The (S)-transaminase mutant and its application in preparation of (R)-3-([1, 1apos; application of-biphenyl]-4-yl)-2-aminopropyl-1-ol
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-02
- Publication Date
- 2026-04-10
AI Technical Summary
然而目前没有直接应用(S)-转氨酶合成光学纯(R)-3-([1,1'-联苯]-4-基)-2-氨基丙-1-醇的研究报道
（1）生产工艺简单，反应条件温和；
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Figure CN121825923A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of enzyme engineering, in particular to (S)-transaminase mutants and their application in the preparation of (R)-3-([1,1'-biphenyl]-4-yl)-2-aminopropan-1-ol. BACKGROUND
[0002] (R)-3-([1,1'-biphenyl]-4-yl)-2-aminopropan-1-ol is a key chiral intermediate of LCZ696. LCZ696 is a dual-acting angiotensin receptor neprilysin inhibitor developed by Novartis, which was approved by FDA on July 7, 2015, and is developed for the treatment of patients with reduced ejection fraction heart failure. LCZ696 is considered to be able to reduce the strain of the failing heart. The safety threshold of cardiovascular drugs is extremely high, and LCZ696 even shows higher safety than conventional drugs, and the industry believes that the outstanding performance of LCZ696 makes it one of the most important progress in the field of cardiology in the past decade. In the next few years, there will be no drug in the cardiovascular field that can rival LCZ696, so its medicinal prospects are broad.
[0003] There are few production methods for (R)-3-([1,1'-biphenyl]-4-yl)-2-aminopropan-1-ol. Generally, chemical synthesis is used, starting from 4-biphenyl aldehyde, which is condensed and cyclized with acetylglycine, ring-opening ester exchange with sodium methoxide / methanol, rhodium-catalyzed asymmetric hydrogenation, sodium borohydride / methanol reduction, and hydrochloric acid deacetyl protecting group. Five-step reaction can chemically synthesize (R)-3-([1,1'-biphenyl]-4-yl)-2-aminopropan-1-ol. However, the above synthesis method faces problems such as high cost, harsh conditions, and difficulty in controlling stereoselectivity. In contrast, biocatalytic synthesis is gradually becoming a more promising alternative, with advantages such as mild reaction conditions, high stereoselectivity, and environmental friendliness. However, there is currently no research report on the direct application of (S)-transaminase to synthesize optically pure (R)-3-([1,1'-biphenyl]-4-yl)-2-aminopropan-1-ol. SUMMARY
[0004] The present application aims at the defects of the existing (R)-3-([1,1'-biphenyl]-4-yl)-2-aminopropan-1-ol synthesis process, and provides (S)-transaminase mutants and their application in the preparation of (R)-3-([1,1'-biphenyl]-4-yl)-2-aminopropan-1-ol.
[0005] The specific technical solutions are as follows: The present application provides, in a first aspect, an (S)-transaminase mutant having 14 or fewer substitutions relative to SEQ ID NO. 2 and less than 100% sequence identity to SEQ ID NO. 2; wherein the substitutions are selected from the group consisting of: T24C, T24G, F25L, F25M, F25V, S26F, S26M, S26R, S26G, S26T, S26A, S26D, S26V, S26W, S26Y, S26E, S26L, T27S, Q32T, Y60S, Y60D, Y60Q, V85T, T88G, T88N, S90L, S90A, S90T, S90V, S163A, S163G, Y164M, R165M, G243A, S327T, S327Q, S327H, L387M, L387Q, L387H, L387T, L387Y, Q420S, Q420H, Q420V, M423R, V436L, T440Q, M441C, M441L, M441V, R442H, R442A, R442N, R442G, R442Q, R442T, and I443L, or combinations thereof.
[0006] The present application provides, in a second aspect, a gene encoding the (S)-transaminase mutant as described.
[0007] The present application provides, in a third aspect, a recombinant vector comprising a gene encoding the (S)-transaminase mutant.
[0008] Further, the recombinant vector is a pET28a plasmid into which a gene encoding an (S)-transaminase mutant is inserted.
[0009] The present application provides, in a fourth aspect, a recombinant microorganism comprising the aforementioned gene or recombinant vector.
[0010] The present application provides, in a fifth aspect, a catalyst comprising the aforementioned (S)-transaminase mutant or recombinant microorganism. The catalyst is an enzyme preparation of the mutant, an immobilized enzyme, or a whole cell or cell lysate of the recombinant microorganism.
