle. The present invention does not require any special shaking method, and preferably uses a shaker or shaker, more preferably a shaker. The shaking culture speed in the present invention is preferably 200-240 rpm, more preferably 220 rpm. The culture temperature in the present invention is preferably 34-40°C, more preferably 37°C. The culture time in the present invention is preferably 5-6 hours, more preferably 5 hours. The present invention employs a combined shaking bottle + shaker culture method, which can satisfy the need to block cellular respiration by increasing the oxygen in the solution.

[0017] Furthermore, after obtaining the primary seed culture solution by combining the above examples, the present invention inoculates the above primary seed culture solution into the seed tank medium to perform secondary seed culture, preferably with an OD 600 = 2 - 3, more preferably an OD 600 = 2.5 for inoculation. The conditions for the secondary seed culture are as follows: temperature: 34 - 40°C, more preferably 37°C; air volume: 0.4 - 0.6 m 3 / h, more preferably 0.5 m 3 / h; rotation speed: 280 - 320 rpm, more preferably 300 rpm; pressure: 0.01 - 0.03 MPa, more preferably 0.02 MPa; pH: adjusted to pH 7.0 ± 0.1 with ammonia water, more preferably adjusted to pH 7.0 with ammonia water; dissolved oxygen content: 20 - 30%, more preferably 25%. The end point of the secondary seed culture process in the present invention is determined by the OD 600 value. Preferably, the secondary seed culture is completed when the OD 600 value reaches 2 - 3, and more preferably, the secondary seed culture is completed when the OD 600 = 2. Specifically, in the present invention, 4 hours after the start of the secondary seed culture, sampling is performed every hour to detect the OD 600 , and the OD 600 value of the system is monitored in real time. The secondary seed culture is a process of amplifying and rapidly growing the bacterial cells to meet the needs of the subsequent fermenter culture. Therefore, it should be understood that all the above selections of the culture conditions are for better amplifying and growing the bacterial cells.

[0018] Furthermore, combining the above examples, the specific composition of the seed tank culture medium in the present invention is preferably 1 wt% to 2 wt% glucose, 1 wt% to 2 wt% potassium dihydrogen phosphate, 0.05 wt% to 0.08 wt% magnesium sulfate, 0.1 wt% to 0.2 wt% citric acid, 0.4 wt% to 0.6 wt% ammonium sulfate, 1000 to 1500 mg / L trace elements, and 0.05 to 0.15 ml / L antifoaming agent. The trace elements in the seed tank culture medium in the present invention preferably consist of 20-30 mg / L of CoCl2.6H2O, 100-200 mg / L of MnSO4.4H2O, 10-20 mg / L of CuCl2.2H2O, 20-40 mg / L of H3BO3, 20-30 mg / L of Na2MoO4.2H2O, 100-150 mg / L of Zn(CH3COO)2.2H2O, and 500-1500 mg / L of F The trace element is e(III)citrate, and more preferably, the trace element is composed of 25 mg / L CoCl2.6H2O, 150 mg / L MnSO4.4H2O, 15 mg / L CuCl2.2H2O, 30 mg / L H3BO3, 25 mg / L Na2MoO4.2H2O, 130 mg / L Zn(CH3COO)2.2H2O, and 1000 mg / L Fe(III)citrate. The trace elements in the seed tank culture medium in this invention have two functions: one is as a component of the microbial cells, and the other is as a component of the enzyme active group or to maintain the enzyme activity. It should be understood that the addition of trace elements in this invention significantly improves the activity and stability of inositol oxidase produced by recombinant engineered bacteria, providing a good foundation for subsequent biofermentation and conversion.

[0019] Furthermore, by combining the above embodiments, there are no special requirements for the type of defoaming agent in the present invention, and commercially available liquid defoaming agents are preferably used. The amount of defoaming agent to be added in the present invention is preferably 0.05 to 0.15 ml / L, and more preferably 0.1 ml / L. The amount of defoaming agent to be added in the present invention can be selectively added depending on the specific situation. For example, if there is too much foam on the liquid surface, the defoaming agent can be added manually as needed. It should be understood that adding too much defoaming agent is inappropriate, and it is appropriate to retain a small amount of foam, otherwise it is likely to cause insufficient oxygen supply.

[0020] After obtaining seed liquid through secondary seed culture, the present invention involves inoculating the seed liquid into a fermentation tank culture medium and performing fermentation culture to obtain a fermented liquid. In the present invention, the temperature of the fermentation culture is preferably 34°C to 40°C, more preferably 37°C, and the airflow is preferably 1.2 to 1.8 m³. 3 / h, more preferably 1.5m 3 The rotation speed is preferably 180-220 rpm, more preferably 200 rpm, the pressure is preferably 0.01-0.03 MPa, more preferably 0.02 MPa, the pH is preferably adjusted to pH=7.0±0.1 with ammonia water, more preferably to pH7.0 with ammonia water, and the dissolved oxygen content is preferably 20%-30%, more preferably 25%. In the present invention, the fermentation reaction time is preferably 36-40 h, more preferably 38 h, and the index for evaluating fermentation is generally OD. 600 This value, when maintaining stability, represents the completion or imminent completion of fermentation, and it should be understood that the 36-40h mentioned above refers to the time for complete fermentation obtained based on production experience. Fermentation culture is a highly developed expression of microbial cells carried out based on the aforementioned seed culture, and in this development, the expression of functional proteins in recombinant engineered bacteria is utilized to produce inositol oxidase, which is then applied to the subsequent inositol conversion process.

[0021] Furthermore, combining the above examples, the specific composition of the fermentation tank culture medium in the present invention is preferably 1 wt% to 2 wt% glucose, 1 wt% to 2 wt% potassium dihydrogen phosphate, 0.05 wt% to 0.08 wt% magnesium sulfate, 0.1 wt% to 0.2 wt% citric acid, 0.4 wt% to 0.6 wt% ammonium sulfate, 1000 to 1500 mg / L trace elements, and 0.05 to 0.15 ml / L antifoaming agent. Trace elements in the fermentation tank culture medium in the present invention The component preferably contains 20-30 mg / L of CoCl 2 .6H 2 O, 100-200 mg / L MnSO4 4 .4H 2 O, 10-20 mg / L CuCl 2 .2H 2 O, H 20-40 mg / L 3 BO 3 , 20-30 mg / L Na 2 MoO 4 .2H 2 O, 100-150 mg / L Zn(CH) 3 COO) 2 .2H 2 O, Fe(III) citrate in a concentration of 500-1500 mg / L, more preferably the trace element is CoCl with a concentration of 25 mg / L. 2 .6H 2 O, 150 mg / L MnSO 4 .4H 2 O, 15 mg / L CuCl 2 .2H 2 O, 30 mg / L H 3 BO 3 , 25 mg / L Na 2 MoO 4 .2H 2 O, 130 mg / L Zn(CH) 3 COO) 2 .2H 2 This is 0, 1000 mg / L Fe(III)citrate. The role of trace elements in the fermentation culture medium in this invention is twofold: one is as a component of the microbial cells, and the other is as a component of the enzyme active group or to maintain the enzyme's activity. It should be understood that the addition of trace elements in this invention, particularly the addition of iron (other trace elements acting as auxiliary groups or activators), significantly improves the activity and stability of inositol oxidase produced by recombinant engineered microorganisms, providing a good foundation for subsequent biofermentation and conversion.

[0023] Furthermore, combining the above embodiments, the present invention further includes controlling the feed rate by adding a feed medium to the reaction system at a constant feed rate after the fermentation reaction has been carried out for a certain period of time. Preferably, the time of feed control is 10 to 14 hours, more preferably 12 hours, after the start of the fermentation reaction. The feed medium preferably contains 500 to 700 g / L of glucose, 1 to 3 g / L of magnesium sulfate, 8 to 12 g / L of nitrogen-containing compounds, and 1000 to 1500 mg / L of trace elements. The trace elements in the feed medium in the present invention are preferably 20-30 mg / L of CoCl2.6H2O, 100-200 mg / L of MnSO4.4H2O, 10-20 mg / L of CuCl2.2H2O, 20-40 mg / L of H3BO3, 20-30 mg / L of Na2MoO4.2H2O, 100-150 mg / L of Zn(CH3COO)2.2H2O, and 500-1500 mg / L of Fe(III)citrate. The nitrogen-containing compound in the feed medium in the present invention is preferably peptone, yeast powder, corn steep liquor powder, and more preferably yeast powder. The main difference between the feed medium in the present invention and the fermentation tank medium described above is its high glucose concentration. This is because the main function of the feed medium is to replenish nutrients and essential trace elements to the microbial cells in the system. In particular, it should be understood that in the present invention, adding nitrogen-containing compounds significantly improves the stability of inositol oxidase produced by the microbial cells and promotes the biological conversion of inositol.

[0024] Furthermore, by combining the above embodiments, the present invention controls induction by adding an inducer during the fermentation reaction process. In the present invention, the timing of adding the inducer is preferably OD 600 When the value reaches 70-80, more preferably OD 600 This occurs when the value is 75. The inducing agent in this invention is preferably a monosaccharide, more preferably arabinose. It should be understood that the action of the inducing agent added in this invention is to initiate the expression of protein genes.

