CN116555024B - Continuous synthesis device and method for unnatural amino acid - Google Patents
Continuous synthesis device and method for unnatural amino acid Download PDFInfo
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- CN116555024B CN116555024B CN202310830069.6A CN202310830069A CN116555024B CN 116555024 B CN116555024 B CN 116555024B CN 202310830069 A CN202310830069 A CN 202310830069A CN 116555024 B CN116555024 B CN 116555024B
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- 239000007787 solid Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- IEHKWSGCTWLXFU-IIBYNOLFSA-N tadalafil Chemical compound C1=C2OCOC2=CC([C@@H]2C3=C([C]4C=CC=CC4=N3)C[C@H]3N2C(=O)CN(C3=O)C)=C1 IEHKWSGCTWLXFU-IIBYNOLFSA-N 0.000 description 1
- 229960000835 tadalafil Drugs 0.000 description 1
- 229910052723 transition metal Inorganic materials 0.000 description 1
- 150000003624 transition metals Chemical class 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/18—Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
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- C—CHEMISTRY; METALLURGY
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention relates to the technical field of synthesis of unnatural amino acid, and provides a continuous synthesis device and method of unnatural amino acid. The continuous synthesizing device comprises: a raw material continuous supply unit; the continuous Grignard reaction unit is provided with a raw material inlet and a product outlet, and the raw material inlet is connected with the raw material continuous supply unit; the continuous quenching purification unit is provided with a to-be-purified object inlet and a purified product outlet, and the to-be-purified object inlet is connected with the product outlet; a saponification reaction unit having an ester inlet and a keto acid product outlet, the ester inlet being connected to the purified product outlet; and a continuous enzyme catalytic reaction unit, which is provided with a keto acid product inlet and an amino acid outlet, wherein the keto acid product inlet is connected with the keto acid product outlet, and the continuous enzyme catalytic reaction unit adopts a packed bed reactor. The device realizes the full-continuous process of the unnatural amino acid, and the products of each step can be connected in a seamless way in a continuous mode, so that the device is a set of full-continuous synthesis system with high productivity, low cost and high production speed.
Description
Technical Field
The invention relates to the technical field of synthesis of unnatural amino acid, in particular to a continuous synthesis device and method of unnatural amino acid.
Background
Unnatural amino acids are widely used in the pharmaceutical, agricultural, food and cosmetic industries. A variety of unnatural amino acids have been used to produce ampicillin and amoxicillin, nateglinide, tadalafil, fluoropentanoate and the like. Unnatural amino acids are also well-suited precursors for nuclear hormone inhibitors, hepatitis c antiviral drugs or cholecystokinin B antagonists and are often used as building blocks for the creation of novel peptides or cyclic peptides.
Unnatural amino acids are usually synthesized chemically, but chemical synthetic routes have many steps, low yields, slow synthesis rates, and difficult product separations. For example, the chiral centers are generally synthesized by conventional methods, such as catalytic asymmetric synthesis, asymmetric hydrogenation or resolution methods, wherein the catalytic methods involve the use of transition metal catalysts; asymmetric hydrogenation requires the use of chiral ligands and hydrogen; the resolution method has the defects of poor economy, no use of half of products, complex operation and the like. Along with the development of enzyme catalysis technology and the excavation of various enzymes, the method for synthesizing unnatural amino acid by enzyme catalysis is mature, and various unnatural amino acids and derivatives thereof can be synthesized by enzyme catalysis technology at present. The enzymatic synthesis of the unnatural amino acid only needs one step, the reaction process is mild, and the yield is high. The preparation of unnatural amino acids usually requires 4-6 steps for protection, by synthesizing keto acids from inexpensive raw materials by chemical synthesis, converting keto acids to single-configuration amino acids by enzymatic catalysis, and chemically attaching functional groups to the amino groups. The current mainstream process is a batch reaction process, but the batch reaction is generally complicated in operation and low in productivity.
Some reports that amino acid (CN 108048317A) can be synthesized by using a continuous reaction process, so that full continuous operations such as Grignard reagent reaction, hydrolysis, extraction and the like are realized to prepare keto acid, and continuous stirring reaction operation is used to realize enzyme catalysis continuous reaction process to prepare amino acid. However, free enzyme cannot be recovered, so that enzyme waste and cost are increased; and the continuous stirring reaction needs stirring, enzyme deactivation is easy to cause, the equipment space utilization rate is low, the capacity lifting space is small, and the energy consumption is high.
Disclosure of Invention
The invention mainly aims to provide a continuous synthesis device and method of unnatural amino acid, which are used for solving the problems of low synthesis efficiency and high cost of unnatural amino acid in the prior art.
In order to achieve the above object, according to one aspect of the present invention, there is provided a continuous synthesis apparatus of unnatural amino acids, comprising: a raw material continuous supply unit; the continuous Grignard reaction unit is provided with a raw material inlet and a product outlet, and the raw material inlet is connected with the raw material continuous supply unit; the continuous quenching purification unit is provided with a to-be-purified object inlet and a purified product outlet, and the to-be-purified object inlet is connected with the product outlet; a saponification reaction unit having an ester inlet and a keto acid product outlet, the ester inlet being connected to the purified product outlet; and a continuous enzyme catalytic reaction unit, which is provided with a keto acid product inlet and an amino acid outlet, wherein the keto acid product inlet is connected with the keto acid product outlet, and the continuous enzyme catalytic reaction unit adopts a packed bed reactor.
Further, the packed bed reactor is filled with immobilized enzymes, wherein the immobilized enzymes comprise any one or more of co-immobilized enzymes of amino acid dehydrogenase and formate dehydrogenase, co-immobilized enzymes of amino acid dehydrogenase and glucose dehydrogenase, amino acid aminotransferase immobilized enzymes, co-immobilized enzymes of amino acid aminotransferase and lactate dehydrogenase and formate dehydrogenase, and co-immobilized enzymes of amino acid aminotransferase and lactate dehydrogenase and glucose dehydrogenase; preferably, the immobilized enzyme is any one or more of cross-linking enzyme and carrier immobilized enzyme;
preferably, the packed bed reactor comprises a first packed bed reactor and a second packed bed reactor, and the reaction liquid flows out of the first packed bed reactor and enters the second packed bed reactor;
preferably, a three-way valve is arranged between the first packed bed reactor and the second packed bed reactor, the three-way valve comprises a first port, a second port and a third port, the first port is connected with the first packed bed reactor, the second port is connected with the second packed bed reactor, the third port is filled with pH regulating liquid, and the third port and the solution in the first port are mixed and then enter the second port; preferably, the mixing is performed in a mixer comprising any one or more of a dynamic stirring mixer and a static microchannel mixer.
Further, the continuous synthesizing apparatus further includes: a continuous upper protecting group reaction unit, which is provided with an amino acid inlet and an amino protecting product outlet, wherein the amino acid inlet is connected with the amino acid outlet;
preferably, the continuous upper protecting group reaction unit further comprises an amino protecting group inlet and an alkali liquor inlet, which are configured to be respectively input with an amino protecting group solution and an alkali liquor;
preferably, the reactor of the continuous upper protecting group reaction unit is a continuous stirred tank reactor.