[0011] The present application provides, in a sixth aspect, use of the (S)-transaminase mutant, or the recombinant microorganism, or the catalyst in the preparation of (R)-3-([1,1'-biphenyl]-4-yl)-2-aminopropan-1-ol.
[0012] The present application provides a method for preparing (R)-3-([1,1'-biphenyl]-4-yl)-2-aminopropan-1-ol, using isopropylamine and 3-([1,1'-biphenyl]-4-yl)-2-ketopropan-1-ol as substrates, pyridoxal phosphate as a cofactor, and the catalyst to perform a transamination reaction to generate (R)-3-([1,1'-biphenyl]-4-yl)-2-aminopropan-1-ol.
[0013] Further, the reaction is carried out in an organic solvent-water mixed solution or an aqueous solution.
[0014] Further preferably, the organic solvent is DMSO.
[0015] More preferably, the amount of the organic solvent added is not more than 20% of the total volume.
[0016] Compared with the prior art, the present application has the following beneficial effects: (1) The production process is simple, and the reaction conditions are mild; (2) The (S)-transaminase mutant has high selectivity, and the product has high optical purity and does not need to be separated; (3) The atomic utilization rate is high, the production process is environmentally friendly, and conforms to the concept of green chemistry; (4) The product separation and purification is simple, and the subsequent processing difficulty is low. BRIEF DESCRIPTION OF DRAWINGS
[0017] Figure 1 It is a schematic diagram of the molecular structure of (R)-3-([1,1'-biphenyl]-4-yl)-2-aminopropan-1-ol.
[0018] Figure 2 It is an equation for synthesizing (R)-3-([1,1'-biphenyl]-4-yl)-2-aminopropan-1-ol by a biosynthetic method.
[0019] Figure 3 It is a high-performance liquid chromatogram of 3-([1,1'-biphenyl]-4-yl)-2-ketopropan-1-ol, (R)-3-([1,1'-biphenyl]-4-yl)-2-aminopropan-1-ol and (S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropan-1-ol standards.
[0020] Figure 4 It is a high-performance liquid chromatogram of the substrate and product in the reaction solution after (R)-3-([1,1'-biphenyl]-4-yl)-2-aminopropan-1-ol is prepared by (S)-transaminase.
[0021] Figure 5 It is a 1H nuclear magnetic resonance spectrum of (R)-3-([1,1'-biphenyl]-4-yl)-2-aminopropan-1-ol. DETAILED DESCRIPTION
[0022] In order for those skilled in the art to better understand the present application, the technical solutions of the present application will be described clearly and completely below in conjunction with specific examples. It should be pointed out that the following detailed description is exemplary and only a part of the embodiments of the present application, but not all the embodiments.
[0023] Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative efforts shall fall within the scope of protection of the present application.
[0024] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the present application belongs. The experimental materials used in the embodiments of the present application are all conventional experimental materials in the art and can be purchased through commercial channels. The experimental methods without detailed conditions are performed according to the conventional experimental methods or according to the operation instructions recommended by the suppliers.
[0025] The reagents used in the catalytic process: isopropylamine, 3-([1,1'-biphenyl]-4-yl)-2-ketoprop-1-ol, pyridoxal phosphate, etc. are all commercially available analytical pure.
[0026] In the present application, the reaction of isopropylamine and 3-([1,1'-biphenyl]-4-yl)-2-ketoprop-1-ol converted into (R)-3-([1,1'-biphenyl]-4-yl)-2-amino prop-1-ol under the catalysis of (S)-transaminase is shown in the following formula:
[0027] In the present application, the concentrations of the substrate and the product in the reaction solution are analyzed by high performance liquid chromatography (HPLC) to monitor the progress of the reaction.
[0028] The HPLC analysis method is as follows: the chromatographic column type is CHIRALPAK AY-3R, the mobile phase is 20 mM ammonium bicarbonate aqueous solution: acetonitrile = 35:65, the flow rate is 0.4 mL / min, the column temperature is 25℃, and the detection wavelength is 256 nm (ultraviolet absorption method). The peak elution conditions are shown in Figures 3-4 , 3-([1,1'-biphenyl]-4-yl)-2-ketoprop-1-ol 24.7 min, (R)-3-([1,1'-biphenyl]-4-yl)-2-amino prop-1-ol 11.8 min or so, and (S)-3-([1,1'-biphenyl]-4-yl)-2-amino prop-1-ol 13.0 min or so.
