CN116970581A - Glucuronic acid preparation and separation method - Google Patents
Glucuronic acid preparation and separation method Download PDFInfo
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- CN116970581A CN116970581A CN202310944297.6A CN202310944297A CN116970581A CN 116970581 A CN116970581 A CN 116970581A CN 202310944297 A CN202310944297 A CN 202310944297A CN 116970581 A CN116970581 A CN 116970581A
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- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 title claims abstract description 56
- 229940097043 glucuronic acid Drugs 0.000 title claims abstract description 55
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 title claims abstract 14
- 238000000926 separation method Methods 0.000 title abstract description 14
- 239000000706 filtrate Substances 0.000 claims abstract description 38
- 239000011347 resin Substances 0.000 claims abstract description 35
- 229920005989 resin Polymers 0.000 claims abstract description 35
- 238000006243 chemical reaction Methods 0.000 claims abstract description 31
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims abstract description 28
- 229960000367 inositol Drugs 0.000 claims abstract description 28
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000007788 liquid Substances 0.000 claims abstract description 27
- 238000001914 filtration Methods 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 22
- 239000000047 product Substances 0.000 claims abstract description 20
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims abstract description 16
- 102000004316 Oxidoreductases Human genes 0.000 claims abstract description 12
- 108090000854 Oxidoreductases Proteins 0.000 claims abstract description 12
- 239000003054 catalyst Substances 0.000 claims abstract description 12
- 238000001694 spray drying Methods 0.000 claims abstract description 12
- 238000002156 mixing Methods 0.000 claims abstract description 9
- 238000003756 stirring Methods 0.000 claims abstract description 9
- 230000035484 reaction time Effects 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 28
- 239000012528 membrane Substances 0.000 claims description 27
- 239000002253 acid Substances 0.000 claims description 11
- 239000007787 solid Substances 0.000 claims description 11
- 239000000919 ceramic Substances 0.000 claims description 9
- 238000001728 nano-filtration Methods 0.000 claims description 9
- 238000000108 ultra-filtration Methods 0.000 claims description 9
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 8
- 239000003729 cation exchange resin Substances 0.000 claims description 8
- 239000012527 feed solution Substances 0.000 claims description 6
- 239000012141 concentrate Substances 0.000 claims description 3
- 238000002955 isolation Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 41
- 238000005406 washing Methods 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 230000000052 comparative effect Effects 0.000 description 7
- 239000012295 chemical reaction liquid Substances 0.000 description 6
- 239000008367 deionised water Substances 0.000 description 6
- 229910021641 deionized water Inorganic materials 0.000 description 6
- 239000000945 filler Substances 0.000 description 6
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 241001052560 Thallis Species 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 206010016952 Food poisoning Diseases 0.000 description 1
- 208000019331 Foodborne disease Diseases 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N Furan Chemical compound C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 229920000715 Mucilage Polymers 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- AEMOLEFTQBMNLQ-WAXACMCWSA-N alpha-D-glucuronic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-WAXACMCWSA-N 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229950002441 glucurolactone Drugs 0.000 description 1
- 150000008134 glucuronides Chemical class 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- -1 salt ions Chemical class 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H7/00—Compounds containing non-saccharide radicals linked to saccharide radicals by a carbon-to-carbon bond
- C07H7/02—Acyclic radicals
- C07H7/033—Uronic acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
- C12Y113/99—Miscellaneous (1.13.99)
- C12Y113/99001—Inositol oxygenase (1.13.99.1), i.e. myo-inositol oxygenase
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Abstract
The invention relates to the technical field of glucuronic acid production, in particular to a glucuronic acid preparation and separation method, which comprises the following steps: (1) Taking inositol feed liquid, adding a whole-cell catalyst for expressing inositol oxidase, stirring and mixing, and controlling the reaction temperature, pH and reaction time to obtain reaction feed liquid for later use; (2) Taking reaction feed liquid, filtering for the first time, filtering for the second time, and collecting secondary filtrate for standby; (3) Taking secondary filtrate, and passing through a resin column to obtain effluent with the conductivity less than 5000us/cm for later use; (4) Taking the effluent liquid with the conductivity less than 5000us/cm in the step (3), concentrating for the first time, and concentrating the collected first concentrated solution for the second time to obtain a second concentrated solution for later use; (5) And taking the second concentrated solution, and carrying out spray drying to obtain the glucuronic acid product. The process can obtain glucuronic acid product with high yield, high yield and high purity.
