Dihydropyrimidine amino hydrolase and application thereof
阅读说明：本技术 一种二氢嘧啶氨基水解酶及其应用 (Dihydropyrimidine amino hydrolase and application thereof ) 是由 张大龙 蒙传珍 于 2021-10-19 设计创作，主要内容包括：本发明提供了一种制备(R)-3-(氨甲酰甲基)-5-甲基己酸的方法,包括如下步骤：以3-异丁基戊二酰亚胺为底物,使用二氢嘧啶氨基水解酶SEQ ID NO:1或者SEQ ID NO:3催化开环反应,得到(R)-3-(氨甲酰甲基)-5-甲基己酸。(The invention provides a method for preparing (R) -3- (carbamoylmethyl) -5-methylhexanoic acid, which comprises the following steps: 3-isobutyl glutarimide is taken as a substrate, dihydropyrimidine amino hydrolase SEQ ID NO:1 or SEQ ID NO:3 is used for catalyzing ring-opening reaction, and (R) -3- (carbamoylmethyl) -5-methylhexanoic acid is obtained.)
1. A process for preparing (R) -3- (carbamoylmethyl) -5-methylhexanoic acid comprising the steps of:
3-isobutyl glutarimide is taken as a substrate, dihydropyrimidine amino hydrolase SEQ ID NO:1 or SEQ ID NO:3 is used for catalyzing ring-opening reaction, and (R) -3- (carbamoylmethyl) -5-methylhexanoic acid is obtained.
2. The method of claim 1, wherein said dihydropyrimidine aminohydrolase is in the form of an enzyme or in the form of a microorganism expressing it.
3. The method of claim 2, wherein the enzyme is an immobilized enzyme.
4. The method of claim 2, wherein the microorganism is selected from the group consisting of bacillus subtilis, pichia pastoris, saccharomyces cerevisiae, and escherichia coli.
5. The method of claim 4, wherein the microorganism is Escherichia coli.
6. The method of claim 5, wherein the nucleotide sequence of the gene encoding dihydropyrimidine aminohydrolase SEQ ID NO. 1 is SEQ ID NO. 2; or the nucleotide sequence of the coding gene of the dihydropyrimidine amino hydrolase SEQ ID NO. 3 is SEQ ID NO. 4.
7. A plasmid comprising the gene of claim 6.
8. The plasmid of claim 7 wherein the plasmid vector is selected from the PET series.
9. The plasmid of claim 8 wherein the plasmid vector is selected from the group consisting of pET22b, pET24a, pET28 a.
10. An Escherichia coli transformed with the plasmid of claim 7.
Technical Field
The invention belongs to the technical field of biocatalysis, relates to a technology for preparing pregabalin chiral intermediate, and particularly relates to a method for preparing (R) -3- (carbamoylmethyl) -5-methylhexanoic acid by utilizing dihydropyrimidine amidohydrolase.
Background
Pregabalin (Pregabalin) is chemically named as (S) -3-aminomethyl-5-methylhexanoic acid, has the following structural formula, is a new generation of gamma-aminobutyric acid (gamma-GABA) receptor agonist developed by Warner-lambert company in the United states, is mainly used for treating peripheral neuralgia and adjuvant therapy of local partial epileptic seizure, wherein the pharmacological activity of S-configuration is 10 times that of R-configuration, so that the synthesis of optically pure chiral Pregabalin has important significance for enhancing the curative effect of medicaments and reducing the side effect caused by inactive enantiomers.
There are many methods for synthesizing pregabalin reported at present, but the methods are mainly chemical asymmetric synthesis methods by asymmetric catalysts and chiral ligands, or chiral resolution of racemic intermediates in the reaction process by using chemical reagents or commercial enzymes. The method for directly obtaining the chiral intermediate by using the high-efficiency enzyme to catalyze the latent chiral compound is the most economic and effective way. The dihydropyrimidine amino hydrolase substrate spectrum is wide, some imides, dihydropyrimidines and hydantoins can be hydrolyzed, but the dihydropyrimidine amino hydrolase substrate spectrum is more prone to hydrolyzing some cyclic imide substances. The chiral intermediate (R) -3- (carbamoylmethyl) -5-methylhexanoic acid of pregabalin can be obtained by hydrolysis of 3-isobutylglutarimide, which is a cyclic imide compound.
Therefore, it is presumed that the chiral intermediate (R) -3- (carbamoylmethyl) -5-methylhexanoic acid can be obtained by directly hydrolyzing 3-isobutylglutarimide with a highly selective and highly active dihydropyrimidine aminohydrolase.
Disclosure of Invention
Based on the above presumptions, the inventors screened various microorganism-derived dihydropyrimidine amidohydrolases (ec.3.5.2.2), including 41 dihydropyrimidine amidohydrolases. The dihydropyrimidine amino hydrolase is expressed by using escherichia coli, 3-isobutyl glutarimide is catalyzed by zymophyte to carry out hydrolysis reaction, the content of (R) -3- (carbamoylmethyl) -5-methylhexanoic acid in reaction liquid is detected, and the target dihydropyrimidine amino hydrolase is screened.
Through screening, two dihydropyrimidine amino hydrolases derived from Pseudomonas fluorescens, SEQ ID NO:1(NCBI accession number: WP-011030900.1) and SEQ ID NO:3(NCBI accession number: WP-011334810.1), were found to be capable of catalyzing the reaction of 3-isobutylglutarimide to produce (R) -3- (carbamoylmethyl) -5-methylhexanoic acid.
