CN112080532A - Method for producing D-p-hydroxyphenylglycine by double-enzyme method - Google Patents
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- LJCWONGJFPCTTL-SSDOTTSWSA-N D-4-hydroxyphenylglycine Chemical compound [O-]C(=O)[C@H]([NH3+])C1=CC=C(O)C=C1 LJCWONGJFPCTTL-SSDOTTSWSA-N 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 27
- 238000006243 chemical reaction Methods 0.000 claims abstract description 75
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 33
- 108090000790 Enzymes Proteins 0.000 claims abstract description 31
- 102000004190 Enzymes Human genes 0.000 claims abstract description 31
- 108090000604 Hydrolases Proteins 0.000 claims abstract description 17
- 102100036238 Dihydropyrimidinase Human genes 0.000 claims abstract description 16
- 108091022884 dihydropyrimidinase Proteins 0.000 claims abstract description 16
- 239000012535 impurity Substances 0.000 claims abstract description 13
- 239000012466 permeate Substances 0.000 claims abstract description 10
- 239000002904 solvent Substances 0.000 claims abstract description 7
- 239000000758 substrate Substances 0.000 claims abstract description 6
- 239000000049 pigment Substances 0.000 claims abstract description 3
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 3
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000012528 membrane Substances 0.000 claims description 8
- 230000000694 effects Effects 0.000 claims description 7
- WJRBRSLFGCUECM-UHFFFAOYSA-N hydantoin Chemical compound O=C1CNC(=O)N1 WJRBRSLFGCUECM-UHFFFAOYSA-N 0.000 claims description 3
- 229940091173 hydantoin Drugs 0.000 claims description 3
- LSHNGTZXQYSYJQ-UHFFFAOYSA-N 2-(n-carbamoyl-4-hydroxyanilino)acetic acid Chemical compound OC(=O)CN(C(=O)N)C1=CC=C(O)C=C1 LSHNGTZXQYSYJQ-UHFFFAOYSA-N 0.000 claims description 2
- 239000012295 chemical reaction liquid Substances 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 230000002860 competitive effect Effects 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 20
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 14
- 238000004128 high performance liquid chromatography Methods 0.000 description 14
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- 238000010170 biological method Methods 0.000 description 5
- 238000001816 cooling Methods 0.000 description 5
- 239000013078 crystal Substances 0.000 description 5
- GSHIDXLOTQDUAV-UHFFFAOYSA-N N-Carbamoyl-2-amino-2-(4-hydroxyphenyl)acetic acid Chemical compound NC(=O)NC(C(O)=O)C1=CC=C(O)C=C1 GSHIDXLOTQDUAV-UHFFFAOYSA-N 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 239000003513 alkali Substances 0.000 description 4
- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 description 4
- 229960003022 amoxicillin Drugs 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- LSQZJLSUYDQPKJ-UHFFFAOYSA-N p-Hydroxyampicillin Natural products O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)C(N)C1=CC=C(O)C=C1 LSQZJLSUYDQPKJ-UHFFFAOYSA-N 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000006555 catalytic reaction Methods 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- MIOPJNTWMNEORI-GMSGAONNSA-N (S)-camphorsulfonic acid Chemical compound C1C[C@@]2(CS(O)(=O)=O)C(=O)C[C@@H]1C2(C)C MIOPJNTWMNEORI-GMSGAONNSA-N 0.000 description 1
- AJSRHILLPJYTMO-UHFFFAOYSA-N 1-(4-hydroxyphenyl)imidazolidine-2,4-dione Chemical compound C1=CC(O)=CC=C1N1C(=O)NC(=O)C1 AJSRHILLPJYTMO-UHFFFAOYSA-N 0.000 description 1
- LJCWONGJFPCTTL-UHFFFAOYSA-N 4-hydroxyphenylglycine Chemical compound OC(=O)C(N)C1=CC=C(O)C=C1 LJCWONGJFPCTTL-UHFFFAOYSA-N 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 108010066099 N-carbamoylamino acid amidohydrolase Proteins 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000007142 ring opening reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 150000003952 β-lactams Chemical class 0.000 description 1
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C273/00—Preparation of urea or its derivatives, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
- C07C273/18—Preparation of urea or its derivatives, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups of substituted ureas
- C07C273/189—Purification, separation, stabilisation, use of additives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
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Abstract
The invention discloses a method for producing D-p-hydroxyphenylglycine by a two-enzyme method, which comprises the following steps: taking DL-p-hydroxyphenylhydantoin as a substrate, and reacting in a solvent under the action of a hydantoinase in an anaerobic environment at the pH of 9.5-11 and the temperature of 40-50 ℃ until the reaction is finished to obtain an intermediate reaction solution; performing ultrafiltration to remove impurities from the intermediate reaction solution, removing proteins and pigments in the intermediate reaction solution, and collecting ultrafiltration permeate; adding N-carbamyl hydrolase into the ultrafiltration permeate, and reacting at the pH of 7.0-8.5 and the temperature of 50-55 ℃ in an oxygen-free environment until the reaction is finished to obtain the D-p-hydroxyphenylglycine. The invention can improve the enzyme conversion efficiency of the substrate and reduce the enzyme dosage used in the production, thereby reducing the cost and improving the competitive advantage of the enzyme method for producing the D-p-hydroxyphenylglycine.