Description
Technical Field
The invention relates to the technical field of glucuronic acid production, in particular to a preparation and separation method of glucuronic acid.
Background
Glucuronic acid (Glucuronic acid), abbreviated as Glucuronic acid, is a compound formed by oxidizing the primary hydroxyl group of glucose into carboxyl group, and is widely distributed in animals and plants. Gum such as acacia gum in the form of a uronic acid in plants is an important component constituting pectin, mucilage and higher polysaccharides. In animals, glucuronic acid and glucuronide form complexes which are stored in tissues. Glucuronic acid with molecular formula of C 6 H 10 O 7 Relative molecular weight 194.14, powder or white needle crystals. The aqueous solution of glucuronic acid is unstable, and 3, 6-glucuronolactone is easily formed to be in a state of interconversion equilibrium, and D-glucuronic acid is easily decarboxylated to generate CO when being heated and in the presence of strong acid 2 Furan and other cleavage products. Glucuronic acid can be combined with in vivo poison to form nontoxic glucuronic acid conjugate for discharge, has liver protecting and toxic substance removing effects, and can be used for treating hepatitis, liver cirrhosis, food and drug poisoning. Glucuronic acid is also an important component of connective tissue and can be used to treat arthritis and collagenous diseases.
Currently, the preparation methods of glucuronic acid mainly comprise a polysaccharide hydrolysis method, a chemical oxidation method, a biological fermentation method and the like, but the methods have some problems: the glucuronic acid obtained by the chemical oxidation method has low yield and purity, and can generate a large amount of byproducts, so that the separation is difficult; the biological fermentation method is adopted, the process is complex, the yield of the obtained glucuronic acid is low, and the separation is difficult; the polysaccharide hydrolysis method has the advantages that the stability of the glycosidic bond connected with uronic acid is high, strong acid-base hydrolysis conditions are needed, the hydrolysis is difficult, the product yield is low, a large amount of wastewater is generated, and the production requirement cannot be met; therefore, in view of the above problems, it is necessary to develop a process for producing glucuronic acid.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: aiming at the defects of the prior art, a preparation and separation method of glucuronic acid is provided, and the preparation and separation method is adopted.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a process for the preparation and isolation of glucuronic acid, said process comprising the steps of:
(1) Taking inositol feed liquid, adding a whole-cell catalyst for expressing inositol oxidase, stirring and mixing, and controlling the reaction temperature, pH and reaction time to obtain reaction feed liquid for later use;
(2) Taking the reaction feed liquid in the step (1), and filtering the primary filtrate collected after primary filtering for secondary filtering, wherein the collected secondary filtrate is reserved;
(3) Taking the secondary filtrate in the step (2), and passing through a resin column to obtain an effluent with the conductivity less than 5000us/cm for later use;
(4) Taking the effluent liquid with the conductivity less than 5000us/cm in the step (3), concentrating for the first time, and concentrating the collected first concentrated solution for the second time to obtain a second concentrated solution for later use;
(5) And (3) taking the second concentrated solution in the step (4), and carrying out spray drying to obtain a glucuronic acid product.
As an improved technical scheme, the inositol feed liquid in the step (1) is added according to the addition amount of 80-150g/L, and the addition amount of the inositol oxidase-expressing whole cell catalyst is such that the OD of the concentration of the inositol in the reaction system is that 600nm 30-50.
As an improved technical scheme, the reaction time in the step (1) is controlled to be 1-3h; the temperature of the reaction is controlled at 32 ℃ and the pH is 7.8 or 7.0 or 6.5; or the temperature of the reaction was controlled to 38℃and the pH was controlled to 7.3 or 6.8.
As an improved technical scheme, ceramic membrane with the pore diameter of 20-100nm is adopted for filtering in the step (2) for the first time; and filtering with ultrafiltration membrane with molecular weight cut-off of 500-1000 Da.
As an improved technical scheme, the secondary filtrate in the step (3) enters the resin column at the flow rate of 1-2 BV/h.
As an improved technical scheme, the resin type in the resin column in the step (3) is weak acid cation exchange resin, and the resin type comprises D111FC or D113SC.