Therefore, the first purpose of the invention is to provide a technical scheme for preparing (R) -3- (carbamoylmethyl) -5-methylhexanoic acid by an enzyme catalysis method.
A process for preparing (R) -3- (carbamoylmethyl) -5-methylhexanoic acid comprising the steps of: 3-isobutyl glutarimide is taken as a substrate, dihydropyrimidine amino hydrolase SEQ ID NO:1 or SEQ ID NO:3 is used for catalyzing ring-opening reaction, and (R) -3- (carbamoylmethyl) -5-methylhexanoic acid is obtained.
Wherein the amino acid sequence of the first dihydropyrimidine amino hydrolase is SEQ ID NO: 1:
MSSRTVIRGGLVITASDEIHADVLIEDGRVAALAATGTPAAEAFTAENVIDASGKYVIPGGVDGHTHMEMPFGGTYAADTFETGTRAAAWGGTTTIVDFAIQSVGHSLREGLDAWHAKAEGNCAIDYGFHMIVSDVNQETLKEMDLLVEEGVTSFKQFMAYPGVFYSDDGQILRAMQRAAENGGLIMMHAENGIAIDVLVEQALARGETDPRFHGEVRKALLEAEATHRAIRLAQVAGAPLYVVHVSATEAVAELTRARDEGLPVFGETCPQYLFLSTDNLAEPDFEGAKYVCSTPLRPKEHQAALWRGLRTNDLQVVSTDHCPFCFSGQKELGRGDFSRIPNGMPGVENRMDLLHQAVVEGHIGRRRWIEIACATPARMFGLYPKKGTIAPGADADIVVYDPHAEQVISAETHHMNVDYSAYEGRRITGRVETVLSRGEPVVTEREYTGRKGHGAYTPRATCQYLT(SEQ ID NO:1)；
the amino acid sequence of the second dihydropyrimidine amino hydrolase is SEQ ID NO: 3:
MSLLIRGATIVTHDESYRADVYCADGVIKAIGENLDIPAGAEVLDGSGQYLMPGGIDPHTHMQLPFMGTVASEDFYSGTAAGLAGGTTSIIDFVIPNPQQSLLEAFHQWRGWAEKSASDYGFHVAITWWSEQVREEMAELVSHHGINSFKHFMAYKNAIMAADDTLVASFERCLELGAVPTVHAENGELVYHLQRKLMAQGITGPEAHPLSRPSQVEGEAASRAIRIAETIGTPLYLVHVSTKEALDEITYARSKGQPVYGEVLAGHLLLDDSVYQHPDWQTAAGYVMSPPFRPRGHQDALWHGLQSGNLHTTATDHCCFCAEQKAAGRDDFSKIPNGTAGIEDRMAVLWDEGVNSGRLSMQDFVALTSTNTAKIFNLYPRKGAIRVGADADLVLWDPQGTRTISAKTHHQQVDFNIFEGKTVTGVPSHTVSQGRVVWADGDLRAERGAGRYIERPAYPAVFDLLSKRAEQHKPTAVKR(SEQ ID NO:3)。
in one embodiment, the dihydropyrimidine aminohydrolase SEQ ID NO 1 or SEQ ID NO 3 described above may be in the form of an enzyme, such as a free or immobilized enzyme, or in the form of its expressing microbial organism.
The above-mentioned microorganism may be any microorganism suitable for expressing the above-mentioned dihydropyrimidinylaminohydrolase, and is selected from, for example, Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, and Escherichia coli.
Preferably, the microorganism is escherichia coli, more preferably escherichia coli BL21(DE 3).
In a second aspect of the present invention, there is provided a gene encoding the above-mentioned dihydropyrimidine aminohydrolase, SEQ ID NO:1 or SEQ ID NO: 3. When the host for the expression of the above-mentioned dihydropyrimidine aminohydrolase is Escherichia coli, the nucleotide sequence of the gene encoding the above-mentioned dihydropyrimidine aminohydrolase, SEQ ID NO:1, may be SEQ ID NO:2, but is not limited thereto; the nucleotide sequence of the gene encoding dihydropyrimidine aminohydrolase SEQ ID NO. 3 may be SEQ ID NO. 4, but is not limited thereto.
The coding gene can be cloned in a plasmid vector for transforming microbial host cells.
In one embodiment, the above plasmid vector is selected from the PET series, for example, but not limited to, PET22b, PET24a, PET28a, and the like.
In a third aspect of the present invention, there is provided Escherichia coli transformed with a plasmid containing the above-mentioned dihydropyrimidine aminohydrolase-encoding gene.
The reaction system of the above enzymatic reaction may be a buffer solution of pH6-9, such as Tris-HCl buffer solution or phosphate buffer solution, but is not limited thereto.
Controlling the pH of the reaction system to be 7.0-9.0, preferably 7.5-8.5, more preferably about 7.5 during the reaction process; the reaction temperature is 25 to 50 ℃, preferably 28 to 48 ℃, more preferably 30 to 45 ℃, more preferably 35 to 40 ℃, and most preferably about 40 ℃.
The dihydropyrimidine amino hydrolase SEQ ID NO 1 and SEQ ID NO 3 screened by the invention can catalyze the substrate 3-isobutyl glutarimide to carry out hydrolysis reaction and generate (R) -3- (carbamoylmethyl) -5-methylhexanoic acid through ring opening, and a new way for producing pregabalin chiral intermediate (R) -3- (carbamoylmethyl) -5-methylhexanoic acid by a biological catalysis method is developed.