Description
Technical Field
The invention relates to the technical field of D-p-hydroxyphenylglycine production, in particular to a method for producing D-p-hydroxyphenylglycine by a two-enzyme method.
Background
D-p-hydroxyphenylglycine is used as a medical intermediate, is an important intermediate of antibiotics and is used for synthesizing side chains of the beta-lactam semisynthetic antibiotics. D-p-hydroxyphenylglycine is used in the synthetic production of the broad spectrum antibiotic amoxicillin (i.e., amoxicillin). Compared with other methods, the method for producing amoxicillin by the enzyme method has great advantages in the aspects of environmental protection, energy consumption, production efficiency and the like, so that the amoxicillin produced by the enzyme method is largely used in the world, and the requirements of the intermediate D-p-hydroxyphenylglycine and the ester thereof are further driven. The production technical route of the D-p-hydroxyphenylglycine mainly comprises a chemical method and a biological method. The chemical method is that firstly, DL-p-hydroxyphenylglycine is chemically synthesized by a chemical synthesis method and is obtained by splitting tartaric acid or camphorsulfonic acid; the biological method is to use p-hydroxy-phenyl-hydantoin as raw material, and to use enzyme to convert and hydrolyze to obtain the product. Compared with chemical method, the biological method has shorter process, only generates single configuration body without resolution by catalyzing ring opening of hydantoin with the specificity of enzyme, and has less pollution. At present, the biological method generally adopts the hydantoinase and the N-carbamyl hydrolase to carry out double-enzyme catalysis simultaneously, and because the method needs to adopt a large amount of biological enzyme catalytic reactions, the cost is higher, the purity of the product is influenced, and the application of the biological method in the production of the D-p-hydroxyphenylglycine is limited.
Disclosure of Invention
Based on the technical problems in the background art, the invention provides a method for producing D-p-hydroxyphenylglycine by a two-enzyme method.
The invention provides a method for producing D-p-hydroxyphenylglycine by a two-enzyme method, which comprises the following steps:
s1, taking DL-p-hydroxy-phenyl-hydantoin as a substrate, reacting in a solvent under the action of a hydantoin enzyme in an anaerobic environment at the pH value of 9.5-11 and the temperature of 40-50 ℃ until the reaction is finished to obtain an intermediate reaction liquid;
s2, performing ultrafiltration impurity removal on the intermediate reaction solution, removing protein and pigment in the intermediate reaction solution, and collecting ultrafiltration permeate;
s3, adding N-carbamyl hydrolase into the ultrafiltration permeating liquid, and reacting under the conditions that the pH value is 7.0-8.5 and the temperature is 50-55 ℃ in an oxygen-free environment until the reaction is finished to obtain the D-p-hydroxyphenylglycine.
Preferably, the usage amount of the hydantoinase is 15-35% of the mass of DL-p-hydroxyphenyl hydantoin; preferably, the enzyme activity of the hydantoinase is 40-80U/g.
Preferably, the dosage of the N-carbamyl hydrolase is 8-25% of the mass of the DL-p-hydroxyphenylhydantoin; preferably, the enzyme activity of the N-carbamyl hydrolase is 60-100U/g.
Wherein U/g refers to micromoles of 1 gram of enzyme converting substrate in 1 minute.
