A prostaglandin reductase mutant and its use in asymmetric reduction of alpha, beta-unsaturated ketone compounds

Abstract

The application discloses a prostaglandin reductase mutant and application thereof in asymmetric reduction of alpha, beta-unsaturated ketone compounds, and belongs to the technical field of biological catalysis and green chemistry. The PTGR2 mutant is obtained through molecular docking virtual screening locking sites, whole-plasmid PCR amplification, DpnI enzyme cutting and transformation verification construction. The PTGR2 mutant can be combined with GDH protein to efficiently catalyze asymmetric reduction of alpha, beta-unsaturated ketone, and through GC-MS determination of product yield and HPLC determination of enantiomeric excess value, the PTGR2 mutant exhibits excellent catalytic activity and stereoselectivity, and provides an efficient and green biological catalysis scheme for asymmetric reduction of alpha, beta-unsaturated ketone compounds.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis and green chemistry technology, specifically relating to a prostaglandin reductase mutant, a method for constructing the mutant, a purification method, a whole-cell co-expression strain, and the application of the mutant in the asymmetric reduction of α,β-unsaturated ketone compounds. Background Technology

[0002] Chirality is a fundamental property of nature. Different enantiomers of pharmaceutically active molecules often exhibit drastically different pharmacological activities, metabolic processes, and toxic side effects in vivo. α,β-unsaturated ketones, as a class of highly reactive organic compounds, possess strong electrophilicity due to their conjugated systems, making them important pharmacophores for the development of covalent inhibitors. Furthermore, this type of skeleton is widely found in natural products such as curcumin. Precise saturation modification of their carbon-carbon double bonds can significantly improve the metabolic stability of the molecules and derive chiral drug intermediates with differentiated activities.

[0003] Therefore, developing efficient and selective asymmetric reduction techniques for α,β-unsaturated ketones has become a core issue in synthetic chemistry and drug development. Currently, methods for achieving asymmetric reduction of these compounds mainly include chemical catalysis and photocatalysis-enzyme co-catalysis. Traditional chemical reduction often relies on noble metal catalysts, which are environmentally unfriendly; while emerging photocatalysis-enzyme co-catalysis can achieve complex radical coupling, research on direct and efficient asymmetric hydrogenation of carbon-carbon double bonds in the α,β-unsaturated ketone skeleton remains relatively limited. In contrast, biocatalysis, with its extremely high substrate specificity, mild reaction conditions, and excellent stereoselectivity, shows great potential in the late-stage structural modification of complex molecules.

[0004] Prostaglandin reductase 2 (PTGR2) belongs to the medium-chain dehydrogenase / reductase (MDR) family and participates in the metabolism of various bioactive molecules in vivo. However, natural PTGR2 generally suffers from bottlenecks such as low catalytic activity, poor substrate tolerance, and difficulty in precisely controlling stereoselectivity when dealing with non-natural industrial enone substrates, especially those with large side chains. Furthermore, the dependence of the enzyme catalysis on cofactors (such as NADPH) leads to high costs, limiting its application in large-scale preparation. Therefore, rationally designing and modifying PTGR2 using protein engineering techniques to reshape its active pocket and improve catalytic efficiency and stereoselectivity for non-natural α,β-unsaturated ketone substrates, and constructing an efficient whole-cell catalytic system by combining it with a highly efficient cofactor in-situ regeneration system, can not only provide a new method for the green synthesis of chiral drug intermediates but also offer a powerful technical solution for the stereochemical diversity of complex molecules. Summary of the Invention

[0005] The purpose of this invention is to address the limitations of natural reductases in catalyzing sterically hindered non-natural enone substrates, including limited activity, insufficient stereochemical control, and high coenzyme recycling costs. This invention provides a prostaglandin reductase mutant and an enzyme cascade catalytic system based on this mutant and glucose dehydrogenase (GDH), along with their applications. This invention utilizes site-directed mutagenesis to obtain the PTGR2 mutant with a spatially reshaped active pocket. This mutant, combined with GDH, constructs a catalytic platform with highly efficient in-situ regeneration capabilities for cofactors. By precisely controlling the substrate binding conformation through the mutant, highly efficient asymmetric reduction of α,β-unsaturated ketones is achieved, along with highly stereoselective control over product configuration. The catalytic system described in this invention offers advantages such as high conversion rate, excellent stereoselectivity, high coenzyme regeneration efficiency, and low production cost, providing a highly efficient biocatalytic scheme for the green synthesis of chiral pharmaceutical intermediates and natural product derivatives.

[0006]

[0007] This invention is achieved through the following technical solution: On the one hand, the present invention systematically mutates the target protein at specific sites, including: mutating tyrosine at position 51 (Y51), cysteine ​​at position 54 (C54), threonine at position 62 (T62), tyrosine at position 64 (Y64), leucine at position 65 (L65), tyrosine at position 259 (Y259), tyrosine at position 265 (Y265), valine at position 289 (V289), and methionine at position 135 (M135) to methionine (M), alanine (A), phenylalanine (F), tryptophan (W), valine (V), arginine (R), leucine (L), and isoleucine (I), respectively, thereby constructing a series of mutant combinations for functional characterization.