[0029] In the present application, the selectivity of (S)-transaminase and its mutants is characterized by ee The specific formula is as follows: ee =|R S| / (R+S)×100% Where R represents the content of (R)-3-([1,1'-biphenyl]-4-yl)-2-aminoprop-1-ol, and S represents the content of (S)-3-([1,1'-biphenyl]-4-yl)-2-aminoprop-1-ol.
[0030] Example 1: Construction of Wild-Type (S)-Transaminase Engineered Bacteria (S)-transaminase genes were retrieved from the NCBI database, and (S)-transaminase (5G0A) with accession number KRF52528.1 was selected, with the base sequence shown in SEQ ID NO.1. The amino acid sequence was converted to a nucleotide sequence through codon optimization, as shown in SEQ ID NO.2. The above nucleotide sequence was then fully synthesized chemically (Qingke Biotechnology) and integrated into the expression vector pET-28a(+). BamHI and HindIII Between the restriction enzyme sites; finally, the constructed plasmid was introduced into Escherichia coli BL21(DE3) host cells to construct an engineered bacterium with high amino substrate recognition (S)-transaminase.
[0031] SEQ ID NO.1:
[0032] SEQ ID NO.2: MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNPKVNAAIKEALDRYGFVWDTYSTDYKAKAAKIIIEDILGDEDWPGKVRFVS TGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSNGNCLKDENGELLSVKYTRRMIENYGPEQVAAVITEV SQGAGSAMPPYEYIPQIRKMTKELGVLWITDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAEFMDRHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQA KNSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSREDIDKAMDALDYALDYLESGEWQQS.
[0033] Example 2: Construction of (S)-transaminase mutant 1. Activation of engineered bacteria and plasmid extraction engineered bacteria ( E. coli BL21(DE3), obtained in Example 1, was activated and cultured using LB medium with the following formulation: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl, dissolved in deionized water and brought to a final volume. The mixture was then sterilized at 121°C for 20 minutes and set aside for use. The solid medium was LB medium with 1-2% agar added.
[0034] The preserved engineered bacterial glycerol tubes were inoculated into test tubes containing 10 mL of LB medium and cultured overnight at 37°C and 220 rpm. After obtaining the cultured bacterial cells, plasmids were extracted according to the instructions of the Axygen plasmid extraction kit. The obtained plasmids can be used directly for point mutagenesis or stored long-term at -20°C.
[0035] 2. Site-directed gene mutation Gene mutations were obtained using whole plasmid PCR.
[0036] Table 1 PCR amplification system (50 μL)
[0037] PCR amplification procedure: (1) Pre-denaturation: 98℃ for 5 min; (2) Denaturation: 98℃ for 30s; Annealing: 60℃ for 30s; Extension: 72℃ for 90s; 30 cycles in total; (3) Post-extension: 72℃ for 10 min; (4) Store at 4°C.
[0038] After PCR amplification, the amplification product was detected by 0.9% agarose gel electrophoresis. The results showed that the amplification product was a single band, approximately 6800 bp in size. The amplification product was purified and recovered using a DNA purification kit. The specific steps were followed according to the kit's instructions. The primers are shown in Table 2.
[0039] Table 2. Mutation primers for different mutants (partial list)
[0040] 3. Construction of mutant engineered bacteria The purified gene fragment was digested with DpnI to remove the template, and then recombined with recombinase; the recombinant product was transformed into... E. coli In BL21(DE3) competent cells, the cells were plated, and single colonies were picked and cultured in LB liquid. Positive transformants were identified by PCR, and the correctness of the mutation sites was verified by sequencing. After verification, sterile glycerol was added to a final concentration of 15%, and the cells were labeled and stored at -80℃ for later use.
[0041] Example 3: Cultivation of bacterial cells and preparation of crude enzyme solution 1. Culture of bacterial cells Liquid culture medium composition: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, dissolved in deionized water and brought to a final volume, then sterilized at 121°C for 20 min.