As an improved technical scheme, the nanofiltration membrane with the molecular weight cut-off of 100-150Da is adopted for concentration in the step (4) for one-time concentration; the solid content of the secondary concentrated solution is 30-50wt%.
As an improved technical scheme, the air inlet temperature in spray drying in the step (5) is 100-140 ℃, the outlet temperature is 70-90 ℃, and the feeding flow is 4.0-6.0mL/min.
After the technical scheme is adopted, the invention has the beneficial effects that:
the invention takes inositol as raw material, adds a whole cell catalyst for expressing inositol oxidase, controls the reaction temperature, pH and time, obtains material liquid containing glucuronic acid after reaction, firstly filters the material liquid by a ceramic membrane (can effectively remove thalli), filters the collected primary filtrate by an ultrafiltration membrane (can effectively remove macromolecular proteins and organic matters), and the collected secondary filtrate passes through a primary resin column (can effectively remove salt ions and pigments), and the collected effluent is concentrated by a nanofiltration membrane for the first time, and then is concentrated by a concentrator to obtain concentrated solution with the solid content of 30-50wt%, and finally obtains glucuronic acid products with high yield and high purity by spray drying.
Drawings
FIG. 1 is a liquid phase detection chart of glucuronic acid in example 4 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
A preparation and separation method of glucuronic acid comprises the following steps:
(1) 60L of inositol feed solution with the concentration of 80g/L is taken and added with a whole cell catalyst (OD of thallus concentration) for expressing inositol oxidase 600nm 30) stirring and mixing, controlling the reaction temperature at 32 ℃ and the pH value at 7.8, and reacting for 3 hours to obtain reaction feed liquid for later use;
(2) Taking 50L of the reaction liquid in the step (1), carrying out primary filtration by adopting a ceramic membrane with the aperture of 20-100nm, carrying out secondary filtration by adopting an ultrafiltration membrane with the molecular weight cut-off of 500-1000Da on the collected primary filtrate, adding water, and carrying out top washing on the collected secondary filtrate for later use;
(3) Taking 55L of the secondary filtrate in the step (2), feeding the secondary filtrate into a resin column (the resin filler is weak acid cation exchange resin, the resin model is D111 FC) according to the flow rate of 1BV/h, and performing top washing with deionized water to obtain effluent with the conductivity of 4500us/cm for later use;
(4) Taking 60L of effluent liquid with the conductivity of 4500us/cm in the step (3), firstly adopting a nanofiltration membrane with the molecular weight cut-off of 100-150Da to perform primary concentration, and then performing thermal concentration (the temperature is 50 ℃ and the vacuum is less than-0.09 MPa) on the collected first concentrated solution through a concentrator to perform secondary concentration to obtain a second concentrated solution with the solid content of 30wt% for later use;
(5) And (3) taking 10L of the second concentrated solution in the step (4), and performing spray drying (the inlet air temperature is 100 ℃, the outlet air temperature is 70 ℃, the feeding flow is 4.0 mL/min) to obtain 2983g of glucuronic acid product, wherein the yield is 96.1%, and the purity is 99.1%.
Example 2
A preparation and separation method of glucuronic acid comprises the following steps:
(1) 60L of inositol feed solution with the concentration of 100g/L is taken and added with a whole cell catalyst (OD with the concentration of thalli) for expressing inositol oxidase 600nm 35) stirring and mixing, controlling the reaction temperature at 32 ℃ and the pH value at 7.0, and reacting for 2.8 hours to obtain reaction feed liquid for standby;
(2) Taking 50L of the reaction liquid in the step (1), carrying out primary filtration by adopting a ceramic membrane with the aperture of 20-100nm, carrying out secondary filtration by adopting an ultrafiltration membrane with the molecular weight cut-off of 500-1000Da on the collected primary filtrate, adding water, and carrying out top washing on the collected secondary filtrate for later use;
(3) Taking 55L of the secondary filtrate in the step (2), feeding the secondary filtrate into a resin column (the resin filler is weak acid cation exchange resin, the resin model is D111 FC) according to the flow rate of 1.3BV/h, and performing top washing with deionized water to obtain an effluent with the conductivity of 4000us/cm for later use;
(4) Taking 60L of effluent liquid with the conductivity of 4000us/cm in the step (3), firstly adopting a nanofiltration membrane with the molecular weight cut-off of 100-150Da to perform primary concentration, and then performing thermal concentration (the temperature is 55 ℃ and the vacuum is less than-0.09 MPa) on the collected first concentrated solution by a concentrator to perform secondary concentration to obtain a second concentrated solution with the solid content of 35wt% for later use;
(5) And (3) taking 10L of the second concentrated solution in the step (4), and performing spray drying (the inlet air temperature is 115 ℃, the outlet air temperature is 75 ℃, the feeding flow is 4.5 mL/min) to obtain 3474g of glucuronic acid product, wherein the yield is 96.4%, and the purity is 99.4%.