Drawings
FIG. 1 is a HPLC detection spectrum after 8 hours of reaction of the substrates in the examples of the present invention.
FIG. 2 is a chiral chromatogram of HPLC-detected product after the catalytic reaction of the embodiment of the present invention.
Detailed Description
The inventors screened the 41 dihydropyrimidine amino hydrolase that could be used for the synthesis of (R) -3- (carbamoylmethyl) -5-methylhexanoic acid. These 41 dihydropyrimidine aminohydrolases include the following NCBI accession/GenBank accession numbers: WP _011030900.1(Streptomyces coelicolor A3(2)), WP _011334810.1(Pseudomonas fluorescens PfO-1), EUB75082.1(Pseudomonas sp.GM41), ADU69230.1(Pantoea sp.At-9b), ABY99529.1(Pseudomonas putida GB-1), ARW18102.1(Komagataeibacter europaeus), ADD28645.1 (Meliothermus ruber DSM 1279), SMC02439.1(Rubrobacter radiobacter DSM 5868), France 90978.1 (Peptophilus asacholibacter DSM20463), SMB99550.1 (Thermenococcus Toyonis Toyobe 36 81913.1 (Corynebacterium 2926), Pseudomonas aeruginosa W05800.1 (Staphylococcus aureus Asp 05800.1), Pseudomonas aeruginosa DSM 05800.1 (Corynebacterium 05800.1), Corynebacterium parvus 05800.1 (Corynebacterium parvus 05800.1), Corynebacterium parvus 363636363672), Corynebacterium parvus 36363636363636363672 (Corynebacterium parvus 05800.1), Corynebacterium parvus 05800.1 (Corynebacterium parvus 05800.1), Corynebacterium parvus 36363636363672), Corynebacterium parvus 3636363672 (Corynebacterium parvus 05800.1), Corynebacterium parvus 363636363672 (Corynebacterium parvus 36363672), Corynebacterium parvus 3636363636363672), Corynebacterium parvus 36363636363636363672 (Corynebacterium parvus 368 (Corynebacterium parvus 36363672), Corynebacterium parvus 3636363636363636368, Corynebacterium parvus 363636363636368 (Corynebacterium parvus 363636363636368), Corynebacterium parvus 36363636363636363636368, Corynebacterium parvus 36363636363636363636363636363636363636363636363636368, Corynebacterium parvus 3636363636363636363672), Corynebacterium parvus 36363636363636363636368 (Corynebacterium parvus 3636363636363636363636363636368, Corynebacterium parvus 368, Corynebacterium parvus 36368, Corynebacterium parvus 3636368 (Corynebacterium parvus 368, Corynebacterium parvus 363636368, Corynebacterium parvus 36368, Corynebacterium parvus 3636363636363636363636363636363636363636363636363636363636363636363636363636368, Corynebacterium parvus 368, Corynebacterium parvus 3636363636363636363636368, Corynebacterium parvus 368, Corynebacterium parvus 3636368, Corynebacterium parvus 36363636363636363636363636363636368, Corynebacterium parvus 368, Corynebacterium parvus 363636368, Corynebacterium parvus 36363636363636363636363636363636363636368, Corynebacterium parvus 36363636363636363636368, 3, Corynebacterium parvus 368, Corynebacterium parvus 3636368, Corynebacterium parvus 368, 3, Corynebacterium parvus 368, Corynebacterium parvus 36363636368, Corynebacterium parvus 363636368, Corynebacterium parvus 36368, Corynebacterium parvus 368, 3, Corynebacterium parvus 368, Corynebacterium parvus 363636368, Corynebacterium parvus 36368, Corynebacterium parvus 368, Corynebacterium parvus 36368, Corynebacterium parvus (Corynebacterium parvus 368, Corynebacterium parvus 3636363636368, Corynebacterium parvus 36363636363636363636363636363636363636363636368, Corynebacterium parvus 368, Corynebacterium parvus 363636363636363636363672, Corynebacterium parvus 368, Corynebacterium parvus 05800.1, Corynebacterium parvus 368, Corynebacterium parvu, AJY49223.1(Halomonas sp.KO116), ADW67334.1 (Granularia tinctoria MP5ACTX9), ADH91532.1(Starkeya novella DSM 506), ADG98040.1 (Segniliarus rotunicus DSM 44985), ACX38503.1(Escherichia coli DH1), ACI50717.1(Gluconacetobacter diazorphism PA 15), ACB68161.1(Burkholderia ambifaria MC40-6), ABR61199.1(Sinorhizobiu WSM419), ABO58457.1(Burkholderia vimentis G4), ABM31567.1(Acidovorax ciferrii AAC00-1), ACU6 (Chionophynesis DSM2588), ACAcidococci 29453.1 (Acidococci DSM 20542), and ABL 25429.1 (Sporotrichia strain 25429.1). Two wild-type dihydropyrimidine aminohydrolase having this function were finally obtained, both of which were derived from Pseudomonas fluorescens, wherein the number of amino acids of dihydropyrimidine aminohydrolase SEQ ID NO. 1 having NCBI accession No. WP _011030900.1 was 467, and the number of amino acids of dihydropyrimidine aminohydrolase SEQ ID NO. 3 having NCBI accession No. WP _011334810.1 was 479.
Because of their definite amino acid sequences, the genes encoding them, expression cassettes and plasmids containing them, and transformants containing the plasmids are readily available to those skilled in the art.
These genes, expression cassettes, plasmids, and transformants can be obtained by genetic engineering construction means well known to those skilled in the art.