According to another aspect of the present application, there is provided a continuous synthesis method of an unnatural amino acid, the continuous synthesis method comprising: taking aqueous solution of keto acid as raw material, adding amino donor, reacting in a packed bed reactor under the action of immobilized enzyme to obtain unnatural amino acid, wherein the keto acid has a structure shown in formula I:
i
Wherein R is selected from the group consisting of substituted or unsubstituted phenyl, substituted or unsubstituted benzyl, substituted or unsubstituted saturated alkyl having a carbon chain length of 2 to 12, substituted or unsubstituted unsaturated alkyl having a carbon chain length of 2 to 12, substituted or unsubstituted cycloalkyl, substituted or unsubstituted C 4 -C 20 Cycloalkyl alkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted C 3 -C 20 Any one of a heterocyclylalkyl group, a substituted or unsubstituted naphthalene ring, and a substituted or unsubstituted biphenyl, each of the cycloalkyl group and the heterocyclic group is independently selected from any one of a three-membered ring, a four-membered ring, a five-membered ring, and a six-membered ring, and a hetero atom of the heterocyclic group is selected from any one of N, S, O; substituents include halogen, C 1 -C 10 Saturated alkyl, C 1 -C 10 Unsaturated alkyl of (C) 1 -C 10 Alkoxy, -CF of (C) 3 and-OCF 3 Any one or more of the following.
Further, the immobilized enzyme includes any one or more of co-immobilized enzymes of amino acid dehydrogenase and formate dehydrogenase, co-immobilized enzymes of amino acid dehydrogenase and glucose dehydrogenase, amino acid aminotransferase immobilized enzyme, co-immobilized enzymes of amino acid aminotransferase and lactate dehydrogenase and formate dehydrogenase, co-immobilized enzymes of amino acid aminotransferase and lactate dehydrogenase and glucose dehydrogenase; preferably, the immobilized enzyme is any one or more of cross-linking enzyme and carrier immobilized enzyme; preferably, the carrier of the carrier immobilized enzyme is any one or more of polymethacrylic acid resin, polystyrene resin, polyacrylonitrile resin, methacrylic acid-styrene-acrylonitrile composite polymer resin, agarose resin, dextran resin, agarose-dextran composite resin, porous glass resin and mesoporous silica; more preferably, the carrier of the carrier immobilized enzyme is any one or more of amino-functional resin, epoxy-functional resin, amino-epoxy hybrid-functional resin, hydrophobic adsorption resin, anion exchange resin, cation exchange resin, IDA ligand affinity resin and NTA ligand affinity resin;
Preferably, the enzyme loading per gram of carrier is 80-200 mg;
preferably, the average particle diameter of the immobilized enzyme is 0.03mm to 2mm.
Further, the amino donor comprises any one or more of ammonium formate, ammonium chloride, ammonium carbonate, ammonium sulfate, ammonium oxalate, alanine, aspartic acid, glutamic acid, phenylalanine, methionine, leucine and isoleucine;
preferably, the amino donor is used in an amount of 1.0 to 4.0 times the molar amount of the keto acid.
Further, the pH value of the reaction liquid in the packed bed reactor is 5.0-10.0;
preferably, the packed bed reactor comprises a first packed bed reactor and a second packed bed reactor, the reaction liquid flows out of the first packed bed reactor, and the reaction liquid enters the second packed bed reactor after the pH value is regulated;
preferably, a three-way valve is arranged between the first packed bed reactor and the second packed bed reactor, the three-way valve comprises a first port, a second port and a third port, the first port is connected with the first packed bed reactor, the second port is connected with the second packed bed reactor, the third port is filled with pH regulating liquid, and the third port and the solution in the first port are mixed and then enter the second port; preferably, the mixing is performed in a mixer comprising any one or more of a dynamic stirring mixer and a static microchannel mixer.
Preferably R is selected from the group consisting of substituted or unsubstituted phenyl, substituted or unsubstituted benzyl, substituted or unsubstituted saturated alkyl having a carbon chain length of 2 to 4, substituted or unsubstituted unsaturated alkyl having a carbon chain length of 2 to 7, substituted or unsubstituted cycloalkyl, substituted or unsubstituted C 4 -C 7 Cycloalkyl alkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted C 3 -C 6 Any one of a heterocyclylalkyl group, a substituted or unsubstituted naphthalene ring, and a substituted or unsubstituted biphenyl, each of the cycloalkyl group and the heterocyclic group is independently selected from any one of a three-membered ring, a four-membered ring, a five-membered ring, and a six-membered ring, and a hetero atom of the heterocyclic group is selected from any one of N, S, O; substituents include halogen, C 1 -C 3 Saturated alkyl, C 1 -C 5 Unsaturated alkyl, methoxy, -CF of (C) 3 and-OCF 3 Any one or more of the following.
Further, an aqueous solution of a keto acid is obtained by: carrying out continuous Grignard reaction on the Grignard reagent and the ester to obtain a keto ester product; continuously quenching and purifying the keto ester product to obtain a purified keto ester product; saponifying the purified keto ester product to obtain an aqueous solution of keto acid;
Preferably, the concentration of the ketoacid in the aqueous solution of the ketoacid is 10 g/L-200 g/L.
Further, the continuous synthesis method further comprises the steps of injecting the unnatural amino acid obtained by the continuous synthesis method of any one of the above, alkali liquor and a compound solution containing amino protecting groups into a continuous stirring tank reactor for reaction to obtain the unnatural amino acid with amino functional group protection, wherein the amino protecting groups comprise any one of tert-butoxycarbonyl, benzyloxycarbonyl and 9-fluorenylmethoxycarbonyl;
preferably, the alkali liquor is selected from any one or more of sodium hydroxide solution, sodium carbonate solution and ammonia water.
By applying the technical scheme of the invention, the continuous synthesis device of the unnatural amino acid realizes a full-continuous process from cheap and easily available starting raw materials to the unnatural amino acid, and products among the steps can be in seamless connection in a continuous mode, so that the continuous synthesis device of the unnatural amino acid is a full-continuous synthesis system of the unnatural amino acid with high productivity, low cost, low energy consumption and high production speed. Especially for the key step of ketoacid ammonification reaction of synthesizing unnatural amino acid, the device can be used for filling immobilized enzyme into the reactor by adopting a packed bed reactor, so that the reaction speed and the production efficiency of ketoacid ammonification can be greatly improved, the packed bed reactor does not need to be stirred, therefore, a reserved space is not needed, the reactor has no dead volume, and the immobilized enzyme is matched with high efficiency, so that the productivity of the packed bed reactor is obvious in improvement effect compared with that of a batch stirring tank reactor under the same volume.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
fig. 1 shows a schematic structural diagram of a continuous synthesizing apparatus provided according to an embodiment of the present application.
Wherein the above figures include the following reference numerals:
111. a grignard reagent continuous generation device; 112. a haloalkane supply device; 113. a magnesium metal supply device; 12. an ester supply device; 20. a continuous grignard reaction unit; 31. an acidic quencher supply means; 32. an extractant supply device; 33. a first continuous quenching purification device; 34. an alkaline neutralization liquid supply device; 35. a second continuous quenching purification device; 41. A continuous immobilized enzyme saponification reaction column; 42. a first pH adjustor supply device; 51. a packed bed reactor; 52. a second pH adjustor supply device; 61. and continuously feeding protecting group reaction units.
Detailed Description
It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The application will be described in detail below with reference to the drawings in connection with embodiments.
As analyzed by the background art of the present application, the prior art has problems of low synthesis efficiency and high cost of unnatural amino acid, and in order to solve the problems, the present application provides a continuous synthesis device and method of unnatural amino acid.