Example 3
A preparation and separation method of glucuronic acid comprises the following steps:
(1) 60L of inositol feed solution with the concentration of 120g/L is taken and added with a whole cell catalyst (OD of thallus concentration) for expressing inositol oxidase 600nm 40) stirring and mixing, controlling the reaction temperature at 38 ℃ and the pH value at 7.3, and reacting for 3 hours to obtain reaction feed liquid for standby;
(2) Taking 50L of the reaction liquid in the step (1), carrying out primary filtration by adopting a ceramic membrane with the aperture of 20-100nm, carrying out secondary filtration by adopting an ultrafiltration membrane with the molecular weight cut-off of 500-1000Da on the collected primary filtrate, adding water, and carrying out top washing on the collected secondary filtrate for later use;
(3) Taking 55L of the secondary filtrate in the step (2), feeding the secondary filtrate into a resin column (the resin filler is weak acid cation exchange resin, the resin model is D113 SC) according to the flow rate of 1.5BV/h, and performing top washing with deionized water to obtain an effluent with the conductivity of 3500us/cm for later use;
(4) Taking 60L of effluent liquid with the conductivity of 3500us/cm in the step (3), firstly adopting a nanofiltration membrane with the molecular weight cut-off of 100-150Da to perform primary concentration, and then performing thermal concentration (the temperature is 60 ℃ and the vacuum is less than-0.09 MPa) on the collected first concentrated solution by a concentrator to perform secondary concentration to obtain a second concentrated solution with the solid content of 40wt% for later use;
(5) And (3) taking 10L of the second concentrated solution in the step (4), and performing spray drying (the inlet air temperature is 120 ℃, the outlet air temperature is 80 ℃, and the feeding flow is 5.0 mL/min) to obtain 3930g of glucuronic acid product, wherein the yield is 95.8%, and the purity is 99.2%.
Example 4
A preparation and separation method of glucuronic acid comprises the following steps:
(1) 60L of inositol feed solution with a concentration of 135g/L is taken and added with a whole cell catalyst (OD of cell concentration) for expressing inositol oxidase 600nm 45) stirring and mixing, controlling the reaction temperature at 38 ℃ and the pH value at 6.8, and reacting for 2.5 hours to obtain reaction feed liquid for standby;
(2) Taking 50L of the reaction liquid in the step (1), carrying out primary filtration by adopting a ceramic membrane with the aperture of 20-100nm, carrying out secondary filtration by adopting an ultrafiltration membrane with the molecular weight cut-off of 500-1000Da on the collected primary filtrate, adding water, and carrying out top washing on the collected secondary filtrate for later use;
(3) Taking 55L of the secondary filtrate in the step (2), feeding the secondary filtrate into a resin column (the resin filler is weak acid cation exchange resin, the resin model is D113 SC) according to the flow rate of 1.5BV/h, and performing top washing with deionized water to obtain an effluent with the conductivity of 3000us/cm for later use;
(4) Taking 60L of effluent liquid with the conductivity of 3000us/cm in the step (3), firstly adopting a nanofiltration membrane with the molecular weight cut-off of 100-150Da to perform primary concentration, and then performing thermal concentration (the temperature is 65 ℃ and the vacuum is less than-0.09 MPa) on the collected first concentrated solution by a concentrator to perform secondary concentration to obtain a second concentrated solution with the solid content of 45wt% for later use;
(5) And (3) taking 10L of the second concentrated solution in the step (4), and performing spray drying (the inlet air temperature is 125 ℃, the outlet air temperature is 85 ℃, the feeding flow is 5.5 mL/min) to obtain 4470g of glucuronic acid product, wherein the yield is 97.1%, and the purity is 99.8%.