Preferably, the ratio of the DL-p-hydroxyphenylhydantoin to the solvent is (15-20) g: 100 mL; preferably, the solvent is water.
Preferably, in the step S2, the ultrafiltration membrane used for removing impurities by ultrafiltration has a cut-off molecular weight of 2500 to 3500 daltons.
Preferably, in step S2, the conditions for removing impurities by ultrafiltration are as follows: the temperature is 30-35 ℃, the film passing pressure is 3-4 mpa, and the flow rate is 15-25 m3/h。
Preferably, in the step S1, the reaction pH is 10-10.5.
Preferably, in the step S3, the reaction pH is 7.5-8.0.
Preferably, in step S1, the reaction is terminated when the concentration of DL-p-hydroxyphenylhydantoin in the reaction system is less than or equal to 0.5%.
Preferably, in step S3, the reaction is terminated when the concentration of N-carbamoyl p-hydroxyphenylglycine in the reaction system is less than or equal to 0.5%.
Preferably, in step S3, after the reaction is finished, extracting a product from the reaction system, where the specific method for extracting the product is as follows: and cooling the reaction system to 20-30 ℃, preferably 20-25 ℃, growing the crystals for 3-5 h, and centrifuging to obtain the D-p-hydroxyphenylglycine product.
Preferably, the hydantoinase and the N-carbamyl hydrolase are purified enzymes.
The invention has the following beneficial effects:
the invention designs a new method for producing D-p-hydroxyphenylglycine by double-enzyme catalysis, utilizes respective characteristics of hydantoinase and N-carbamyl hydrolase to carry out enzyme conversion step by step under different reaction conditions, exerts the maximum conversion capability of the two enzymes, and improves the conversion efficiency, thereby improving the enzyme conversion efficiency of a substrate, reducing the enzyme dosage used in production, reducing the cost, improving the product purity, and improving the competitive advantage of producing D-p-hydroxyphenylglycine by an enzyme method.
Drawings
FIG. 1 is a schematic process flow diagram of the process of the present invention.
Detailed Description
The technical solution of the present invention will be described in detail below with reference to specific examples.
In the following examples and comparative examples, the enzyme activity of the hydantoinase used was 60U/g, and the enzyme activity of the N-carbamoylase was 80U/g.
Example 1
A method for producing D-p-hydroxyphenylglycine by a two-enzyme method comprises the following steps:
s1, adding 18kg of DL-p-hydroxy-phenyl-hydantoin and 100L of water into a 200L reaction kettle, stirring until the DL-p-hydroxy-phenyl-hydantoin and the 100L of water are dissolved, introducing nitrogen into the reaction kettle for 1h, then adding 4kg of hydantoinase, reacting for 45h under the conditions that the pH is 10.0-10.5 and the temperature is 42 ℃ in an oxygen-free environment, detecting the concentration of the DL-p-hydroxy-phenyl-hydantoin in a reaction system by HPLC (high performance liquid chromatography) to be 0.3%, and finishing the reaction to obtain an intermediate reaction solution;
s2, adding the intermediate reaction solution into an ultrafiltration system with an ultrafiltration membrane cut-off molecular weight of 2500 daltons to carry out ultrafiltration impurity removal, and collecting ultrafiltration permeate, wherein the conditions of ultrafiltration impurity removal are as follows: the temperature is 30-35 ℃, the membrane pressure is 2.5mpa, and the flow rate is 20m3/h；
S3, adding the ultrafiltration permeate into a reaction kettle with the volume of 200L, introducing nitrogen into the reaction kettle for 1h, then adding 2.7kg of N-carbamyl hydrolase, reacting for 72h under the conditions that the pH value is 7.5-8.0 and the temperature is 53 ℃ in an oxygen-free environment, detecting the concentration of N-carbamyl p-hydroxyphenylglycine in the reaction system to be 0.2% by HPLC (high performance liquid chromatography), finishing the reaction, cooling the reaction system to 24 ℃, growing crystals for 4h, centrifuging and drying to obtain 14.11kg of D-p-hydroxyphenylglycine, wherein the product yield is 78.4%, the optical rotation of the product is 158.4, the alkali absorption is 0.035, and the enzyme dosage is 0.372kg/kg (w/w).
