CN114934082B - Method for synthesizing (S) -5-methyl-2-pyrrolidone by continuous flow biocatalysis - Google Patents
Method for synthesizing (S) -5-methyl-2-pyrrolidone by continuous flow biocatalysis Download PDFInfo
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- CN114934082B CN114934082B CN202210493972.3A CN202210493972A CN114934082B CN 114934082 B CN114934082 B CN 114934082B CN 202210493972 A CN202210493972 A CN 202210493972A CN 114934082 B CN114934082 B CN 114934082B
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- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
- 150000004040 pyrrolidinones Chemical class 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 108010048818 seryl-histidine Proteins 0.000 description 1
- 108010071207 serylmethionine Proteins 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 108010020532 tyrosyl-proline Proteins 0.000 description 1
- 150000003952 β-lactams Chemical class 0.000 description 1
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Abstract
The invention discloses a method for synthesizing (S) -5-methyl-2-pyrrolidone by continuous flow biocatalysis, which uses amine dehydrogenase to catalyze asymmetric reduction of levulinic acid to prepare optical 4-aminopentanoic acid, and synthesizes (S) -5-methyl-2-pyrrolidone by intramolecular cyclization of intermediate optical 4-aminopentanoic acid, wherein in enzymatic reaction, formate dehydrogenase provides coenzyme circulation, and the enzymatic reaction occurs in a microreactor. Compared with the prior art, the preparation method provided by the invention uses continuous flow biocatalysis to prepare chiral (S) -5-methyl-2-pyrrolidone, has the remarkable advantages of simple process, high optical purity of products, space-time yield and environmental friendliness, and has a good industrial application prospect.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for synthesizing (S) -5-methyl-2-pyrrolidone by continuous flow biocatalysis.
Background
Lactams are key compounds in organic chemistry, as they are found in many biologically active products, and can also serve as valuable intermediates for more complex structures including synthetic polymers. In addition to the β -lactams (2-azetidinones), which constitute the most important class of current antibiotics, the synthesis of γ -lactams (2-pyrrolidone) and δ -lactams (2-piperidone) has also received attention for several years. Secondly, the lactam is a natural product fragment widely existing in the nature, and researches show that the lactam is mostly existing on land and in marine organisms (bacteria, fungi, blue algae and sponges). Natural source lactam derivatives are reported to have very remarkable and wide biological activities such as insecticidal, herbicidal, antiviral, antitumor, anti-inflammatory and the like. Therefore, it would be of great economic value to find a lactam synthesis route with high yield and high stereoselectivity.
The 5-methyl-2-pyrrolidone is an important chemical and intermediate for the synthesis of medicines, pesticides and the like, and can be prepared from biomass renewable resource derivatives levulinic acid and esters thereof through catalytic hydrogenation and reductive amination. The common synthesis method is to catalyze levulinic acid (ester) under the action of a metal catalyst by taking hydrogen as a hydrogen source and ammonia or organic amine as a nitrogen source.
For example, shilling et al uses hydrogen as a hydrogen source, ammonia as a nitrogen source, diatomite-supported nickel as a catalyst, and performs reductive amination on levulinic acid to synthesize 5-methyl-2-pyrrolidone at 200 ℃ in a yield of 87% (Shilling, wilburL. Pyrrolidinones: U.S. Pat. No. 3,235,562[ P ] 1966.2.15.).
In 2017, king et al studied the reaction route and mechanism of preparing 5-methyl-2-pyrrolidone by directly reductive amination of levulinic acid with ammonium formate as a hydrogen source and N, N-dimethylformamide as a solvent, and optimized the reaction conditions, but had moderate stereoselectivity (biomass chemistry engineering, 2017,51 (02): 19-25.).
Chinese patent publication No. CN1764376a discloses a process for producing 5-methyl-N-aryl-2-pyrrolidone, 5-methyl-N-cycloalkyl-2-pyrrolidone, and 5-methyl-N-alkyl-2-pyrrolidone by reductive amination of levulinic acid with nitro compounds using an optionally supported metal catalyst, also requiring hydrogen as a reducing agent, resulting in poor stereoselectivity of the product, even below 10%.