According to an exemplary embodiment of the present application, there is provided a continuous synthesis apparatus of unnatural amino acids, the synthesis apparatus comprising: a raw material continuous supply unit; the continuous Grignard reaction unit is provided with a raw material inlet and a product outlet, and the raw material inlet is connected with the raw material continuous supply unit; the continuous quenching purification unit is provided with a to-be-purified object inlet and a purified product outlet, and the to-be-purified object inlet is connected with the product outlet; a saponification reaction unit having an ester inlet and a keto acid product outlet, the ester inlet being connected to the purified product outlet; and a continuous enzyme catalytic reaction unit, which is provided with a keto acid product inlet and an amino acid outlet, wherein the keto acid product inlet is connected with the keto acid product outlet, and the continuous enzyme catalytic reaction unit adopts a packed bed reactor.
The continuous synthesis device of the unnatural amino acid realizes the full-continuous process from cheap and easily available starting materials to the unnatural amino acid, and products among the steps can be connected in a seamless way in a continuous mode, so that the continuous synthesis device of the unnatural amino acid is a full-continuous synthesis system of the unnatural amino acid with high productivity, low cost, low energy consumption and high production speed. Especially for the key step of ketoacid ammonification reaction of synthesizing unnatural amino acid, the device can be used for filling immobilized enzyme into the reactor by adopting a packed bed reactor, so that the reaction speed and the production efficiency of ketoacid ammonification can be greatly improved, the packed bed reactor does not need to be stirred, therefore, a reserved space is not needed, the reactor has no dead volume, and the immobilized enzyme is matched with high efficiency, so that the productivity of the packed bed reactor is obvious in improvement effect compared with that of a batch stirring tank reactor under the same volume.
In a preferred embodiment of the present application, preferably as shown in fig. 1, the above-mentioned raw material continuous supply unit includes a grignard reagent supply device and an ester supply device 12, the grignard reagent supply device being connected to the raw material inlet through a second automatic feed pump to continuously supply the grignard reagent to the continuous grignard reaction unit 20; the ester supply 12 is connected to the feed inlet via a third automatic feed pump to continuously supply esters to the continuous grignard reaction unit 20. According to the application, the Grignard reagent and the ester are respectively supplied by the second automatic material-spraying pump and the third automatic material-spraying pump, and accurate material supply is realized by utilizing the automatic material-spraying pumps.
The second automatic material-beating pump, the third automatic material-beating pump and the following respective automatic material-beating pumps are all automatically controllable, wherein the pump not only comprises a pump for providing power, but also comprises a detection device for detecting the material-beating rate, for example, the detection device is used for reacting the material-beating rate by detecting the weight change of the raw material and adjusting the material-beating rate according to the detection result, the structure of the automatic material-beating pumps is inherent to the automatic material-beating pumps in the prior art, and a person skilled in the art can select a specific automatic material-beating pump suitable for the application according to the prior art, and the description is omitted herein.
In order to further improve the safety of the continuous synthesis apparatus of the present application, it is preferable that the grignard reagent supply device includes a grignard reagent continuous generation device 111, a haloalkane supply device 112, and a magnesium metal supply device 113, as shown in fig. 1, the grignard reagent continuous generation device 111 is connected to a raw material inlet through a second automatic feed pump to continuously supply the grignard reagent to the continuous grignard reaction unit 20; the halogenated alkane supply device 112 is connected with the grignard reagent continuous generation device 111 through a first automatic feed pump to continuously supply halogenated alkane to the grignard reagent continuous generation device 111; and the magnesium metal supply device 113 is connected to the grignard reagent continuous generation device 111 to continuously supply magnesium metal to the grignard reagent continuous generation device 111. The continuous grignard reagent production device is connected with the continuous format reaction unit, so that the grignard reagent is immediately used for subsequent reaction after preparation, accumulation of a large amount of grignard reagent is avoided, and potential safety hazards caused by accumulation of the grignard reagent are further reduced.
It is known to those skilled in the art that magnesium metal used in the preparation of the grignard reagent is magnesium powder or magnesium dust, and in order to improve the supply efficiency and stability of magnesium metal, the magnesium metal supply device 113 is preferably a screw conveyor. In order to better adapt to the characteristics of the solid-liquid reaction for preparing the grignard reagent, the grignard reagent continuous forming device 111 is preferably a continuous stirring reactor. The stirring effect in the continuous stirring reactor is utilized to increase the contact effect of the halogenated alkane and the magnesium metal in the continuous reaction, thereby improving the reaction efficiency.
In another preferred embodiment of the present application, as shown in fig. 1, the above-mentioned continuous quenching and purifying unit includes an acidic quenching agent supply device 31, an extracting agent supply device 32, a first continuous quenching and purifying device 33, an alkaline neutralization liquid supply device 34, and a second continuous quenching and purifying device 35, the first continuous quenching and purifying device 33 having an acidic quenching agent inlet, an extracting agent inlet, a primary purifying object outlet, and an object to be purified inlet, the acidic quenching agent supply device 31 and the acidic quenching agent inlet being connected by a fourth automatic material pump to continuously supply the acidic quenching agent to the first continuous quenching and purifying device 33, the extracting agent supply device 32 and the extracting agent inlet being connected by a fifth automatic material pump to continuously supply the extracting agent to the first continuous quenching and purifying device 33; the second continuous quenching and purifying device 35 has an alkaline neutralization liquid inlet, a primary purified product inlet, and a purified product outlet, and the alkaline neutralization liquid supply device 34 and the alkaline neutralization liquid inlet are connected by a sixth automatic feed pump to continuously supply alkaline neutralization liquid to the second continuous quenching and purifying device 35, and the primary purified product outlet and the primary purified product inlet are connected by a seventh automatic feed pump.
The product system of the continuous Grignard reaction unit 20 is subjected to acid quenching and extraction simultaneously by using the first continuous quenching and purification device 33; then the second continuous quenching and purifying device 35 is used for neutralizing and washing the acid quenching agent in the primary purified product to finish the purifying effect, thereby avoiding the deterioration caused by long-time accumulation of the product.
In order to realize the whole-course continuity of the continuous synthesizing apparatus of the present application, the above-mentioned saponification reaction unit is preferably a continuous immobilized enzyme saponification reaction unit, and preferably the continuous immobilized enzyme saponification reaction unit includes a continuous immobilized enzyme saponification reaction column 41 for carrying out an enzyme saponification reaction and a first pH adjuster supply device 42 connected to the continuous immobilized enzyme saponification reaction column 41.
The saponification reaction unit can be realized by adopting a batch reaction unit, for example, naOH solution is firstly used for reacting with a ketoacid organic phase generated by the reaction to generate corresponding sodium salt to be dissolved in a water phase, then the solution is separated, the pH of the aqueous solution of the sodium ketoacid is regulated to be slightly acidic by hydrochloric acid solution, and then the ketoacid is extracted into the organic phase by using a solvent. The continuous immobilized enzyme saponification reaction unit is preferably adopted to realize continuous enzyme saponification reaction, so that the continuous synthesis device can carry out integral continuous production, and further plays the advantage of continuous production. In addition, the application further preferably adopts the continuous immobilized enzyme saponification reaction column 41, so that the saponification reaction process is more convenient and quicker, simultaneously materials are saved, and the generation of three wastes is reduced. And the first pH regulator supply device 42 is used for regulating the pH value of the system in the reaction process of the organic phase and the immobilized enzyme.