[0025] Furthermore, by combining the above embodiments, the feed control in the present invention is carried out in stages, specifically, within 0 to 3 hours from the start of feeding, the feed rate is preferably controlled to 600 to 700 g / h, more preferably to 650 g / h; from 3 hours from the start of feeding until the addition of the inducer, the feed rate is preferably controlled to 900 to 1100 g / h, more preferably to 1000 g / h; and after the addition of the inducer, the feed rate is preferably controlled to 700 to 800 g / h, more preferably to 750 g / h. However, experiments have verified that when the feed rate is controlled to 650 g / h within 0 to 3 hours from the start of feed control, controlled to 1000 g / h from 3 hours from the start of feeding until the addition of the inducer, and controlled to 750 g / h after the addition of the inducer, a high production of inositol oxidase and stable technical effects can be achieved. The time starting point for feed control is generally understood to be that when continuous expression of bacterial cells and nutrient deficiency due to medium consumption cause a significant decrease in the enzyme production and yield of the product, feed control of the reaction system can ensure timely replenishment of nutrients to the bacterial cells and guarantee efficient production of inositol oxidase. Combined with the aforementioned improvement of enzyme activity by trace elements in the fermenter medium, this can achieve an overall improvement in the enzyme activity, stability, and concentration of inositol oxidase, providing a good foundation for inositol conversion.

[0026] After obtaining a fermentation liquid by fermentation culture, the present invention obtained moist microbial cells by centrifuging the fermentation liquid. The centrifugal separation process of the present invention preferably employs a centrifuge, with a rotation speed of preferably 14,000 to 18,000 rpm, more preferably 16,000 rpm, and a centrifugal separation time of preferably 15 to 25 min, more preferably 20 min.

[0027] In another embodiment, the present invention may further involve membrane filtration of the fermentation liquid. The present invention does not require any special properties for the membrane, as long as it can achieve the corresponding filtration blocking function, and preferably it is a ceramic membrane, and the pore size of the membrane is preferably controlled to 50 to 100 nm, more preferably to 80 nm.

[0028] After obtaining the aforementioned wet bacterial cells, the present invention adds the wet bacterial cells to a reaction solution and inverts them to obtain D-glucuronic acid. The reaction solution is a reaction solution containing inositol, preferably containing inositol in a mass fraction of 4 to 7 wt% and boric acid in a concentration of 40 to 60 mM, and more preferably containing inositol in a mass fraction of 5 wt% and boric acid in a concentration of 50 mM.

[0029] In another embodiment, the reaction solution preferably comprises inositol in a mass fraction of 4-7 wt%, phosphate in a concentration of 20-40 mM, and Fe in a concentration of 2-4 mM. 2+ It contains, more preferably, inositol in a mass fraction of 5 wt%, phosphate in a concentration of 30 mM, and Fe in a concentration of 3 mM. 2+ It contains, and the phosphate is preferably potassium dihydrogen phosphate.

[0030] Furthermore, by combining the above examples, the amount of wet bacterial cells added in the present invention is preferably 25 to 35 g / L, more preferably 30 g / L.

[0031] Furthermore, combining the above embodiments, the present invention involves adding wet bacterial cells to the reaction solution and then performing inversion. During the inversion process, the pH is preferably adjusted to 7-9, more preferably to 8, the dissolved oxygen level is preferably controlled to 40% or more, more preferably to 50%, and the inversion time is preferably 6-8 hours, more preferably 7 hours.

[0032] Furthermore, combining the above embodiments, the present invention further includes continuing to add a certain amount of inositol to the reaction system for a certain period of time after the start of the conversion reaction. In the present invention, the time at which inositol is added is preferably 1.5 to 2 hours after the start of conversion, more preferably 2 hours after the start of conversion. In the present invention, the mass fraction of the added inositol is preferably 4 to 6 wt%, more preferably 4.5 wt%. The inositol is preferably in the form of an aqueous solution, the mass concentration of the aqueous inositol solution is preferably 12 to 15 wt%, more preferably 13 wt%, the method of adding the aqueous inositol solution is preferably flow addition, and the addition time is preferably 1.5 to 2.5 hours, more preferably 2 hours.

[0033] Furthermore, combining the above embodiments, the present invention further includes filtering and collecting the inversion solution after the conversion is complete to obtain bacterial cells for repeated use. In the present invention, the filtration is preferably performed using a ceramic membrane, and the pore size of the ceramic membrane is preferably 50 to 100 nm, more preferably 80 nm. In the present invention, repeated use may involve filtering to obtain wet bacterial cells, and then adding the wet bacterial cells back to the inositol reaction solution to directly perform inositol conversion.

[0034] To better illustrate the proposed D-glucuronic acid production technology of the present invention, the present invention further provides the following specific examples. It should be understood that the raw materials used in the following examples are all commercially available unless otherwise specified.

[0035] Example 1 This embodiment provides a method for producing D-glucuronic acid, the steps being S1 to S6 below.

[0036] S1. A single colony of recombinant engineered fungus is inoculated into 300 ml of LB medium and cultured with shaking for 5.5 hours at 37°C and 220 rpm to obtain a primary seed solution. S2.OD 600When the ratio reaches 2.2, the primary seed solution is inoculated into 30 L of seed tank medium and cultured, OD 600 When the result is 2.12, the secondary seed culture is completed, the inoculation amount is 2%, and seed liquid is obtained. The composition of the 30L seed tank culture medium is: The mixture consists of 1 wt% glucose, 1 wt% potassium dihydrogen phosphate, 0.05 wt% magnesium sulfate, 0.15 wt% citric acid, 0.5 wt% ammonium sulfate, 20 mg / L CoCl₂₆H₂O, 120 mg / L MnSO₄₆H₂O, 15 mg / L CuCl₂₂H₂O, 30 mg / L H₃BO₃, 25 mg / L Na₂MoO₄₂H₂O, 120 mg / L Zn(CH₃COO)₂₂H₂O, 1 g / L Fe(III) citrate, and 0.1 ml / L of antifoaming agent, with the remainder being water. The conditions for secondary seed culture are: Temperature: 37℃, Air volume: 0.5m 3 The parameters are: / h, rotation speed: 300 rpm, pressure: 0.02 MPa, pH: adjusted to pH 7 with ammonia water, dissolved oxygen content: 20%. S3. The seed solution was inoculated into 100 L of fermentation tank culture medium and fermented for 36 hours to obtain the fermented liquid, with an inoculation amount of 10%. The composition of the culture medium in a 100L fermentation tank is: The solution consists of 1 wt% glucose, 1 wt% potassium dihydrogen phosphate, 0.06 wt% magnesium sulfate, 0.2 wt% citric acid, 0.5 wt% ammonium sulfate, 20 mg / L CoCl₂₆H₂O, 120 mg / L MnSO₄₆H₂O, 12 mg / L CuCl₂₂H₂O, 25 mg / L H₃BO₃, 25 mg / L Na₂MoO₄₂H₂O, 130 mg / L Zn(CH₃COO)₂₂H₂O, 0.8 g / L Fe(III) citrate, and 0.1 ml / L of antifoaming agent, with the remainder being water. The conditions for fermentation culture are: Temperature: 37℃, Air volume: 1.5m 3 The parameters are: / h, rotation speed: 200 rpm, pressure: 0.02 MPa, pH: adjusted to pH 7 with ammonia water, dissolved oxygen content: 25%, S4. Add feed medium to the reaction system in step S3 to control the feed rate, OD 600When the value reaches 75, arabinose (an inducer) is added to induce further growth. The composition of the feed medium is, by mass per liter of feed medium, The solution consists of 600 g / L glucose, 2 g / L magnesium sulfate, 10 g / L yeast powder, 25 mg / L CoCl2.6H2O, 150 mg / L MnSO4.4H2O, 15 mg / L CuCl2.2H2O, 30 mg / L H3BO3, 25 mg / L Na2MoO4.2H2O, 120 mg / L Zn(CH3COO)2.2H2O, and 1000 mg / L Fe(III)citrate, with the remainder being water. After culturing in the culture tank for approximately 12 hours, the dissolved oxygen level increased significantly, the pH rose, and feeding was initiated. The feed rate was controlled as follows: within 0-3 hours of the start of feeding, the feed rate was controlled to 700 g / h; from 3 hours after the start of feeding until the addition of the inducer, the feed rate was controlled to 900 g / h; and after the addition of the inducer, the feed rate was controlled to 800 g / h to control the OD of the fermentation liquid. 600 We obtained = 150.3, S5. The fermentation liquid is centrifuged at 18,000 rpm for 25 minutes to obtain wet microbial cells. The wet bacterial cells at a concentration of 6.25 g / L are added to a reaction solution containing 5 wt% inositol by mass and 40 mM boric acid. D-glucuronic acid was obtained by inversion for 6 hours under conditions of pH=8 and dissolved oxygen content of 40%.

[0037] Following the steps described above, the molar conversion rate of inositol was over 98.4%, the content of the obtained D-glucuronic acid was 53.0 g / L, and the inositol oxidase was successfully recycled.

[0038] Example 2 This embodiment provides a method for producing D-glucuronic acid, the steps being S1 to S8 below.