Example 5
A preparation and separation method of glucuronic acid comprises the following steps:
(1) 60L of inositol feed liquid with the concentration of 140g/L is taken and added with whole cells for expressing inositol oxidaseCatalyst (OD of cell concentration) 600nm 48), stirring and mixing, controlling the reaction temperature at 32 ℃ and the pH value at 6.5, and reacting for 2 hours to obtain reaction feed liquid for later use;
(2) Taking 50L of the reaction liquid in the step (1), carrying out primary filtration by adopting a ceramic membrane with the aperture of 20-100nm, carrying out secondary filtration by adopting an ultrafiltration membrane with the molecular weight cut-off of 500-1000Da on the collected primary filtrate, adding water, and carrying out top washing on the collected secondary filtrate for later use;
(3) Taking 55L of the secondary filtrate in the step (2), feeding the secondary filtrate into a resin column (the resin filler is weak acid cation exchange resin, the resin model is D113 SC) according to the flow rate of 2BV/h, and performing top washing with deionized water to obtain an effluent with the conductivity of 4300us/cm for later use;
(4) Taking 60L of effluent with the conductivity of 4300us/cm in the step (3), firstly adopting a nanofiltration membrane with the molecular weight cut-off of 100-150Da to perform primary concentration, and then performing thermal concentration (the temperature is 68 ℃ and the vacuum is < -0.09 MPa) on the collected first concentrated solution by a concentrator to perform secondary concentration to obtain a second concentrated solution with the solid content of 58wt% for later use;
(5) And (3) taking 10L of the second concentrated solution in the step (4), and performing spray drying (the inlet air temperature is 130 ℃, the outlet air temperature is 88 ℃, the feeding flow is 5.8 mL/min) to obtain 4732g of glucuronic acid product, wherein the yield is 96.5%, and the purity is 99.3%.
Example 6
A preparation and separation method of glucuronic acid comprises the following steps:
(1) 60L of inositol feed solution with the concentration of 150g/L is taken and added with a whole cell catalyst (OD with the concentration of thalli) for expressing inositol oxidase 600nm 50) stirring and mixing, controlling the reaction temperature at 38 ℃ and the pH value at 7.3, and reacting for 1.5h to obtain reaction feed liquid for standby;
(2) Taking 50L of the reaction liquid in the step (1), carrying out primary filtration by adopting a ceramic membrane with the aperture of 20-100nm, carrying out secondary filtration by adopting an ultrafiltration membrane with the molecular weight cut-off of 500-1000Da on the collected primary filtrate, adding water, and carrying out top washing on the collected secondary filtrate for later use;
(3) Taking 55L of the secondary filtrate in the step (2), feeding the secondary filtrate into a resin column (the resin filler is weak acid cation exchange resin, the resin model is D113 SC) according to the flow rate of 1BV/h, and performing top washing with deionized water to obtain effluent with the conductivity of 3300us/cm for later use;
(4) Taking 60L of effluent liquid with the conductivity of 3300us/cm in the step (3), firstly adopting a nanofiltration membrane with the molecular weight cut-off of 100-150Da to perform primary concentration, and then performing thermal concentration (the temperature is 70 ℃ and the vacuum is less than-0.09 MPa) on the collected first concentrated solution through a concentrator to perform secondary concentration to obtain a second concentrated solution with the solid content of 50wt% for later use;
(5) And (3) taking 10L of the second concentrated solution in the step (4), and performing spray drying (the inlet air temperature is 140 ℃, the outlet air temperature is 90 ℃, the feeding flow is 6.0 mL/min) to obtain 4865g of glucuronic acid product, wherein the yield is 95.3%, and the purity is 99.4%.
The corresponding preparation methods of the whole cell catalysts for expressing inositol oxidase in examples 1 to 6 have been disclosed in the prior art and will not be repeated here.
In order to better demonstrate that the process of the present invention allows to obtain a high yield, a high yield and a high purity glucuronic acid product compared to the prior art, the following comparative examples are given with reference to example 4, in particular in the following:
comparative example 1
Unlike example 4, the secondary filtrate in step (3) was first subjected to a cationic resin column (resin model D67), and the collected effluent was then subjected to a macroporous resin column (resin model LS-109D) to collect an effluent having a conductivity of 6800us/cm, and the remaining operations were the same; 3630g of glucuronic acid product is obtained in the step (5), the yield is 79.2% and the purity is 87.6%.