In order to optimally express dihydropyrimidine aminohydrolase SEQ ID NO:1 or SEQ ID NO:3 in a microbial host such as E.coli host most commonly used in genetic engineering, the present invention codon optimizes the gene expressed thereby.
Codon optimization is one technique that can be used to maximize protein expression in an organism by increasing the translation efficiency of a gene of interest. Different organisms often show a special preference for one of several codons encoding the same amino acid due to mutation tendencies and natural selection. For example, in rapidly growing microorganisms such as E.coli, the optimized codons reflect the composition of their respective pools of genomic tRNA's. Thus, in a fast growing microorganism, the low frequency codons of an amino acid can be replaced with codons for the same amino acid but with a high frequency. Thus, expression of optimized DNA sequences is improved in fast growing microorganisms.
Through codon optimization, the coding gene of the dihydropyrimidine amino hydrolase SEQ ID NO. 1 can be SEQ ID NO. 2, and the coding gene of the dihydropyrimidine amino hydrolase SEQ ID NO. 3 can be SEQ ID NO. 4.
When used as a biocatalyst for the production of (R) -3- (carbamoylmethyl) -5-methylhexanoic acid, the dihydropyrimidine amidohydrolase of the present invention, as well as the added glucose dihydropyrimidine amidohydrolase, may be in the form of an enzyme or in the form of a bacterial cell. The enzyme forms comprise free enzyme and immobilized enzyme, including purified enzyme, crude enzyme, fermentation liquor, enzyme immobilized by a carrier and the like; the form of the thallus comprises a viable thallus and a dead thallus.
Pure dihydropyrimidine aminohydrolase, SEQ ID NO:1 and SEQ ID NO:3, used for catalyzing hydrolysis reactions, can generally be used only once, so that the production cost of (R) -3- (carbamoylmethyl) -5-methylhexanoic acid is relatively high. In order to improve the reaction process economy, it is necessary to reuse the dihydropyrimidine aminohydrolase.
As is well known in the field of biological catalysis, compared with a free enzyme method, the application of an immobilized enzyme technology has the advantages of simplified production process, improved production efficiency and the like. Meanwhile, the enzyme can be used for multiple times, and the stability of the enzyme is improved, so that the productivity of unit enzyme is effectively improved; and secondly, the immobilized enzyme is easily separated from the substrate and the product, the purification process is simplified, the yield is high, and the product quality is good.
The invention adopts ion exchange resin as an immobilization carrier to respectively immobilize dihydropyrimidine amino hydrolase SEQ ID NO 1 and SEQ ID NO 3, and can be repeatedly used for hydrolytic ring-opening reaction of a substrate 3-isobutyl glutarimide.
The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
Examples
In the examples, the addition, content and concentration of various substances are mentioned, wherein the percentages refer to mass percentages unless otherwise indicated.
Materials and methods
The whole gene synthesis, primer synthesis and sequencing in the examples were performed by Jinzhi Biotechnology, Inc., Suzhou.
The molecular biological experiments in the examples include plasmid construction, digestion, ligation, competent cell preparation, transformation, culture medium preparation, and the like, and were mainly performed with reference to "molecular cloning laboratory Manual" third edition (J. SammBruk, D.W. Lassel (America), translation of Huang Peigang, science publishers, Beijing, 2002). The specific experimental conditions can be determined by simple experiments if necessary.
PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the supplier of the plasmid or DNA template. If necessary, it can be adjusted by simple experiments.
LB culture medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH 7.2. (20 g/L agar powder was additionally added to LB solid medium.)
TB culture medium: 24g/L yeast extract, 12g/L tryptone, 16.43g/L K2HPO4·3H2O、2.31g/L KH2PO45g/L of glycerol, and the pH value is 7.0-7.5. (20 g/L agar powder was additionally added to TB solid medium.)
Substrate and product HPLC detection methods:
1. content detection
Chromatograph: shimadzu LC-2010
Column: c18 column
Mobile phase: acetonitrile: water (20:80, v/v; pH 2.5, pH adjusted with phosphoric acid)
Flow rate: 1.0ml/min
Inspection tester, UV 210
Column oven: at 30 ℃.
2. Chiral ee detection
Chromatograph: shimadzu LC-2010
Column: chiralpack AD-RH column (Daicel)
Mobile phase: acetonitrile: water (50:50, v/v; pH 2.5, pH adjusted with phosphoric acid)
Flow rate: 0.5ml/min
Inspection tester, UV 210
Column oven: at 30 ℃.
Example 1: construction of recombinant E.coli expressing dihydropyrimidine amino hydrolase
1.1 according to Pseudomonas fluorescens derived dihydropyrimidine amino hydrolase amino acid sequence SEQ ID NO:1(NCBI accession number WP _011030900.1), suitable for Escherichia coli expression of codon optimization, optimization of gene sequence is SEQ ID NO: 2. The gene sequence was synthesized as a whole, enzyme cleavage sites Nde I and XhoI were designed at both ends, and subcloned into the corresponding site on the vector pET24a (purchased from Novagen) to obtain the recombinant plasmid pET24 a-IH-1. The constructed recombinant plasmid pET24a-IH-1 is transformed into escherichia coli BL21(DE3) competence by an electrical transformation method to obtain recombinant escherichia coli BL21(DE3)/pET24a-IH-1 for expressing dihydropyrimidine amino hydrolase SEQ ID NO: 1.
1.2 with reference to step 1.1, recombinant E.coli BL21(DE3)/pET24a-IH-2 expressing dihydropyrimidine aminohydrolase, SEQ ID NO:3, was constructed.