[0039] S1. A single colony of recombinant engineered fungus is inoculated into 300 ml of LB medium and cultured with shaking at 37°C and 200 rpm for 5.5 hours to obtain a primary seed solution. S2.OD600 When the ratio reaches 2.26, the primary seed solution is inoculated into 30 L of seed tank culture medium and cultured. 600 When the result is 2.12, the secondary seed culture is completed, the inoculation amount is 2%, and seed liquid is obtained. The composition of the 30L seed tank culture medium is: The mixture consists of 2 wt% glucose, 2 wt% potassium dihydrogen phosphate, 0.05 wt% magnesium sulfate, 0.15 wt% citric acid, 0.5 wt% ammonium sulfate, 20 mg / L CoCl₂₆H₂O, 120 mg / L MnSO₄₆H₂O, 15 mg / L CuCl₂₂H₂O, 30 mg / L H₃BO₃, 25 mg / L Na₂MoO₄₂H₂O, 120 mg / L Zn(CH₃COO)₂₂H₂O, 0.8 g / L Fe(III) citrate, and 0.1 ml / L of antifoaming agent, with the remainder being water. The conditions for secondary seed culture are: Temperature: 37℃, Air volume: 0.5m 3 The parameters are: / h, rotation speed: 300 rpm, pressure: 0.02 MPa, pH: adjusted to pH 7 with ammonia water, dissolved oxygen content: 20%. S3. The seed solution was inoculated into 100 L of fermentation tank culture medium and fermented for 36 hours to obtain the fermented liquid, with an inoculation amount of 10%. The composition of the culture medium in a 100L fermentation tank is: The mixture consists of 1 wt% glucose, 2 wt% potassium dihydrogen phosphate, 0.08 wt% magnesium sulfate, 0.1 wt% citric acid, 0.5 wt% ammonium sulfate, 20 mg / L CoCl₂₆H₂O, 120 mg / L MnSO₄₆H₂O, 12 mg / L CuCl₂₂H₂O, 25 mg / L H₃BO₃, 25 mg / L Na₂MoO₄₂H₂O, 130 mg / L Zn(CH₃COO)₂₂H₂O, 0.8 g / L Fe(III) citrate, and 0.08 ml / L of antifoaming agent, with the remainder being water. The conditions for fermentation culture are: Temperature: 37℃, Air volume: 1.5m 3 The parameters are: / h, rotation speed: 200 rpm, pressure: 0.02 MPa, pH: adjusted to pH 7 with ammonia water, dissolved oxygen content: 25%, S4. Add feed medium to the reaction system in step S3 to control the feed rate, OD 600When the value reaches 70, arabinose (an inducer) is added to induce further induction. The composition of the feed medium is: The solution consists of 600 g / L glucose, 2 g / L magnesium sulfate, 10 g / L yeast powder, 25 mg / L CoCl2.6H2O, 150 mg / L MnSO4.4H2O, 15 mg / L CuCl2.2H2O, 30 mg / L H3BO3, 25 mg / L Na2MoO4.2H2O, 120 mg / L Zn(CH3COO)2.2H2O, and 1000 mg / L Fe(III)citrate, with the remainder being water. After culturing in the culture tank for approximately 12 hours, the dissolved oxygen level increased significantly, the pH rose, and feeding was initiated. The feed rate was controlled as follows: within 0-3 hours of the start of feeding, the feed rate was controlled to 700 g / h; from 3 hours after the start of feeding until the addition of the inducer, the feed rate was controlled to 900 g / h; and after the addition of the inducer, the feed rate was controlled to 700 g / h to control the OD of the fermentation liquid. 600 We obtained = 151.4, S5. The fermentation liquid is filtered through a 50 nm ceramic membrane to obtain wet microbial cells. The wet bacterial cells at a concentration of 6.25 g / L are added to a reaction solution containing 7 wt% inositol by mass and 50 mM boric acid. Under conditions of pH=8 and dissolved oxygen content of 40%, the process was converted for 6 hours to obtain D-glucuronic acid. S7. Two hours after the start of conversion, an aqueous solution of inositol with a mass concentration of 15 wt% was added to the reaction system by flow addition for 2 hours, and the mass of the added inositol was 5 wt% of the total mass of the reaction system. S8. The inversion solution was filtered through an 80 nm ceramic membrane, collected, and used repeatedly to obtain bacterial cells.

[0040] Following the steps described above, the molar conversion rate of inositol was over 85.7%, the D-glucuronic acid content was 89.3 g / L, and the inositol oxidase was successfully recycled.

[0041] Example 3 S1. A single colony of recombinant engineered fungus is inoculated into 300 ml of LB medium and cultured with shaking at 37°C and 220 rpm for 5 hours to obtain a primary seed solution. S2.OD 600 When the value reaches 2.18, the primary seed solution is inoculated into 30 L of seed tank culture medium and cultured, OD 600 When the result is 2.08, the secondary seed culture is completed, the inoculation amount is 2%, and seed liquid is obtained. The composition of the 30L seed tank culture medium is: The mixture consists of 1 wt% glucose, 1 wt% potassium dihydrogen phosphate, 0.05 wt% magnesium sulfate, 0.15 wt% citric acid, 0.5 wt% ammonium sulfate, 20 mg / L CoCl₂₆H₂O, 120 mg / L MnSO₄₆H₂O, 15 mg / L CuCl₂₂H₂O, 30 mg / L H₃BO₃, 25 mg / L Na₂MoO₄₂H₂O, 120 mg / L Zn(CH₃COO)₂₂H₂O, 1 g / L Fe(III) citrate, and 0.1 ml / L of antifoaming agent, with the remainder being water. The conditions for secondary seed culture are: Temperature: 37℃, Air volume: 0.5m 3 The parameters are: / h, rotation speed: 300 rpm, pressure: 0.02 MPa, pH: adjusted to pH 7 with ammonia water, dissolved oxygen content: 20%. S3. The seed solution was inoculated into 100 L of fermentation tank culture medium and fermented for 36 hours to obtain the fermented liquid, with an inoculation amount of 10%. The composition of the culture medium in a 100L fermentation tank is: The mixture consists of 1 wt% glucose, 2 wt% potassium dihydrogen phosphate, 0.08 wt% magnesium sulfate, 0.1 wt% citric acid, 0.5 wt% ammonium sulfate, 20 mg / L CoCl₂₆H₂O, 120 mg / L MnSO₄₆H₂O, 12 mg / L CuCl₂₂H₂O, 25 mg / L H₃BO₃, 25 mg / L Na₂MoO₄₂H₂O, 130 mg / L Zn(CH₃COO)₂₂H₂O, 0.8 g / L Fe(III) citrate, and 0.08 ml / L of antifoaming agent, with the remainder being water. The conditions for fermentation culture are: Temperature: 37℃, Air volume: 1.8m 3The settings are: / h, rotation speed: 220 rpm, pressure: 0.03 MPa, pH: adjusted to pH 7 with ammonia water, dissolved oxygen content: 25%, S4. Add feed medium to the reaction system in step S3 to control the feed rate, OD 600 When the value reaches 80, arabinose (an inducer) is added to induce further induction. The composition of the feed medium is, by mass per liter of feed medium, The solution consists of 600 g / L glucose, 2 g / L magnesium sulfate, 10 g / L yeast powder, 25 mg / L CoCl2.6H2O, 150 mg / L MnSO4.4H2O, 15 mg / L CuCl2.2H2O, 30 mg / L H3BO3, 25 mg / L Na2MoO4.2H2O, 120 mg / L Zn(CH3COO)2.2H2O, and 1000 mg / L Fe(III)citrate, with the remainder being water. After culturing in the culture tank for approximately 12 hours, the dissolved oxygen level increased significantly, the pH rose, and feeding was initiated. The feed rate was controlled as follows: within 0-3 hours of the start of feeding, the feed rate was controlled to 650 g / h; from 3 hours after the start of feeding until the addition of the inducer, the feed rate was controlled to 1000 g / h; and after the addition of the inducer, the feed rate was controlled to 750 g / h to control the OD of the fermentation liquid. 600 We obtained = 152.3, S5. The fermentation liquid is filtered through a 75 nm ceramic membrane to obtain wet microbial cells. The aforementioned wet bacterial cells containing S6.30 g / L are treated with 5 wt% inositol, 30 mM phosphate, and 3 mM Fe. 2+ Add to the reaction solution containing, Under conditions of pH=8 and dissolved oxygen content of 50%, the mixture was converted for 8 hours to obtain D-glucuronic acid. S7. 1.8 hours after the start of conversion, an aqueous solution of inositol with a mass concentration of 12 wt% was added to the reaction system by flow addition for 1.5 hours, and the mass of the added inositol was 5 wt% of the total mass of the reaction system. S8. The inversion solution was filtered through a 100 nm ceramic membrane, collected, and used repeatedly to obtain bacterial cells.

[0042] Following the steps described above, the molar conversion rate of inositol was over 95.7%, the D-glucuronic acid content was 83.2 g / L, and the inositol oxidase was successfully recycled.

[0043] Example 4 This embodiment provides a method for producing D-glucuronic acid, the steps being S1 to S8 below.