Comparative example 2
Unlike example 4, the second concentrate in step (4) had a solids content of 60wt% and the remainder was the same; 3510g of glucuronic acid product was obtained in step (5) in a yield of 78.4% and a purity of 81.2%.
Comparative example 3
Unlike example 4, the second concentrate in step (4) had a solids content of 70wt% and the remainder was the same; 2980g of glucuronic acid product was obtained in step (5) in 67.3% yield and 78.8% purity.
Comparative example 4
Unlike example 4, the secondary filtrate in step (3) was fed into the resin column at a flow rate of 2BV/h, and the rest was the same; 3440g of glucuronic acid product is obtained in the step (5), the yield is 77.4%, and the purity is 83.5%.
Comparative example 5
Unlike example 4, the temperature of the reaction in step (1) was controlled at 40℃and the pH was controlled at 8.5, and the rest of the operations were the same; 3720g of glucuronic acid product was obtained in the step (5), the yield was 83.6%, and the purity was 85.9%.
Comparative example 6
Unlike example 4, the temperature of the reaction in step (1) was controlled at 30℃and the pH was controlled at 7.5, and the rest of the operations were the same; 3330g of glucuronic acid product was obtained in step (5) in a yield of 75.0% and a purity of 84.1%.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (8)
1. A process for the preparation and isolation of glucuronic acid, characterized in that said process comprises the steps of:
(1) Taking inositol feed liquid, adding a whole-cell catalyst for expressing inositol oxidase, stirring and mixing, and controlling the reaction temperature, pH and reaction time to obtain reaction feed liquid for later use;
(2) Taking the reaction feed liquid in the step (1), and filtering the primary filtrate collected after primary filtering for secondary filtering, wherein the collected secondary filtrate is reserved;
(3) Taking the secondary filtrate in the step (2), and passing through a resin column to obtain an effluent with the conductivity less than 5000us/cm for later use;
(4) Taking the effluent liquid with the conductivity less than 5000us/cm in the step (3), concentrating for the first time, and concentrating the collected first concentrated solution for the second time to obtain a second concentrated solution for later use;
(5) And (3) taking the second concentrated solution in the step (4), and carrying out spray drying to obtain a glucuronic acid product.
2. The method for preparing and separating glucuronic acid according to claim 1, wherein: the inositol feed solution in the step (1) is added according to the addition amount of 80-150g/L, and the addition amount of the inositol oxidase-expressing whole cell catalyst is such that the OD of the concentration of the inositol in the reaction system is that of the inositol 600nm 30-50.
3. The method for preparing and separating glucuronic acid according to claim 1, wherein: the reaction time in the step (1) is controlled to be 1-3h; the temperature of the reaction is controlled at 32 ℃ and the pH is 7.8 or 7.0 or 6.5; or the temperature of the reaction was controlled to 38℃and the pH was controlled to 7.3 or 6.8.
4. The method for preparing and separating glucuronic acid according to claim 1, wherein: in the step (2), a ceramic membrane with the aperture of 20-100nm is adopted for filtering in the primary filtering; and filtering with ultrafiltration membrane with molecular weight cut-off of 500-1000 Da.
5. The method for preparing and separating glucuronic acid according to claim 1, wherein: and (3) feeding the secondary filtrate in the step (3) into a resin column at a flow rate of 1-2 BV/h.
6. The method for preparing and separating glucuronic acid according to claim 1, wherein: the resin type in the resin column in the step (3) is a weak acid cation exchange resin, and the resin type includes D111FC or D113SC.
7. The method for preparing and separating glucuronic acid according to claim 1, wherein: concentrating the primary concentrate in the step (4) by adopting a nanofiltration membrane with the molecular weight cut-off of 100-150 Da; the solid content of the secondary concentrated solution is 30-50wt%.
8. The method for preparing and separating glucuronic acid according to claim 1, wherein: the air inlet temperature in the spray drying in the step (5) is 100-140 ℃, the outlet temperature is 70-90 ℃, and the feeding flow is 4.0-6.0mL/min.
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