The Chinese patent publication No. CN110615754A discloses a preparation method of 5-methyl-2-pyrrolidone, which takes biomass derivative levulinic acid as a starting material, ammonium formate as a hydrogen source and a nitrogen source, a supported bimetallic catalyst as a hydrogenation catalyst, and adopts a one-pot method in water to synthesize the 5-methyl-2-pyrrolidone, wherein the supported metal of the supported bimetallic catalyst is bimetallic composed of two noble metals, bimetallic composed of one noble metal and one non-noble metal A or B, or bimetallic composed of one non-noble metal A and one non-noble metal B. The method has the advantages that the levulinic acid conversion rate can reach 100%, and the yield of the 5-methyl-2-pyrrolidone can reach more than 94%.
In 2018 Angela et al mainly described a biosynthetic route to gamma-and delta-lactams by reductive amination of ketoester substrates (isopropylamine IPA as amine donor) by catalytic action of transaminases, followed by intramolecular self-cyclization of the intermediate product to form the lactam. All substrates tested were subjected to preparation experiments with a conversion of the product of 66% -89% and an ee value of greater than 91%. This paper describes a simple one-pot two-step process for converting different gamma-and delta-keto esters to the corresponding optically active lactams. This strategy is based on a selective biotransformation reaction which allows the spontaneous intramolecular cyclization of the amino ester intermediate in its own aqueous medium without the addition of external reagents, but the first step of reductive amination requires the addition of an amine donor, so that the overall reaction is by-product and has a low yield (Advanced Synthesis & Catalysis,2018,360 (4): 686-695).
In summary, many methods for synthetically obtaining 5-methyl-2-pyrrolidone have been reported, which can be obtained by chemical or biocatalytic methods, but chemical methods have many disadvantages, such as: the diastereoselectivity is poor, the reaction environment is not mild, and byproducts are more. Transaminases catalyze the reductive amination of ketoester substrates, require the provision of amine donors, and have low enzymatic activity.
Disclosure of Invention
Aiming at the current situation that a plurality of biological methods exist for preparing the optical activity chiral (S) -5-methyl-2-pyrrolidone, the invention provides a method for synthesizing (S) -5-methyl-2-pyrrolidone by continuous flow biological catalysis.
The invention mainly uses amine dehydrogenase AmDH after directed evolution as a catalyst to catalyze the asymmetric reduction reaction of levulinic acid, the reaction is carried out in a micro-reactor, two injection pumps work simultaneously, and then (S) -5-methyl-2-pyrrolidone is prepared by intramolecular cyclization of an intermediate product.
The aim of the invention can be achieved by the following technical scheme:
one of the technical schemes of the invention is as follows: a method for continuous flow biocatalytic synthesis of (S) -5-methyl-2-pyrrolidone is provided, wherein an amine dehydrogenase is used for catalyzing asymmetric reduction of levulinic acid to prepare optical 4-aminopentanoic acid, and then the intermediate optical 4-aminopentanoic acid is subjected to intramolecular cyclization to synthesize (S) -5-methyl-2-pyrrolidone, and in an enzymatic reaction, a formate dehydrogenase provides coenzyme circulation, and the enzymatic reaction occurs in a microreactor.
In one embodiment of the invention, the solution I and the solution II are continuously introduced into a micro-reactor in proportion for reaction, and (S) -5-methyl-2-pyrrolidone is obtained through cyclization and purification after biocatalysis reaction; the first solution contains amine dehydrogenase, formate dehydrogenase, and NAD + The second solution contains substrate levulinic acid.
In one embodiment of the invention, the first and second solutions involved in the reaction are prepared using an ammonium formate/ammonia buffer solution having a concentration of 3m and a ph of 6.5;
in one embodiment of the invention, the volumetric activity ratio of the amine dehydrogenase to the formate dehydrogenase is 1:1.5.
In one embodiment of the invention, NAD in the reaction liquid + The concentration of (C) was 0.2mmol/L.
In one embodiment of the invention, the micro-reactor is a continuous flow micro-channel reactor, the continuous flow micro-channel reactor is formed by connecting 1-20 micro-reactor chips, and comprises two feeding ports and a discharging port, wherein the two feeding ports are respectively used for injecting a first raw material and a second raw material so as to carry out continuous mixing reaction, and the discharging port is used for discharging after the reaction.
In one embodiment of the invention, the continuous flow micro-reactor is formed by connecting 3 micro-reactor chips, the 3 micro-reactor chips are respectively arranged into 3 layers, the first layer micro-reactor chip is provided with two feeding holes, the third layer micro-reactor chip is provided with a discharging hole, the second layer micro-reactor chip is connected with the first layer micro-reactor chip and the third layer micro-reactor chip, each micro-reactor chip is provided with a plurality of mixed reaction cavities for liquid mixed reaction, and raw materials I and II respectively enter and are mixed from the two feeding holes and sequentially pass through each mixed reaction cavity of the first layer micro-reactor chip, each mixed reaction cavity of the second layer micro-reactor chip and each mixed reaction cavity of the third layer micro-reactor chip, and then flow out from the discharging hole of the third layer micro-reactor chip.