In some exemplary embodiments of the present application, in order to further increase the ammonification efficiency of the keto acid product, the packed bed reactor 51 is filled with immobilized enzymes including any one or more of co-immobilized enzymes of amino acid dehydrogenase and formate dehydrogenase, co-immobilized enzymes of amino acid dehydrogenase and glucose dehydrogenase, amino acid aminotransferase immobilized enzymes, co-immobilized enzymes of amino acid aminotransferase and lactate dehydrogenase and formate dehydrogenase, co-immobilized enzymes of amino acid aminotransferase and lactate dehydrogenase and glucose dehydrogenase.
In some exemplary embodiments of the present application, as shown in fig. 1, the continuous enzyme catalytic reaction unit is a packed bed reactor 51, and a second pH adjuster supply device 52 is provided to adjust the pH of the reaction solution in the packed bed reactor 51.
The packed bed reactor 51 may be one or a plurality of packed bed reactors 51 connected in series, and may include a plurality of packed bed reactors 51 in order to facilitate accurate adjustment of the pH in the packed bed reactor 51 and further improve the reaction efficiency when the pH value in the reaction liquid is greatly changed and it is difficult to obtain a desired conversion rate in a range of the change. In some embodiments of the present application, the packed bed reactor 51 comprises a first packed bed reactor from which the reaction liquid flows out and a second packed bed reactor into which the reaction liquid flows out; preferably, a three-way valve is arranged between the first packed bed reactor and the second packed bed reactor, the three-way valve comprises a first port, a second port and a third port, the first port is connected with the first packed bed reactor, the second port is connected with the second packed bed reactor, the third port is filled with pH regulating liquid, the third port is mixed with the solution in the first port and then enters the second port, and the pH regulating liquid and the reaction liquid coming out of the first packed bed reactor can be fully mixed and then enter the second packed bed reactor by arranging the three-way valve between the two packed bed reactors.
In order to uniformly mix the pH adjusting liquid with the reaction liquid flowing out of the first packed bed reactor, prevent local peracid or over-alkali, reduce the reaction rate, and even cause irreversible damage to the enzyme, it is preferable that the mixing is performed in a mixer, and the specific kind of mixer may be selected from the prior art, such as any one or more of a dynamic stirring mixer and a static microchannel mixer.
When an unnatural amino acid is used as a reaction precursor, it is generally necessary to protect the amino group, and thus products protected with an amino function are widely used. In some exemplary embodiments of the present application, the above-mentioned apparatus for continuously synthesizing unnatural amino acid further comprises: the continuous upper protecting group reaction unit 61 is provided with an amino acid inlet and an amino protecting product outlet, wherein the amino acid inlet is connected with the amino acid outlet, so that the full continuous production from simple raw materials to amino functional group protection is realized. In some embodiments of the present application, the continuous upper protecting group reaction unit 61 further includes an amino protecting group inlet and a lye inlet configured to allow the input of an amino protecting group solution and a lye, respectively, to increase the reaction efficiency. In some embodiments of the present application, the reactor of the continuous upper protecting group reaction unit 61 is a continuous stirred tank reactor, which can significantly improve the reaction efficiency of the reaction at this step.
According to another exemplary embodiment of the present application, there is provided a continuous synthesis method of an unnatural amino acid, comprising: taking an aqueous solution of a keto acid as a raw material, adding an amino donor, and reacting in a packed bed reactor 51 under the action of immobilized enzyme to obtain an unnatural amino acid, wherein the keto acid has a structure shown in formula I:
i
Wherein R is selected from the group consisting of substituted or unsubstituted phenyl, substituted or unsubstituted benzyl, substituted or unsubstituted saturated alkyl having a carbon chain length of 2 to 12, substituted or unsubstituted unsaturated alkyl having a carbon chain length of 2 to 12, substituted or unsubstituted cycloalkyl, substituted or unsubstituted C 4 -C 20 Cycloalkyl alkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted C 3 -C 20 Any one of a heterocyclylalkyl group, a substituted or unsubstituted naphthalene ring, and a substituted or unsubstituted biphenyl, each of the cycloalkyl group and the heterocyclic group is independently selected from any one of a three-membered ring, a four-membered ring, a five-membered ring, and a six-membered ring, and a hetero atom of the heterocyclic group is selected from any one of N, S, O; when having substituents, the number of substituents may be one or plural, and the plural substituents may be the same or different, and the substituents include halogen, C 1 -C 10 Saturated alkyl, C 1 -C 10 Unsaturated alkyl of (C) 1 -C 10 Alkoxy, -CF of (C) 3 and-OCF 3 Any one or more of the following.
Aiming at the key step of keto acid ammonification reaction for synthesizing the unnatural amino acid, the continuous synthesis method of the unnatural amino acid can fill the immobilized enzyme into the reaction kettle by adopting a packed bed reactor, and can greatly improve the reaction speed and the production efficiency of keto acid ammonification. The packed bed reactor does not need stirring, so that a reserved space is not needed, the reactor has no dead volume, and the reactor is matched with high-efficiency immobilized enzyme, so that the productivity of the synthesis method is obvious in lifting effect compared with that of a batch stirred tank reactor under the same volume. In addition, the service life of the enzyme can be greatly prolonged by the mode of combining the immobilized enzyme and the packed bed reactor, so that the production cost of the unnatural amino acid is obviously reduced. Because the dosage of the enzyme in the packed bed reactor is large, the reaction speed can be improved by tens of times or hundreds of times, and the reaction speed and the production efficiency can be improved. In addition, the packed bed reactor does not need stirring, so that a reserved space is not needed, the reactor has no dead volume, and the catalytic efficiency of the enzyme is high, so that the productivity of the packed bed reactor is obviously improved under the same volume.
In some embodiments of the application, R is selected from the group consisting of substituted or unsubstituted phenyl, substituted or unsubstituted benzyl, substituted or unsubstituted saturated alkyl having a carbon chain length of 2-7, substituted or unsubstituted unsaturated alkyl having a carbon chain length of 2-7, substituted or unsubstituted cycloalkyl, substituted or unsubstituted C 4 -C 10 Cycloalkyl alkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted C 3 -C 10 Any one of a heterocyclylalkyl group, a substituted or unsubstituted naphthalene ring, and a substituted or unsubstituted biphenyl, each of the cycloalkyl group and the heterocyclic group is independently selected from any one of a three-membered ring, a four-membered ring, a five-membered ring, and a six-membered ring, and a hetero atom of the heterocyclic group is selected from any one of N, S, O; substituents include halogen, C 1 -C 3 Saturated alkyl, C 1 -C 5 Unsaturated alkyl of (C) 1 -C 5 Alkoxy, -CF of (C) 3 and-OCF 3 Any one or more of the above methods can be used to obtain the corresponding amino acid in a high yield.
In some embodiments of the application, R is selected from the group consisting of substituted or unsubstituted phenyl, substituted or unsubstituted benzyl, substituted or unsubstituted saturated alkyl having a carbon chain length of 2-4, substituted or unsubstituted unsaturated alkyl having a carbon chain length of 2-7, substituted or unsubstituted cycloalkyl, substituted or unsubstituted C 4 -C 7 Cycloalkyl alkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted C 3 -C 6 Any one of a heterocyclylalkyl group, a substituted or unsubstituted naphthalene ring, and a substituted or unsubstituted biphenyl ring, the cycloalkyl group and the heterocyclic group each being independently selected from a three-membered ring, a four-membered ring, a five-membered ring, and a six-membered ringAny one of hetero atoms of the heterocyclic group is selected from any one of N, S, O; substituents include halogen, C 1 -C 3 Saturated alkyl, C 1 -C 5 Unsaturated alkyl, methoxy, -CF of (C) 3 and-OCF 3 Any one or more of the following.
