Method for producing D-glucuronic acid and production process of 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 the priority of a Chinese patent filed with the China National Intellectual Property Administration on February 28, 2023, with an application number of 202310177817.5 and an application title of "Process for Producing Glucuronolactone", and a Chinese patent filed with the China National Intellectual Property Administration on March 22, 2023, with an application number of 202310283516.0 and an application title of "Method for Producing D-Glucuronic Acid by Biotransformation", and all of their contents are incorporated herein by reference.

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

Background Art

[0003] Inositol, also known as cyclohexanehexol, hexahydroxycyclohexane, cyclohexane sugar alcohol, myo-inositol, and non-rotating inositol, has a molecular formula of C6H 12 O6, a molecular weight of 180, belongs to one of the B vitamins, and there are a total of 9 isomers due to the different orientations of the hydroxy groups relative to the ring plane, 7 of which are non-rotating optical isomers and 2 are rotating optical isomers (left-handed and right-handed). Inositol is widely distributed in animals and plants and is a growth factor for animals and microorganisms. It is mainly used for the treatment of diseases such as cirrhosis, hepatitis, fatty liver, and too high blood cholesterol. Since inositol is an essential substance for the growth of humans, animals, and microorganisms, it has important applications in fields such as feed, medicine, and food.

[0004] D-Glucuronic acid (D-glucuronic acid) is also known as D-glucuronic acid (GlcUA), with a molecular formula of C6H 10It is O7 and has a molecular weight of 194.14. As a biogenic detoxifying agent, D-glucuronic acid binds to endogenous and exogenous toxic substances containing groups such as hydroxy groups, amino groups, carboxyl groups, and mercapto groups in the livers of animals, improves the water solubility of the toxic substances, and is finally excreted outside the body in the form of D-glucuronic acid esters, D-glucuronates or complexes, enabling a detoxifying effect. In addition, D-glucuronic acid has been proven to have the function of preventing and treating skin inflammation and reducing the concentrations of blood cholesterol and triglycerides, and is widely applied in the beverage, food and cosmetics industries. Glucuronolactone, a D-glucuronic acid derivative, is commonly called hepatone and is a liver detoxifying agent and an immunomodulator, and can be used for the treatment of liver diseases, the detoxification of food and drugs, and the adjuvant treatment of rheumatoid arthritis. Through the above applications, the annual demand for D-glucuronic acid is gradually increasing.

[0005] The conventional methods for producing D-glucuronic acid mainly include the polysaccharide hydrolysis method and the chemical oxidation catalyst method. The polysaccharide hydrolysis method refers to the process of obtaining D-glucuronic acid by hydrolyzing uronic acid-containing polysaccharides. For example, obtaining D-glucuronic acid from the water-soluble part of sunflower membrane hemicellulose, hydrolyzing cotton and cellulose with a base, extracting holocellulose with hot water, and producing D-glucuronic acid by methods such as oxidizing cellulose with an aqueous chlorine solution. However, in the polysaccharide hydrolysis method, the glycosidic bond connecting with uronic acid is generally highly stable and difficult to be hydrolyzed. Therefore, it is necessary to use strong acids and strong bases in the hydrolysis process. Under the conditions of strong acids and strong bases, the D-glucuronic acid of the product is often decomposed, resulting in defects such as low oxidation selectivity, many by-products, and low product yield, and it cannot meet the production needs. The chemical oxidation method refers to oxidizing saccharides and their derivatives with inorganic reagents to produce D-glucuronic acid. The application of producing D-glucuronic acid by the nitric acid oxidation method is the most extensive. The process of this method is to first oxidize starch with concentrated nitric acid to obtain a crude starch oxidation solution, then hydrolyze the crude starch oxidation solution by heating and pressurizing under acidic conditions, concentrate the obtained hydrolysis solution under reduced pressure, add acetic acid for esterification, and finally produce glucuronolactone by freeze crystallization. However, this method has defects such as relatively low total yield (about 10%), high energy consumption, low selectivity, and serious environmental pollution, so it cannot meet the production needs.

[0006] With the improvement of people's understanding of microorganisms, the research on producing D-glucuronic acid by the biocatalyst method is increasing day by day. Inositol oxidase is an enzyme that promotes the conversion of inositol to D-glucuronic acid. However, due to the low stability of this enzyme, the phenomenon of incomplete conversion occurs during the conversion process, and it is easy to cause problems such as low concentration and conversion rate of the product when finally producing D-glucuronic acid with 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 with the main component being 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 allowed to stand for 24 hours or more. After standing to complete the esterification, alcohol is added and stirred for crystallization. The temperature is lowered to 10-15°C in 0-24 hours, to below 0°C in 24-48 hours, and to below 8°C in 48-60 hours. In the above technical solution, the total time for cooling and 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 due to 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, resulting in fine glucuronolactone crystal particles obtained by the reaction, which are 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 overcome the problems that in this field, due to the low stability of inositol oxidase, incomplete conversion occurs during the conversion process, ultimately resulting in low product concentration and conversion rate when producing D-glucuronic acid by inositol oxidase, and the problems that in the prior art, the production process of glucuronolactone is complicated, the production time is long, the efficiency is low, and the crystal particles of glucuronolactone are fine and difficult to filter. The present invention provides a method for producing D-glucuronic acid and a method for producing glucuronolactone using D-glucuronic acid.

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, wherein 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. The method of the present invention controls the parameters of the fermentation process and the conversion process, particularly controls the components of the medium (seed tank medium and fermenter medium) and adopts feed control in the fermentation process, thereby promoting the excellent expression of the engineering bacterium, obtaining inositol oxidase with high activity, high stability and high content in the wet bacterial cells, further promoting the conversion rate of inositol in the conversion process, and improving the concentration of D-glucuronic acid in the product. 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, Add concentrated phosphoric acid to the D-glucuronic acid solution, and while stirring, carry out an esterification reaction at a reaction temperature of 40°C to 80°C, followed by crystallization to obtain crude glucuronolactone.

[0012] Compared with the prior art, the production process of glucuronolactone in the present invention uses concentrated phosphoric acid instead of the mixed acid of phosphoric acid and sulfuric acid in the prior art, and by controlling the concentration of phosphoric acid and the reaction temperature, while stirring, an esterification reaction is carried out with the D-glucuronic acid solution to produce glucuronolactone. When the technical solution of the present invention is adopted to produce glucuronolactone, the total time from the reaction to the completion of crystallization is less than 6 hours, significantly reducing the reaction crystallization time, shortening the production cycle, and improving the production efficiency. In addition, 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 produced glucuronolactone has the advantages of large crystal particles, easy filtration, and high product yield.

Brief Description of the Drawings

[0013]

Figure 1

Modes for Carrying Out the Invention

[0014] In order to more clearly illustrate the technical problems, technical solutions and beneficial effects to be solved by the present invention, the present invention will be described in more detail below in combination with specific examples. It should be understood that the specific examples described here are only for interpreting the present invention and do not limit the present invention.

[0015] 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 fermentation tank 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.

[0016] The present invention cultures a recombinant engineering bacterium to obtain a seed solution. In the present invention, the culture of the recombinant engineering bacterium includes two steps: primary seed culture and secondary seed culture. The primary seed culture includes inoculating the recombinant engineering bacterium into an LB medium and culturing for 5 - 6 h under the conditions of 200 - 240 rpm and 34°C - 40°C. The recombinant engineering bacterium in the present invention is purchased externally, and the source of purchase is the Institute of Microbiology, Chinese Academy of Sciences. This bacterium has already been patent-applied for and obtained patent approval, and the application number is 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 engineering bacterium itself, but inositol oxidase produced by the expression of the functional protein of the engineering bacterium. In the present invention, there is no special requirement for the LB medium. The LB medium is a medium known in the technical field, and any commercially available LB medium can be selected in this case. The present invention has no special requirement for the container / holder for primary seed culture, and any corresponding container or holder capable of realizing shaking culture can be used. Preferably, it is carried out using a shaking flask. In the present invention, there is no special requirement for the shaking method. Preferably, it is carried out using a shaker or a vibrator, and more preferably a shaker. In the present invention, the speed of shaking culture is preferably 200 - 240 rpm, and more preferably 220 rpm. In the present invention, the culture temperature is preferably 34 - 40°C, and more preferably 37°C. In the present invention, the culture time is preferably 5 - 6 h, and more preferably 5 h. The present invention adopts a combined culture method of a shaking flask + shaker. Such a method can meet the need to block cell 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 aqueous ammonia, more preferably adjusted to pH 7.0 with aqueous ammonia; 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 1 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 of the above selection of culture conditions are for better amplifying and growing the bacterial cells.

[0018] Furthermore, by combining the above embodiments, in the present invention, the specific composition of the seed tank medium is preferably 1 wt% - 2 wt% glucose, 1 wt% - 2 wt% potassium dihydrogen phosphate, 0.05 wt% - 0.08 wt% magnesium sulfate, 0.1 wt% - 0.2 wt% citric acid, 0.4 wt% - 0.6 wt% ammonium sulfate, 1000 - 1500 mg / L of trace elements, and 0.05 - 0.15 ml / L of antifoaming agent. The trace elements in the seed tank medium in the present invention preferably have components 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 Fe(III)citrate. More preferably, the trace elements have components of 25 mg / L of CoCl2·6H2O, 150 mg / L of MnSO4·4H2O, 15 mg / L of CuCl2·2H2O, 30 mg / L of H3BO3, 25 mg / L of Na2MoO4·2H2O, 130 mg / L of Zn(CH3COO)2·2H2O, and 1000 mg / L of Fe(III)citrate. The functions of the trace elements in the seed tank medium in the present invention are twofold. One is a component that constitutes the bacterial cells, and the other is to serve as a component of the enzyme active group or maintain the activity of the enzyme. It should be understood that by adding the trace elements in the present invention, the activity and stability of the inositol oxidase produced by the recombinant engineering bacteria are greatly improved, providing a good foundation for subsequent biotransformation and fermentation.