In one embodiment of the present invention, the method of feeding sample into the microreactor by using two injection pumps is adopted, the pump 1 injects substrate levulinic acid (using ammonium formate/ammonia buffer) into the microreactor, and the pump 2 injects amine dehydrogenase, formate dehydrogenase, NAD into the microreactor + The liquids of the two pumps meet in the microreactor so that an enzymatic reaction takes place.
In one embodiment of the invention, the amino acid sequence of the amine dehydrogenase is shown as SEQ ID No.2, and the nucleotide sequence corresponding to the amine dehydrogenase is shown as SEQ ID No. 1. The amine dehydrogenase of the invention can catalyze the asymmetric reduction of levulinic acid to prepare optically pure 4-aminopentanoic acid. The amine dehydrogenase AmDH of the invention is a mutant obtained by directed evolution, and the enzyme is NADH dependent.
In one embodiment of the present invention, the chemical structure of the (S) -5-methyl-2-pyrrolidone is as follows:
in one embodiment of the present invention, the method for synthesizing (S) -5-methyl-2-pyrrolidone by intramolecular cyclization of intermediate optical 4-aminopentanoic acid is: a certain amount of solid EDCI is added into a reaction liquid containing the intermediate optical 4-aminopentanoic acid, and the mixture is magnetically stirred for 2 hours at room temperature for the self-cyclization of the intermediate optical 4-aminopentanoic acid to form lactam.
In one embodiment of the invention, the enzymatic reaction may be carried out as follows in an exemplary manner: in ammonium formate/ammonia buffer at pH 6.5, in formate dehydrogenase and NAD + In the presence of the amine dehydrogenase AmDHThe asymmetric reduction of levulinic acid is catalyzed in the following.
In such applications, the concentration of the substrate in the reaction solution may be 20mmol/L. The amine dehydrogenase may be used in an amount of 1 to 500U/L depending on the reaction system used. Enzymatic levulinic acid asymmetric reduction, NADH is oxidized to NAD + To carry out the cyclic regeneration of the coenzyme NADH, formate dehydrogenase is additionally added to the reaction system. The reaction conversions and enantiomeric excess values (ee) of the products can be analyzed by gas chromatography, preferably using a CP-Chirasil-Dex CB column, 25m 0.25mm 0.39 μm specification, by the following analysis of the conversion and ee values: the initial column temperature is 110 ℃,20 ℃/min is raised to 150 ℃, 2min is kept, 5 ℃/min is raised to 160 ℃, and 5min is kept.
After the enzymatic reaction is completed, the reaction solution is cooled to room temperature, a certain amount of solid EDCI is added into the reaction solution, and the mixture is magnetically stirred at room temperature for 2 hours for the self cyclization of the intermediate amino acid to form lactam. After cyclization, the reaction solution was sufficiently extracted with 10 volumes of ethyl acetate, and the ethyl acetate phase was collected by high-speed centrifugation, and the ethyl acetate was removed by vacuum spin evaporation to obtain a crude lactam product. The crude product contains a small amount of EDCI and levulinic acid, and thus it is required to be purified by a silica gel column. The mobile phase is dichloromethane, methanol=1:20, and then the organic solvent is removed by vacuum rotary evaporation, so that the pure product of the lactam is obtained.
The specific synthetic route for (S) -5-methyl-2-pyrrolidone is as follows:
the invention provides a method for synthesizing 5-methyl-2-pyrrolidone by continuous flow biocatalysis, which comprises the steps of carrying out reductive amination on substrate levulinic acid by using amine dehydrogenase, recycling NADH coenzyme factor by using free ammonia as an amine donor, generating by-products only by water, and finally synthesizing an optically pure product 5-methyl-2-pyrrolidone by intramolecular cyclization. In the invention, injection pumps are used for injecting samples into the microreactor, and two pumps are used for injecting different substances so as to enable the substances to perform enzymatic reaction in the microreactor.