In order to further improve the reaction efficiency of the keto acid ammonification reaction, it is preferable that the immobilized enzyme includes any one or more of Amino Acid Dehydrogenase (AADH), glucose Dehydrogenase (GDH), formate Dehydrogenase (FDH), amino acid aminotransferase (DAAT), and Lactate Dehydrogenase (LDH), that is, the enzyme functioning in the immobilized enzyme is one or a combination of more of the above enzymes.
The immobilized enzyme is an independent immobilized enzyme or a co-immobilized enzyme. In some embodiments of the present application, co-immobilized enzymes comprising any one of glucose dehydrogenase and formate dehydrogenase and amino acid dehydrogenase, co-immobilized enzymes comprising amino acid aminotransferase, lactate dehydrogenase and formate dehydrogenase, co-immobilized enzymes comprising amino acid aminotransferase, lactate dehydrogenase and glucose dehydrogenase, co-immobilized enzymes comprising any one of glucose dehydrogenase and formate dehydrogenase and amino acid aminotransferase, lactate dehydrogenase, can act synergistically to further increase the rate of reaction and the conversion rate of raw materials. In some embodiments of the application, a better catalytic effect is also achieved using an immobilized enzyme alone of the amino acid aminotransferase.
The immobilized enzyme is one or more of cross-linking enzyme and carrier immobilized enzyme. Wherein, the cross-linking enzyme is solid enzyme which is insoluble in water and other solvents and is obtained by adding high molecular polymer and cross-linking agent for cross-linking after enzyme is precipitated under the action of precipitant. The precipitant includes, but is not limited to, ammonium sulfate, ethanol, acetonitrile, polyethylene glycol, etc., and the polymer and the crosslinking agent may be selected from the prior art, and the present application will not be described in detail herein.
In some embodiments of the present application, the carrier of the immobilized enzyme is any one or more of polymethacrylic acid resin, polystyrene resin, polyacrylonitrile resin, methacrylic acid-styrene-acrylonitrile composite polymer resin, agarose resin, dextran resin, agarose-dextran composite resin, porous glass resin and mesoporous silica, and the catalytic effect of the immobilized enzyme using the carrier is obviously improved. The carrier material can be modified by any one or more of amino functional group resin, epoxy functional group resin, amino-epoxy hybrid functional group resin, hydrophobic adsorption resin, anion exchange resin, cation exchange resin, IDA ligand affinity resin and NTA ligand affinity resin, so that the conversion rate and the reaction rate of raw materials can be further improved. Preferably, the enzyme loading amount per gram of carrier is 80-200 mg, and the catalytic efficiency is high.
The immobilization method of the carrier immobilized enzyme can be selected from the prior art, wherein the covalent immobilization method or the adsorption immobilization method is adopted, so that the process is simple, the carrier immobilized enzyme is convenient to apply to a packed bed reactor, and the catalytic efficiency is high.
In order to further improve the catalytic efficiency of the immobilized enzyme in the packed bed, it is preferable that the average particle diameter of the immobilized enzyme is 0.03mm to 2mm, and the particle diameter is too large to facilitate the exertion of the enzyme activity, and the particle diameter is too small, so that the flow of the reaction solution from the immobilized enzyme in the packed bed becomes difficult, and the retention time becomes long to facilitate the improvement of the reaction efficiency.
The above amino donor may be selected from the prior art, for example, from any one or more of ammonium formate, ammonium chloride, ammonium carbonate, ammonium sulfate, ammonium oxalate, alanine, aspartic acid, glutamic acid, phenylalanine, methionine, leucine, isoleucine. In some preferred embodiments of the application, the amino donor is used in an amount of 1.0 to 4.0 molar to the keto acid, which is advantageous for improving the reaction efficiency.
In order to further increase the activity of the enzyme and thereby increase the reaction rate and the yield of the target product, the pH of the reaction solution in the packed bed reactor 51 is preferably 5.0 to 10.0. In some embodiments of the application, the aqueous solution of the keto acid starting material and the amino donor is added with an acid or base to bring the pH of the solution within the preferred ranges described above, and then enters the packed bed reactor 51 to avoid affecting the activity of the enzyme by local peracid or over-base within the packed bed. In some embodiments of the present application, due to the selection of the immobilized enzyme species, for example, when GDH is contained in the immobilized enzyme, the pH value changes greatly during the reaction, and in order to achieve continuous reaction and online pH value regulation, the packed bed reactor 51 comprises a first packed bed reactor and a second packed bed reactor, and the reaction solution flows out of the first packed bed reactor, and enters the second packed bed reactor after pH value regulation. In some preferred embodiments, a three-way valve is provided between the first packed bed reactor and the second packed bed reactor, the three-way valve comprising a first port, a second port and a third port, wherein the first port is connected to the first packed bed reactor, the second port is connected to the second packed bed reactor, the third port is filled with a pH adjusting liquid, the third port and the solution in the first port are mixed and enter the second port, and the adjustment of the pH value is achieved in a very simple manner.
In order to uniformly mix the pH adjusting liquid with the reaction liquid flowing out of the first packed bed reactor, prevent local peracid or over-alkali, reduce the reaction rate, and even cause irreversible damage to the enzyme, it is preferable that the mixing is performed in a mixer, and the specific kind of mixer may be selected from the prior art, such as any one or more of a dynamic stirring mixer and a static microchannel mixer.
In some exemplary embodiments of the application, the aqueous solution of a keto acid is obtained by: carrying out continuous Grignard reaction on the Grignard reagent and the ester to obtain a keto ester product; continuously quenching and purifying the keto ester product to obtain a purified keto ester product; and (3) performing saponification reaction on the purified keto ester product to obtain an aqueous solution of keto acid. Thereby realizing the continuous production of the whole process from the ester to the unnatural amino acid. The above-mentioned continuous method for preparing an aqueous ketonic acid solution may be selected according to the prior art, and will not be described in detail herein.
In a preferred embodiment of the present application, in order to fully exploit the advantages of the continuous grignard reaction, the retention time of the grignard reagent and the ester is preferably 5-60 min, the reaction temperature is-70 to-15 ℃, preferably-70 to-60 ℃, more preferably the continuous grignard reaction is carried out in a continuous coil reactor, and the molar ratio of the ester to the grignard reagent is 0.8-1.5:1.
In order to further improve the safety of the continuous synthesis method of the present application, it is preferable that the continuous synthesis method further includes a process of continuously supplying a grignard reagent, the process of continuously supplying a grignard reagent including: continuously reacting a haloalkane with magnesium to continuously obtain a grignard reagent, preferably continuously reacting in a continuously stirred reactor, preferably haloalkane being any one or more of straight-chain haloalkane having 3 to 5 carbon atoms, branched-chain haloalkane having 3 to 5 carbon atoms, and halocycloalkane having 5 to 7 carbon atoms; preferably, the molar ratio of magnesium to haloalkane is 1.05 to 1.3:1. By continuously providing the grignard reagent, accumulation of a large amount of grignard reagent is avoided, and potential safety hazards caused by accumulation of the grignard reagent are reduced.
In yet another preferred embodiment of the present application, the continuous quench purification process described above comprises: continuously conveying the ketoester product to a first continuous quenching and purifying device 33, and continuously adding an acid quenching agent and an extracting agent into the first continuous quenching and purifying device 33 to perform first continuous quenching and purifying on the ketoester product to obtain a primary purified product; continuously conveying the primary purified product to a second continuous quenching and purifying device 35, and continuously adding an alkaline neutralizing liquid into the second continuous quenching and purifying device 35 to neutralize and wash the acid quenching agent in the primary purified product to obtain the purified keto ester product. Acid quenching and extraction of the ketoester product of the continuous grignard reaction unit 20 using the first continuous quenching and purification device 33; and then the second continuous quenching and purifying device 35 is used for neutralizing and washing the acid quenching agent in the primary purified product to finish the purifying effect.
