CN118064412A - Amidase, preparation method and application thereof - Google Patents
Amidase, preparation method and application thereof Download PDFInfo
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- CN118064412A CN118064412A CN202211431050.6A CN202211431050A CN118064412A CN 118064412 A CN118064412 A CN 118064412A CN 202211431050 A CN202211431050 A CN 202211431050A CN 118064412 A CN118064412 A CN 118064412A
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- Prior art keywords
- amidohydrolase
- preparation
- carbamoylmethyl
- substrate
- pregabalin
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- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 108700023418 Amidases Proteins 0.000 title claims description 7
- 102000005922 amidase Human genes 0.000 title claims description 7
- 102000004092 Amidohydrolases Human genes 0.000 claims abstract description 29
- 108090000531 Amidohydrolases Proteins 0.000 claims abstract description 29
- AYXYPKUFHZROOJ-ZETCQYMHSA-N pregabalin Chemical compound CC(C)C[C@H](CN)CC(O)=O AYXYPKUFHZROOJ-ZETCQYMHSA-N 0.000 claims abstract description 25
- NPDKTSLVWGFPQG-SSDOTTSWSA-N (3r)-3-(2-amino-2-oxoethyl)-5-methylhexanoic acid Chemical compound CC(C)C[C@H](CC(N)=O)CC(O)=O NPDKTSLVWGFPQG-SSDOTTSWSA-N 0.000 claims abstract description 24
- 238000006243 chemical reaction Methods 0.000 claims abstract description 23
- 229960001233 pregabalin Drugs 0.000 claims abstract description 22
- 102000004190 Enzymes Human genes 0.000 claims abstract description 18
- 108090000790 Enzymes Proteins 0.000 claims abstract description 18
- FNAQPQLVCOZGRH-UHFFFAOYSA-N 4-(2-methylpropyl)piperidine-2,6-dione Chemical compound CC(C)CC1CC(=O)NC(=O)C1 FNAQPQLVCOZGRH-UHFFFAOYSA-N 0.000 claims abstract description 17
- 102000004157 Hydrolases Human genes 0.000 claims abstract description 16
- 108090000604 Hydrolases Proteins 0.000 claims abstract description 16
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 25
- 239000000758 substrate Substances 0.000 claims description 16
- 241000588724 Escherichia coli Species 0.000 claims description 13
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
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- 150000007523 nucleic acids Chemical class 0.000 claims description 8
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- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 6
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 4
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- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 3
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 3
- 108010093096 Immobilized Enzymes Proteins 0.000 claims description 2
- 241001052560 Thallis Species 0.000 claims description 2
- 125000003368 amide group Chemical group 0.000 claims description 2
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- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 4
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- 230000014759 maintenance of location Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- SWFXGBPZRVILCJ-QMMMGPOBSA-N (3R)-3-(2-hydroxyethyl)-5-methylhexanamide Chemical compound C(N)(=O)C[C@@H](CCO)CC(C)C SWFXGBPZRVILCJ-QMMMGPOBSA-N 0.000 description 3
- NPDKTSLVWGFPQG-UHFFFAOYSA-N 3-(2-amino-2-oxoethyl)-5-methylhexanoic acid Chemical compound CC(C)CC(CC(N)=O)CC(O)=O NPDKTSLVWGFPQG-UHFFFAOYSA-N 0.000 description 3
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- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 description 2
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 206010010904 Convulsion Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000193385 Geobacillus stearothermophilus Species 0.000 description 2
- 241000589540 Pseudomonas fluorescens Species 0.000 description 2
- 239000012670 alkaline solution Substances 0.000 description 2
- 230000003556 anti-epileptic effect Effects 0.000 description 2
- 239000001961 anticonvulsive agent Substances 0.000 description 2
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- 238000006555 catalytic reaction Methods 0.000 description 2
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- -1 cyclic imine Chemical class 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
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- 238000004128 high performance liquid chromatography Methods 0.000 description 2
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- 239000007924 injection Substances 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 208000004296 neuralgia Diseases 0.000 description 2
- 239000002858 neurotransmitter agent Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- HYHLWVJLJXARGY-UHFFFAOYSA-N 3-(aminomethyl)benzamide Chemical compound NCC1=CC=CC(C(N)=O)=C1 HYHLWVJLJXARGY-UHFFFAOYSA-N 0.000 description 1
- AYXYPKUFHZROOJ-UHFFFAOYSA-N 3-(azaniumylmethyl)-5-methylhexanoate Chemical compound CC(C)CC(CN)CC(O)=O AYXYPKUFHZROOJ-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 238000007167 Hofmann rearrangement reaction Methods 0.000 description 1
- 206010061334 Partial seizures Diseases 0.000 description 1
- 206010036376 Postherpetic Neuralgia Diseases 0.000 description 1