[0019] Furthermore, by combining the above embodiments, there are no special requirements for the type of defoaming agent in the present invention, and preferably a commercially available liquid defoaming agent is adopted. The addition amount of the defoaming agent in the present invention is preferably 0.05 - 0.15 ml / L, more preferably 0.1 ml / L. The addition of the defoaming agent in the present invention can be selectively added according to specific situations. For example, if there are too many bubbles on the liquid surface, the defoaming agent can be manually added according to the situation. It is inappropriate that the addition amount of the defoaming agent is too much, and it is appropriate to retain a small amount of bubbles. Otherwise, it should be understood that oxygen supply shortage is likely to occur.

[0020] After obtaining the seed liquid through secondary seed culture, the present invention inoculates the seed liquid into the fermentation tank medium for fermentation culture to obtain a fermentation broth. In the present invention, the temperature of the fermentation culture is preferably 34°C - 40°C, more preferably 37°C, the air volume is preferably 1.2 - 1.8 m 3 / h, more preferably 1.5 m 3 / h, 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 adjusted to pH 7.0 with ammonia water, and the dissolved oxygen content is preferably 20% - 30%, more preferably 25%. In the present invention, the time of the fermentation reaction is preferably 36 - 40 h, preferably 38 h. The index for evaluating the fermentation is generally the OD 600 value. When stability is maintained, it represents the completion or immediately before the completion of fermentation. It should be understood that the above 36 - 40 h refers to the time of complete fermentation obtained based on production experience. The fermentation culture is a high-level expression of the bacterial cells carried out based on the above seed culture. In this appearance, inositol oxidase is produced by utilizing the expression of the functional protein in the recombinant engineering bacteria, and is applied to the subsequent inositol conversion process.

[0021] Furthermore, by combining the above embodiments, in the present invention, the specific composition of the fermentation tank medium is preferably 1 wt% - 2 wt% glucose, 1 wt% - 2 wt% potassium dihydrogen phosphate, 0.05 wt% - 0.08 wt% magnesium sulfate, 0.1 wt% - 0.2 wt% citric acid, 0.4 wt% - 0.6 wt% ammonium sulfate, 1000 - 1500 mg / L of trace elements, and 0.05 - 0.15 ml / L of antifoaming agent. The trace elements in the fermentation tank medium in the present invention preferably have components 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 Fe(III)citrate. More preferably, the trace elements have components of 25 mg / L of CoCl2·6H2O, 150 mg / L of MnSO4·4H2O, 15 mg / L of CuCl2·2H2O, 30 mg / L of H3BO3, 25 mg / L of Na2MoO4·2H2O, 130 mg / L of Zn(CH3COO)2·2H2O, and 1000 mg / L of Fe(III)citrate. The trace elements in the fermentation tank medium in the present invention have two functions. One is a component constituting the bacterial cells, and the other is to serve as a component of the enzyme active group or maintain the activity of the enzyme. It should be understood that by adding the trace elements in the present invention, especially the addition of iron element therein (the other trace elements play the role of auxiliary groups or activators), the activity and stability of the inositol oxidase produced by the recombinant engineering bacteria are greatly improved, providing a good foundation for subsequent biotransformation and fermentation.

[0022] Furthermore, by combining the above examples, it can be seen that the present invention does not require any special requirements for the type of defoaming agent, and preferably uses a commercially available liquid defoaming agent. The amount of defoaming agent added in the present invention is preferably 0.05 to 0.15 ml / L, more preferably 0.1 ml / L. The defoaming agent can be added selectively according to the specific situation. For example, if there is too much foam on the liquid surface, defoaming agent can be added manually according to the situation. It should be understood that adding too much defoaming agent is inappropriate, and that it is appropriate to retain a small amount of foam, otherwise it is likely to cause an insufficient oxygen supply.

[0023] Furthermore, by combining the above-mentioned examples, the present invention further includes carrying out feed control by adding a feed medium to the reaction system at a constant feed rate after a fermentation reaction has been carried out for a certain period of time. Preferably, the time point for the 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 a nitrogen-containing compound, and 1000 to 1500 mg / L of trace elements. The trace elements in the feed medium of the present invention are preferably 20-30 mg / L CoCl2.6H2O, 100-200 mg / L MnSO4.4H2O, 10-20 mg / L CuCl2.2H2O, 20-40 mg / L H3BO3, 20-30 mg / L Na2MoO4.2H2O, 100-150 mg / L Zn(CH3COO)2.2H2O, and 500-1500 mg / L Fe(III) citrate. The nitrogen-containing compounds in the feed medium of the present invention are preferably peptone, yeast powder, or corn steep liquor powder, more preferably yeast powder. It should be understood that the feed medium of the present invention is primarily distinguished from the fermenter medium by its high glucose concentration. This is because the main function of the feed medium is to provide nutrients and essential trace elements to the bacterial cells in the system. In particular, the addition of nitrogen-containing compounds in the present invention significantly improves the stability of the inositol oxidase produced by the bacterial cells, thereby facilitating the biological conversion of inositol.

[0024] Furthermore, by combining the above embodiments, the present invention performs induction control by adding an inducer during the fermentation reaction process. In the present invention, the time point for adding the inducer is preferably when the OD 600 value reaches 70 - 80, more preferably when OD 600 = 75. The inducer in the present invention is preferably a monosaccharide, more preferably arabinose. It should be understood that the action of the inducer added in the present invention is to initiate the expression of the protein gene.

[0025] Furthermore, regarding the feed control in the present invention by combining the above embodiments, it is carried out step by step. Specifically, within 0 - 3 h from the start of feeding, the control of the feed rate is preferably 600 - 700 g / h, more preferably 650 g / h. From 3 h after the start of feeding until the addition of the inducer, the control of the feed rate is preferably 900 - 1100 g / h, more preferably 1000 g / h. After the addition of the inducer, the control of the feed rate is preferably 700 - 800 g / h, more preferably 750 g / h. However, through experiments, it has been verified that when the feed rate is controlled at 650 g / h within 0 - 3 h from the start of feed control, at 1000 g / h from 3 h after the start of feeding until the addition of the inducer, and at 750 g / h after the addition of the inducer, the production amount of inositol oxidase is high and stable technical effects can be achieved. The starting point of the feed control generally refers to the case where due to the continuous expression of the bacterial cells and the nutrient deficiency caused by the consumption of the medium, the enzyme production amount and yield of the product are significantly reduced. By performing feed control on the reaction system, nutrients can be timely supplemented to the bacterial cells, and the efficient production of inositol oxidase can be guaranteed. Combining with the improvement of enzyme activity by trace elements in the above-mentioned fermentation tank medium, the overall improvement of the enzyme activity, stability, and concentration of inositol oxidase can be realized, and a good foundation for the conversion of inositol can be provided.

[0026] After obtaining the fermentation broth by fermentation culture, the present invention centrifuged the fermentation broth to obtain wet bacterial cells. The centrifugation process of the present invention preferably employs a centrifuge, the rotation speed is preferably 14000 - 18000 rpm, more preferably 16000 rpm, and the centrifugation time is preferably 15 - 25 min, more preferably 20 min.

[0027] In another embodiment, the present invention may further subject the fermentation broth to membrane filtration treatment. The present invention has no special requirements for the membrane, as long as the corresponding filtration and blocking function can be realized. Preferably, it is a ceramic membrane, and the pore size of the membrane is preferably controlled to be 50 - 100 nm, more preferably 80 nm.

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

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

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

[0031] Furthermore, by combining the above embodiments, in the present invention, wet bacterial cells were added to the reaction solution and then conversion was carried out. During the conversion process, preferably the pH was adjusted to 7 - 9, more preferably to 8, preferably the dissolved oxygen content was controlled at 40% or more, more preferably the dissolved oxygen content was controlled at 50%, and preferably the conversion time was 6 - 8 h, more preferably 7 h.

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

[0033] Furthermore, by combining the above embodiments, after the conversion is completed, the present invention further includes filtering the conversion solution, collecting it to obtain bacterial cells, and providing them for repeated use. In the present invention, for the above filtration, preferably a ceramic membrane is used for filtration, and the pore size of the ceramic membrane is preferably 50 - 100 nm, more preferably 80 nm. In the present invention, the repeated use means that after filtering to obtain wet bacterial cells, the wet bacterial cells can be directly added to the inositol reaction solution again to carry out the conversion of inositol.

[0034] To better illustrate the production technology solution of D - glucuronic acid 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 raw materials unless otherwise specified.

[0035] Example 1 This example provides a method for producing D-glucuronic acid, and the steps are as follows: S1 to S6 below.