1.3 with reference to step 1.1, recombinant strains expressing dihydropyrimidine aminohydrolase (GenBank accession number or NCBI accession number WP _011334810.1, EUB75082.1, ADU69230.1, ABY99529.1, ARW18102.1, ADD28645.1, SMC02439.1, SMB90978.1, ADO 90978.1, AEW 90978.1, AEV 90978.1, OCK 90978.1, EED 90978.1, APA 90978.1, CDM 90978.1, ADP 90978.1, ARW 90978.1, APR 90978.1, ODP 90978.1, ACL 90978.1, OAZ 90978.1, KXA 90978.1, KXB 90978.1, ADW 90978.1, ADH 90978.1, ADG 90978.1, ACX 90978.1, ACI 90978.1, ACB 90978.1, ABpER 90978.1, ABO 90978.1, ABM 90978.1, ACU 90978.1, ACV 90978.1, ABX 90978.1, ERBL 90978.1) of other microbial origin were constructed respectively (E.coli 90978.1-90978.1)/E.coli 90978.1 (E.coli 90978.1-90978.1)/90978.1-90978.1 (E90978.1-90978.1)/E90978.1-90978.1).
Example 2: screening for dihydropyrimidine amino hydrolase
2.1 fermentation of the strains
Individual colonies of each dihydropyrimidine aminohydrolase-expressing strain were picked up and inoculated into 3mL of LB medium containing 50. mu.g/mL of kanamycin sulfate, and cultured overnight at 37 ℃ and 200 rpm. Transferring into 200mL TB medium at a inoculum size of 1 v/v%, culturing at 37 deg.C and 250rpm to OD6000.6-0.8, 0.5mM IPTG was added and cultured overnight at 28 ℃ and 200 rpm. Then, centrifugation is carried out for 10min at 4 ℃ and 10000rpm, and thalli are collected and frozen for later use.
2.2 catalysis of the Synthesis of (R) -3- (carbamoylmethyl) -5-methylhexanoic acid
Reaction system: 1L of 0.1M Tris-HCl buffer solution (pH7.5), 10 wt% of substrate 3-isobutyl glutarimide and 5% w/v of freeze-thaw bacteria, reacting at 30 ℃ for 24 hours, detecting the concentration of a substrate sample and a product by HPLC, and screening a strain in which the product (R) -3- (carbamoylmethyl) -5-methylhexanoic acid appears.
HPLC detection proves that (R) -3- (carbamoylmethyl) -5-methylhexanoic acid absorption peaks appear in reaction liquid catalyzed by BL21(DE3)/pET24a-IH-1 and BL21(DE3)/pET24 a-IH-2.
Example 3: extraction and immobilization of dihydropyrimidine amidohydrolases
3.1 shake flask fermentation of dihydropyrimidine amidohydrolase expressing strains: respectively preparing liquid culture medium TB (pH7.2), subpackaging in 500mL triangular shake flasks with the liquid filling amount of 100mL, and then heating and sterilizing in an autoclave at 121 ℃ for 20 min. Several colonies were picked from LB plates of the engineered bacteria BL21(DE3)/pET24a-IH-1 or BL21(DE3)/pET24a-IH-2 with an inoculating loop, inoculated into TB flasks, 100. mu.g/mL kanamycin was added to the TB medium before inoculation, and shake-cultured at 37 ℃ and 220rpm until OD6005-6, 0.2mM IPTG was added and induced at 28 ℃ for about 24 h.
3.2 extraction and separation of enzyme: 50ml of fermentation liquor is taken and put into a centrifuge tube; centrifuging at 8000rpm to remove supernatant to obtain thallus, adding purified water according to thallus 200g/L for resuspension, cooling suspended thallus with ice bath, performing ultrasonication (voltage 400W, ultrasonication time 3s, interval time 5s, and working times 80 times) to obtain crude enzyme solution, and placing in ice bath for use.
The crude enzyme solution is purified by Ni-NTG affinity chromatography column (Nanjing Jinruis biology, product number: L00250/L00250-C). The Ni-NTG affinity chromatography column was pre-equilibrated with buffer, washed with 10 column volumes of wash buffer (50mM pH8.0 Tris-HCl, 300mM NaCl, 50mM imidazole), after which the protein of interest was eluted from the Ni column with elution buffer (50mM pH8.0 Tris-HCl, 300mM NaCl, 250mM imidazole).
3.3 enzyme immobilization: using epoxy type ion exchange resinLX-1000EP immobilizes the enzyme by the following method: the immobilized carrier was repeatedly washed with 0.05M phosphate buffer (pH8.0)LX-1000EP (Xian lan Xiao Ke)Technical limited company). According to the enzyme protein: the carrier ratio is about 1: 12-15, placing in 0.1M potassium phosphate buffer (pH8.0), and shaking at 20-25 deg.C and 150 rpm; stopping shaking after 1min, detecting and adjusting pH to about 8.0, and continuing shaking for 18 h; stopping shaking, and standing for 24h at the same temperature; removing supernatant, and shaking with 0.02M potassium phosphate buffer (pH8.0) at 20-25 deg.C for 2 min; removing supernatant, and washing with the same buffer solution for 45 min; removing supernatant, washing with 0.02M potassium phosphate buffer (pH8.0), and vacuum-pumping to obtain immobilized enzyme, and storing at 2-8 deg.C.