- 206010041250 Social phobia Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000009098 adjuvant therapy Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 125000004202 aminomethyl group Chemical group [H]N([H])C([H])([H])* 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 1
- 230000003178 anti-diabetic effect Effects 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 238000011914 asymmetric synthesis Methods 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
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- 206010015037 epilepsy Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
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- 235000019253 formic acid Nutrition 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 208000021722 neuropathic pain Diseases 0.000 description 1
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- 230000000707 stereoselective effect Effects 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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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- 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
- C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
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Abstract
The invention provides an amide hydrolase, a preparation method and application thereof. The amino acid sequence of the amidohydrolase is shown as SEQ ID NO. 1. The amide hydrolase of the invention can efficiently and exclusively catalyze 3-isobutyl glutarimide to generate single chiral (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid, the conversion rate of the amide hydrolase of the invention can reach 99.5%, the ee value can reach 100%, the enzyme consumption is low, and the reaction time is shorter. The invention has important significance for producing pregabalin with high optical purity.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and relates to an amide hydrolase, a preparation method and application thereof, in particular to a synthesis method of an amide hydrolase and (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid.
Background
Pregabalin (Pregabalin), chemical name: (S) -3- (aminomethyl) -5-methylhexanoic acid (CAS number: 148553-50-8), english name: (S) -3- (Aminomethyl) -5-methylhexanioc acid is a representative product in the fields of treatment of neuropathic pain and antiepileptic, and can be used for the adjuvant treatment of neuralgia caused by antidiabetic, postherpetic neuralgia, and human local seizure epilepsy and social anxiety disorder.
The pregabalin has a structure similar to neurotransmitter gamma-aminobutyric acid (GABA), inhibits the release of a series of neurotransmitters by inhibiting the inflow of calcium ions, enables the hyperexcitable neurons to return to a normal state, cannot be metabolized by P450 in cytochromes, does not interact with other medicines, has good drug susceptibility, and can be combined with the existing antiepileptic medicines. Pregabalin was approved for the treatment of partial seizures in adults in the united states and europe since 2004, and is considered a very promising drug in the market. Therefore, the development of the synthesis process research of pregabalin has wide market prospect and great economic value.
3- (Aminomethyl) -5-methylhexanoic acid has 2 configurations, the pregabalin of S configuration has pharmacological activity, and the activity of R configuration is only 1/10 of that of S configuration, so how to simply and efficiently obtain the S configuration with high optical purity is a key point and a difficult point for synthesizing pregabalin.
(R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid (CAS number: 181289-33-8) is obtained by Hofmann rearrangement to obtain (S) -3- (aminomethyl) -5-methylhexanoic acid (pregabalin), which is a key intermediate of pregabalin, and its preparation method can be mainly divided into stereoselective synthesis method, racemate resolution method (chemical resolution, microbial enzyme resolution), desymmetric synthesis method, etc., and the chemical resolution method is the main method of current industrial production, and although the process of this method is mature, the reaction period is long, the raw material cost is high, the utilization rate is low, and the production route has pollution. The microbial enzyme method has the advantages of high specificity and high efficiency, and the biological enzyme catalysis has the advantages of mild condition, environmental friendliness and the like, and has great research value.
Chinese patent CN114164198a discloses the directed engineering of amidase BsHase from bacillus stearothermophilus (Bacillus stearothermophilus SD-1) to obtain mutants, which are subjected to hydrolysis of 3-isobutylglutarimide to produce (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid. Wherein the chiral purity of the product prepared by the mutant M63AL65HC317T can reach 98.5%.
Patent CN114686465A discloses that the hydrolase derived from pseudomonas fluorescens (Pseudomonas fluorescens) exhibits excellent (R) - (-) -3- (carbamoylmethyl) -5-methylhexanol product selectivity, and is genetically engineered to obtain the hydrolase of sequence 1 after modification, which can reach a conversion rate of 99.3% and an ee value of 100% after 36 hours of reaction at a substrate concentration of 300 g/L.