[0008] Furthermore, the site-directed mutagenesis specifically includes: mutating tyrosine at position 64 to phenylalanine, leucine, valine, and isoleucine (Y64F, Y64L, Y64V, Y64I); mutating methionine at position 135 to alanine and phenylalanine (M135A, M135F); mutating tyrosine at position 259 to alanine (Y259A); mutating tyrosine at position 265 to tryptophan, leucine, valine, and isoleucine (Y265W, Y265L, Y265V, Y265I); and mutating valine at position 289 to alanine (V289A).

[0009] Further, the site-directed mutation refers to a combination mutation, specifically including: simultaneously mutating tyrosine at position 51 and position 265 to alanine (Y51A / Y265A); simultaneously mutating tyrosine at position 259 and position 265 to alanine (Y259A / Y265A); mutating tyrosine at position 51 to methionine and simultaneously mutating tyrosine at position 265 to arginine (Y51M / Y265R); simultaneously mutating tyrosine at positions 51, 259, and 265 to alanine (Y51A / Y259A / Y265A); mutating tyrosine at position 51 to phenylalanine and simultaneously mutating threonine at position 62 to phenylalanine (Y51F / T62F); and further mutating position 2... The following mutations were performed: Tyrosine at position 65 was mutated to alanine (Y51F / T62F / Y265A); tyrosine at positions 51 and 265 were simultaneously mutated to phenylalanine and alanine (Y51F / Y265A); threonine at position 62 and tyrosine at position 265 were mutated to phenylalanine and alanine (T62F / Y265A); tyrosine at positions 51, 259, and 265 were mutated to phenylalanine, alanine, and alanine (Y51F / Y259A / Y265A); tyrosine at position 51 was mutated to alanine, and threonine at position 62 was mutated to phenylalanine (Y51A / T62F); threonine at position 62 and tyrosine at position 259 were mutated to phenylalanine and alanine (T62F / Y265A). F / Y259A); Mutate tyrosine at positions 64 and 265 to phenylalanine simultaneously (Y64F / Y265F); Mutate threonine at position 62, tyrosine at position 259, and tyrosine at position 265 to phenylalanine, alanine, and alanine respectively (T62F / Y259A / Y265A); Mutate tyrosine at positions 51 and 259 to phenylalanine and alanine respectively (Y51F / Y259A); Mutate threonine at position 62 and tyrosine at position 259 to phenylalanine simultaneously (T62F / Y259F); Mutate tyrosine at positions 51 and 265 to phenylalanine simultaneously (Y51F / Y265F); Mutate tyrosine at positions 51 and 64 to phenylalanine simultaneously (Y51F / Y64F); Mutate tyrosine at position 64 to phenylalanine, and simultaneously mutate tyrosine at position 265 to alanine (Y64F / Y265A); Mutate tyrosine at positions 64 and 259 to phenylalanine respectively (Y64F / Y259F); Mutate threonine at position 62 and tyrosine at position 64 to phenylalanine simultaneously (T62F / Y64F); Mutate tyrosine at positions 62 and 265 to phenylalanine respectively (T62F / Y265F); Mutate tyrosine at positions 51, threonine at position 62, and tyrosine at position 64 to phenylalanine simultaneously (Y51F / T62F / Y64F); Mutate tyrosine at positions 51, 62, and 265 to phenylalanine simultaneously (Y51F / T62F / Y265F).Simultaneously mutate tyrosine at positions 51, 64, and 265 to phenylalanine (Y51F / Y64F / Y265F); simultaneously mutate threonine at position 62, and tyrosine at positions 64 and 265 to phenylalanine (T62F / Y64F / Y265F); mutate threonine at position 62 and tyrosine at position 64 to phenylalanine, and mutate tyrosine at position 265 to alanine (T62F / Y64F / Y265A); mutate tyrosine at position 51 to alanine, and mutate tyrosine at position 265 to phenylalanine (Y51A / Y265F); mutate tyrosine at positions 51 and 64 to phenylalanine, and mutate tyrosine at position 265 to alanine (Y51F / Y64F / Y265A); mutate tyrosine at position 51 to alanine, and mutate tyrosine at position 64 to phenylalanine (Y51F / Y64F / Y265A); mutate tyrosine at position 51 to alanine, and mutate tyrosine at position 64 to phenylalanine (Y51F / Y64F / Y265A). The following mutations were performed: 51A / Y64F); mutating tyrosine at positions 259 and 265 to phenylalanine simultaneously (Y259F / Y265F); mutating tyrosine at position 51 to phenylalanine, threonine at position 62 to tryptophan, tyrosine at position 64 to tryptophan, and tyrosine at position 265 to phenylalanine (Y51F / T62W / Y64W / Y265F); mutating tyrosine at position 51 to phenylalanine and cysteine ​​at position 54 to tryptophan (Y51F / C54W); mutating threonine at position 62 to tryptophan and tyrosine at position 64 to phenylalanine (T62W / Y64F); and mutating tyrosine at position 51 to phenylalanine, threonine at position 62 to tryptophan, and tyrosine at position 265 to phenylalanine (Y51F / T62W / Y265F).