Compared with the prior art, the invention has the positive progress effects that:
the invention provides a method for synthesizing (S) -5-methyl-2-pyrrolidone by continuous flow biocatalysis, which can be used for catalyzing asymmetric reduction of levulinic acid in a micro-reactor to generate corresponding optical pure 4-aminopentanoic acid, and synthesizing chiral lactam by intramolecular cyclization, and has the advantages of mild reaction condition, high catalytic efficiency, high enzymatic catalytic conversion rate in a short time, good optical purity of products, ee value higher than 99 percent and good industrial application prospect.
Compared with the traditional mechanical stirring preparation method, the method carries out enzyme catalysis in the micro-reactor, and can achieve higher conversion rate in shorter time. Meanwhile, the amine dehydrogenase AmDH subjected to directed evolution is high in catalytic activity and excellent in stereoselectivity.
Detailed Description
The individual reaction or detection conditions described in the summary of the invention can be combined or modified according to common general knowledge in the art and can be verified by experiments. The invention will be further illustrated by way of examples, it being understood that the examples set forth, while indicating preferred embodiments of the invention, are given by way of illustration only and are not intended to limit the invention to the examples set forth.
The sources of materials in the following examples are:
syringe pumps were purchased from beijing star dazhi development.
Expression plasmid pET28a was purchased from Novagen.
E.coli DH 5. Alpha. And E.coli BL21 (DE 3) competent cells, 2X Taq PCR MasterMix, agarose gel DNA recovery kit were purchased from Beijing Tiangen Biochemical technology Co.
The restriction enzymes EcoR I and Hind III are both commercially available products from the company New England Biolabs (NEB).
Unless otherwise indicated, the specific experiments in the following examples were performed according to methods and conditions conventional in the art, or following the commercial specifications of the kit.
The adopted 3D three-dimensional continuous flow micro-reactor is formed by connecting 3 micro-reactor chips, the 3 micro-reactor chips are respectively arranged into 3 layers, the first layer micro-reactor chip is provided with two feed inlets, the third layer micro-reactor chip is provided with a discharge outlet, the second layer micro-reactor chip is connected with the first layer micro-reactor chip and the third layer micro-reactor chip, each layer micro-reactor chip is provided with a plurality of mixed reaction cavities for liquid mixed reaction, and after the solution I and the solution II enter and are mixed from the two feed inlets, the solution I and the solution II pass through each mixed reaction cavity of the first layer micro-reactor chip, each mixed reaction cavity of the second layer micro-reactor chip and each mixed reaction cavity of the third layer micro-reactor chip in sequence and then flow out from the discharge outlet of the third layer micro-reactor chip, and the mixed reaction cavities on the first layer micro-reactor chip, the second layer micro-reactor chip and the third layer micro-reactor chip are all used as places for continuous mixed reaction.
Wherein, two feed inlets are all connected with longer entry passageway, are feeding runner 1 and feeding runner 2 respectively, and wherein, feeding runner 1 divide into two, form the design of cross with feeding runner 2, are favorable to improving the mixing efficiency of two fluids.
In the invention, each micro-reactor chip is provided with a plurality of mixing reaction chambers for liquid mixing reaction, and different mixing reaction chambers are communicated through pipelines. In the invention, the layer-by-layer cascade mixed reaction cavity further improves the microcosmic mixing strength of reactants, and is beneficial to strengthening the reaction process.
In the invention, the whole microreactor chip is in a serpentine arrangement form, a zigzag arrangement form or a zigzag arrangement form, and the mixing reaction cavity on the microreactor chip is in a heart-shaped structure.
The material of the microreactor chip is one of glass, ceramic, corrosion-resistant alloy or fluorine-containing polymer.
The invention provides a method for synthesizing (S) -5-methyl-2-pyrrolidone by continuous flow biocatalysis, which uses amine dehydrogenase to catalyze asymmetric reduction of levulinic acid to prepare optical 4-aminopentanoic acid, and synthesizes (S) -5-methyl-2-pyrrolidone by intramolecular cyclization of intermediate optical 4-aminopentanoic acid, wherein in enzymatic reaction, formate dehydrogenase provides coenzyme circulation, and the enzymatic reaction occurs in a microreactor.
Ammonium formate/ammonia buffer solution (3M, pH 6.5) solution one (amine dehydrogenase lyophilized enzyme powder, formate dehydrogenase lyophilized enzyme powder, NAD) + ) Solution two (levulinic acid 40 mM) prepared by ammonium formate/ammonia water buffer solution (3M, pH 6.5) enters a micro-reactor mixing zone through a feeding flow channel 1 and a feeding flow channel 2 respectively, and is uniformly mixed instantaneously, and two different materials are ensured not to be contacted with each other before entering the micro-reactor mixing zone; after the enzyme solution and the substrate solution enter the micro-channel reactor, through homogenization and microcosmic control of temperature and concentration in each unit reactor, the catalytic reaction can be efficiently and rapidly carried out in the tube side of the micro-reactor, finally, the product 4-aminopentanoic acid is generated, and the product is discharged into 1mol/L sulfuric acid solution from a discharge hole to terminate the reaction.