[0036] S1. Inoculate a single colony of recombinant engineering bacteria into 300 ml of LB medium, and perform shaking culture at 37 °C and 220 rpm for 5.5 h to obtain a primary seed solution. S2. When OD 600 = 2.2, inoculate the primary seed solution into 30 L of seed tank medium for culture. When OD 600 = 2.12, complete the secondary seed culture. The inoculation amount is 2%, and a seed solution is obtained. The composition of the 30 L seed tank medium is Glucose 1 wt%, potassium dihydrogen phosphate 1 wt%, magnesium sulfate 0.05 wt%, citric acid 0.15 wt%, ammonium sulfate 0.5 wt%, 20 mg / L of CoCl2·6H2O, 120 mg / L of MnSO4·4H2O, 15 mg / L of CuCl2·2H2O, 30 mg / L of H3BO3, 25 mg / L of Na2MoO4·2H2O, 120 mg / L of Zn(CH3COO)2·2H2O, 1 g / L of Fe(III) citrate, antifoaming agent 0.1 ml / L, and the rest is water. The conditions for the secondary seed culture are Temperature: 37 °C, air volume: 0.5 m 3 / h, rotation speed: 300 rpm, pressure: 0.02 MPa, pH: adjusted to pH 7 with ammonia water, dissolved oxygen content: 20%. S3. Inoculate the seed solution into 100 L of fermentation tank medium for 36 h of fermentation culture to obtain a fermentation broth. The inoculation amount is 10%. The composition of the 100 L fermentation tank medium is Glucose 1 wt%, potassium dihydrogen phosphate 1 wt%, magnesium sulfate 0.06 wt%, citric acid 0.2 wt%, ammonium sulfate 0.5 wt%, 20 mg / L of CoCl2·6H2O, 120 mg / L of MnSO4·4H2O, 12 mg / L of CuCl2·2H2O, 25 mg / L of H3BO3, 25 mg / L of Na2MoO4·2H2O, 130 mg / L of Zn(CH3COO)2·2H2O, 0.8 g / L of Fe(III) citrate, antifoaming agent 0.1 ml / L, and the rest is water. The conditions for fermentation culture are Temperature: 37 °C, air volume: 1.5 m 3 / h, rotation speed: 200 rpm, pressure: 0.02 MPa, pH: adjusted to pH 7 with aqueous ammonia, dissolved oxygen content: 25%, and S4. Add the feed medium to the reaction system of step S3 for feed control. When OD 600 = 75, add arabinose (inducer) for induction, and The composition of the feed medium is by mass per liter of the feed medium Glucose 600 g / L, magnesium sulfate 2 g / L, yeast powder 10 g / L, 25 mg / L of CoCl2·6H2O, 150 mg / L of MnSO4·4H2O, 15 mg / L of CuCl2·2H2O, 30 mg / L of H3BO3, 25 mg / L of Na2MoO4·2H2O, 120 mg / L of Zn(CH3COO)2·2H2O, 1000 mg / L of Fe(III) citrate, and the rest is water. When cultured in a culture tank for about 12 h, the dissolved oxygen increased significantly, the pH increased, and the feed was started. The addition rate of feed control was controlled at a feed rate of 700 g / h within 0 - 3 h from the start of feeding, at a feed rate of 900 g / h from 3 h after the start of feeding to the addition of the inducer, and at a feed rate of 800 g / h after the addition of the inducer. The OD of the fermentation broth 600 = 150.3 was obtained, and S5. Centrifuge the fermentation broth at 18000 rpm for 25 min to obtain wet bacterial cells, and S6. Add 25 g / L of the wet bacterial cells to a reaction solution containing inositol with a mass fraction of 5 wt% and boric acid with a concentration of 40 mM, and convert it under the conditions of pH = 8 and a dissolved oxygen content of 40% for 6 h to obtain D-glucuronic acid.

[0037] Through the above steps, the molar conversion rate of inositol is 98.4% or more, the content of the obtained D-glucuronic acid is 53.0 g / L, and the inositol oxidase can be recycled.

[0038] Example 2 This example provides a method for producing D-glucuronic acid, and the steps are as follows: S1 to S8 below.

[0039] S1. Inoculate a single colony of recombinant engineering bacteria into 300 ml of LB medium, and perform shaking culture at 37 °C and 200 rpm for 5.5 h to obtain a primary seed solution. S2. When OD 600 = 2.26, inoculate the primary seed solution into 30 L of seed tank medium for culture. When OD 600 = 2.12, complete the secondary seed culture. The inoculation amount is 2%, and a seed solution is obtained. The composition of the 30 L seed tank medium is Glucose 2 wt%, potassium dihydrogen phosphate 2 wt%, magnesium sulfate 0.05 wt%, citric acid 0.15 wt%, ammonium sulfate 0.5 wt%, 20 mg / L of CoCl2·6H2O, 120 mg / L of MnSO4·4H2O, 15 mg / L of CuCl2·2H2O, 30 mg / L of H3BO3, 25 mg / L of Na2MoO4·2H2O, 120 mg / L of Zn(CH3COO)2·2H2O, 0.8 g / L of Fe(III)citrate, antifoaming agent 0.1 ml / L, and the rest is water. The conditions for the secondary seed culture are Temperature: 37 °C, air volume: 0.5 m 3 / h, rotation speed: 300 rpm, pressure: 0.02 MPa, pH: adjusted to pH 7 with ammonia water, dissolved oxygen content: 20%. S3. Inoculate the seed solution into 100 L of fermentation tank medium for 36 h of fermentation culture to obtain a fermentation broth. The inoculation amount is 10%. The composition of the 100 L fermentation tank medium is Glucose 1 wt%, potassium dihydrogen phosphate 2 wt%, magnesium sulfate 0.08 wt%, citric acid 0.1 wt%, ammonium sulfate 0.5 wt%, 20 mg / L of CoCl2·6H2O, 120 mg / L of MnSO4·4H2O, 12 mg / L of CuCl2·2H2O, 25 mg / L of H3BO3, 25 mg / L of Na2MoO4·2H2O, 130 mg / L of Zn(CH3COO)2·2H2O, 0.8 g / L of Fe(III)citrate, antifoaming agent 0.08 ml / L, and the rest is water. The conditions for fermentation culture are Temperature: 37 °C, air volume: 1.5 m 3 / h, rotation speed: 200 rpm, pressure: 0.02 MPa, pH: adjusted to pH 7 with aqueous ammonia, dissolved oxygen content: 25%, and S4. Add the feed medium to the reaction system of step S3 for feed control. When OD 600 = 70, add arabinose (inducer) for induction, and The composition of the feed medium is Glucose 600 g / L, magnesium sulfate 2 g / L, yeast powder 10 g / L, 25 mg / L of CoCl₂·6H₂O, 150 mg / L of MnSO₄·4H₂O, 15 mg / L of CuCl₂·2H₂O, 30 mg / L of H₃BO₃, 25 mg / L of Na₂MoO₄·2H₂O, 120 mg / L of Zn(CH₃COO)₂·2H₂O, 1000 mg / L of Fe(III) citrate, and the rest is water. When cultured in a culture tank for about 12 h, the dissolved oxygen increased significantly, the pH increased, and the feed was started. The addition rate for feed control was to control the feed rate at 700 g / h within 0 - 3 h from the start of feeding, control the feed rate at 900 g / h from 3 h after the start of feeding until the addition of the inducer, and control the feed rate at 700 g / h after the addition of the inducer, and the OD of the fermentation broth 600 = 151.4 was obtained, and S5. Filter the fermentation broth through a 50 nm ceramic membrane to obtain wet bacterial cells, and S6. Add 25 g / L of the wet bacterial cells to a reaction solution containing inositol with a mass fraction of 7 wt% and boric acid with a concentration of 50 mM, Convert under the conditions of pH = 8 and dissolved oxygen content of 40% for 6 h to obtain D-glucuronic acid, and S7. After 2 h from the start of conversion, add an aqueous inositol solution with a mass concentration of 15 wt% to the reaction system in a flow addition manner. The addition time is 2 h, and the mass of the added inositol is 5 wt% of the total mass of the aforementioned reaction system. S8. Filter the conversion solution through an 80 nm ceramic membrane, collect it to obtain bacterial cells, and use them repeatedly.

[0040] After the above steps, the molar conversion rate of inositol is 85.7% or more, the content of the obtained D-glucuronic acid is 89.3 g / L, and the inositol oxidase can be recycled.

[0041] Example 3 S1. Inoculate a single colony of recombinant engineering bacteria into 300 ml of LB medium, and perform shaking culture at 37 °C and 220 rpm for 5 h to obtain a primary seed solution. S2. When OD 600 = 2.18, inoculate the primary seed solution into 30 L of seed tank medium for culture. When OD 600 = 2.08, complete the secondary seed culture with an inoculation amount of 2% to obtain a seed solution. The composition of the 30 L seed tank medium is Glucose 1 wt%, potassium dihydrogen phosphate 1 wt%, magnesium sulfate 0.05 wt%, citric acid 0.15 wt%, ammonium sulfate 0.5 wt%, 20 mg / L of CoCl2·6H2O, 120 mg / L of MnSO4·4H2O, 15 mg / L of CuCl2·2H2O, 30 mg / L of H3BO3, 25 mg / L of Na2MoO4·2H2O, 120 mg / L of Zn(CH3COO)2·2H2O, 1 g / L of Fe(III) citrate, antifoaming agent 0.1 ml / L, and the rest is water. The conditions for the secondary seed culture are Temperature: 37 °C, air volume: 0.5 m 3 / h, rotation speed: 300 rpm, pressure: 0.02 MPa, pH: adjusted to pH 7 with ammonia water, dissolved oxygen content: 20%. S3. Inoculate the seed solution into 100 L of fermentation tank medium for fermentation culture for 36 h to obtain a fermentation broth with an inoculation amount of 10%. The composition of the 100 L fermentation tank medium is Glucose 1 wt%, potassium dihydrogen phosphate 2 wt%, magnesium sulfate 0.08 wt%, citric acid 0.1 wt%, ammonium sulfate 0.5 wt%, 20 mg / L CoCl2.6H2O, 120 mg / L MnSO4.4H2O, 12 mg / L CuCl2.2H2O, 25 mg / L H3BO3, 25 mg / L Na2MoO4.2H2O, 130 mg / L Zn(CH3COO)2.2H2O, 0.8 g / L Fe(III) citrate, antifoaming agent 0.08 ml / L, and the remainder was water. The fermentation conditions are: Temperature: 37℃, Air volume: 1.8m 3 / h, rotation speed: 220 rpm, pressure: 0.03 MPa, pH: adjusted to pH 7 with ammonia water, dissolved oxygen content: 25%, S4. Feed medium is added to the reaction system in step S3 to control the feed rate. 600 When the ratio reaches 80, arabinose (inducer) is added to induce the reaction. The composition of the feed medium is, by mass per liter of feed medium: The mixture was 600g / L glucose, 2g / L magnesium sulfate, 10g / L yeast flour, 25mg / L CoCl2.6H2O, 150mg / L MnSO4.4H2O, 15mg / L CuCl2.2H2O, 30mg / L H3BO3, 25mg / L Na2MoO4.2H2O, 120mg / L Zn(CH3COO)2.2H2O, 1000mg / L Fe(III) citrate, and the remainder was water. After culturing for about 12 hours in the culture tank, the dissolved oxygen concentration and pH increased significantly, and feeding was initiated. The feed rate was controlled to 650 g / h within 0-3 hours of the start of feeding, and then to 1000 g / h from 3 hours after the start of feeding until the addition of the inducer. After the addition of the inducer, the feed rate was controlled to 750 g / h. The OD of the fermentation liquid was 600 = 152.3, S5. The fermentation liquid is filtered through a 75 nm ceramic membrane to obtain wet microbial cells; S6. 30 g / L of the wet bacterial cells were mixed with a mass fraction of 5 wt% inositol, a concentration of 30 mM phosphate, and a concentration of 3 mM Fe. 2+added to the reaction solution containing, under the conditions of pH = 8 and dissolved oxygen content of 50%, it was converted for 8 h to obtain D-glucuronic acid, 1.8 h after the start of conversion, an aqueous inositol solution with a mass concentration of 12 wt% was added to the reaction system in a flow-added manner. The addition time was 1.5 h, and the mass of the added inositol was 5 wt% of the total mass of the aforementioned reaction system, S8. The conversion solution was filtered through a 100 nm ceramic membrane, collected to obtain thalli, and provided for repeated use.