Example 2
A method for producing D-p-hydroxyphenylglycine by a two-enzyme method comprises the following steps:
s1, adding 18kg of DL-p-hydroxy-phenyl-hydantoin and 100L of water into a 200L reaction kettle, stirring until the DL-p-hydroxy-phenyl-hydantoin and the 100L of water are dissolved, introducing nitrogen into the reaction kettle for 1h, then adding 3kg of hydantoinase, reacting for 54h under the conditions that the pH is 10.0-10.5 and the temperature is 45 ℃ in an oxygen-free environment, detecting the concentration of the DL-p-hydroxy-phenyl-hydantoin in a reaction system by HPLC (high performance liquid chromatography) to be 0.2%, and finishing the reaction to obtain an intermediate reaction solution;
s2, adding the intermediate reaction solution into an ultrafiltration system with an ultrafiltration membrane cut-off molecular weight of 2500 daltons to carry out ultrafiltration impurity removal, and collecting ultrafiltration permeate, wherein the conditions of ultrafiltration impurity removal are as follows: the temperature is 30-35 ℃, the membrane pressure is 2.5mpa, and the flow rate is 25m3/h；
S3, adding the ultrafiltration permeate into a reaction kettle with the volume of 200L, introducing nitrogen into the reaction kettle for 1h, then adding 1.5kg of N-carbamyl hydrolase, reacting for 83h under the conditions that the pH value is 7.5-8.0 and the temperature is 50 ℃ in an oxygen-free environment, detecting the concentration of N-carbamyl p-hydroxyphenylglycine in the reaction system to be 0.4% by HPLC (high performance liquid chromatography), finishing the reaction, cooling the reaction system to 26 ℃, growing crystals for 3h, centrifuging and drying to obtain 11.85kg of D-p-hydroxyphenylglycine products, wherein the product yield is 79%, the optical rotation product degree is 158.7, and the alkali absorption is 0.031. The amount of enzyme used was 0.25kg/kg (w/w).
Example 3
A method for producing D-p-hydroxyphenylglycine by a two-enzyme method comprises the following steps:
s1, adding 18kg of DL-p-hydroxy-phenyl-hydantoin and 100L of water into a 200L reaction kettle, stirring until the DL-p-hydroxy-phenyl-hydantoin and the 100L of water are dissolved, introducing nitrogen into the reaction kettle for 1h, then adding 6kg of hydantoinase, reacting for 26h under the conditions that the pH is 10.0-10.5 and the temperature is 47 ℃ in an oxygen-free environment, detecting the concentration of the DL-p-hydroxy-phenyl-hydantoin in a reaction system by HPLC (high performance liquid chromatography) to be 0.4%, and finishing the reaction to obtain an intermediate reaction solution;
s2, adding the intermediate reaction solution into an ultrafiltration system with an ultrafiltration membrane cut-off molecular weight of 3500 Dalton for ultrafiltration impurity removal, and collecting ultrafiltration permeate, wherein the conditions of ultrafiltration impurity removal are as follows: the temperature is 30-35 ℃, the membrane pressure is 2.5mpa, and the flow rate is 24m3/h；
S3, adding the ultrafiltration permeate into a reaction kettle with the volume of 200L, introducing nitrogen into the reaction kettle for 1h, then adding 4kg of N-carbamyl hydrolase, reacting for 56h under the conditions of pH 7.5-8.0 and temperature of 55 ℃ in an oxygen-free environment, detecting the concentration of N-carbamyl p-hydroxyphenylglycine in the reaction system to be 0.4% by HPLC (high performance liquid chromatography), finishing the reaction, cooling the reaction system to 20 ℃, growing crystals for 5h, centrifuging and drying to obtain 15.9kg of D-p-hydroxyphenylglycine product, wherein the product yield is 79%, the product optical rotation is 158.8, and the alkali absorption is 0.026. The enzyme dosage was 0.556kg/kg (w/w).