[0044] S1. A single colony of recombinant engineered fungus is inoculated into 300 ml of LB medium and cultured with shaking for 5.5 hours at 35°C and 220 rpm to obtain a primary seed solution. S2.OD 600 When the value reaches 2, the primary seed solution is inoculated into 30 L of seed tank medium and cultured, OD 600 When the result is 2.4, the secondary seed culture is completed, the inoculation amount is 2%, and seed liquid is obtained. The composition of the 30L seed tank culture medium is: The mixture consists of 2 wt% glucose, 1 wt% potassium dihydrogen phosphate, 0.05 wt% magnesium sulfate, 0.15 wt% citric acid, 0.6 wt% ammonium sulfate, 20 mg / L CoCl₂₆H₂O, 120 mg / L MnSO₄₆H₂O, 15 mg / L CuCl₂₂H₂O, 30 mg / L H₃BO₃, 25 mg / L Na₂MoO₄₂H₂O, 120 mg / L Zn(CH₃COO)₂₂H₂O, 0.8 g / L Fe(III) citrate, and 0.1 ml / L of antifoaming agent, with the remainder being water. The conditions for secondary seed culture are: Temperature: 37℃, Air volume: 0.5m 3 The settings are: / h, rotation speed: 280 rpm, pressure: 0.015 MPa, pH: adjusted to pH 7 with ammonia water, dissolved oxygen content: 30%. S3. The seed solution was inoculated into 100 L of fermentation culture medium and fermented for 38 hours to obtain the fermented liquid, with an inoculation amount of 10%. The composition of the culture medium in a 100L fermentation tank is: The solution consists of 1 wt% glucose, 1 wt% potassium dihydrogen phosphate, 0.06 wt% magnesium sulfate, 0.2 wt% citric acid, 0.5 wt% ammonium sulfate, 20 mg / L CoCl₂₆H₂O, 120 mg / L MnSO₄₆H₂O, 12 mg / L CuCl₂₂H₂O, 25 mg / L H₃BO₃, 25 mg / L Na₂MoO₄₂H₂O, 130 mg / L Zn(CH₃COO)₂₂H₂O, 0.8 g / L Fe(III) citrate, and 0.1 ml / L of antifoaming agent, with the remainder being water. The conditions for fermentation culture are: Temperature: 37℃, Air volume: 1.5m 3 The parameters are: / h, rotation speed: 200 rpm, pressure: 0.02 MPa, pH: adjusted to pH 7 with ammonia water, dissolved oxygen content: 25%, S4. Add feed medium to the reaction system in step S3 to control the feed rate, OD 600 When the value reaches 75, arabinose (an inducer) is added to induce further growth. The composition of the feed medium is, by mass per liter of feed medium, The solution consists of 500 g / L glucose, 3 g / L magnesium sulfate, 8 g / L yeast powder, 25 mg / L CoCl2.6H2O, 150 mg / L MnSO4.4H2O, 15 mg / L CuCl2.2H2O, 30 mg / L H3BO3, 25 mg / L Na2MoO4.2H2O, 120 mg / L Zn(CH3COO)2.2H2O, and 1000 mg / L Fe(III)citrate, with the remainder being water. After culturing in the culture tank for approximately 10 hours, the dissolved oxygen level increased significantly, the pH rose, and feeding was initiated. The feed rate was controlled as follows: within 0-3 hours of the start of feeding, the feed rate was controlled to 700 g / h; from 3 hours after the start of feeding until the addition of the inducer, the feed rate was controlled to 1000 g / h; and after the addition of the inducer, the feed rate was controlled to 700 g / h to control the OD of the fermentation liquid. 600 We obtained = 156.8, S5. The fermentation liquid is filtered through a 100 nm ceramic membrane to obtain wet microbial cells. The aforementioned wet bacterial cells containing 6.35 g / L of S are treated with inositol at a mass fraction of 4 wt%, phosphate at a concentration of 20 mM, and Fe at a physical concentration of 4 mM. 2+Add to the reaction solution containing, Under conditions of pH=7 and dissolved oxygen content of 50%, the process was converted for 7 hours to obtain D-glucuronic acid. S7. Two hours after the start of conversion, an aqueous solution of inositol with a mass concentration of 15 wt% was added to the reaction system by flow addition for 2 hours, and the mass of the added inositol was 4 wt% of the total mass of the reaction system. S8. The inversion solution was filtered through a 100 nm ceramic membrane, collected, and used repeatedly to obtain bacterial cells.

[0045] Following the steps described above, the molar conversion rate of inositol was over 96.8%, the D-glucuronic acid content was 86.5 g / L, and the inositol oxidase was successfully recycled.

[0046] Example 5 This embodiment provides a method for producing D-glucuronic acid, the steps being S1 to S8 below.

[0047] S1. A single colony of recombinant engineered fungus is inoculated into 300 ml of LB medium and cultured with shaking at 37°C and 240 rpm for 6 hours to obtain a primary seed solution. S2.OD 600 When the value reaches 2, the primary seed solution is inoculated into 30 L of seed tank medium and cultured, OD 600 When the value reaches 2, the secondary seed culture is completed, the inoculation amount is 2%, and seed liquid is obtained. The composition of the 30L seed tank culture medium is: The mixture consists of 1 wt% glucose, 1 wt% potassium dihydrogen phosphate, 0.05 wt% magnesium sulfate, 0.15 wt% citric acid, 0.5 wt% ammonium sulfate, 20 mg / L CoCl₂₆H₂O, 120 mg / L MnSO₄₆H₂O, 15 mg / L CuCl₂₂H₂O, 30 mg / L H₃BO₃, 25 mg / L Na₂MoO₄₂H₂O, 120 mg / L Zn(CH₃COO)₂₂H₂O, 1 g / L Fe(III) citrate, and 0.1 ml / L of antifoaming agent, with the remainder being water. The conditions for secondary seed culture are: Temperature: 37℃, Air volume: 0.5m3 The parameters are: / h, rotation speed: 300 rpm, pressure: 0.02 MPa, pH: adjusted to pH 7 with ammonia water, dissolved oxygen content: 20%. S3. The seed solution was inoculated into 100 L of fermentation tank culture medium and fermented for 36 hours to obtain the fermented liquid, with an inoculation amount of 10%. The composition of the culture medium in a 100L fermentation tank is: The mixture consists of 1 wt% glucose, 2 wt% potassium dihydrogen phosphate, 0.08 wt% magnesium sulfate, 0.1 wt% citric acid, 0.5 wt% ammonium sulfate, 20 mg / L CoCl₂₆H₂O, 120 mg / L MnSO₄₆H₂O, 12 mg / L CuCl₂₂H₂O, 25 mg / L H₃BO₃, 25 mg / L Na₂MoO₄₂H₂O, 130 mg / L Zn(CH₃COO)₂₂H₂O, 0.8 g / L Fe(III) citrate, and 0.08 ml / L of antifoaming agent, with the remainder being water. The conditions for fermentation culture are: Temperature: 37℃, Air volume: 1.8m 3 The settings are: / h, rotation speed: 220 rpm, pressure: 0.03 MPa, pH: adjusted to pH 7 with ammonia water, dissolved oxygen content: 25%, S4. Add feed medium to the reaction system in step S3 to control the feed rate, OD 600 When the value reaches 80, arabinose (an inducer) is added to induce further induction. The composition of the feed medium is, by mass per liter of feed medium, The solution consists of 700 g / L glucose, 1 g / L magnesium sulfate, 12 g / L yeast powder, 25 mg / L CoCl2.6H2O, 150 mg / L MnSO4.4H2O, 15 mg / L CuCl2.2H2O, 30 mg / L H3BO3, 25 mg / L Na2MoO4.2H2O, 120 mg / L Zn(CH3COO)2.2H2O, 1000 mg / L Fe(III)citrate, with the remainder being water. After culturing in the culture tank for approximately 14 hours, the dissolved oxygen level increased significantly, the pH rose, and feeding was initiated. The feed rate was controlled as follows: within 0-3 hours of the start of feeding, the feed rate was controlled to 700 g / h; from 3 hours after the start of feeding until the addition of the inducer, the feed rate was controlled to 900 g / h; and after the addition of the inducer, the feed rate was controlled to 750 g / h to control the OD of the fermentation liquid. 600 We obtained = 153.1, S5. The fermentation liquid is centrifuged at 16,000 rpm for 20 minutes to obtain wet microbial cells. The wet bacterial cells at a concentration of 6.30 g / L are added to a reaction solution containing 7 wt% inositol by mass and 40 mM boric acid. Under conditions of pH=8 and dissolved oxygen content of 50%, the process was converted for 7 hours to obtain D-glucuronic acid. S7. Two hours after the start of conversion, an aqueous solution of inositol with a mass concentration of 15 wt% was added to the reaction system by flow addition for 2 hours, and the mass of the added inositol was 4.5 wt% of the total mass of the reaction system. S8. The inversion solution was filtered through a 50 nm ceramic membrane, collected, and used repeatedly to obtain bacterial cells.

[0048] Following the steps described above, the molar conversion rate of inositol was 86.2% or higher, the content of the obtained D-glucuronic acid was 85.1 g / L, and the inositol oxidase was successfully recycled.

[0049] Example 6 This embodiment provides a method for producing D-glucuronic acid, the steps being S1 to S8 below.