The amino acid sequence of the amine dehydrogenase is shown as SEQ ID No.2, and the nucleotide sequence corresponding to the amine dehydrogenase is shown as SEQ ID No. 1.
The chemical structure of the (S) -5-methyl-2-pyrrolidone is shown as follows:
the method for synthesizing (S) -5-methyl-2-pyrrolidone by intramolecular cyclization of intermediate optical 4-aminopentanoic acid comprises the following steps: a certain amount of solid EDCI is added into a reaction liquid containing the intermediate optical 4-aminopentanoic acid, and the mixture is magnetically stirred for 2 hours at room temperature for the self-cyclization of the intermediate optical 4-aminopentanoic acid to form lactam.
In one embodiment of the invention, the enzymatic reaction may be carried out as follows in an exemplary manner: formic acid at pH 6.5In ammonium/ammonia buffer, in formate dehydrogenase and NAD + In the presence of the amine dehydrogenase AmDH, catalyzing an asymmetric reduction of the levulinic acid. In such applications, the concentration of the substrate in the reaction solution may be 20mmol/L. The amine dehydrogenase may be used in an amount of 1 to 500U/L depending on the reaction system used. Enzymatic levulinic acid asymmetric reduction, NADH is oxidized to NAD + To carry out the cyclic regeneration of the coenzyme NADH, formate dehydrogenase is additionally added to the reaction system. The reaction conversions and enantiomeric excess values (ee) of the products can be analyzed by gas chromatography, preferably using a CP-Chirasil-Dex CB column, 25m 0.25mm 0.39 μm specification, by the following analysis of the conversion and ee values: the initial column temperature is 110 ℃,20 ℃/min is raised to 150 ℃, 2min is kept, 5 ℃/min is raised to 160 ℃, and 5min is kept.
After the enzymatic reaction is completed, the reaction solution is cooled to room temperature, a certain amount of solid EDCI is added into the reaction solution, and the mixture is magnetically stirred at room temperature for 2 hours for the self cyclization of the intermediate amino acid to form lactam. After cyclization, the reaction solution was sufficiently extracted with 10 volumes of ethyl acetate, and the ethyl acetate phase was collected by high-speed centrifugation, and the ethyl acetate was removed by vacuum spin evaporation to obtain a crude lactam product. The crude product contains a small amount of EDCI and levulinic acid, and thus it is required to be purified by a silica gel column. The mobile phase is dichloromethane, methanol=1:20, and then the organic solvent is removed by vacuum rotary evaporation, so that the pure product of the lactam is obtained.
The specific synthetic route for (S) -5-methyl-2-pyrrolidone is as follows:
EXAMPLE 1 continuous flow biocatalytic levulinic acid Synthesis of (S) -5-methyl-2-pyrrolidone
The whole enzymatic experiment is carried out in a microreactor, two injection pumps are needed to be communicated into the microreactor, the substrate levulinic acid is injected into the microreactor by the pump 1, and the amine dehydrogenase, the formate dehydrogenase and the NAD are injected into the microreactor by the pump 2 + Two pumpsIs brought into contact with the liquid in the microreactor so that an enzyme-catalyzed reaction takes place.
The activity volumes of the amine dehydrogenase and the formate dehydrogenase in the solution I are 3.5U/mL and 5.25U/mL and NAD respectively + The concentration of levulinic acid in the solution II was 40mmol/L, and the solution I and the solution II were both prepared from an ammonium formate/ammonia buffer solution (3M, pH 6.5). The reaction is carried out at the reaction temperature of 40 ℃, the flow rates of the first solution and the second solution are respectively 0.044mL/min and 0.044mL/min, after the reaction is finished, the substrate conversion rate is 72.8% by using liquid chromatography, and the ee (S) value can be higher than 99%. After the enzymatic reaction is completed, the reaction solution is cooled to room temperature, a certain amount of solid EDCI is added into the reaction solution, and the mixture is magnetically stirred at room temperature for 2 hours for the self cyclization of the intermediate amino acid to form lactam. After cyclization, the reaction solution was sufficiently extracted with 10 volumes of ethyl acetate, and the ethyl acetate phase was collected by high-speed centrifugation, and the ethyl acetate was removed by vacuum spin evaporation to obtain a crude lactam product. The crude product contains a small amount of EDCI and levulinic acid, and thus it is required to be purified by a silica gel column. The mobile phase is dichloromethane, methanol=1:20, and then the organic solvent is removed by vacuum rotary evaporation, so that the pure product of the lactam is obtained.