[0042] Through the above steps, the molar conversion rate of inositol was 95.7% or more, the content of the obtained D-glucuronic acid was 83.2 g / L, and the inositol oxidase could be recycled.

[0043] Example 4 This example provides a method for producing D-glucuronic acid. The steps are as follows: S1 to S8.

[0044] S1. A single colony of recombinant engineering bacteria was inoculated into 300 ml of LB medium and cultured with shaking for 5.5 h at 35 °C and 220 rpm to obtain a primary seed solution, S2. OD 600 When it reached 2, the primary seed solution was inoculated into a 30 L seed tank medium for culture. When OD 600 reached 2.4, the secondary seed culture was completed. The inoculation amount was 2%, and a seed solution was obtained. The composition of the 30 L seed tank medium was glucose 2 wt%, potassium dihydrogen phosphate 1 wt%, magnesium sulfate 0.05 wt%, citric acid 0.15 wt%, ammonium sulfate 0.6 wt%, 20 mg / L of CoCl2·6H2O, 120 mg / L of MnSO4·4H2O, 15 mg / L of CuCl2·2H2O, 30 mg / L of H3BO3, 25 mg / L of Na2MoO4·2H2O, 120 mg / L of Zn(CH3COO)2·2H2O, 0.8 g / L of Fe(III) citrate, antifoaming agent 0.1 ml / L, and the rest was water. The conditions for the secondary seed culture were Temperature: 37°C, air volume: 0.5 m 3 / h, rotation speed: 280 rpm, pressure: 0.015 MPa, pH: adjusted to pH 7 with aqueous ammonia, dissolved oxygen content: 30%, S3. Inoculate the seed solution into a 100 L fermentation tank medium and perform fermentation culture for 38 h to obtain a fermentation broth, with an inoculation amount of 10%, The composition of the 100 L fermentation tank medium is Glucose 1 wt%, potassium dihydrogen phosphate 1 wt%, magnesium sulfate 0.06 wt%, citric acid 0.2 wt%, ammonium sulfate 0.5 wt%, 20 mg / L of CoCl2·6H2O, 120 mg / L of MnSO4·4H2O, 12 mg / L of CuCl2·2H2O, 25 mg / L of H3BO3, 25 mg / L of Na2MoO4·2H2O, 130 mg / L of Zn(CH3COO)2·2H2O, 0.8 g / L of Fe(III) citrate, antifoaming agent 0.1 ml / L, and the rest is water, The conditions for fermentation culture are Temperature: 37°C, air volume: 1.5 m 3 / h, rotation speed: 200 rpm, pressure: 0.02 MPa, pH: adjusted to pH 7 with aqueous ammonia, dissolved oxygen content: 25%, S4. Add the feed medium to the reaction system in step S3 and perform feed control. When OD 600 = 75, add arabinose (inducer) for induction, The composition of the feed medium is in terms of mass per liter of the feed medium, Glucose 500 g / L, magnesium sulfate 3 g / L, yeast powder 8 g / L, 25 mg / L of CoCl2·6H2O, 150 mg / L of MnSO4·4H2O, 15 mg / L of CuCl2·2H2O, 30 mg / L of H3BO3, 25 mg / L of Na2MoO4·2H2O, 120 mg / L of Zn(CH3COO)2·2H2O, 1000 mg / L of Fe(III) citrate, and the rest is water, When cultured in the culture tank for about 10 h, the dissolved oxygen increased significantly, the pH increased, and the feed was started. The addition rate of feed control was controlled at 700 g / h within 0 - 3 h after the start of feeding, and from 3 h after the start of feeding to the addition of the inducer, the feed rate was controlled at 1000 g / h. After the addition of the inducer, the feed rate was controlled at 700 g / h, and the OD of the fermentation broth 600 = 156.8 was obtained, S5. Filter the fermentation broth through a 100 nm ceramic membrane to obtain wet bacterial cells, S6. Add 35 g / L of the wet bacterial cells to a reaction solution containing inositol with a mass fraction of 4 wt%, phosphate with a concentration of 20 mM, and Fe with a concentration of 4 mM 2+ and add it to the reaction solution, Under the conditions of pH = 7 and a dissolved oxygen content of 50%, convert for 7 h to obtain D-glucuronic acid, S7. 2 h after the start of conversion, add an aqueous inositol solution with a mass concentration of 15 wt% to the reaction system in a flow-added manner. The addition time is 2 h, and the mass of inositol added is 4 wt% of the total mass of the aforementioned reaction system, S8. Filter the conversion solution through a 100 nm ceramic membrane, collect it to obtain bacterial cells, and provide them for repeated use.

[0045] Through the above steps, the molar conversion rate of inositol is 96.8% or more, the content of D-glucuronic acid obtained is 86.5 g / L, and inositol oxidase can be recycled.

[0046] Example 5 This example provides a method for producing D-glucuronic acid. The steps are as follows: S1 - S8.

[0047] S1. Inoculate a single colony of recombinant engineering bacteria into 300 ml of LB medium, and shake and culture at 37 °C and 240 rpm for 6 h to obtain a primary seed solution, S2. When OD 600 = 2, inoculate the primary seed solution into a 30 L seed tank medium for culture. When OD 600 = 2, complete the secondary seed culture. The inoculation amount is 2%, and a seed solution is obtained, The composition of the 30 L seed tank medium is 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 CoCl₂·6H₂O, 120 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, 1 g / L Fe(III) citrate, 0.1 ml / L antifoaming agent, with the balance being water, The conditions for the secondary seed culture are Temperature: 37 °C, air flow rate: 0.5 m 3 / h, rotation speed: 300 rpm, pressure: 0.02 MPa, pH: adjusted to pH 7 with aqueous ammonia, dissolved oxygen content: 20%, S3. Inoculate the seed solution into the 100 L fermentation tank medium and perform fermentation culture for 36 h to obtain a fermentation broth, with an inoculation amount of 10%, The composition of the 100 L fermentation tank medium is 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 CoCl₂·6H₂O, 120 mg / L MnSO₄·4H₂O, 12 mg / L CuCl₂·2H₂O, 25 mg / L H₃BO₃, 25 mg / L Na₂MoO₄·2H₂O, 130 mg / L Zn(CH₃COO)₂·2H₂O, 0.8 g / L Fe(III) citrate, 0.08 ml / L antifoaming agent, with the balance being water, The conditions for the fermentation culture are Temperature: 37 °C, air flow rate: 1.8 m 3 / h, rotation speed: 220 rpm, pressure: 0.03 MPa, pH: adjusted to pH 7 with aqueous ammonia, dissolved oxygen content: 25%, S4. Add the feed medium to the reaction system of step S3 and perform feed control. When OD 600 = 80, add arabinose (inducer) for induction, The composition of the feed medium is in terms of mass per liter of the feed medium, The mixture was 700g / L glucose, 1g / L magnesium sulfate, 12g / L yeast flour, 25mg / L CoCl2.6H2O, 150mg / L MnSO4.4H2O, 15mg / L CuCl2.2H2O, 30mg / L H3BO3, 25mg / L Na2MoO4.2H2O, 120mg / L Zn(CH3COO)2.2H2O, 1000mg / L Fe(III) citrate, and the remainder was water. After culturing for about 14 hours in the culture tank, the dissolved oxygen concentration and pH increased significantly, and feeding was initiated. The feed rate was controlled to 700 g / h within 0 to 3 hours of the start of feeding, and then to 900 g / h from 3 hours after the start of feeding until the addition of the inducer. After the addition of the inducer, the feed rate was controlled to 750 g / h. 600 =153.1, S5. The fermentation liquid is centrifuged at 16,000 rpm for 20 minutes to obtain wet cells. S6.30 g / L of the wet bacterial cells was added to a reaction solution containing inositol at a mass fraction of 7 wt% and boric acid at a concentration of 40 mM; After 7 hours of conversion under conditions of pH=8 and dissolved oxygen content of 50%, D-glucuronic acid was obtained. S7. 2 hours after the start of conversion, add an aqueous solution of inositol with a mass concentration of 15 wt% to the reaction system by flow addition, the addition time is 2 hours, and the mass of inositol added is 4.5 wt% of the total mass of the reaction system; S8. The inverted solution was filtered through a 50 nm ceramic membrane and collected to obtain bacterial cells for repeated use.