[0050] S1. A single colony of recombinant engineered fungus is inoculated into 300 ml of LB medium and cultured with shaking at 40°C and 240 rpm for 6 hours to obtain a primary seed solution. S2.OD 600 When the value reaches 2, the primary seed solution is inoculated into 30 L of seed tank medium and cultured, OD 600 When the result is 2.14, the secondary seed culture is completed, the inoculation amount is 2%, and seed liquid is obtained. The composition of the 30L seed tank culture medium is: The mixture consists of 2 wt% glucose, 1 wt% potassium dihydrogen phosphate, 0.05 wt% magnesium sulfate, 0.15 wt% citric acid, 0.6 wt% ammonium sulfate, 20 mg / L CoCl₂₆H₂O, 120 mg / L MnSO₄₆H₂O, 15 mg / L CuCl₂₂H₂O, 30 mg / L H₃BO₃, 25 mg / L Na₂MoO₄₂H₂O, 120 mg / L Zn(CH₃COO)₂₂H₂O, 0.8 g / L Fe(III) citrate, and 0.1 ml / L of antifoaming agent, with the remainder being water. The conditions for secondary seed culture are: Temperature: 37℃, Air volume: 0.5m 3 The settings are: / h, rotation speed: 320 rpm, pressure: 0.03 MPa, pH: adjusted to pH 7 with ammonia water, dissolved oxygen content: 30%. S3. The seed solution was inoculated into 100 L of fermentation culture medium and fermented for 40 hours to obtain the fermented liquid, with an inoculation amount of 10%. The composition of the culture medium in a 100L fermentation tank is: The solution consists of 1 wt% glucose, 1 wt% potassium dihydrogen phosphate, 0.06 wt% magnesium sulfate, 0.2 wt% citric acid, 0.5 wt% ammonium sulfate, 20 mg / L CoCl₂₆H₂O, 120 mg / L MnSO₄₆H₂O, 12 mg / L CuCl₂₂H₂O, 25 mg / L H₃BO₃, 25 mg / L Na₂MoO₄₂H₂O, 130 mg / L Zn(CH₃COO)₂₂H₂O, 0.8 g / L Fe(III) citrate, and 0.1 ml / L of antifoaming agent, with the remainder being water. The conditions for fermentation culture are: Temperature: 37℃, Air volume: 1.5m 3 The parameters are: / h, rotation speed: 200 rpm, pressure: 0.02 MPa, pH: adjusted to pH 7 with ammonia water, dissolved oxygen content: 25%, S4. Add feed medium to the reaction system in step S3 to control the feed rate, OD 600 When the value reaches 75, arabinose (an inducer) is added to induce further growth. The composition of the feed medium is, by mass per liter of feed medium, The solution consists of 600 g / L glucose, 3 g / L magnesium sulfate, 8 g / L yeast powder, 25 mg / L CoCl2.6H2O, 150 mg / L MnSO4.4H2O, 15 mg / L CuCl2.2H2O, 30 mg / L H3BO3, 25 mg / L Na2MoO4.2H2O, 120 mg / L Zn(CH3COO)2.2H2O, 1000 mg / L Fe(III)citrate, with the remainder being water. After culturing in the culture tank for approximately 11.5 hours, the dissolved oxygen level increased significantly, the pH rose, and feeding was initiated. The feed rate was controlled as follows: within 0-3 hours of the start of feeding, the feed rate was controlled to 700 g / h; from 3 hours after the start of feeding until the addition of the inducer, the feed rate was controlled to 1100 g / h; and after the addition of the inducer, the feed rate was controlled to 800 g / h to control the OD of the fermentation liquid. 600 We obtained = 161.2, S5. The fermentation liquid is centrifuged at 16,000 rpm for 15 minutes to obtain wet microbial cells. The wet bacterial cells at a concentration of 6.35 g / L are added to a reaction solution containing 4 wt% inositol by mass and 60 mM boric acid. Under conditions of pH=7 and dissolved oxygen content of 50%, the mixture was converted for 8 hours to obtain D-glucuronic acid. S7. 1.5 hours after the start of conversion, an aqueous solution of inositol with a mass concentration of 12 wt% was added to the reaction system by flow addition for 2 hours, and the mass of the inositol was 4.5 wt% of the total mass of the reaction system. S8. The inversion solution was filtered through a 70 nm ceramic membrane, collected, and used repeatedly to obtain bacterial cells.

[0051] Following the steps described above, the molar conversion rate of inositol was over 97.6%, the D-glucuronic acid content was 85.4 g / L, and the inositol oxidase was successfully recycled.

[0052] Comparative Example 1 In Comparative Example 1, the nitrogen-containing compound in the feed medium was replaced with an equal amount of trace element, while the other reaction conditions remained unchanged.

[0053] Ultimately, the OD of the fermented liquid obtained in step S4 is 600 The ratio was 122.3, the molar conversion rate of inositol was 56.8% or higher, and the content of the obtained D-glucuronic acid was 54.7 g / L.

[0054] This was presumed to be because the deficiency of nitrogen-containing compounds made the inositol oxides produced during the fermentation process unstable, leading to a decrease in the inositol conversion rate.

[0055] Comparative Example 2 In Comparative Example 2, the trace elements in the seed tank culture medium were replaced with glucose in equal amounts, while the other reaction conditions remained unchanged.

[0056] Ultimately, the molar conversion rate of inositol was over 53.6%, and the resulting D-glucuronic acid content was 42.7 g / L.

[0057] This was presumed to be because the deficiency of trace elements led to a deficiency in bacterial cell components, slowing down cell growth, while the lack of key trace element support for enzyme activity groups caused a decrease in enzyme activity, ultimately leading to a decrease in the inositol conversion rate.

[0058] Comparative Example 3 In Comparative Example 3, the trace elements in the fermentation culture medium were replaced with glucose in equal amounts, while the other reaction conditions remained unchanged.

[0059] Ultimately, the molar conversion rate of inositol was over 45.2%, and the resulting D-glucuronic acid content was 34.7 g / L.

[0060] Compared to Comparative Example 2, the molar conversion rate of inositol and the D-glucuronic acid content decreased further because the number of microbial cells was larger during the fermentation process, resulting in a greater demand for trace elements.

[0061] Comparative Example 4 In Comparative Example 4, the trace elements in the feed medium were replaced with an equivalent amount of nitrogen-containing compound, while the other reaction conditions remained unchanged.

[0062] Ultimately, the molar conversion rate of inositol was over 68.2%, and the resulting D-glucuronic acid content was 56.4 g / L.

[0063] Compared to Comparative Examples 2 and 3, the relatively small decrease in the molar conversion rate of inositol and the D-glucuronic acid content is likely because nitrogen-containing compounds themselves belong to organic substances that contain small amounts of trace elements, and these trace elements may have partially acted as a supplement to the trace elements, thereby improving the activity of some of the enzymes.

[0064] Comparative Example 5 In Comparative Example 5, in comparison to Example 2, Fe(III)citrate in the trace elements of the seed tank medium, fermentation tank medium, and feed medium was replaced with an equal amount of CoCl2.6H2O, while the other reaction conditions remained unchanged.

[0065] Ultimately, the molar conversion rate of inositol was 36.8% or higher, and the resulting D-glucuronic acid content was 22.5 g / L.

[0066] This was hypothesized because iron, a trace element, plays a crucial role in the activation of inositol oxidase, and therefore plays a more significant role in enzyme activity compared to other trace elements.

[0067] Comparative Example 6 In Comparative Example 6, feed control was not employed during the fermentation process, and other reaction conditions remained unchanged.

[0068] Ultimately, the molar conversion rate of inositol was 0, and the resulting D-glucuronic acid content was 0.

[0069] This is because, without feed control, protein expression was not induced even when the inducing agent was added afterward, and the inositol conversion reaction could not proceed.

[0070] Comparative Example 7 In Comparative Example 7, the feed rate used in the fermentation process was changed from stepwise to a constant rate of 650 g / h throughout the entire process, while other reaction conditions remained unchanged.

[0071] Ultimately, the molar conversion rate of inositol was over 87%, and the resulting D-glucuronic acid content was 67.2 g / L.

[0072] It was hypothesized that the addition of the inducer caused the fermentation process in the reaction system to be less gradual before and after the inducer's addition. If the inducer was always added at the same feed rate, it would not be possible to satisfy the requirement of maintaining a gradual fermentation process. If the reaction accelerated rapidly after the induction of the inducer, it would lead to a situation where nutrients were deficient, resulting in a decrease in the reaction rate and a reduction in the product.

[0073] Furthermore, after producing D-glucuronic acid through the above process, it is possible to further convert the D-glucuronic acid into a D-glucuronic acid derivative, namely glucuronolactone, commonly known as "Kantairaku." Ta.

[0074] To solve the above technical problems, the embodiments of the present invention further provide a glucuronolactone production process, The process includes the step of adding concentrated phosphoric acid to a D-glucuronic acid solution, stirring, and carrying out an esterification reaction at a constant reaction temperature to crystallize and obtain the crude product glucuronolactone.

[0075] In the above proposed technology, concentrated phosphoric acid was used instead of the mixed acid of phosphoric acid and sulfuric acid used in the conventional technology, and by controlling the concentration of phosphoric acid and the reaction temperature, glucuronolactone was produced by esterification reaction with a D-glucuronic acid solution. By adopting the above proposed technology, glucuronolactone was produced, and because the reaction system has good fluidity, the reaction was carried out with stirring, and the total time from reaction to completion of crystallization was less than 6 hours, significantly shortening the reaction crystallization time, shortening the production cycle, and improving production efficiency. Furthermore, in the esterification reaction system of the present invention, only concentrated phosphoric acid is used, the temperature of the reaction system can be controlled, and the glucuronolactone produced has advantages such as large crystal particles, ease of filtration, and high product yield.