Comparative example
Adding 18kg of DL-p-hydroxyphenylhydantoin and 100L of water into a reaction kettle with the volume of 200L, stirring, introducing nitrogen into the reaction kettle for 1h, then adding 12kg of hydantoinase and 8kg of N-carbamyl hydrolase, reacting for 104h under the conditions that the pH is controlled to be 7.5-8.0 and the temperature is 45 ℃ in an oxygen-free environment, detecting the concentration of the DL-p-hydroxyphenylhydantoin in a reaction system to be 0.3% by HPLC (high performance liquid chromatography), detecting the concentration of the N-carbamyl-p-hydroxyphenylglycine to be 0.5%, finishing the reaction, cooling the reaction system to 24 ℃, growing crystals for 4h, centrifuging and drying to obtain 13.86kg of D-p-hydroxyphenylglycine product, wherein the product yield is 77%, the product optical rotation is 157.3, and the alkali absorption is 0.062. The amount of enzyme used was 1.111kg/kg (w/w).
As can be seen from the above examples and comparative examples, the invention adopts hydantoinase and N-carbamyl hydrolase to carry out stepwise conversion under appropriate conditions, and can obtain a D-p-hydroxyphenylglycine product with the yield of 78-80%; in order to achieve the same conversion effect, the enzyme dosage of the existing method for carrying out enzyme conversion by using hydantoinase and N-carbamyl hydrolase is far higher than that of the invention. Therefore, the invention reduces the dosage of enzyme, improves the purity of the product, reduces the production cost and improves the product quality.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. A method for producing D-p-hydroxyphenylglycine by a two-enzyme method is characterized by comprising the following steps:
s1, taking DL-p-hydroxy-phenyl-hydantoin as a substrate, reacting in a solvent under the action of a hydantoin enzyme in an anaerobic environment at the pH value of 9.5-11 and the temperature of 40-50 ℃ until the reaction is finished to obtain an intermediate reaction liquid;
s2, performing ultrafiltration impurity removal on the intermediate reaction solution, removing protein and pigment in the intermediate reaction solution, and collecting ultrafiltration permeate;
s3, adding N-carbamyl hydrolase into the ultrafiltration permeating liquid, and reacting under the conditions that the pH value is 7.0-8.5 and the temperature is 50-55 ℃ in an oxygen-free environment until the reaction is finished to obtain the D-p-hydroxyphenylglycine.
2. The method for producing D-p-hydroxyphenylglycine by the two-enzyme method according to claim 1, wherein the amount of the hydantoinase is 15-35% of the mass of DL-p-hydroxyphenylhydantoin; preferably, the enzyme activity of the hydantoinase is 40-80U/g.
3. The method for producing D-p-hydroxyphenylglycine by the two-enzyme method according to claim 1 or 2, wherein the amount of the N-carbamyl hydrolase is 8-25% of the mass of the DL-p-hydroxyphenylhydantoin; preferably, the enzyme activity of the N-carbamyl hydrolase is 60-100U/g.
4. The method for producing D-p-hydroxyphenylglycine by a two-enzyme method according to any one of claims 1 to 3, wherein the ratio of DL-p-hydroxyphenylhydantoin to the solvent is (15 to 20) g: 100 mL; preferably, the solvent is water.
5. The method for producing D-p-hydroxyphenylglycine by the two-enzyme method according to any one of claims 1 to 4, wherein in the step S2, the ultrafiltration membrane used for ultrafiltration and impurity removal has a cut-off molecular weight of 2500 to 3500 Dalton; the conditions of ultrafiltration impurity removal are as follows: the temperature is 30-35 ℃, the film passing pressure is 3-4 mpa, and the flow rate is 15-25 m3/h。
6. The method for producing D-p-hydroxyphenylglycine by the two-enzyme method according to any one of claims 1 to 5, wherein the reaction is carried out at a pH of 10 to 10.5 in step S1.
7. The method for producing D-p-hydroxyphenylglycine according to any one of claims 1 to 6, wherein the reaction is carried out at a pH of 7.5 to 8.0 in step S3.
8. The process for producing D-p-hydroxyphenylglycine according to any one of claims 1 to 7, wherein the reaction is terminated when the concentration of DL-p-hydroxyphenylhydantoin in the reaction system is 0.5% or less in step S1.
9. The process for producing D-p-hydroxyphenylglycine by the two-enzyme method according to any one of claims 1 to 8, wherein the reaction is terminated when the concentration of N-carbamoyl-p-hydroxyphenylglycine in the reaction system is 0.5% or less in step S3.
10. The process for producing D-p-hydroxyphenylglycine according to any one of claims 1 to 9, wherein the hydantoinase and the N-carbamoyl hydrolase are purified enzymes.
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| Title |
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