[0048] After the above steps, the molar conversion rate of inositol was 86.2% or more, the content of D-glucuronic acid obtained was 85.1 g / L, and the inositol oxidase could be recycled.

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

[0050] S1. Inoculate a single colony of the recombinant engineering bacteria into 300 ml of LB medium, and perform shaking culture at 40 °C and 240 rpm for 6 h to obtain a primary seed solution. S2. When OD 600 = 2, inoculate the primary seed solution into 30 L of seed tank medium for culture. When OD 600 = 2.14, complete the secondary seed culture with an inoculation amount of 2% to obtain a seed solution. The composition of the 30 L seed tank medium is Glucose 2 wt%, potassium dihydrogen phosphate 1 wt%, magnesium sulfate 0.05 wt%, citric acid 0.15 wt%, ammonium sulfate 0.6 wt%, 20 mg / L of CoCl2·6H2O, 120 mg / L of MnSO4·4H2O, 15 mg / L of CuCl2·2H2O, 30 mg / L of H3BO3, 25 mg / L of Na2MoO4·2H2O, 120 mg / L of Zn(CH3COO)2·2H2O, 0.8 g / L of Fe(III) citrate, antifoaming agent 0.1 ml / L, and the rest is water. The conditions for the secondary seed culture are Temperature: 37 °C, air volume: 0.5 m 3 / h, rotation speed: 320 rpm, pressure: 0.03 MPa, pH: adjusted to pH 7 with ammonia water, dissolved oxygen content: 30%. S3. Inoculate the seed solution into 100 L of fermentation tank medium for 40 h of fermentation culture to obtain a fermentation broth with an inoculation amount of 10%. The composition of the 100 L fermentation tank medium is Glucose 1 wt%, potassium dihydrogen phosphate 1 wt%, magnesium sulfate 0.06 wt%, citric acid 0.2 wt%, ammonium sulfate 0.5 wt%, 20 mg / L of CoCl2·6H2O, 120 mg / L of MnSO4·4H2O, 12 mg / L of CuCl2·2H2O, 25 mg / L of H3BO3, 25 mg / L of Na2MoO4·2H2O, 130 mg / L of Zn(CH3COO)2·2H2O, 0.8 g / L of Fe(III) citrate, antifoaming agent 0.1 ml / L, and the rest is water. The conditions for the fermentation culture are Temperature: 37 °C, air volume: 1.5 m 3 / h, Rotation speed: 200 rpm, Pressure: 0.02 MPa, pH: Adjusted to pH 7 with aqueous ammonia, Dissolved oxygen content: 25%, S4. Add the feed medium to the reaction system of Step S3 to perform feed control. When OD 600 = 75, add arabinose (inducer) for induction, The composition of the feed medium is by mass per liter of the feed medium, Glucose 600 g / L, Magnesium sulfate 3 g / L, Yeast powder 8 g / L, 25 mg / L of CoCl2·6H2O, 150 mg / L of MnSO4·4H2O, 15 mg / L of CuCl2·2H2O, 30 mg / L of H3BO3, 25 mg / L of Na2MoO4·2H2O, 120 mg / L of Zn(CH3COO)2·2H2O, 1000 mg / L of Fe(III) citrate, and the rest is water, After culturing in the culture tank for about 11.5 h, the dissolved oxygen increased significantly, the pH increased, and the feed was started. The addition rate of the feed control was controlled at a feed rate of 700 g / h within 0 - 3 h from the start of the feed, at a feed rate of 1100 g / h from 3 h after the start of the feed to the addition of the inducer, and at a feed rate of 800 g / h after the addition of the inducer. The OD of the fermentation broth 600 = 161.2 was obtained, S5. Centrifuge the fermentation broth at 16000 rpm for 15 min to obtain wet bacterial cells, S6. Add 35 g / L of the wet bacterial cells to a reaction solution containing inositol with a mass fraction of 4 wt% and boric acid with a concentration of 60 mM, Convert under the conditions of pH = 7 and a dissolved oxygen content of 50% for 8 h to obtain D-glucuronic acid, S7. 1.5 h after the start of the conversion, add an aqueous inositol solution with a mass concentration of 12 wt% to the reaction system in a flow-added manner. The addition time is 2 h, and the mass of the aforementioned inositol is 4.5 wt% of the total mass of the aforementioned reaction system, S8. Filter the conversion solution through a 70 nm ceramic membrane, collect it to obtain bacterial cells, and supply them for repeated use.

[0051] After the above steps, the molar conversion rate of inositol was 97.6% or more, the content of the obtained D-glucuronic acid was 85.4 g / L, and the inositol oxidase could be recycled.

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

[0053] Finally, as a result, the OD of the fermentation broth obtained in Step S4 600 = 122.3, the molar conversion rate of inositol was 56.8% or more, and the content of the obtained D-glucuronic acid was 54.7 g / L.

[0054] This was presumably because the inositol oxide produced during the fermentation process became unstable due to the lack of nitrogen-containing compounds, causing a decrease in the inositol conversion rate.

[0055] Comparative Example 2 Compared with Example 2, in Comparative Example 2, the trace elements in the seed tank medium were replaced with an equal amount of glucose, and other reaction conditions remained unchanged.

[0056] Finally, as a result, the molar conversion rate of inositol was 53.6% or more, and the content of the obtained D-glucuronic acid was 42.7 g / L.

[0057] This was presumably because the lack of trace elements caused a lack of cell components, resulting in slow cell growth. On the other hand, the lack of the assistance of key trace elements for the enzyme active group caused a decrease in enzyme activity, ultimately leading to a decrease in the inositol conversion rate.

[0058] Comparative Example 3 Compared with Example 2, in Comparative Example 3, the trace elements in the fermentation tank medium were replaced with an equal amount of glucose, and other reaction conditions remained unchanged.

[0059] Finally, as a result, the molar conversion rate of inositol was 45.2% or more, and the content of the obtained D-glucuronic acid was 34.7 g / L.

[0060] Compared with Comparative Example 2, the further decrease in the molar conversion rate of inositol and the content of D-glucuronic acid was because the number of bacterial cells was larger in the fermentation process and the demand for trace elements was also larger.

[0061] Comparative Example 4 Compared with Example 2, in Comparative Example 4, the trace elements in the feed medium were replaced with an equal amount of nitrogen-containing compounds, and the other reaction conditions remained unchanged.

[0062] Finally, as a result, the molar conversion rate of inositol was 68.2% or more, and the content of the obtained D-glucuronic acid was 56.4 g / L.

[0063] Compared with Comparative Examples 2 and 3, the relatively small decrease in the molar conversion rate of inositol and the content of D-glucuronic acid was because the nitrogen-containing compound itself belongs to an organic substance containing a small amount of trace elements, so a part of the trace elements may have partially played a role in supplementing the trace elements and improving the activity of some enzymes.

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

[0065] Finally, as a result, the molar conversion rate of inositol was 36.8% or more, and the content of the obtained D-glucuronic acid was 22.5 g / L.

[0066] It was speculated that this was because the iron element in the trace elements played an important role in the activation of inositol oxidase and played a more important role in the activity of the enzyme compared with other trace elements.

[0067] Comparative Example 6 Compared with Example 2, in Comparative Example 6, no feed control was adopted during the fermentation process, and other reaction conditions remained unchanged.

[0068] Finally, as a result, the molar conversion rate of inositol was 0, and the content of D-glucuronic acid obtained was 0.

[0069] This is because when feed control is not performed, even if an inducer is added later, protein expression is not induced, and the conversion reaction of inositol cannot proceed.

[0070] Comparative Example 7 Compared with Example 3, in Comparative Example 7, during the fermentation process, the feed rate of the adopted feed control was not performed step by step, and it was changed to be added at a constant rate of 650 g / h throughout the process, and other reaction conditions remained unchanged.

[0071] Finally, as a result, the molar conversion rate of inositol was 87% or more, and the content of D-glucuronic acid obtained was 67.2 g / L.

[0072] This is presumed to have caused the fermentation process in the reaction system not to be gentle before and after the addition of the inducer due to the addition of the inducer. If it is always added at the same feed rate, the requirement to maintain a gentle fermentation process cannot be met. If the reaction accelerates suddenly after the addition of the inducer, it will cause a situation where nutrients are insufficient, thereby causing the reaction rate to decelerate and the product to decrease.