The extractant, the acid quencher and the alkaline neutralizing solution used in the application can refer to the corresponding substances adopted in the purification in the prior art, and in order to reduce the purification cost and improve the purification effect as much as possible, the extractant is preferably any one or more of tetrahydrofuran, 2-methyltetrahydrofuran, ethyl acetate and methylene dichloride; preferably, the acid quencher comprises an aqueous ammonium chloride solution of an acid, the acid is any one or more of hydrochloric acid, sulfuric acid and acetic acid, and preferably, the molar concentration of the acid in the acid quencher is 1-3 mol/L; more preferably, the volume ratio of the acid quencher to the keto ester product is 3-8:1; preferably, the alkaline neutralization solution is any one or more of sodium carbonate aqueous solution, sodium bicarbonate aqueous solution, potassium bicarbonate aqueous solution, sodium hydroxide solution, sodium dihydrogen phosphate solution, disodium hydrogen phosphate solution, potassium dihydrogen phosphate solution and dipotassium hydrogen phosphate solution.
In order to realize the whole-course continuity of the continuous synthesis method, the saponification reaction is preferably continuous immobilized enzyme saponification reaction, the purification keto ester product is preferably continuous immobilized enzyme saponification reaction in a chromatographic column, the adsorbent of the chromatographic column is preferably one or more of white carbon black, diatomite, silica gel, active carbon, clay, zeolite and magnesium silicate, and the adsorbent is more preferably silica-based powder adsorbent (white carbon black, diatomite powder, clay and zeolite) and active carbon. The enzyme immobilized on the chromatographic column is selected from any one or more of lipase produced by any one of the following strains: rhizopus lutescens (Thermomyces lanuginous), mucor miehei, pseudomonas fluorescens (Pseudomonas fluorescens), aspergillus niger (Aspergillus niger), rhizopus oryzae (Rhizopus oryzae), candida lipolytica (Candida Rugosa), burkholderia (Burkholderia sp.), candida Rugosa (Candida Rugosa) and Rhizopus (Rhizopus sp.); further preferred are immobilized enzymes selected from any one or more of porcine pancreatic lipase, S.lanuginosus (Thermomyces lanuginous), mucor miehei (Mucor miehei), pseudomonas fluorescens (Pseudomonas fluorescens), aspergillus niger (Aspergillus niger), rhizomucor miehei (Rhizopusoyzae), candida lipolytica (Candida Rugosa), rhizopus (Rhizopus sp.). Thereby the saponification reaction process is more convenient and quicker, simultaneously the materials are saved, and the generation of three wastes is reduced.
Preferably, the reaction temperature of the continuous immobilized enzyme saponification reaction is 25-40 ℃, preferably 30-35 ℃, and the retention time is 2-20 h. By controlling the temperature and the time, the reaction is more convenient and rapid, the materials are saved, and the generation of three wastes is reduced.
The product solution obtained by the saponification reaction can be directly used as a keto acid raw material to enter the packed bed reactor 51 for reaction without any concentration or purification and separation, so that the production efficiency is improved, the process is simplified, and the energy consumption is reduced. In some preferred embodiments of the application, the concentration of ketoacid in the aqueous solution of the ketoacid as starting material is in the range of 10 to 200 g/L.
When an unnatural amino acid is used as a reaction precursor, it is generally necessary to protect the amino group, and therefore, the unnatural amino acid product in which the amino functional group is protected is widely used. In some embodiments of the present application, the above-mentioned continuous synthesis method further includes that the unnatural amino acid product obtained by any one of the above-mentioned continuous synthesis methods is injected into a continuous stirred tank reactor with alkali liquor and a compound solution containing an amino protecting group for reaction without post-treatment separation, so as to obtain an unnatural amino acid with amino functional group protection, where the amino protecting group includes any one of tert-butoxycarbonyl, benzyloxycarbonyl and 9-fluorenylmethoxycarbonyl.
The amount of the above-mentioned amino protecting group-containing compound may be determined according to the stoichiometric ratio of the reaction, and in some embodiments of the present application, the molar amount of the amino protecting group is 2 to 6 times the molar amount of the amino acid in order to further improve the efficiency of the reaction.
In some embodiments of the application, the alkali liquor is selected from any one or more of sodium hydroxide solution, sodium carbonate solution and ammonia water, preferably the alkali liquor is used in an amount which is 4-10 times of the molar amount of amino acid, which is beneficial to improving the reaction rate and the conversion rate of target products.
The specific method for separating and purifying the unnatural amino acid material with amino functional group protection obtained by the reaction can be easily determined by those skilled in the art, and will not be described in detail herein.
The advantageous effects that can be achieved by the present application will be further described below with reference to examples and comparative examples.
The enzymes and sources of carriers used in the following examples and comparative examples are shown in the following table.
TABLE 1
Examples 1 to 18
Dissolving amino donor in keto acid water solution, wherein the concentrations of amino donor and keto acid are shown in Table 2, adjusting pH of water solution to the value shown in Table 2, adding coenzyme NAD (P) + A mixed solution was obtained, and the mixed solution was continuously injected into a packed bed reactor 51 (PBR) by a plunger pump at a reaction temperature of 30℃and an average particle diameter of the immobilized enzyme packed in the PBR was 0.2mm as shown in Table 2 below. When the immobilized enzyme is a carrier immobilized enzyme, the enzyme loading per gram of carrier is 100 mg, and the types of the carriers are shown in Table 2 below. Taking the AADH1 and GDH co-immobilized enzyme of example 1 as an example, the preparation method is as follows: activating 1 g amino carrier with glutaraldehyde water solution with concentration of 4 mL of 2% v/v at room temperature for 1 h, filtering to remove supernatant; washing the carrier with 0.1M phosphate buffer; the enzyme solution was added to the washed carrier, stirred at 20℃for 20 h, the supernatant was filtered off, the immobilized enzyme was washed with 0.1M phosphate buffer solution, and the washing solution was filtered off.
TABLE 2
When the number of the packed bed reactors is more than 2, the two packed bed reactors are connected through a three-way valve, the acid liquid or alkali liquid and the reaction liquid flowing out of the previous packed bed reactor are mixed in a mixer, and the pH value is adjusted to the value in the table 2.
The retention time, conversion, runnability time of the immobilized enzyme, and productivity of the unnatural amino acid synthesis of examples 1 to 18 are shown in Table 3 below. Wherein, the reactor productivity refers to the yield (g) of the target amino acid per unit space (L) per unit time (day, d) in the reactor.
TABLE 3 Table 3
From the experimental results of the above examples, it was found that at a suitable flow rate (retention time) there was almost no raw material remaining in the solution collected at the outlet of the packed bed reactor, and the conversion was >95%. Can be operated continuously for more than 5 days.
Example 19
The only difference from example 1 is that the immobilized enzyme carrier was LXECR8309, and the immobilization method and enzyme loading were the same as in example 1.
Correspondingly, the retention time of the reaction was 8min, the keto acid conversion was 99, the activity of the enzyme was significantly reduced after 120. 120 h runs, and the reactor productivity was 120 g/(L.d).