[0076] Furthermore, by combining the above examples, the embodiments of the present invention are further limited to selecting a solid content of 50 wt% to 70 wt% in the D-glucuronic acid solution. Measurements show that this solid content corresponds to 20 to 30 Baume degrees, the viscosity of the D-glucuronic acid solution is approximately 8 to 12 mPa·s, and the fluidity is good. Exemplarily, the solid content of the D-glucuronic acid solution can be selected from 60 wt% to 70 wt%. Specifically, the solid content of the D-glucuronic acid solution may be 50 wt%, 55 wt%, 60 wt%, 65 wt%, or 70 wt%. With this solid content, the viscosity of the D-glucuronic acid solution is appropriate, and in the esterification reaction process in which the D-glucuronic acid solution reacts with concentrated phosphoric acid, the stirring speed can generally be controlled to 80 to 120 rpm so that the system has high fluidity. In the reaction system and reaction conditions of the embodiment of the present invention, it was possible to obtain glucuronolactone crystals by direct crystallization without the need to add ethanol, and the particle size of the product was large, reaching 20 to 30 mesh.

[0077] Furthermore, combining the above embodiments, the embodiments of the present invention further limit the mass concentration of the concentrated phosphoric acid to 70 wt% to 85 wt%. Exemplarily, the mass concentration of the concentrated phosphoric acid can be selected from 80 wt% to 85 wt%. Specifically, the mass concentration of the concentrated phosphoric acid can be selected from 70 wt%, 75 wt%, 80 wt%, or 85 wt%. Through testing, the inventors verified that by limiting the mass concentration of concentrated phosphoric acid to a certain range, the esterification reaction between concentrated phosphoric acid and D-glucuronic acid solution could proceed smoothly. Furthermore, when the mass concentration of concentrated phosphoric acid was selected as 70 wt% to 85 wt% and the solid content of the D-glucuronic acid solution was set to 50 wt% to 70 wt%, the maximum esterification reaction rate could be achieved at a reaction temperature of 40°C to 80°C. At this temperature, the yield of glucuronolactone was highest, the glucuronolactone content in the crude product could reach 95 wt% or more, and the crystal particle size was uniform, the particle size was large, and it was easily filtered.

[0078] Furthermore, combining the above examples, the embodiment of the present invention further limits the amount of concentrated phosphoric acid added to 10 wt% to 50 wt% of the mass of solids in the D-glucuronic acid solution, and the esterification reaction time is 1 to 2 hours.

[0079] According to the above technical proposal, when the amount of concentrated phosphoric acid added is 10 wt% to 50 wt% of the mass of solids in the D-glucuronic acid solution, the esterification reaction time can be further shortened, the reaction efficiency can be improved, and the yield of the product can be increased. Under the above reaction conditions, the esterification reaction can generally be completed if the reaction time is 1 to 2 hours, significantly shortening the reaction cycle and improving the reaction efficiency.

[0080] Furthermore, combining the above examples, the embodiment of the present invention further includes evaporating water from the product of the esterification reaction after the esterification reaction and before crystallization at a temperature of 40°C to 80°C and a vacuum pressure of ≤-0.09 MPa, wherein the volume of evaporated water was 30% to 50% of the volume of the reaction solution.

[0081] Furthermore, the crystallization process involves dynamically gradient cooling the product of the esterification reaction at a rate of 5-10°C / h to crystallize it, with a crystallization completion temperature of 5-15°C, thereby obtaining a crude glucuronolactone.

[0082] According to the above proposed technology, the fluidity of the reaction system is good throughout the esterification and crystallization process, eliminating the need to add ethanol. By dynamically controlling the gradient cooling of the esterification reaction product at a rate of 5-10°C / h, glucuronolactone crystals can be directly crystallized, and the crystal particles can be large, reaching 20-30 mesh. At the same time, the system obtained after the crystallization is a solid-liquid mixture, making it easy to separate and filter the glucuronolactone. The crystallization process is simple to operate, and the total time for the esterification reaction and crystallization can be reduced to a minimum of 6 hours. This overcomes the long crystallization time in the double acid reaction process and the safety problems caused by the addition of ethanol during the crystallization process in conventional technology. Simultaneously, the glucuronolactone content in the crude product obtained by adopting the above proposed technology was ≥95 wt%, and the crystallization rate was ≥80%.

[0083] Furthermore, in the embodiments of the present invention, the D-glucuronic acid solution may be prepared from commercially available D-glucuronic acid, produced by reacting inositol raw material solution with inositol oxidase, or produced by the production methods of Examples 1 to 6 of the present invention. stomach.

[0084] Furthermore, combining the above embodiments, the filtration in the embodiment of the present invention includes filtering the inverted liquid through a ceramic membrane with a separation pore size of 20 to 100 nm and then collecting the ceramic membrane filtrate, and filtering the ceramic membrane filtrate through an ultrafiltration membrane with a pore size of 5000 to 20000 Da and collecting the ultrafiltration solution. Filtration with a ceramic membrane and filtration with an ultrafiltration membrane enable the purification of D-glucuronic acid products, allowing for rapid filtration and removal of impurities, resulting in high separation efficiency and excellent impurity removal effectiveness.

[0085] Furthermore, combining the above embodiments, the embodiments of the present invention further include desalting the filtrate with a cation exchange resin after filtering the inversion solution and before concentration, to obtain a desalted solution with an conductivity of <7000 us / cm. Exemplarily, a strongly acidic cation exchange resin can be selected. Unlike D-glucuronic acid solutions prepared with commercially available D-glucuronic acid, it should be understood that a certain amount of salt is added in the process of converting inositol to prepare a D-glucuronic acid solution, and the presence of salt may adversely affect the subsequent crystallization process. Therefore, crystals are more likely to be obtained by desalting, and by controlling the desalting conductivity to be as low as possible. Verification by testing showed that by controlling the desalting conductivity to <7000 us / cm, crystals with uniform particle size and large particle size were obtained.

[0086] Furthermore, combining the above examples, the embodiment of the present invention further includes, after filtering the inverted liquid and before concentrating it, adsorbing the filtrate with a macroporous adsorption resin to decolorize it and then collecting the decolorized liquid. Exemplarily, LS-108 or LS-109D can be selected as the macroporous adsorption resin, and further decolorizing the D-glucuronic acid solution obtained by the reaction of inositol and inositol oxidase further improves the purity of the D-glucuronic acid solution and facilitates the subsequent esterification reaction.

[0087] Furthermore, the concentration process includes concentrating the liquid to be concentrated using a nanofiltration membrane with a pore size of 150 to 300 Da, collecting a nanofiltration concentrate with a solid content of 10 wt% to 15 wt%, and concentrating the nanofiltration concentrate in a concentrator to obtain a D-glucuronic acid concentrate with a solid content of 50 wt% to 70 wt%.

[0088] According to the above technical proposal, the embodiment of the present invention performs a two-step dehydration operation on the obtained D-glucuronic acid solution before the esterification reaction to reduce or avoid the loss of solubility of glucuronolactone in water, thereby improving the crystal yield of glucuronolactone.

[0089] To better illustrate the technical concept of the present invention, the present invention further provides the following specific examples to further illustrate a method for producing glucuronolactone using D-glucuronic acid. It should be understood that the raw materials used in the following examples are all commercially available unless otherwise specified, and among them, D-glucuronic acid may be produced by the above production process, provided that the performance requirements such as the solid content and viscosity of D-glucuronic acid after processing are met.

[0090] In the following examples, the operating parameters of the ceramic membrane, nanofiltration membrane, and ultrafiltration membrane used are as follows.

[0091] [Table 1]

[0092] Example 7 This embodiment provides a glucuronolactone production process and includes the following steps S1 and S2.

[0093] S1. A 5.8 L D-glucuronic acid solution (viscosity 12.4 mPa·s) with a solid content of 73 wt% was to which concentrated phosphoric acid with a mass concentration of 70 wt% was added. The amount of concentrated phosphoric acid added was 58% of the mass of solids in the D-glucuronic acid solution. The esterification reaction was carried out at a reaction temperature of 80°C and a stirring speed of 120 rpm for 1 hour.

[0094] S2. The product of the esterification reaction was crystallized by dynamic gradient cooling at a rate of 5°C / h, and the crystallization completion temperature was 5°C, yielding crude glucuronolactone. The crude glucuronolactone was filtered by suction, the solid was washed with 2.0 L of anhydrous ethanol, and vacuum dried to obtain 3161 g of white crystals.

[0095] Liquid chromatography analysis revealed that the glucuronolactone content in the white crystals was 95.3 wt%, the crystallization rate was 82.3%, and the glucuronolactone particle size was 20-25 mesh.

[0096] Example 8 This embodiment provides a glucuronolactone production process and includes the following steps S1 to S3.

[0097] S1. A 6.4 L D-glucuronic acid solution (viscosity 8.1 mPa·s) with a solid content of 50 wt% was to which concentrated phosphoric acid with a mass concentration of 72 wt% was added. The amount of concentrated phosphoric acid added was 22% of the mass of solids in the D-glucuronic acid solution. The esterification reaction was carried out at a reaction temperature of 40°C and a stirring speed of 80 rpm for 2 hours.