[0073] Note that after producing D-glucuronic acid by the above process, D-glucuronic acid can be further converted into a D-glucuronic acid derivative, i.e., glucuronolactone, which is commonly known as hepatone. The prior art mainly uses a composite acid reagent composed of D-glucuronic acid, phosphoric acid, and sulfuric acid to carry out an esterification reaction to produce glucuronolactone. However, in the case of the composite acid esterification reaction, since sulfuric acid is present, when water is present or generated in the reaction system, a large amount of heat is released from the reaction system, and sulfuric acid has strong oxidizing properties. If the temperature is too high during the reaction process, the carbonization of the reaction product will become serious, thereby causing a decrease in the yield. Based on this, in order to avoid the generation of water during the reaction process, it is necessary to control the reaction system to be carried out at a relatively high concentration. Usually, the Baumé degree of the D-glucuronic acid concentrated solution reaches 46-47 Baumé degrees. At this concentration, the fluidity of the system is almost lost. On the other hand, the esterification reaction needs to be strictly controlled to be carried out at a relatively low temperature of 35 °C or below, and it is not necessary to raise the temperature and concentrate during the process. In this way, the time required for the reaction becomes long. Generally, the total production time from the reaction to crystallization exceeds 60 h. On the other hand, because the concentration of the reaction system is too high, the fluidity of the reaction system is low, and ethanol must be added during crystallization. Ethanol is a flammable and explosive chemical. When used during production, there is a certain safety risk. In addition, in the reaction system, the crystal particles of the produced reaction product, glucuronolactone, are fine, the particle size of the particles is generally larger than 50 mesh, and it is difficult to filter. At the same time, the temperature drop and stirring of the reaction system are also strictly controlled, and it is necessary to lower the temperature to -8 °C. In this way, the operation of the entire production process becomes complicated, and the production efficiency is extremely low.

[0074] In order to solve the above technical problems, an embodiment of the present invention further provides a production process of glucuronolactone, including the step of adding concentrated phosphoric acid to a D-glucuronic acid solution, carrying out an esterification reaction at a certain reaction temperature while stirring, crystallizing, and obtaining crude glucuronolactone.

[0075] In the above technical solution, concentrated phosphoric acid is used instead of the mixed acid of phosphoric acid and sulfuric acid in the prior art, and by controlling the concentration of phosphoric acid and the reaction temperature, an esterification reaction is carried out with a D-glucuronic acid solution to produce glucuronolactone. By adopting the above technical solution to produce glucuronolactone, since the fluidity of the reaction system is good, it is carried out while stirring, and the total time from the reaction to the completion of crystallization is less than 6 h, greatly shortening the reaction crystallization time, shortening the production cycle, and improving the production efficiency. Further, in the esterification reaction system of the present invention, only concentrated phosphoric acid is used, the temperature of the reaction system is controllable, and the produced glucuronolactone has the advantages of large crystal particles, easy filtration, and high product yield.

[0076] Furthermore, by combining the above examples, in the examples of the present invention, it is further limited that the solid content of the D-glucuronic acid solution can be selected from 50 wt% to 70 wt%. By measurement, the solid content corresponds to 20 to 30 Baumé degrees, the viscosity of the D-glucuronic acid solution is about 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%. At this solid content, the D-glucuronic acid solution has an appropriate viscosity. In the esterification reaction process of reacting the D-glucuronic acid solution with concentrated phosphoric acid, the rotation speed of stirring can generally be controlled at 80 to 120 rpm so that the system has high fluidity. Under the reaction system and reaction conditions of the examples of the present invention, there is no need to add ethanol, and glucuronolactone crystals can be directly obtained by crystallization, and the particle size of the product is large and can reach 20 mesh to 30 mesh.

[0077] Furthermore, by 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 as 70 wt%, 75 wt%, 80 wt% or 85 wt%. The inventor verified by tests that by only limiting the mass concentration of the concentrated phosphoric acid within a certain range, the esterification reaction between the concentrated phosphoric acid and the D-glucuronic acid solution can proceed smoothly. Moreover, when the mass concentration of the concentrated phosphoric acid is selected to be 70 wt% to 85 wt% and combined with the solid content of the D-glucuronic acid solution being 50 wt% to 70 wt%, the maximum esterification reaction rate can be achieved at a reaction temperature of 40°C to 80°C. At this time, the yield of the obtained glucuronolactone is the highest, the content of glucuronolactone in the crude glucuronolactone can reach 95 wt% or more, and it is found that the crystal particle size is uniform, the particle size is large, and it is easy to filter.

[0078] Furthermore, by combining the above embodiments, the embodiments of the present invention further limit the addition amount of the concentrated phosphoric acid to 10 wt% to 50 wt% of the mass of the solid content in the D-glucuronic acid solution, and the esterification reaction time is 1 h to 2 h.

[0079] According to the above technical solution, when the addition amount of the concentrated phosphoric acid is 10 wt% to 50 wt% of the mass of the solid content 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 improved. Under the above reaction conditions, generally, if the reaction time is 1 h to 2 h, the esterification reaction can be completed, the reaction cycle is significantly shortened, and the reaction efficiency is improved.

[0080] Furthermore, by combining the above embodiments, the embodiments of the present invention further include evaporating water from the product of the esterification reaction at a temperature of 40°C to 80°C and a vacuum degree of ≤ -0.09 MPa before the crystallization after the esterification reaction, and the volume of the evaporated water is 30% to 50% of the volume of the reaction solution.

[0081] Furthermore, the crystallization includes dynamically gradient-cooling the product of the esterification reaction at a rate of 5 to 10 °C / h for crystallization, with the crystallization end temperature being 5 to 15 °C, to obtain crude glucuronolactone.

[0082] According to the above technical solution, throughout the esterification crystallization process, since the fluidity of the reaction system is good, there is no need to add ethanol. By controlling the product of the esterification reaction to be dynamically gradient-cooled at a rate of 5 to 10 °C / h, glucuronolactone crystals can be directly crystallized, and the crystal particles are large and can reach 20 to 30 mesh. At the same time, the system obtained after the above crystallization is a solid-liquid mixing system, and it is easy to separate and filter glucuronolactone. The crystallization process is simple in operation, and the total time of the esterification reaction and crystallization may be shortened to 6 hours at the shortest. In the prior art, the crystallization time in the double acid reaction process is long, and the safety problems caused by adding ethanol in the crystallization process are overcome. At the same time, the content of glucuronolactone in the crude glucuronolactone obtained by adopting the above technical solution is ≥ 95 wt%, and the crystallization rate is ≥ 80%.

[0083] Furthermore, in the embodiments of the present invention, the D-glucuronic acid solution may be prepared from commercially available D-glucuronic acid, may be produced by reacting an inositol raw material solution with inositol oxidase, or may further be produced by the production methods of Examples 1 to 6 of the present invention. Exemplarily, when the D-glucuronic acid solution is produced by the reaction of an inositol raw material solution with inositol oxidase, specifically, it includes preliminarily preparing an inositol raw material solution and adding inositol oxidase to the inositol raw material solution for conversion. For example, the conversion reaction conditions between inositol and inositol oxidase may refer to a reaction temperature of 30 to 40°C and a reaction pH value of 8.0 to 9.0. The conversion solution is filtered and concentrated in sequence to obtain a D-glucuronic acid concentrate with a solid content of 50 wt% to 70 wt%. Furthermore, according to the inventor's tests, the D-glucuronic acid concentrate corresponded to a Baume degree with the above solid content of 20 to 30 and had a viscosity of 9 to 10 mPa·s. Since a concentrated phosphoric acid esterification reaction system is adopted, the D-glucuronic acid concentrate only needs to correspond to a concentration of 20 to 30 Baume degrees. It is easy to obtain the D-glucuronic acid concentrate at this concentration, with low concentrated energy consumption, no need to replenish the acid again during the crystallization process, short time, and further shortened reaction time.

[0084] Furthermore, by combining the above embodiments, in the filtration of the embodiments of the present invention, the conversion solution is filtered through a ceramic membrane with a separation pore diameter of 20 to 100 nm, and then the ceramic membrane filtrate is collected. The ceramic membrane filtrate is filtered through an ultrafiltration membrane with a pore diameter of 5000 to 20000 Da, and the ultrafiltration filtrate is collected. Through filtration by the ceramic membrane and filtration by the ultrafiltration membrane, purification of the D-glucuronic acid product can be realized, impurities therein can be quickly filtered and removed, and it has the advantages of high separation efficiency and excellent impurity removal effect.

[0085] Furthermore, by combining the above embodiments, in the embodiments of the present invention, after filtering the conversion solution and before concentration, the filtrate is desalted with a cation exchange resin to obtain a desalted solution with a conductivity of <7000 us / cm. Exemplarily, a strongly acidic cation exchange resin can be selected as the cation exchange resin. It should be understood that, unlike the D-glucuronic acid solution prepared with commercially available D-glucuronic acid, in the process of converting inositol to prepare the D-glucuronic acid solution, a certain amount of salt is added, and the presence of the salt may have an adverse effect on the subsequent crystallization process. Therefore, by desalting and controlling the desalting conductivity to be smaller, crystals are more likely to be obtained. Verification by tests showed that by controlling the desalting conductivity to <7000 us / cm, crystals with uniform particle size and large particle size could be obtained.

[0086] Furthermore, by combining the above embodiments, in the embodiments of the present invention, after filtering the conversion solution and before concentration, the filtrate is adsorbed with a macroporous adsorption resin for decolorization, and then the decolorized solution is collected. Exemplarily, LS-108 or LS-109D can be selected as the macroporous adsorption resin, and further, the D-glucuronic acid solution obtained by the reaction of inositol and inositol oxidase is decolorized to further improve the purity of the D-glucuronic acid solution and facilitate the subsequent esterification reaction.

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

[0088] According to the above technical solution, in the embodiments of the present invention, before the esterification reaction, a two-stage dehydration operation is performed on the obtained D-glucuronic acid solution to reduce or avoid the dissolution loss of glucuronolactone in water, thereby improving the crystal yield of glucuronolactone.

[0089] To better explain the technical solution of the present invention, the present invention further provides the following specific examples to further explain the 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 raw materials unless otherwise specified. Among them, D-glucuronic acid may also be produced by the above production process as long as it meets the performance requirements such as the solid content and viscosity of D-glucuronic acid after treatment.

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

[0091]

Table 1

[0092] Example 7 This example provides a production process for glucuronolactone, including the following steps S1 and S2.