Example 4: enzymatic synthesis of (R) -3- (carbamoylmethyl) -5-methylhexanoic acid
Reaction system (1L): 0.1M Tris-HCl buffer (pH7.5)1L, 10 wt% substrate 3-isobutylglutarimide, 10 wt% immobilized dihydropyrimidine aminohydrolase SEQ ID NO:1, pH7.5 corrected with 6M NaOH. After 8 hours of reaction at 30 ℃, 100 μ l of the reaction solution was centrifuged at 12000rpm for 5 minutes, the supernatant was ultrafiltered, and the filtrate was subjected to HPLC to detect the conversion of the product, as shown in fig. 1, at least 60% of the substrate was converted into the product after 8 hours of reaction, and the conversion rate reached 95% or more after 12 hours.
The results of the catalytic reaction of the immobilized dihydropyrimidine aminohydrolase, SEQ ID NO 3, are similar to those described above.
Example 5: enzymatic synthesis of (R) -3- (carbamoylmethyl) -5-methylhexanoic acid
Reaction system (1L): 1L of 0.1M Tris-HCl buffer (pH7.5), 10 wt% of substrate 3-isobutylglutarimide, 10 wt% of immobilized dihydropyrimidine aminohydrolase SEQ ID NO:1, pH8.5 corrected with 6M NaOH. Stirring and reacting for 24 hours at 40 ℃, filtering to obtain reaction liquid, distilling the reaction liquid under reduced pressure, concentrating until about 1/5 volumes of the reaction liquid remain, cooling to 0-5 ℃, stirring and crystallizing lh, filtering, and drying to obtain the target product compound. The chiral purity of (R) -3- (carbamoylmethyl) -5-methylhexanoic acid was determined by HPLC. As shown in FIG. 2, the ee value of the R-configuration in the crystal of (R) -3- (carbamoylmethyl) -5-methylhexanoic acid as the product was 99.5% or more. Shows that the immobilized dihydropyrimidine amino hydrolase SEQ ID NO 1 or SEQ ID NO 3 has high stereoselectivity and can catalyze and prepare pregabalin intermediate (R) -3- (carbamoylmethyl) -5-methylhexanoic acid with high chiral purity.
The catalytic reaction results of the immobilized dihydropyrimidine aminohydrolase, SEQ ID NO:3, were similar, and (R) -3- (carbamoylmethyl) -5-methylhexanoic acid of high optical purity was also obtained.
The experiments show that the dihydropyrimidine amino hydrolase SEQ ID NO:1 and SEQ ID NO:3 can catalyze 3-isobutyl glutarimide to carry out ring-opening reaction to obtain (R) -3- (carbamoylmethyl) -5-methylhexanoic acid, both have high stereoselectivity, and can be used for preparing pregabalin intermediates with high chiral purity.
Sequence listing
<110> Hangzhou enzyme causes Biotechnology Ltd
<120> dihydropyrimidine amino hydrolase and application thereof
<130> SHPI2110434
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 467
<212> PRT
<213> Pseudomonas fluorescens
<400> 1
Met Ser Ser Arg Thr Val Ile Arg Gly Gly Leu Val Ile Thr Ala Ser
1 5 10 15
Asp Glu Ile His Ala Asp Val Leu Ile Glu Asp Gly Arg Val Ala Ala
20 25 30
Leu Ala Ala Thr Gly Thr Pro Ala Ala Glu Ala Phe Thr Ala Glu Asn
35 40 45
Val Ile Asp Ala Ser Gly Lys Tyr Val Ile Pro Gly Gly Val Asp Gly
50 55 60
His Thr His Met Glu Met Pro Phe Gly Gly Thr Tyr Ala Ala Asp Thr
65 70 75 80
Phe Glu Thr Gly Thr Arg Ala Ala Ala Trp Gly Gly Thr Thr Thr Ile
85 90 95
Val Asp Phe Ala Ile Gln Ser Val Gly His Ser Leu Arg Glu Gly Leu
100 105 110
Asp Ala Trp His Ala Lys Ala Glu Gly Asn Cys Ala Ile Asp Tyr Gly
115 120 125
Phe His Met Ile Val Ser Asp Val Asn Gln Glu Thr Leu Lys Glu Met
130 135 140
Asp Leu Leu Val Glu Glu Gly Val Thr Ser Phe Lys Gln Phe Met Ala
145 150 155 160
Tyr Pro Gly Val Phe Tyr Ser Asp Asp Gly Gln Ile Leu Arg Ala Met
165 170 175
Gln Arg Ala Ala Glu Asn Gly Gly Leu Ile Met Met His Ala Glu Asn