Therefore, the method screens the amidohydrolase from different sources, widens the source of the amidohydrolase, enlarges the size of the enzyme library, is favorable for developing an excellent pregabalin synthesis process, and has important significance for developing a route for synthesizing pregabalin with green and environment-friendly property and low cost so as to improve the market competitiveness of the product.
Disclosure of Invention
In order to solve the defects of preparation of pregabalin and intermediates thereof by a biological enzyme catalysis method in the prior art, the invention provides an amide hydrolase, a preparation method and application thereof. The amide hydrolase of the invention can efficiently and exclusively catalyze 3-isobutyl glutarimide to generate single chiral (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid, the conversion rate of the amide hydrolase of the invention can reach 99.5%, the ee value can reach 100%, the enzyme consumption is low, and the reaction time is shorter. The invention has important significance for producing pregabalin with high optical purity.
The first aspect of the invention provides an amidase, the amino acid sequence of which is shown as SEQ ID NO. 1.
In a second aspect the invention provides an isolated nucleic acid encoding an amidohydrolase according to the first aspect of the invention.
Preferably, the nucleotide sequence of the nucleic acid is shown as SEQ ID NO. 2.
In a third aspect the invention provides a recombinant expression vector comprising a nucleic acid according to the second aspect of the invention.
In a fourth aspect, the present invention provides a transformant comprising a nucleic acid according to the second aspect of the present invention or a recombinant expression vector according to the third aspect of the present invention.
Preferably, the host cell of the transformant is E.coli (ESCHERICHIA COLI), e.g., E.coli DH 5. Alpha.
In a fifth aspect the present invention provides a method of preparing an amidohydrolase according to the first aspect of the invention, the method comprising culturing a transformant according to the fourth aspect of the invention under conditions suitable for expression of the amidohydrolase.
In a sixth aspect the invention provides a kit comprising an amidohydrolase according to the first aspect of the invention.
In a preferred embodiment, the kit further comprises a buffer solution, preferably Tris-HCl buffer, or an alkaline solution, preferably ammonia, sodium hydroxide, sodium carbonate or sodium bicarbonate.
In a preferred embodiment, the kit further comprises a substrate such as 3-isobutylglutarimide.
In a seventh aspect the present invention provides a process for the amidohydrolysis of a substrate, the process comprising providing at least one substrate and an amidohydrolase according to the first aspect of the invention, and contacting the substrate with the amidohydrolase under conditions such that the substrate is amidohydrolysed to produce at least one amidohydrolase product, wherein the substrate is a substrate having an amide group.
In an eighth aspect, the present invention provides a process for the preparation of (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid, said process comprising the steps of: the (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid is obtained by catalyzing 3-isobutylglutarimide with an amidase according to the first aspect of the present invention.
In a preferred embodiment, the amidohydrolase is in the form of an amidohydrolase cell, a crude enzyme solution, a pure enzyme solution, or an immobilized enzyme.
In a preferred embodiment, the working concentration of 3-isobutylglutarimide is 50-400g/L, preferably 100g/L, 150g/L, 200g/L, 250g/L, 300g/L, 350g/L or 400g/L.
The working concentration refers to the initial concentration of the substance in the entire reaction system.
In a preferred embodiment, the amidohydrolase comprises 1 to 15%, preferably 3%, 5%, 8% or 12% by volume of the system.
The volume percentage is the percentage of the amidohydrolase crude enzyme liquid in the whole reaction system.
The crude enzyme solution is prepared by wet thalli and buffer solution according to the following ratio of 1:10 (g: mL) homogenized. The ratio of the homogenized wet bacterial cells to the buffer solution can be adjusted without affecting the amount of the enzyme bacterial cells.
The buffer may be conventional in the art, for example a 0.1M PB (pH 7.6) buffer.
In a preferred embodiment, the reaction is carried out at a pH of 7 to 8, preferably 7.5.
In a preferred embodiment, the pH is controlled by a buffer solution and an alkaline solution.
Preferably, the buffer solution is Tris-HCl buffer solution.
Preferably, the alkali liquor is ammonia water, sodium hydroxide, sodium carbonate or sodium bicarbonate.
In a preferred embodiment, the reaction is carried out with shaking or stirring.
Preferably, the reaction is carried out at a rotational speed of 200-500rpm, for example 250rpm, 300rpm, 400rpm or 450rpm.