[0093] S1. Concentrated phosphoric acid with a mass concentration of 70 wt% was added to 5.8 L of a D-glucuronic acid solution (viscosity 12.4 mPa·s) with a solid content of 73 wt%. The addition amount of the concentrated phosphoric acid was 58% of the mass of the solid content 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 dynamically gradient-cooled at a rate of 5 °C / h for crystallization, and the crystallization end temperature was 5 °C to obtain crude glucuronolactone. The crude glucuronolactone was suction-filtered, and the solid was washed with 2.0 L of absolute ethanol and vacuum-dried to obtain 3161 g of white crystals.

[0095] After passing through a liquid chromatogram test, the content of glucuronolactone in the white crystals was 95.3 wt%, the crystallization rate was 82.3%, and the particle size of glucuronolactone was 20 - 25 mesh.

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

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

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

[0099] S3. The product of the esterification reaction after being treated in step S2 was dynamically gradient-cooled at a rate of 10 °C / h for crystallization, and the crystallization end temperature was 10 °C to obtain crude glucuronolactone. The crude glucuronolactone was suction-filtered, and the solid was washed with 2.0 L of absolute ethanol and vacuum-dried to obtain 2453 g of white crystals.

[0100] After passing through a liquid chromatogram test, the content of glucuronolactone in the white crystals was 95.7 wt%, the crystallization rate was 84.5%, and the particle size of glucuronolactone was 25 - 30 mesh.

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

[0102] S1. Concentrated phosphoric acid with a mass concentration of 84 wt% was added to 6.0 L of a D-glucuronic acid solution (viscosity 11.8 mPa·s) with a solid content of 70 wt%. The addition amount of the concentrated phosphoric acid was 48% of the mass of the solid content in the D-glucuronic acid solution. An 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. For the product of the esterification reaction, water was evaporated at a temperature of 40 °C and a vacuum degree of -0.09 MPa, and the volume of the evaporated water was 30% of the volume of the reaction solution.

[0104] S3. The product of the esterification reaction after being treated in Step S2 was dynamically gradient-cooled at a rate of 5 °C / h for crystallization, and the final crystallization temperature was 10 °C, obtaining crude glucuronolactone. The crude glucuronolactone was suction-filtered, the solid was washed with 2.0 L of absolute ethanol, and vacuum-dried to obtain 3361 g of white crystals.

[0105] After passing through a liquid chromatogram test, the content of glucuronolactone in the white crystals was 97.7 wt%, the crystallization rate was 88.2%, and the particle size of glucuronolactone was 20 - 25 mesh.

[0106] Example 10 This example provides a production process of glucuronolactone, including the following steps S1 - S4.

[0107] S1. An inositol raw material solution was preliminarily prepared, and inositol oxidase was added to the inositol raw material solution for conversion to obtain a conversion solution. The specific steps include 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 5 μg of functional protein: 2 mmol of L-cysteine: 20 mmol of inositol: 1 mmol of ferrous sulfate, and in the reaction system, the initial concentration of each component was 100 μg / mL of functional protein, 2 mmol / L of L-cysteine, 0.3 mol / L of inositol, and Fe 2+ 1 mmol / L. The buffer system was a 50 mM Tris-HCl buffer with a pH of 8.0. The conversion temperature was 37 °C, the reaction pH was 8.0, and the reaction time was 6 - 8 h.

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

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

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

[0111] S4. The product of the esterification reaction after being treated in step S3 was dynamically gradient-cooled at a rate of 8 °C / h for crystallization. The crystallization end temperature was 10 °C, and crude glucuronolactone was obtained. The crude glucuronolactone was suction-filtered, the solid was washed with 2.0 L of absolute ethanol, and vacuum-dried to obtain 1895 g of white crystals.

[0112] After passing through the liquid chromatography test, the content of glucuronolactone in the white crystal was 98.6 wt%, the crystallization rate was 86.4%, and the particle size of glucuronolactone was 25 - 30 mesh.

[0113] Example 11 This example provides a production process of glucuronolactone, including the following steps S1 - S4.

[0114] S1. Prepare a preliminary inositol raw material solution, add inositol oxidase to the inositol raw material solution for conversion to obtain a conversion solution. The specific steps include the following. Add functional protein, L - cysteine, inositol and ferrous sulfate to the buffer system. The mixing ratio of functional protein, L - cysteine, inositol and ferrous sulfate is 1×10 5 μg of functional protein: 2 mmol of L - cysteine: 20 mmol of inositol: 1 mmol of ferrous sulfate. In the reaction system, the initial concentration of each component is 100 μg / mL of functional protein, 2 mmol / L of L - cysteine, 0.3 mol / L of inositol, Fe 2+ 1 mmol / L. The buffer system is a 50 mM Tris - HCl buffer with pH 8.0. The conversion temperature is 37°C, the reaction pH is 8.0, and the reaction time is 30 min.

[0115] 33.9L of the converted liquid with a solid content of 6.1wt% was passed through a ceramic membrane with a pore size of 100nm, and the ceramic membrane filtrate was collected. The operating temperature was 35°C, the pressure in the membrane was 0.5MPa, the pressure out of the membrane was 0.2MPa, and the membrane flow rate was 80L / h. The ceramic membrane filtrate was then filtered again through an ultrafiltration membrane with a pore size of 20,000Da. The operating temperature was 35°C, the pressure in the membrane was 0.3MPa, the pressure out of the membrane was 0.2MPa, and the membrane flow rate was 150L / h. The ultrafiltration liquid was collected. The ultrafiltrate was desalted using a strong acidic cation exchange resin to obtain a desalted solution. The conductivity of the desalted solution was 5000 μs / cm. The desalted solution was then decolorized by adsorption using an LS-108D macroporous adsorption resin. The decolorized solution was then collected and 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 flow rate was 40 L / h. The nanofiltration concentrate had a solids content of 15 wt%. The nanofiltration concentrate was then concentrated using a concentrator to obtain 3.4 L of D-glucuronic acid concentrate (viscosity 8.3 MPa·s) with a solids content of 53 wt%.

[0116] S2. Concentrated phosphoric acid with a mass concentration of 74 wt% was added to 3.4 L of a D-glucuronic acid concentrated solution with a solid content of 53 wt%, and the amount of concentrated phosphoric acid added was 11% of the mass of the solid content 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] S2. The water content of the esterification reaction product was evaporated at a temperature of 60°C and a vacuum pressure of -0.09 MPa, and the volume of evaporated water was 45% of the volume of the reaction solution.

[0118] S3. The esterification product after step S2 was crystallized by dynamically decreasing the temperature at a rate of 10°C / h until the crystallization temperature reached 10°C, yielding crude glucuronolactone. The crude glucuronolactone was filtered by suction, and the solid was washed with 2.0 L of absolute ethanol and dried under vacuum to yield 1401 g of white crystals.

[0119] After passing through the liquid chromatography test, the content of glucuronolactone in the white crystals was 96.1 wt%, the crystallization rate was 85.7%, and the particle size of glucuronolactone was 20 - 25 mesh.

[0120] Example 12 This example provides a production process of glucuronolactone, including the following steps S1 - S4.

[0121] S1. Prepare a preliminary inositol raw material solution, add inositol oxidase to the inositol raw material solution for conversion to obtain a conversion solution. The specific steps include the following. Add functional protein, L - cysteine, inositol, and ferrous sulfate to the buffer system. The mixing ratio of functional protein, L - cysteine, inositol, and ferrous sulfate is 1×10 5 μg of functional protein: 2 mmol of L - cysteine: 20 mmol of inositol: 1 mmol of ferrous sulfate. In the reaction system, the initial concentration of each component is 100 μg / mL of functional protein, 2 mmol / L of L - cysteine, 0.3 mol / L of inositol, and Fe 2+ 1 mmol / L. The buffer system is a 50 mM Tris - HCl buffer at pH 8.0. The reaction temperature is 37°C, the reaction pH is 8.0, and the reaction time is 30 min.

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

[0123] S2. Add 0.48 L of concentrated phosphoric acid with a mass concentration of 79 wt% to 3.9 L of the D - glucuronic acid concentrated solution with a solid content of 62 wt%, and carry out an esterification reaction at a reaction temperature of 70°C and a stirring rotation speed of 110 rpm for 1.5 hours.

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

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

[0126] According to the detection by liquid chromatography, the content of glucuronolactone in the above white crystals was 98.1 wt%, the crystallization rate was 89.7%, and the particle size of glucuronolactone was 20 - 25 mesh.

[0127] Comparative Example 8 Compared with Example 7, concentrated phosphoric acid with a mass concentration of 70 wt% was equivalently replaced with concentrated sulfuric acid with a mass concentration of 70 wt%, and other reaction conditions remained unchanged.

[0128] According to the test, crystalline glucuronolactone could not be obtained in the reaction system, and the system directly became a black viscous liquid.

[0129] Comparative Example 9 Compared with Example 7, concentrated phosphoric acid with a mass concentration of 70 wt% was equivalently replaced 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%, and the mass ratio of concentrated sulfuric acid to concentrated phosphoric acid in the mixed acid was 1:1, and other reaction conditions remained unchanged.

[0130] Under such conditions, the reaction system turned black, a small amount of crystals were obtained, and the color of the crystals became gray. According to the liquid chromatogram test, the content of glucuronolactone was 62.3 wt%, the crystallization rate was 21.4%, and the particle size was >50 mesh.

[0131] Since the water content in the reaction system was high, it was speculated that sulfuric acid generated heat during the reaction process, leading to an increase in the carbonization rate of the product.

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

[0133] According to the liquid chromatogram test, the content of glucuronolactone produced under this condition was 65.3 wt%, the crystallization rate was 30.4%, the appearance was powdery, and the particle size was >50 mesh.