180 185 190
Gly Ile Ala Ile Asp Val Leu Val Glu Gln Ala Leu Ala Arg Gly Glu
195 200 205
Thr Asp Pro Arg Phe His Gly Glu Val Arg Lys Ala Leu Leu Glu Ala
210 215 220
Glu Ala Thr His Arg Ala Ile Arg Leu Ala Gln Val Ala Gly Ala Pro
225 230 235 240
Leu Tyr Val Val His Val Ser Ala Thr Glu Ala Val Ala Glu Leu Thr
245 250 255
Arg Ala Arg Asp Glu Gly Leu Pro Val Phe Gly Glu Thr Cys Pro Gln
260 265 270
Tyr Leu Phe Leu Ser Thr Asp Asn Leu Ala Glu Pro Asp Phe Glu Gly
275 280 285
Ala Lys Tyr Val Cys Ser Thr Pro Leu Arg Pro Lys Glu His Gln Ala
290 295 300
Ala Leu Trp Arg Gly Leu Arg Thr Asn Asp Leu Gln Val Val Ser Thr
305 310 315 320
Asp His Cys Pro Phe Cys Phe Ser Gly Gln Lys Glu Leu Gly Arg Gly
325 330 335
Asp Phe Ser Arg Ile Pro Asn Gly Met Pro Gly Val Glu Asn Arg Met
340 345 350
Asp Leu Leu His Gln Ala Val Val Glu Gly His Ile Gly Arg Arg Arg
355 360 365
Trp Ile Glu Ile Ala Cys Ala Thr Pro Ala Arg Met Phe Gly Leu Tyr
370 375 380
Pro Lys Lys Gly Thr Ile Ala Pro Gly Ala Asp Ala Asp Ile Val Val
385 390 395 400
Tyr Asp Pro His Ala Glu Gln Val Ile Ser Ala Glu Thr His His Met
405 410 415
Asn Val Asp Tyr Ser Ala Tyr Glu Gly Arg Arg Ile Thr Gly Arg Val
420 425 430
Glu Thr Val Leu Ser Arg Gly Glu Pro Val Val Thr Glu Arg Glu Tyr
435 440 445
Thr Gly Arg Lys Gly His Gly Ala Tyr Thr Pro Arg Ala Thr Cys Gln
450 455 460
Tyr Leu Thr
465
<210> 2
<211> 1404
<212> DNA
<213> Artificial sequence ()
<400> 2
atgagcagcc gtaccgttat tcgtggtggt ctggttatta ccgcaagtga tgaaattcat 60
gccgatgtgc tgattgaaga tggtcgtgtt gcagcactgg cagcaaccgg tacaccggca 120
gcagaagcat ttaccgcaga aaatgttatt gatgccagcg gcaaatatgt tattccaggt 180
ggtgttgatg gtcataccca catggaaatg ccgtttggtg gcacctatgc agcagatacc 240
tttgaaaccg gtacgcgtgc agcagcatgg ggtggcacca ccaccattgt tgattttgca 300
attcagagcg ttggtcatag cctgcgtgaa ggtctggatg catggcatgc aaaagccgaa 360
ggtaattgtg caattgatta tggctttcac atgattgtga gcgacgttaa tcaagaaacc 420
ctgaaagaaa tggatctgct ggttgaagaa ggtgtgacca gctttaaaca gtttatggca 480
tatccgggtg tgttctatag tgatgatggt cagattctgc gtgcaatgca gcgtgcagcc 540
gaaaatggtg gcctgattat gatgcatgcg gaaaatggta ttgccattga tgttctggtt 600
gaacaggcac tggcacgtgg tgaaaccgat ccgcgttttc atggtgaagt tcgtaaagca 660
ctgctggaag ccgaagcaac ccatcgtgca attcgtctgg cacaggttgc gggtgcaccg 720
ctgtatgttg ttcatgttag cgcaaccgaa gcagttgcag aactgacccg tgcacgtgat 780
gaaggcctgc cggtttttgg cgaaacctgt ccgcagtacc tgtttctgag caccgataat 840
ctggccgaac cggattttga aggtgcaaaa tatgtttgta gcacaccgct gcgtccgaaa 900
gaacatcagg cagcactgtg gcgtggtctg cgtaccaatg atctgcaggt tgttagcacc 960
gatcattgtc cgttttgttt tagcggtcag aaagaattag gtcgcggtga ttttagccgt 1020
attccgaatg gtatgcctgg tgttgaaaat cgtatggacc tgctgcatca ggccgttgtg 1080
gaaggtcata ttggtcgtcg tcgttggatt gaaattgcat gtgcaacacc ggcacgtatg 1140
tttggtctgt atccgaaaaa aggcaccatt gcaccgggtg cagatgcaga tattgttgtt 1200
tatgatccgc atgccgaaca ggttattagc gcagaaaccc atcacatgaa tgttgattat 1260
agcgcctatg aaggtcgtcg tattaccggt cgtgtggaaa ccgttctgag ccgtggtgaa 1320
ccggttgtta ccgaacgtga atataccggt cgcaaaggtc atggtgcata tacaccgcgt 1380
gcaacctgtc agtatctgac ctaa 1404
<210> 3
<211> 479
<212> PRT
<213> Pseudomonas fluorescens
<400> 3
Met Ser Leu Leu Ile Arg Gly Ala Thr Ile Val Thr His Asp Glu Ser
1 5 10 15
Tyr Arg Ala Asp Val Tyr Cys Ala Asp Gly Val Ile Lys Ala Ile Gly
20 25 30
Glu Asn Leu Asp Ile Pro Ala Gly Ala Glu Val Leu Asp Gly Ser Gly
35 40 45
Gln Tyr Leu Met Pro Gly Gly Ile Asp Pro His Thr His Met Gln Leu
50 55 60
Pro Phe Met Gly Thr Val Ala Ser Glu Asp Phe Tyr Ser Gly Thr Ala
65 70 75 80
Ala Gly Leu Ala Gly Gly Thr Thr Ser Ile Ile Asp Phe Val Ile Pro
85 90 95