In a preferred embodiment, the reaction is carried out at a temperature of from 30 to 50℃and preferably at 40 ℃.
According to a ninth aspect of the present invention there is provided a process for the preparation of pregabalin comprising the step of preparing (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid according to the process according to the eighth aspect of the present invention.
Preferably, the above preparation method further comprises the step of converting (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid into pregabalin by Huffman rearrangement.
In a tenth aspect, the present invention provides the use of an amidohydrolase according to the first aspect of the invention for the preparation of pregabalin or an intermediate thereof, or for the preparation of a formulation for the production of pregabalin or an intermediate thereof, said intermediate being (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that:
The method selects the step from cyclic imine to monoamide as a starting point, and screens a large number of different microbial sources of amidohydrolase to obtain the amidohydrolase which can efficiently and specifically catalyze 3-isobutyl glutarimide to generate single chiral (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid, and the specific conversion from cyclic imine to R-monoamide is realized by a biological method, so that the reaction step is simplified, the utilization rate of raw materials is improved, the conversion rate of a substrate can reach 99.5%, the ee value can reach 100%, the enzyme consumption is low, and the reaction time is shorter. The R-monoamide prepared by the invention can be utilized to further finally obtain the pregabalin with high optical purity. The invention has important significance for producing pregabalin with high optical purity.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which should not be construed as limiting the scope of the invention.
3-Isobutylglutarimide: CAS number: 916982-10-0, 3-isobutylglutarimide used in the examples was MACKLIN product.
3- (Carbamoylmethyl) -5-methylhexanoic acid: CAS number: 181289-15-6, purchased from Shanghai Bi to pharmaceutical technologies Co., ltd.
(R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid: CAS number: 181289-33-8; the (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid standard used in the examples is Shanghai Bide pharmaceutical product.
High performance liquid chromatography detection method (liquid chromatography) of 3-isobutyl glutarimide and (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid:
Diamonsil C.mu.m, 250X 4.6 mm; buffer salt: 10mM ammonium formate (formic acid adjusted pH to 3.0); mobile phase: acetonitrile: buffer salt = 20:80; flow rate: 1.0ml/min; sample injection amount: 10 μl; column temperature: 35 ℃.
The retention time of the control 3-isobutylglutarimide is as follows: 22.338min; the retention time of the control (R) - (-) -3- (carbamoylmethyl) -5-methylhexanol was 6.807min.
The chiral purity of the (R) - (-) -3- (carbamoylmethyl) -5-methylhexanol is firstly detected by high performance liquid chromatography under the following detection conditions:
Chromatographic column: CHIRALPAK IA,5um,4.6 x 250mm; mobile phase: n-hexane: ethanol: tfa=880:120:1.5; flow rate: 0.5ml/min; concentration: 2mg/ml; sample injection amount: 10 μl; column temperature: 25 ℃; wavelength: 210nm.
The retention time of the racemate 3- (carbamoylmethyl) -5-methylhexanoic acid of the control is 16.098min and 17.732min, and the retention time of the (R) -3- (carbamoylmethyl) -5-methylhexanoic acid of the control is 16.051min.
The expression plasmids pET28a, E.coli DH5 alpha and E.coli BL21 (DE 3) competent cells, 2X Taq PCR MasterMix, agarose gel DNA recovery kit were purchased from Beijing Ding Guo Changchun biotechnology Limited company.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified. The quantitative tests in the following examples were all set up in triplicate and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.
EXAMPLE 1 hydrolase library screening
3-Isobutyl glutarimide is used as a substrate, and a hydrolase library is subjected to high-throughput screening to detect the enzyme with the capability of catalyzing and generating (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid. Screening conditions: 10 g/L3-isobutylglutarimide, hydrolase enzyme solution, 50mM Tris-Cl (pH 7.5), at 40℃for 24 hours.
The results show that the amidase (UniProtKB: Q44184.1) derived from Agrobacterium tumefaciens (Agrobacterium tumefaciens) has (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid selectivity. Then, the A105V-P140A mutant is obtained by engineering transformation, the amino acid sequence is shown as SEQ ID NO. 1, and the encoding nucleotide sequence is shown as SEQ ID NO. 2. The mutant gene is connected with pET28a plasmid to obtain pET28a recombinant expression plasmid, and the recombinant expression plasmid is transformed into E.coli (E.coli) DH5 alpha competent cells to obtain recombinant transformant.