Example 20
The only difference from example 1 is that the immobilized enzyme carrier was LX1000EP, and the immobilization method and enzyme loading were the same as in example 1.
Correspondingly, the retention time of the reaction is 120min, the keto acid conversion rate is 70%, the activity of the enzyme is obviously reduced after 12h of operation, and the productivity of the reactor is 170 g/(L.d).
Example 21
The difference from example 1 is that the pH of the reaction solution was adjusted without adding an acid or a base, the pH of the reaction solution was varied in the range of 4.0 to 6.0, the retention time was 120min, the keto acid conversion was 65%, and the activity was remarkably decreased after the operation of 10 h.
Example 22
The difference from example 4 is that only one packed bed reactor is used, i.e. no online adjustment of the pH is made, with a pH variation in the range 4.0-8.5.
Correspondingly, the retention time of the reaction was 100min, the keto acid conversion was 85%, and the activity of the enzyme was significantly reduced after running 16. 16 h.
Examples 23 to 28
The amino acid product solutions prepared in examples 1 and 5 above were directly and continuously fed into a Continuous Stirred Tank Reactor (CSTR) without post-treatment separation, and an amino protecting group (as shown in table 4) solution and a 2N NaOH solution (N represents mol/L, the same applies hereinafter) were continuously fed through two additional pumps and mixed in the CSTR at a stirring speed of 300 rpm. The molar equivalent of the amino protecting group is 3 times that of the amino acid, and the molar equivalent of the alkali liquor is 4 times that of the amino acid. The flow rate was adjusted so that the conversion at the outlet was >90%.
TABLE 4 Table 4
Example 29
The preparation of 2-cyclopentylglycine was carried out using a continuous reaction apparatus shown in FIG. 1.
The halogenated alkane supply device 112 is adopted to continuously supply the brominated cyclopentane to the Grignard reagent continuous generation device 111 through the first automatic material-beating pump, and the magnesium metal supply device 113 is adopted to continuously supply the magnesium chips to the Grignard reagent continuous generation device 111, wherein the brominated cyclopentane is dissolved in tetrahydrofuran, the Grignard reagent is dissolved in tetrahydrofuran, the using amount of the solvent is 15 times that of the brominated cyclopentane, and the using amount of the Grignard reagent magnesium chips is 1.0eq.
The grignard reagent prepared by the grignard reagent continuous generation device 111 and diethyl oxalate react in a continuous coil reactor to generate keto ester, the retention time is 25 min, wherein the diethyl oxalate is dissolved in tetrahydrofuran, the dosage is 3 times of the volume of the diethyl oxalate, and the dosage of the diethyl oxalate is 1.0eq.
The product system produced by the continuous coil reactor was quenched with a mixed solution of 5N hydrochloric acid and 3N ammonium chloride in a first continuous quenching and extraction apparatus and continuously extracted with 2-methyltetrahydrofuran, and the resulting initially purified product was continuously neutralized and washed with sodium bicarbonate solution in a second continuous quenching and purification apparatus 35 to yield a purified ketoester product having a purity of 92% and a yield of 86%.
The obtained purified keto ester product is subjected to saponification reaction by a continuous immobilized enzyme saponification reaction column 41 to generate keto acid, wherein the reaction temperature is 30 ℃, the reaction time is 0.5 and h, the enzyme adopted is porcine pancreatic lipase, and the adsorbent in the chromatographic column is white carbon black.
The concentration of ketoacid in the ketoacid product produced by the continuous immobilized enzyme saponification reaction column 41 is 60 g/L, the concentration of the amino donor ammonium chloride is 48 g/L in the ketoacid aqueous solution, 5N NaOH is added to adjust the pH value of the aqueous solution to 9.0, and coenzyme NAD is added + A mixed solution was obtained, and the mixed solution was continuously injected into the packed bed reactor 51 (PBR) by a plunger pump, and the immobilized enzyme packed in the PBR was passed through the same procedure as in example 1. The reaction was carried out in 3 packed bed reactors 51 for a retention time of 8min, a keto acid conversion of 99% and an amino acid yield of 90%. The service life of the enzyme is 186 h, the enzyme productivity is 96g/g, namely, 96g of 2-cyclopentylglycine can be obtained by catalysis of each g of immobilized enzyme (excluding a carrier), and the reactor productivity is 650 g/(L.d).
Comparative example 1
The difference from example 29 is that the keto acid product produced by the continuous immobilized enzyme saponification reaction column is treated as follows: and (3) carrying out film concentration through a continuous film separator, and carrying out enzyme catalysis on the concentrated keto acid in a continuous stirring reactor to generate amino acid, wherein the adopted enzyme is free enzyme of AADH1 and GDH, the stirring speed of the enzyme catalysis reaction in the continuous stirring reactor is 60r/min, the enzyme catalysis reaction temperature is 30 ℃, the retention time is 30h, the keto acid conversion rate is 99%, and the yield of the amino acid is 83%. The service life of the enzyme is 30h, the enzyme productivity is 20g/g, and the reactor productivity is 72 g/(L.d).
Comparative example 2
The difference from example 29 is that the prepared keto acid product was added to the reaction vessel at once, then the amino donor was added, after mixing, 5N NaOH solution was added to adjust the pH to 8.5, then the free enzymes of AADH1 and GDH and the coenzyme were added, the temperature was kept at 30℃and the reaction was carried out at a stirring speed of 200 rpm for 40 h. The conversion of keto acid was 99%, the yield of amino acid was 85%, the service life of the enzyme was 40. 40 h, and the reactor productivity was 26 g/(L.d).
From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: aiming at the key step of keto acid ammonification reaction for synthesizing the unnatural amino acid, the continuous synthesis method of the unnatural amino acid can fill the immobilized enzyme into the reaction kettle by adopting a packed bed reactor, and can greatly improve the reaction speed and the production efficiency of keto acid ammonification. The packed bed reactor does not need stirring, so that a reserved space is not needed, the reactor has no dead volume, and the reactor is matched with high-efficiency immobilized enzyme, so that the productivity of the synthesis method is obvious in lifting effect compared with that of a batch stirred tank reactor under the same volume. In addition, the service life of the enzyme can be greatly prolonged by the mode of combining the immobilized enzyme and the packed bed reactor, so that the production cost of the unnatural amino acid is obviously reduced.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (13)
1. A continuous synthesis apparatus for unnatural amino acids, comprising:
a raw material continuous supply unit;
a continuous grignard reaction unit having a feedstock inlet and a product outlet, the feedstock inlet being connected to the feedstock continuous supply unit;
the continuous quenching purification unit is provided with a to-be-purified object inlet and a purified product outlet, and the to-be-purified object inlet is connected with the product outlet;
a saponification reaction unit having an ester inlet and a keto acid product outlet, the ester inlet being connected to the purified product outlet; and
a continuous enzyme catalytic reaction unit, which is provided with a keto acid product inlet and an amino acid outlet, wherein the keto acid product inlet is connected with the keto acid product outlet, the continuous enzyme catalytic reaction unit adopts a packed bed reactor, immobilized enzyme is filled in the packed bed reactor,
And the packed bed reactor comprises a first packed bed reactor and a second packed bed reactor, the reaction liquid flows out from the first packed bed reactor and enters the second packed bed reactor, a three-way valve is arranged between the first packed bed reactor and the second packed bed reactor and comprises a first port, a second port and a third port, wherein the first port is connected with the first packed bed reactor, the second port is connected with the second packed bed reactor, the third port is filled with pH regulating liquid, and the third port is mixed with the solution in the first port and then enters the second port.