Asn Pro Gln Gln Ser Leu Leu Glu Ala Phe His Gln Trp Arg Gly Trp
100 105 110
Ala Glu Lys Ser Ala Ser Asp Tyr Gly Phe His Val Ala Ile Thr Trp
115 120 125
Trp Ser Glu Gln Val Arg Glu Glu Met Ala Glu Leu Val Ser His His
130 135 140
Gly Ile Asn Ser Phe Lys His Phe Met Ala Tyr Lys Asn Ala Ile Met
145 150 155 160
Ala Ala Asp Asp Thr Leu Val Ala Ser Phe Glu Arg Cys Leu Glu Leu
165 170 175
Gly Ala Val Pro Thr Val His Ala Glu Asn Gly Glu Leu Val Tyr His
180 185 190
Leu Gln Arg Lys Leu Met Ala Gln Gly Ile Thr Gly Pro Glu Ala His
195 200 205
Pro Leu Ser Arg Pro Ser Gln Val Glu Gly Glu Ala Ala Ser Arg Ala
210 215 220
Ile Arg Ile Ala Glu Thr Ile Gly Thr Pro Leu Tyr Leu Val His Val
225 230 235 240
Ser Thr Lys Glu Ala Leu Asp Glu Ile Thr Tyr Ala Arg Ser Lys Gly
245 250 255
Gln Pro Val Tyr Gly Glu Val Leu Ala Gly His Leu Leu Leu Asp Asp
260 265 270
Ser Val Tyr Gln His Pro Asp Trp Gln Thr Ala Ala Gly Tyr Val Met
275 280 285
Ser Pro Pro Phe Arg Pro Arg Gly His Gln Asp Ala Leu Trp His Gly
290 295 300
Leu Gln Ser Gly Asn Leu His Thr Thr Ala Thr Asp His Cys Cys Phe
305 310 315 320
Cys Ala Glu Gln Lys Ala Ala Gly Arg Asp Asp Phe Ser Lys Ile Pro
325 330 335
Asn Gly Thr Ala Gly Ile Glu Asp Arg Met Ala Val Leu Trp Asp Glu
340 345 350
Gly Val Asn Ser Gly Arg Leu Ser Met Gln Asp Phe Val Ala Leu Thr
355 360 365
Ser Thr Asn Thr Ala Lys Ile Phe Asn Leu Tyr Pro Arg Lys Gly Ala
370 375 380
Ile Arg Val Gly Ala Asp Ala Asp Leu Val Leu Trp Asp Pro Gln Gly
385 390 395 400
Thr Arg Thr Ile Ser Ala Lys Thr His His Gln Gln Val Asp Phe Asn
405 410 415
Ile Phe Glu Gly Lys Thr Val Thr Gly Val Pro Ser His Thr Val Ser
420 425 430
Gln Gly Arg Val Val Trp Ala Asp Gly Asp Leu Arg Ala Glu Arg Gly
435 440 445
Ala Gly Arg Tyr Ile Glu Arg Pro Ala Tyr Pro Ala Val Phe Asp Leu
450 455 460
Leu Ser Lys Arg Ala Glu Gln His Lys Pro Thr Ala Val Lys Arg
465 470 475
<210> 4
<211> 1440
<212> DNA
<213> Artificial sequence ()
<400> 4
atgagcctgc tgattcgtgg tgcaaccatt gttacccatg atgaaagcta tcgtgccgat 60
gtttattgtg cagatggtgt gattaaagcc attggcgaaa atctggatat tcctgccggt 120
gccgaagttc tggatggtag cggtcagtat ctgatgcctg gtggtattga tccgcataca 180
cacatgcagc tgccgtttat gggcaccgtt gcaagcgaag atttctatag cggcaccgca 240
gcaggtctgg caggcggtac aaccagcatt attgattttg ttattccgaa tccgcagcaa 300
agcctgctgg aagcatttca tcagtggcgt ggttgggcag aaaaaagcgc aagcgattat 360
ggttttcatg ttgcaattac ctggtggtca gaacaggttc gtgaagaaat ggcagaactg 420
gttagccatc atggcattaa cagctttaaa cacttcatgg cctataaaaa cgcaatcatg 480
gcagcagatg ataccctggt tgccagcttt gaacgttgtc tggaactggg tgcagttccg 540
accgttcatg cagaaaatgg tgaactggtt tatcatttac agcgtaaact gatggcacag 600
ggtattaccg gtccggaagc acatccgctg agccgtccga gccaggttga aggtgaagca 660
gcaagccgtg caattcgtat tgcagaaacc attggtacac cgctgtatct ggttcatgtt 720
agcaccaaag aagcactgga cgaaatcacc tatgcacgta gcaaaggtca gccggtttat 780
ggtgaagtgc tggcaggtca tttactgctg gatgatagcg tttatcagca tccggattgg 840
cagaccgcag ccggttatgt tatgagccct ccgtttcgtc cgcgtggtca tcaggatgca 900
ctgtggcatg gtctgcagag cggtaatctg cataccaccg caaccgatca ttgttgtttt 960
tgtgccgaac agaaagcagc cggtcgtgat gattttagca aaattccgaa tggtacagcc 1020
ggtattgaag atcgtatggc agttctgtgg gatgaaggtg ttaatagcgg tcgtctgagc 1080
atgcaggatt ttgttgcact gaccagcacc aataccgcca aaatctttaa tctgtatccg 1140
cgtaaaggtg ccattcgtgt tggtgcagat gcagatctgg tgctgtggga cccgcagggc 1200
acccgtacca ttagcgcaaa aacccatcat cagcaggttg attttaacat ctttgaaggt 1260
aaaaccgtta ccggtgttcc gagccatacc gttagccagg gtcgtgttgt ttgggcagat 1320
ggggatctgc gtgcagaacg tggtgcaggt cgttatattg aacgtccggc atatccggca 1380
gtttttgatc tgctgagcaa acgtgcggaa cagcataaac cgaccgcagt taaacgttag 1440