[0042] After activating the engineered bacteria from Examples 1 and 2 by streak plating, single colonies were inoculated into 5 mL of LB broth containing 50 μg / mL kanamycin and cultured overnight at 37°C with shaking. Then, 2% of the inoculum was transferred to 50 mL of fresh LB broth containing 50 μg / mL kanamycin and cultured at 37°C with shaking until OD (outlet capacity) was reached. 600When the concentration reaches approximately 0.8, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM, and induce culture at 18°C for 18-24 h. After culture, centrifuge the culture at 4000 rpm for 10 min, discard the supernatant, collect the bacterial cells, and wash the cells twice with 10 mM pH 8.0 phosphate buffer. Store at -80°C for later use.
[0043] 2. Preparation of crude enzyme solution After the culture was completed, the bacterial cells collected were suspended four times in 100 mM pH 8.0 phosphate buffer and sonicated 30 times at 400W, with each sonication lasting 3 seconds and a 7-second interval. The lysate was then centrifuged at 12000 rpm at 4℃ for 10 minutes to remove the precipitate. The supernatant obtained was the crude enzyme solution containing (S)-transaminase.
[0044] Example 4: Preparation of (R)-3-([1,1'-biphenyl]-4-yl)-2-aminoprop-1-ol from (S)-transaminase mutant in water-DMSO solution Following the method described in Example 3, engineered bacteria expressing (S)-transaminase and its mutants of (R)-3-([1,1'-biphenyl]-4-yl)-2-aminoprop-1-ol were cultured to obtain crude enzyme solution. The reaction system (1 L) contained 500 mM isopropylamine, 50 mM 3-([1,1'-biphenyl]-4-yl)-2-ketoprop-1-ol, 50 μM pyridoxal phosphate, 250 mL of crude enzyme solution, and 20% DMSO (v / v). The reaction was carried out at 40 °C for 9 h, after which the concentrations of substrate and product were measured. ee Values. Data at the end of the reaction are shown in Table 3.
[0045] Table 3. Preparation of (R)-3-([1,1'-biphenyl]-4-yl)-2-aminoprop-1-ol by different mutants ee Value and final conversion rate
[0046] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.
Claims
1. A (S)-transaminase mutant, characterized in that, The mutant has 14 or fewer substitutions relative to SEQ ID NO. 2 and has less than 100% sequence identity with SEQ ID NO. 2; wherein the substitutions are selected from: T24C, T24G, F25L, F25M, F25V, S26F, S26M, S26R, S26G, S26T, S26A, S26D, S26V, S26W, S26Y, S26E, S26L, T27S, Q32T, Y60S, Y60D, Y60Q, V85T, T88G, T88N, S90L, S90A, S90T, S90V, S1 63A, S163G, Y164M, R165M, G243A, S327T, S327Q, S327H, L387M, L387Q, L387H, L387T, L387Y, Q420S, Q420H, Q420V, M423R, V436L, T440Q, M441C, M441L, M441V, R442H, R442A, R442N, R442G, R442Q, R442T and I443L, or combinations thereof.
2. A gene encoding the (S)-transaminase mutant as described in claim 1.
3. A recombinant vector, characterized in that, The recombinant vector contains the gene described in claim 2.
4. A recombinant microorganism, characterized in that, The recombinant microorganism comprises the gene of claim 2 or the recombinant vector of claim 3.
5. A catalyst, characterized in that, The catalyst comprises the (S)-transaminase mutant of claim 1 or the recombinant microorganism of claim 4; The catalyst is an enzyme preparation of the mutant, an immobilized enzyme, or a whole cell or cell lysate of the recombinant microorganism.
6. The use of the (S)-transaminase mutant as described in claim 1, or the recombinant microorganism as described in claim 4, or the catalyst as described in claim 5 in the preparation of (R)-3-([1,1'-biphenyl]-4-yl)-2-aminoprop-1-ol.
7. A method for preparing (R)-3-([1,1'-biphenyl]-4-yl)-2-aminoprop-1-ol, characterized in that, Using isopropylamine and 3-([1,1'-biphenyl]-4-yl)-2-ketoprop-1-ol as substrates, pyridoxal phosphate as a cofactor, and the catalyst described in claim 5, a transamine reaction is carried out to generate (R)-3-([1,1'-biphenyl]-4-yl)-2-aminoprop-1-ol.
8. The method as described in claim 7, characterized in that, The reaction is carried out in an organic solvent-water mixture or an aqueous solution.
9. The method as described in claim 7, characterized in that, The organic solvent is DMSO, and the amount of organic solvent added does not exceed 20% of the total volume.