[0098] S2. Water was evaporated from the esterification reaction product at a temperature of 80°C and a vacuum of -0.09 MPa. The volume of evaporated water was 50% of the volume of the reaction solution.

[0099] S3. The esterification product after treatment in step S2 was crystallized by dynamic gradient cooling at a rate of 10°C / h, and the crystallization completion temperature was 10°C, yielding crude glucuronolactone. The crude glucuronolactone was filtered by suction, the solid was washed with 2.0 L of anhydrous ethanol, and vacuum dried to obtain 2453 g of white crystals.

[0100] Liquid chromatography analysis revealed that the glucuronolactone content in the white crystals was 95.7 wt%, the crystallization rate was 84.5%, and the glucuronolactone particle size was 25-30 mesh.

[0101] Example 9 This embodiment provides a glucuronolactone production process and includes the following steps S1 to S3.

[0102] S1. To a 6.0 L D-glucuronic acid solution (viscosity 11.8 mPa·s) with a solid content of 70 wt%, concentrated phosphoric acid with a mass concentration of 84 wt% was added. The amount of concentrated phosphoric acid added was 48% of the mass of solids in the D-glucuronic acid solution. The esterification reaction was carried out at a reaction temperature of 78°C and a stirring speed of 120 rpm for 1.5 hours.

[0103] S2. Water was evaporated from the product of the esterification reaction at a temperature of 40°C and a vacuum of -0.09 MPa. The volume of evaporated water was 30% of the volume of the reaction solution.

[0104] S3. The esterification product after treatment in step S2 was crystallized by dynamic gradient cooling at a rate of 5°C / h until the crystallization was completed at 10°C, yielding crude glucuronolactone. The crude glucuronolactone was filtered by suction, the solid was washed with 2.0 L of anhydrous ethanol, and vacuum dried to obtain 3361 g of white crystals.

[0105] Liquid chromatography testing revealed that the glucuronolactone content in the white crystals was 97.7 wt%, the crystallization rate was 88.2%, and the glucuronolactone particle size was 20-25 mesh.

[0106] Example 10 This embodiment provides a glucuronolactone production process and includes the following steps S1 to S4.

[0107] S1. The inositol raw material solution was pre-prepared, and inositol oxidase was added to the inositol raw material solution to convert it and obtain the converted solution. The specific steps included the following: Functional protein, L-cysteine, inositol, and ferrous sulfate were added to the buffer system, and the mixing ratio of the functional protein, L-cysteine, inositol, and ferrous sulfate was 1 × 10⁻⁶. 5 The reaction system consisted of μg of functional protein, 2 mmol of L-cysteine, 20 mmol of inositol, and 1 mmol of ferrous sulfate. In the reaction system, the initial concentrations of each component were 100 μg / mL of functional protein, 2 mmol / L of L-cysteine, 0.3 mol / L of inositol, and Fe 2+ The concentration was 1 mmol / L. The buffer system was a 50 mM Tris-HCl buffer at pH 8.0. The conversion temperature was 37°C, the reaction pH was 8.0, and the reaction time was 6–8 hours.

[0108] 47.3 L of inverted liquid with a solid content of 5.6 wt% was passed through a ceramic membrane with a pore size of 20 nm in sequence at an operating temperature of 35°C, with a pressure entering the membrane of 0.6 MPa and exiting the membrane of 0.4 MPa, and a membrane flow rate of 80 L / h. The ceramic membrane filtrate was collected after filtration. The ceramic membrane filtrate was filtered again through an ultrafiltration membrane with a pore size of 5000 Da at an operating temperature of 35°C, with a pressure entering the membrane of 0.7 MPa and exiting the membrane of 0.6 MPa, and a membrane flow rate of 150 L / h. The ultrafiltrate was collected. The ultrafiltrate was desalted with a strongly acidic cation exchange resin to obtain a desalted solution, the conductivity of which was 6900 us / cm. The desalted solution was adsorbed with an LS-109 D macroporous adsorption resin to decolorize it, and the decolorized solution was collected. The decolorized solution was concentrated using a nanofiltration membrane with a pore size of 150 Da, the operating temperature was 35°C, the pressure entering the membrane was 3.0 MPa, the pressure exiting the membrane was 2.5 MPa, and the membrane passage rate was 40 L / h. The nanofiltration concentrate was collected. The nanofiltration concentrate had a solid content of 10 wt%, and the nanofiltration concentrate was concentrated in a concentrator to obtain a 3.9 L D-glucuronic acid concentrate (viscosity 10.5 mpa·s) with a solid content of 62 wt%.

[0109] S2. To a 3.9 L D-glucuronic acid solution with a solid content of 62 wt%, concentrated phosphoric acid with a mass concentration of 76 wt% was added. The amount of concentrated phosphoric acid added was 30% of the mass of solids in the D-glucuronic acid solution. The esterification reaction was carried out at a reaction temperature of 60°C and a stirring speed of 100 rpm for 1.5 hours.

[0110] S3. Water was evaporated from the product of the esterification reaction at a temperature of 70°C and a vacuum of -0.09 MPa. The volume of evaporated water was 35% of the volume of the reaction solution.

[0111] S4. The esterification product after treatment in step S3 was crystallized by dynamic gradient cooling at a rate of 8°C / h until the crystallization was completed at 10°C, yielding crude glucuronolactone. The crude glucuronolactone was filtered by suction, the solid was washed with 2.0 L of anhydrous ethanol, and vacuum dried to obtain 1895 g of white crystals.

[0112] Liquid chromatography testing revealed that the glucuronolactone content in the white crystals was 98.6 wt%, the crystallization rate was 86.4%, and the glucuronolactone particle size was 25-30 mesh.

[0113] Example 11 This embodiment provides a glucuronolactone production process and includes the following steps S1 to S4.

[0114] S1. The inositol raw material solution was pre-prepared, and inositol oxidase was added to the inositol raw material solution to convert it and obtain the converted solution. The specific steps included the following: Functional protein, L-cysteine, inositol, and ferrous sulfate were added to the buffer system, and the mixing ratio of the functional protein, L-cysteine, inositol, and ferrous sulfate was 1 × 10⁻⁶. 5 The reaction system consisted of μg of functional protein, 2 mmol of L-cysteine, 20 mmol of inositol, and 1 mmol of ferrous sulfate. In the reaction system, the initial concentrations of each component were 100 μg / mL of functional protein, 2 mmol / L of L-cysteine, 0.3 mol / L of inositol, and Fe 2+ The concentration was 1 mmol / L. The buffer system was a 50 mM Tris-HCl buffer at pH 8.0. The conversion temperature was 37°C, the reaction pH was 8.0, and the reaction time was 30 min.

[0115] 33.9 L of inverted liquid with a solid content of 6.1 wt% was passed through a ceramic membrane with a pore size of 100 nm, and the ceramic membrane filtrate was collected. The operating temperature was 35°C, the pressure entering the membrane was 0.5 MPa, the pressure exiting the membrane was 0.2 MPa, and the membrane flow rate was 80 L / h. The ceramic membrane filtrate was filtered again through an ultrafiltration membrane with a pore size of 20000 Da. The operating temperature was 35°C, the pressure entering the membrane was 0.3 MPa, the pressure exiting the membrane was 0.2 MPa, and the membrane flow rate was 150 L / h, and the ultrafiltration solution was collected. The ultrafiltrate was desalted with a strongly acidic cation exchange resin to obtain a desalted solution, the conductivity of which was 5000 us / cm. The desalted solution was adsorbed with an LS-108D macroporous adsorption resin to decolorize it, and the decolorized solution was collected. The decolorized solution was concentrated using a nanofiltration membrane with a pore size of 300 Da, the operating temperature was 35°C, the pressure entering the membrane was 2.5 MPa, the pressure exiting the membrane was 2.0 MPa, and the membrane passage rate was 40 L / h. The nanofiltration concentrate was collected. The nanofiltration concentrate had a solid content of 15 wt%, and the nanofiltration concentrate was concentrated in a concentrator to obtain a 3.4 L D-glucuronic acid concentrate (viscosity 8.3 mpa·s) with a solid content of 53 wt%.

[0116] S2. To a 3.4 L D-glucuronic acid concentrate with a solid content of 53 wt%, concentrated phosphoric acid with a mass concentration of 74 wt% was added. The amount of concentrated phosphoric acid added was 11% of the mass of solids in the D-glucuronic acid solution. The esterification reaction was carried out at a reaction temperature of 45°C and a stirring speed of 85 rpm for 2 hours.

[0117] S3 The water in the product of the esterification reaction was evaporated at a temperature of 60°C and a vacuum of -0.09 MPa. The volume of evaporated water was 45% of the volume of the reaction solution.

[0118] S4 .step S3The product of the esterification reaction after treatment was crystallized by dynamic gradient cooling at a rate of 10°C / h, with a crystallization completion temperature of 10°C, yielding crude glucuronolactone. The crude glucuronolactone was filtered by suction, the solid was washed with 2.0 L of anhydrous ethanol, and vacuum dried to obtain 1401 g of white crystals.