The sequence information related to the invention is shown in the following table:
After streaking and activating engineering bacteria plates containing mutant genes, single colonies are selected and inoculated into a resistant LB medium containing 50 mug/mL kanamycin, shake-cultured for 4 hours at 37 ℃, transferred into 150mL of fresh TB liquid medium also containing 50 mug/mL kanamycin according to the inoculum size of 1v/v%, cultured at about 0.8 at 37 ℃ and 220rpm, added with 0.1mM IPTG with the final concentration for 16 hours at 25 ℃, centrifuged at 4500rpm for 20 minutes (centrifuge: eppendorf Centrifuge 5810R) after the culture is finished, the supernatant is discarded, and the bacteria are collected and stored in an ultralow temperature refrigerator at-20 ℃ for standby.
EXAMPLE 2 preparation of recombinant E.coli hydrolase enzyme preparation
Recombinant E.coli wet cell expressing hydrolase in example 1 was mixed with 0.1M PB (pH 7.6) according to a 1:10 (g: mL) and evenly mixing, homogenizing and crushing at 4 ℃ to release intracellular amidohydrolase, centrifuging at 4000rpm for 20min, and taking the supernatant to obtain crude enzyme liquid.
EXAMPLE 3 asymmetric Synthesis of (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid
The total volume of the reaction system is 100mL, wherein, 3-isobutyl glutarimide is 300g/L, recombinant escherichia coli crude enzyme solution is 5% (v/v), 50mM Tris-HCl (pH 7.6) is used as buffer solution, 2 times of ammonia water is used for dilution in the reaction process to control the pH of the reaction system to 7.5, the reaction temperature is 40 ℃, the oscillation reaction is carried out for 24 hours, the conversion rate reaches 99.5%, and the ee value is 100%.
Claims (10)
1. The amidohydrolase is characterized in that the amino acid sequence of the amidohydrolase is shown as SEQ ID NO. 1.
2. An isolated nucleic acid encoding the amidohydrolase of claim 1; preferably, the nucleotide sequence of the nucleic acid is shown as SEQ ID NO. 2.
3. A recombinant expression vector comprising the nucleic acid of claim 2.
4. A transformant comprising the nucleic acid of claim 2 or the recombinant expression vector of claim 3; preferably, the host cell of the transformant is E.coli (ESCHERICHIA COLI) such as E.coli DH 5. Alpha.
5. A method of making the amidohydrolase of claim 1, comprising culturing the transformant of claim 4 under conditions suitable for expression of the amidohydrolase.
6. A kit comprising the amidohydrolase of claim 1; preferably, the kit further comprises 3-isobutylglutarimide, a buffer solution and/or an alkali solution.
7. A method for the amidohydrolysis of a substrate, the method comprising providing at least one substrate and the amidohydrolase of claim 1, and contacting the substrate with the amidohydrolase under conditions such that the substrate is amidohydrolyzed to produce at least one amidohydrolyzate; wherein the substrate is a substrate having an amide group.
8. A process for the preparation of (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid, characterized in that it comprises the following steps: catalyzing 3-isobutylglutarimide with the amidase according to claim 1 to obtain (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid;
preferably, the preparation method satisfies one or more of the following conditions:
(1) The amidohydrolase exists in the form of amidohydrolase thalli, crude enzyme liquid, pure enzyme liquid or immobilized enzyme;
(2) The working concentration of the 3-isobutyl glutarimide is 50-400g/L, preferably 300g/L;
(3) The amide hydrolase accounts for 1-15% of the volume of the system, preferably 5%;
(4) The reaction is carried out at a pH of 7 to 8, preferably at a pH of 7.5;
(5) The pH is controlled by a buffer solution and an alkali solution, wherein the buffer solution is preferably Tris-HCl buffer solution, and the alkali solution is preferably ammonia water, sodium hydroxide, sodium carbonate or sodium bicarbonate;
(6) The reaction is carried out with shaking or stirring, preferably at a speed of 200-500rpm;
(7) The reaction is carried out at a temperature of 30-50 c, for example 40 c.
9. A process for the preparation of pregabalin comprising the step of preparing (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid according to the process of claim 8.
10. Use of an amidohydrolase according to claim 1 in the preparation of pregabalin or an intermediate thereof, or in the preparation of a formulation for the production of pregabalin or an intermediate thereof, said intermediate being (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid.
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