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

[0135] According to the test, almost no glucuronolactone was obtained under this condition.

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

[0137] According to the liquid chromatogram test, the content of glucuronolactone produced under this condition was 85.1 wt%, the crystallization rate 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 to 12 and Comparative Examples 8 to 12 of the present invention were as follows.

[0139] Mobile phase: 10 mmol / L aqueous formic acid solution, Chromatographic column: Calcium column (300 * 7.7 or similar column), Flow rate: 0.5 ml / min, Detector: Differential detector, Column temperature: 55 °C, Detector temperature: 45°C, Solvent: 10 mmol / L aqueous formic acid solution, Concentration of standard product: 1.0 mg / mL (D-glucuronolactone [CAS No.] 32449-92-6, calculated as C6H8O6, content 99.9%, National Institutes for Food and Drug Control), Concentration of test sample: 1.0 mg / mL, Chromatograph conditions: Operate with 100% 10 mmol / L aqueous formic acid solution for 30 min.

[0140] Here, taking the liquid chromatography diagram of Example 10 as an example, 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 content of glucuronolactone 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 mixing a D-glucuronic acid solution with a certain concentration and concentrated phosphoric acid with a certain concentration, the viscosity of the system can reach 8 - 12 mPa·s. In this case, by stirring, the two can react, and there is no need to add ethanol. Under the condition that the total time from reaction to crystallization is 6 hours or less, coarse crystals with uniform particle size and a particle size range reaching 20 mesh - 30 mesh can be obtained. It has advantages such as short crystallization time, high production efficiency, large crystal particles, easy filtration, and high product yield.

[0142] The above are only preferred embodiments of the present invention and do not limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in each of the foregoing embodiments, or perform equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should all be included within the protection scope of the present invention.

Claims

1. (1) culturing a recombinant engineering bacterium to obtain a seed solution; (2) inoculating the seed solution into a fermentation tank 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, wherein step (2) includes performing feed control and induction control after inoculating the seed solution into the fermentation tank medium and culturing, and is a method for producing D-glucuronic acid, characterized in that.

2. The culturing process of the seed solution in step (1) is step A of primary seed culture in which the recombinant engineering bacterium is inoculated into an LB medium and cultured at 200-240 rpm and 34-40 °C for 5-6 h; OD ６００ When OD = 2 - 3, inoculate and culture in the seed tank medium, and when OD ６００ reaches 2 - 3, complete the seed culture to obtain the secondary seed culture for obtaining the seed solution, including step B, The specific composition of the seed tank medium is 1 wt% - 2 wt% glucose, 1 wt% - 2 wt% potassium dihydrogen phosphate, 0.05 wt% - 0.08 wt% magnesium sulfate, 0.1 wt% - 0.2 wt% citric acid, 0.4 wt% - 0.6 wt% ammonium sulfate, 1000 - 1500 mg / L of trace elements, 0.05 - 0.15 ml / L of antifoaming agent, and the balance is water; The conditions for the secondary seed culture Temperature: 34 to 40 °C, air volume: 0.4 to 0.6 m ３ / h, rotation speed: 280 to 320 rpm, pressure: 0.01 to 0.03 MPa, pH: adjusted to pH 7.0 ± 0.1 with aqueous ammonia, dissolved oxygen content: 20 to 30%, The method for producing D-glucuronic acid according to claim 1, characterized in that.

3. The fermentation culture in the step (2) ends when OD ６００ is stably maintained after 36 to 40 h, and the fermentation broth is obtained, The specific composition of the fermentation tank medium is 1 wt% - 2 wt% glucose, 1 wt% - 2 wt% potassium dihydrogen phosphate, 0.05 wt% - 0.08 wt% magnesium sulfate, 0.1 wt% - 0.2 wt% citric acid, 0.4 wt% - 0.6 wt% ammonium sulfate, 1000 - 1500 mg / L of trace elements, 0.05 - 0.15 ml / L of antifoaming agent, and the balance is water; The fermentation culture conditions Temperature: 34 to 40 °C, air volume: 1.2 to 1.8 m ３ / h, rotation speed: 180 to 220 rpm, pressure: 0.01 to 0.03 MPa, pH: adjusted to pH 7.0 ± 0.1 with aqueous ammonia, dissolved oxygen content: 20 to 30%, The method for producing D-glucuronic acid according to claim 1, characterized in that.

4. The feed control is to perform feed control by adding a feed medium to the reaction system after 10 - 14 hours of fermentation culture. The feed medium contains 500 - 700 g / L of glucose, 1 - 3 g / L of magnesium sulfate, 8 - 12 g / L of nitrogen-containing compound, 1000 - 1500 mg / L of trace elements, and the balance is water. The method for producing D-glucuronic acid according to claim 1 or 3.

5. The induction control is OD ６００ The method for producing D-glucuronic acid according to claim 1 or 3, characterized in that an inducer is added when ６００ reaches 70 to 80.

6. Controlling the feed rate according to the stage includes controlling the feed rate to 600 - 700 g / h within 0 - 3 h from the start of feeding, controlling the feed rate to 900 - 1100 g / h from 3 h after the start of feeding until the addition of the inducer, and controlling the feed rate to 700 - 800 g / h after the addition of the inducer. The method for producing D-glucuronic acid according to claim 5 is characterized by this.

7. In the step (3), the centrifugation conditions are centrifuging for 15 - 25 min under the condition of 14000 - 18000 rpm, and the pore size of the filtration membrane used for the above membrane filtration is 50 - 100 nm. The method for producing D-glucuronic acid according to claim 1 is characterized by this.

8. The reaction solution contains inositol with a mass fraction of 4 - 7 wt% and boric acid with a concentration of 40 - 60 mM. Alternatively, the reaction solution contains inositol with a mass fraction of 4 to 7 wt%, phosphate with a concentration of 20 to 40 mM, and Fe with a concentration of 2 to 4 mM ２＋ and Based on the total volume of the reaction solution, the addition amount of the wet bacterial cells is 25 - 35 g / L. After the step of adding the wet bacterial cells to the reaction solution, it further includes adjusting the pH to 7 - 9 and controlling the dissolved oxygen amount to 40% or more. The conversion time is 6 - 8 h. The method for producing D-glucuronic acid according to claim 1 is characterized by this.

9. It further includes continuously adding inositol with a mass fraction of 4 - 6 wt% to the reaction system 1.5 - 2 h after the start of the conversion. The inositol is in the form of an aqueous solution with a mass concentration of 12 - 15 wt%, the addition method is flow addition, and the time is 1.5 - 2.5 h. The method for producing D-glucuronic acid according to claim 8 is characterized by this.

10. After the step (4), it further includes filtering the conversion solution, collecting it to obtain bacterial cells, and providing them for repeated use. The method for producing D-glucuronic acid according to claim 1 is characterized by this.

11. The production process of glucuronolactone includes adding concentrated phosphoric acid to the D-glucuronic acid solution and performing an esterification reaction at a reaction temperature of 40°C - 80°C while stirring, followed by crystallization to obtain crude glucuronolactone.

12. 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 addition amount of the concentrated phosphoric acid is 10 wt% to 50 wt% of the mass of the solid content in the D-glucuronic acid solution, and the esterification reaction time is 1 h to 2 h. The process for producing glucuronolactone according to claim 11 is characterized in that.

13. After the esterification reaction and before the crystallization, The method further includes evaporating water from the product of the esterification reaction at a temperature of 40°C to 80°C and a degree of vacuum of ≤ -0.09 MPa, and the volume of the evaporated water is 30% to 50% of the volume of the reaction solution. The process for producing glucuronolactone according to claim 11 is characterized in that.

14. The crystallization includes dynamically gradient-cooling the product of the esterification reaction at a rate of 5 to 10°C / h for crystallization, with the final crystallization temperature being 5 to 15°C, to obtain crude glucuronolactone. The glucuronolactone content in the crude glucuronolactone is ≥ 95 wt% or more, and the crystallization rate is ≥ 80% or more. The process for producing glucuronolactone according to claim 11 is characterized in that.

15. The D-glucuronic acid solution is produced by reacting an inositol raw material solution with inositol oxidase. The process for producing glucuronolactone according to claim 11 is characterized in that.

16. The reaction between the inositol raw material solution and inositol oxidase is The inositol raw material solution preparation step of adding inositol oxidase to the inositol raw material solution for conversion, filtering the conversion solution in sequence, concentrating it, and obtaining a D-glucuronic acid concentrate with a solid content of 50 wt% to 70 wt%. The process for producing glucuronolactone according to claim 15 is characterized in that.

17. The filtration is Filtering the conversion solution with a ceramic membrane having a separation pore diameter of 20 to 100 nm and collecting the ceramic membrane filtrate, Filtering the ceramic membrane filtrate with an ultrafiltration membrane having a pore diameter of 5000 to 20000 Da and collecting the ultrafiltration filtrate. The process for producing glucuronolactone according to claim 16 is characterized in that.

18. The process for producing glucuronolactone according to claim 16, further comprising: after filtering the conversion solution and before concentration, desalting the filtrate with a cation exchange resin to obtain a desalted solution having a conductivity of <7000 us / cm.

19. The process for producing glucuronolactone according to claim 18, further comprising: after adsorbing and decolorizing the desalted solution with a macroporous adsorption resin, collecting the decolorized solution.

20. The concentration concentrating the liquid to be concentrated using a nanofiltration membrane with a pore size of 150 - 300 Da, and collecting a nanofiltration concentrate having a solid content of 10 wt% - 15 wt%; concentrating the nanofiltration concentrate with a concentrator to obtain a D-glucuronic acid concentrate having a solid content of 50 wt% - 70 wt%, the process for producing glucuronolactone according to claim 16.

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