The sequence related to the invention is as follows:
SEQ ID No.1:
SEQ ID No.2:
the previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Sequence listing
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<120> a method for continuous flow biocatalytic synthesis of (S) -5-methyl-2-pyrrolidone
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Gly Ile Ala Lys Met Ile Leu Ser Lys Lys Gly Met Glu Ile Val Gly
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Tyr Thr Glu Lys Val Phe Pro Leu Ile Lys Leu Ala Val Glu Asn Gly
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His Leu Glu Leu Ala Lys Glu Ile Asp Arg Leu Ala Arg Glu Asn Gly
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Leu Ile Ile Ala Leu Thr Gly Val Cys Val Asp Val Asp Ser Ile Lys
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Ala Ala Arg Ile Asn Asp Leu Ser Pro Phe Gly Lys Ala Val Met Glu
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Pro Gly Leu Val Thr Met Leu Asp Leu Pro Val Pro Arg Ala Ile Met
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Gly Asp Ala Arg Asp Met Ile Arg Arg Arg
340 345
Claims (4)
1. A method for synthesizing (S) -5-methyl-2-pyrrolidone by continuous flow biocatalysis is characterized in that an amine dehydrogenase is used for catalyzing asymmetric reduction of levulinic acid to prepare optical 4-aminopentanoic acid, and then the intermediate optical 4-aminopentanoic acid is synthesized by intramolecular cyclizationS) -5-methyl-2-pyrrolidone, in an enzymatic reaction, formate dehydrogenase provides a coenzyme cycle, the enzymatic reaction of which takes place in a microreactor;
the micro-reactor is a continuous flow micro-channel reactor, the continuous flow micro-channel reactor is formed by connecting 3 micro-reactor chips, the 3 micro-reactor chips are respectively arranged into 3 layers, the first layer micro-reactor chip is provided with two feed inlets, the third layer micro-reactor chip is provided with a discharge outlet, the second layer micro-reactor chip is connected with the first layer micro-reactor chip and the third layer micro-reactor chip, each micro-reactor chip is provided with a plurality of mixing reaction chambers for liquid mixing reaction, and raw materials I and II respectively enter and are mixed through the two feed inlets and sequentially pass through each mixing reaction chamber of the first layer micro-reactor chip, each mixing reaction chamber of the second layer micro-reactor chip and each mixing reaction chamber of the third layer micro-reactor chip, and then flow out from the discharge outlet of the third layer micro-reactor chip;
continuously introducing the first solution and the second solution into a micro-reactor in proportion for reaction, and cyclizing and purifying after biocatalysis reaction to obtain the catalystS) -5-methyl-2-pyrrolidone; the first solution contains amine dehydrogenase, formate dehydrogenase, and NAD + The volume capacity ratio of the amine dehydrogenase to the formate dehydrogenase is 1:1.5, and the solution II contains substrate levulinic acid;
the amino acid sequence of the amine dehydrogenase is shown as SEQ ID No. 2;
the%S) The chemical structure of the-5-methyl-2-pyrrolidone is shown below:
。
2. the method for the biocatalytic synthesis of (S) -5-methyl-2-pyrrolidone according to claim 1, wherein the solution I and the solution II participating in the reaction are prepared by using ammonium formate/ammonia water buffer solution with a concentration of 3M and a pH of 6.5.
3. The method of continuous flow biocatalytic synthesis of (S) -5-methyl-2-pyrrolidone according to claim 1, wherein NAD is present in the solution one + The concentration of (C) was 0.2mmol/L.
4. The method for synthesizing (S) -5-methyl-2-pyrrolidone by continuous flow biocatalysis according to claim 1, wherein the intramolecular cyclization synthesis of the intermediate optical 4-aminopentanoic acidS) The method for preparing the-5-methyl-2-pyrrolidone comprises the following steps: solid EDCI was added to the reaction solution containing the intermediate optical 4-aminopentanoic acid and magnetically stirred at room temperature for self-cyclization of the intermediate optical 4-aminopentanoic acid to form the lactam.
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