[0119] Liquid chromatography analysis revealed that the glucuronolactone content in the white crystals was 96.1 wt%, the crystallization rate was 85.7%, and the glucuronolactone particle size was 20-25 mesh.

[0120] Example 12 This embodiment provides a glucuronolactone production process and includes the following steps S1 to S4.

[0121] S1. The inositol raw material solution was pre-prepared, and inositol oxidase was added to the inositol raw material solution to convert it and obtain the converted solution. The specific steps included the following: Functional protein, L-cysteine, inositol, and ferrous sulfate were added to the buffer system, and the mixing ratio of the functional protein, L-cysteine, inositol, and ferrous sulfate was 1 × 10⁻⁶. 5 The reaction system consisted of μg of functional protein, 2 mmol of L-cysteine, 20 mmol of inositol, and 1 mmol of ferrous sulfate. In the reaction system, the initial concentrations of each component were 100 μg / mL of functional protein, 2 mmol / L of L-cysteine, 0.3 mol / L of inositol, and Fe 2+ The concentration was 1 mmol / L. The buffer system was a 50 mM Tris-HCl buffer at pH 8.0. The reaction temperature was 37°C, the reaction pH was 8.0, and the reaction time was 30 min.

[0122] 42.2 L of invert solution with a solid content of 6.5 wt% was taken and concentrated under reduced pressure at a temperature of 70°C and a vacuum of -0.09 MPa to obtain 3.9 L of D-glucuronic acid concentrate, which had a solid content of 62 wt% (viscosity 11.4 MPa·s).

[0123] S2. To 3.9 L of D-glucuronic acid concentrate with a solid content of 62 wt%, 0.48 L of concentrated phosphoric acid with a mass concentration of 79 wt% was added, and the esterification reaction was carried out at a reaction temperature of 70°C and a stirring speed of 110 rpm for 1.5 hours.

[0124] S3. Water was evaporated from the product of the esterification reaction at a temperature of 70°C and a vacuum of -0.09 MPa. The volume of evaporated water was 39% of the volume of the reaction solution.

[0125] S4. The esterification product after treatment in step S3 was crystallized by dynamic gradient cooling at a rate of 10°C / h, and the crystallization completion temperature was 10°C, yielding crude glucuronolactone. The crude glucuronolactone was filtered by suction, the solid was washed with 2.0 L of anhydrous ethanol, and vacuum dried to obtain 1968 g of white crystals.

[0126] Liquid chromatography revealed that the glucuronolactone content in the white crystals was 98.1 wt%, the crystallization rate was 89.7%, and the glucuronolactone particle size was 20-25 mesh.

[0127] Comparative Example 8 Compared to Example 7, concentrated phosphoric acid at a mass concentration of 70 wt% was replaced with an equal amount of concentrated sulfuric acid at a mass concentration of 70 wt%, while all other reaction conditions remained unchanged.

[0128] Tests revealed that crystalline glucuronolactone could not be obtained in this reaction system; instead, the system directly became a black, viscous liquid.

[0129] Comparative Example 9 In comparison with Example 7, concentrated phosphoric acid with a mass concentration of 70 wt% was substituted in equal amounts with a mixed acid of concentrated sulfuric acid with a mass concentration of 70 wt% and concentrated phosphoric acid with a mass concentration of 70 wt%. The mass ratio of concentrated sulfuric acid to concentrated phosphoric acid in the mixed acid was 1:1, and the other reaction conditions remained unchanged.

[0130] Under these conditions, the reaction system turned black, a small amount of crystals were obtained, and the color of the crystals was gray. Liquid chromatography revealed that the glucuronolactone content was 62.3 wt%, the crystallization rate was 21.4%, and the particle size was >50 mesh.

[0131] It was presumed that the high water content in the reaction system caused sulfuric acid to generate heat during the reaction process, leading to an increase in the carbonization rate of the product.

[0132] Comparative Example 10 Compared to Example 7, the esterification reaction temperature was adjusted to 35°C, while the other reaction conditions remained unchanged.

[0133] Liquid chromatography analysis revealed that the glucuronolactone content produced under these conditions was 65.3 wt%, the crystallinity was 30.4%, the appearance was powdery, and the particle size was >50 mesh.

[0134] Comparative Example 11 Compared to Example 7, concentrated phosphoric acid at a mass concentration of 70 wt% was substituted with an equal amount of acetic acid, while other reaction conditions remained unchanged.

[0135] The tests showed that almost no glucuronolactone was obtained under those conditions.

[0136] Comparative Example 12 Compared to Example 10, the conductivity of the desalted solution was 7500 us / cm, and other conditions remained unchanged.

[0137] Liquid chromatography analysis revealed that the glucuronolactone content produced under these conditions was 85.1 wt%, the crystallinity was 51.3%, the appearance was powdery, and the particle size was >50 mesh.

[0138] Test example Furthermore, the test conditions for the liquid chromatogram tests in Examples 7-12 and Comparative Examples 8-12 of the present invention were as follows.

[0139] Mobile phase: 10 mmol / L formic acid aqueous solution, Chromatography column: Calcium column (300*7.7 or similar column), Flow rate: 0.5ml / min, Detector: Differential detector, Column temperature: 55℃, Detector temperature: 45°C, Solvent: 10 mmol / L aqueous solution of formic acid, Standard concentration: 1.0 mg / mL (calculated using D-glucuronolactone [CAS No.] 32449-92-6, C6H8O6, content 99.9%, China Food and Drug Administration). Concentration of the test sample: 1.0 mg / mL Chromatography conditions: Operate for 30 minutes in a 100% 10 mmol / L formic acid aqueous solution.

[0140] Here, we will explain using the liquid chromatography diagram of Example 10 as an example, and the test results are shown in Figure 1. As can be seen from Figure 1, the peak at 16.383 min is the glucuronolactone peak, the peak at 24 min is the system peak, and the glucuronolactone content was 98.6%.

[0141] As can be seen from the data of the above examples, comparative examples, and test examples, under the conditions of the examples of the present invention, by blending a D-glucuronic acid solution of a certain concentration with concentrated phosphoric acid of a certain concentration, the viscosity of the system can reach 8 to 12 mPa·s. In this case, the two can be reacted by stirring, and there is no need to add ethanol. Under conditions where the total time from reaction to crystallization is 6 hours or less, a crude crystal with a uniform particle size and a particle size range of 20 to 30 mesh can be obtained. This has advantages such as a short crystallization time, high production efficiency, large crystal particles, ease of filtration, and high product yield.

Claims

1. A glucuronolactone production process comprising the steps of adding concentrated phosphoric acid to a D-glucuronic acid solution, stirring, and carrying out an esterification reaction at a reaction temperature of 40°C to 80°C to crystallize the product, wherein the solid content of the D-glucuronic acid solution is 50 wt% to 70 wt%, the mass concentration of the concentrated phosphoric acid is 70 wt% to 85 wt%, the amount of concentrated phosphoric acid added is 10 wt% to 50 wt% of the mass of solids in the D-glucuronic acid solution, the esterification reaction time is 1 h to 2 h, the crystallization is carried out by dynamically gradient cooling the product of the esterification reaction at a rate of 5 to 10°C / h, the crystallization completion temperature is 5 to 15°C, and the glucuronolactone content in the crude glucuronolactone is ≥95 wt%, and the crystallization rate is ≥80%.

2. After the esterification reaction and before the crystallization, The glucuronolactone production process according to claim 1, further comprising evaporating water from the product of the esterification reaction at a temperature of 40°C to 80°C and a vacuum pressure of ≤-0.09 MPa, wherein the volume of evaporated water is 30% to 50% of the volume of the reaction solution.

3. The glucuronolactone production process according to claim 1, characterized in that the D-glucuronic acid solution is produced by reacting an inositol raw material solution with inositol oxidase.

4. The reaction between the inositol raw material solution and inositol oxidase is as follows: The glucuronolactone production process according to claim 3, comprising an inositol raw material solution preliminary production step, inositol oxidase being added to the inositol raw material solution to invert it, the inverted solution being filtered sequentially and concentrated to obtain a D-glucuronic acid concentrate having a solid content of 50 wt% to 70 wt%.

5. The aforementioned filtration is The aforementioned conversion liquid is filtered through a ceramic membrane with a separation pore size of 20 to 100 nm, and the ceramic membrane filtrate is collected. The glucuronolactone production process according to claim 4, characterized by comprising filtering the ceramic membrane filtrate with an ultrafiltration membrane having a pore size of 5,000 to 20,000 Da, and collecting the ultrafiltration solution.

6. The glucuronolactone production process according to claim 4, further comprising filtering the inverted liquid and, before concentrating it, desalting the filtrate with a cation exchange resin to obtain a desalted liquid having an electrical conductivity of <7000 us / cm.

7. The glucuronolactone production process according to claim 6, further comprising adsorbing the desalted liquid with a macroporous adsorption resin to decolorize it, and then collecting the decolorized liquid.

8. The aforementioned concentration The method involves concentrating the target liquid using a nanofiltration membrane with a pore size of 150 to 300 Da, and collecting the nanofiltration concentrate with a solid content of 10 wt% to 15 wt%, The glucuronolactone production process according to claim 4, characterized by comprising: concentrating the nanofiltration concentrate in a concentrator to obtain a D-glucuronic acid concentrate having a solid content of 50 wt% to 70 wt%.

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