2. The continuous synthesis apparatus according to claim 1, wherein the immobilized enzyme comprises any one or more of co-immobilized enzyme of amino acid dehydrogenase and formate dehydrogenase, co-immobilized enzyme of amino acid dehydrogenase and glucose dehydrogenase, amino acid aminotransferase immobilized enzyme, co-immobilized enzyme of amino acid aminotransferase and lactate dehydrogenase and formate dehydrogenase, co-immobilized enzyme of amino acid aminotransferase and lactate dehydrogenase and glucose dehydrogenase.
3. The continuous synthesis apparatus of claim 1, wherein the solutions within the third port and the first port are mixed in a mixer comprising any one or more of a dynamic stirring mixer and a static microchannel mixer.
4. A continuous synthesis apparatus according to any one of claims 1 to 3, further comprising:
the continuous upper protecting group reaction unit is provided with an amino acid inlet and an amino protecting product outlet, wherein the amino acid inlet is connected with the amino acid outlet, the continuous upper protecting group reaction unit further comprises an amino protecting group inlet and an alkali liquor inlet, an amino protecting group solution and an alkali liquor can be respectively input through configuration, and the reactor of the continuous upper protecting group reaction unit is a continuous stirring tank reactor.
5. A continuous synthesis method of unnatural amino acids, characterized in that the continuous synthesis apparatus according to any one of claims 1 to 4 is employed and that the continuous synthesis method comprises: taking an aqueous solution of a keto acid as a raw material, adding an amino donor, and reacting in a packed bed reactor under the action of immobilized enzyme to obtain the unnatural amino acid, wherein the keto acid has a structure shown in a formula I:
i
Wherein R is selected from the group consisting of substituted or unsubstituted phenyl, substituted or unsubstituted benzyl, substituted or unsubstituted saturated alkyl having a carbon chain length of 2 to 12, substituted or unsubstituted unsaturated alkyl having a carbon chain length of 2 to 12, substituted or unsubstituted cycloalkyl, substituted or unsubstituted C 4 -C 20 Cycloalkyl alkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted C 3 -C 20 Any one of a heterocyclylalkyl group, a substituted or unsubstituted naphthalene ring, and a substituted or unsubstituted biphenyl, each of the cycloalkyl group and the heterocyclic group is independently selected from any one of a three-membered ring, a four-membered ring, a five-membered ring, and a six-membered ring, and a heteroatom of the heterocyclic group is selected from any one of N, S, O; substituents include halogen, C 1 -C 10 Saturated alkyl, C 1 -C 10 Unsaturated alkyl of (C) 1 -C 10 Alkoxy group of (C),-CF 3 and-OCF 3 Any one or more of the following.
6. The continuous synthesis method according to claim 5, wherein the immobilized enzyme comprises any one or more of co-immobilized enzyme of amino acid dehydrogenase and formate dehydrogenase, co-immobilized enzyme of amino acid dehydrogenase and glucose dehydrogenase, amino acid aminotransferase immobilized enzyme, co-immobilized enzyme of amino acid aminotransferase and lactate dehydrogenase and formate dehydrogenase, co-immobilized enzyme of amino acid aminotransferase and lactate dehydrogenase and glucose dehydrogenase.
7. The continuous synthesis method according to claim 6, wherein the immobilized enzyme is any one or more of a cross-linking enzyme and a carrier immobilized enzyme; the carrier of the carrier immobilized enzyme is any one or more of polymethacrylic acid resin, polystyrene resin, polyacrylonitrile resin, methacrylic acid-styrene-acrylonitrile composite polymer resin, agarose resin, dextran resin, agarose-dextran composite resin, porous glass resin and mesoporous silica, the loading amount of the enzyme on each gram of the carrier is 80-200 mg, and the average particle size of the immobilized enzyme is 0.03-2 mm.
8. The continuous synthesis method according to claim 7, wherein the carrier of the carrier-immobilized enzyme is any one or more of amino-functional resin, epoxy-functional resin, amino-epoxy hybrid-functional resin, hydrophobic adsorption resin, anion exchange resin, cation exchange resin, IDA ligand affinity resin, NTA ligand affinity resin.
9. The continuous synthesis method according to claim 5, wherein the amino donor comprises any one or more of ammonium formate, ammonium chloride, ammonium carbonate, ammonium sulfate, ammonium oxalate, alanine, aspartic acid, glutamic acid, phenylalanine, methionine, leucine, and isoleucine, and the amino donor is used in an amount of 1.0 to 4.0 times the molar amount of the keto acid.
10. The continuous synthesis process according to claim 5, wherein the pH of the reaction solution in the packed bed reactor is from 5.0 to 10.0;
and/or the concentration of the ketoacid in the aqueous solution of the ketoacid is 10 g/L-200 g/L.
11. The continuous synthesis process according to claim 5, wherein R is selected from the group consisting of substituted or unsubstituted phenyl, substituted or unsubstituted benzyl, substituted or unsubstituted saturated alkyl having a carbon chain length of 2 to 4, substituted or unsubstituted unsaturated alkyl having a carbon chain length of 2 to 7, substituted or unsubstituted cycloalkyl, substituted or unsubstituted C 4 -C 7 Cycloalkyl alkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted C 3 -C 6 Any one of a heterocyclylalkyl group, a substituted or unsubstituted naphthalene ring, and a substituted or unsubstituted biphenyl, each of the cycloalkyl group and the heterocyclic group is independently selected from any one of a three-membered ring, a four-membered ring, a five-membered ring, and a six-membered ring, and a heteroatom of the heterocyclic group is selected from any one of N, S, O; substituents include halogen, C 1 -C 3 Saturated alkyl, C 1 -C 5 Unsaturated alkyl, methoxy, -CF of (C) 3 and-OCF 3 Any one or more of the following.
12. The continuous synthesis process according to any one of claims 5 to 11, wherein the aqueous solution of a keto acid is obtained by:
carrying out continuous Grignard reaction on the Grignard reagent and the ester to obtain a keto ester product;
continuously quenching and purifying the keto ester product to obtain a purified keto ester product;
and (3) carrying out saponification reaction on the purified keto ester product to obtain an aqueous solution of keto acid.
13. The continuous synthesis method according to any one of claims 5 to 11, further comprising injecting the unnatural amino acid obtained by the continuous synthesis method according to any one of claims 5 to 12 with a lye, a solution of a compound containing an amino protecting group, which includes any one of t-butoxycarbonyl, benzyloxycarbonyl and 9-fluorenylmethoxycarbonyl, into a continuous stirred tank reactor for reaction to obtain an unnatural amino acid having an amino functional group protection; the alkali liquor is selected from one or more of sodium hydroxide solution, sodium carbonate solution and ammonia water.
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| CN115155462A (en) * | 2022-07-27 | 2022-10-11 | 沈阳化工研究院有限公司 | Method and device for preparing 2, 4-diaminoanisole by continuous catalytic hydrogenation |
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| CN103880754A (en) * | 2012-12-21 | 2014-06-25 | 西藏海思科药业集团股份有限公司 | Alkaline amino acid ester salt of propofol |
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| CN114369583A (en) * | 2022-01-12 | 2022-04-19 | 凯莱英生命科学技术(天津)有限公司 | Immobilized enzyme and application thereof in continuous production |
| CN115155462A (en) * | 2022-07-27 | 2022-10-11 | 沈阳化工研究院有限公司 | Method and device for preparing 2, 4-diaminoanisole by continuous catalytic hydrogenation |
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