[0010] On the other hand, the present invention provides a method for designing and constructing a prostaglandin reductase (PTGR2) mutant.

[0011] 1. Molecular docking virtual screening: Using AutoDock Vina software, a search frame was defined for the active pocket near the cofactor NADPH of the PTGR2 wild-type protein crystal structure (PDB ID: 2ZB3) to perform molecular docking virtual screening. The calculated binding free energy (… ΔG _bind As a comprehensive evaluation criterion for docking configurations, the following is set: ΔG _bind <-6.5kcal·mol -1 To determine the effective binding threshold, conformations that meet the criteria are sorted by energy to select the dominant binding conformation with the lowest energy, thereby locking onto the amino acid residue sites.

[0012] 2. Construction of mutant library: The original PTGR2 template plasmid was extracted, and specific primers with the target mutation site were designed for full plasmid amplification via PCR. The amplification products were digested with DpnI restriction endonuclease to remove the original methylated template plasmid, and the PCR product bands were verified by agarose gel electrophoresis.

[0013] 3. Plasmid transformation and validation: The digested product was introduced into the plasmid using a thermal excitation method. E. coli Recombination circularization was performed in DH5α competent cells. Plasmids were extracted and sequenced, and mutant plasmids with correct sequencing results were screened.

[0014] On the other hand, the present invention provides a method for protein expression, purification and enzyme reaction verification of the above-mentioned PTGR2 mutant.

[0015] Furthermore, the protein expression and validation are carried out through the following steps: 1. Heterologous expression: The correctly sequenced mutant plasmid is introduced into the target cell via thermal excitation. E. coli BL21(DE3) competent cells. The mutant protein expression was induced by adding IPTG and cooling in a medium containing the appropriate antibiotic.

[0016] 2. Protein purification and identification: Bacterial cells were collected by centrifugation and sonicated. The target protein was purified using affinity chromatography. The purified protein was concentrated by ultrafiltration, and the relative molecular mass and purity of the target protein were determined by SDS-PAGE protein electrophoresis.

[0017] 3. Two-enzyme coupled reaction: A cascade reduction system was constructed by coupling purified PTGR2 mutant protein with GDH protein. After the reaction, the product yield and enantiomeric excess were determined by gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC). Ee ), to evaluate the catalytic activity and stereoselectivity of the mutants.

[0018] In another aspect, the present invention also provides the application of the prostaglandin reductase mutant and enzyme cascade reduction system in catalyzing asymmetric reduction reactions of α,β-unsaturated ketones.

[0019] The beneficial effects of this invention are as follows: This invention constructs a highly efficient biocatalytic platform for α,β-unsaturated ketones through two rounds of directed evolution and spatial reshaping of prostaglandin reductase 2 (PTGR2), which has the following significant beneficial effects: 1. A significant leap forward in catalytic performance and stereoselectivity: This invention utilizes semi-rational design to lock in the core residues of the active pocket and construct a mutant library. The high-performance mutants Y51F / T62F / Y265F obtained through screening address the pain points of low activity and poor selectivity of wild-type enzymes on non-natural substrates: taking model substrate 1a as an example, the yield is significantly increased from 30% to 87%; stereoselectivity ( Ee Value) by 6% S Configuration) increased to 80% S (Configuration), achieving a breakthrough in catalytic efficiency.

[0020] 2. A highly efficient and low-cost cofactor regeneration cycle system was constructed: This invention employs a PTGR2 and GDH dual-enzyme cascade system. Utilizing inexpensive glucose to drive NADPH regeneration effectively avoids the direct addition of expensive coenzymes, significantly reducing the cost of the catalytic reaction. Simultaneously, by optimizing screening indicators, dual optimization of substrate 1a yield and stereoselectivity was achieved, enhancing the overall synergistic effect of the cascade system. Attached Figure Description

[0021] Figure 1 In Example 1, the docking of 1a with the PTGR2 protein molecule was demonstrated using PyMOL, showing the protein cavity and interacting amino acid residues. Figure 2 Agarose gel images of the mutants Y51F / T62F / Y265F, Y51F / T62F and Y51F after PCR reaction in Example 1; Figure 3 This is the first half of the sequencing results of the Y51F / T62F / Y265F mutant plasmid in Example 1; Figure 4 This is the latter half of the sequencing results of the Y51F / T62F / Y265F mutant plasmid in Example 1; Figure 5 SDS-PAGE gel images of the PTGR2 protein mutants Y51F, Y51F / T62F, and Y51F / T62F / Y265F in Example 2; Figure 6 This is an SDS-PAGE gel image of the GDH protein in Example 2; Figure 7 For raw material 1a in Example 3 1 H-NMR; Figure 8 For product 2a in Example 3 1 H-NMR; Figure 9 The external standard curve for product 2a in Example 3; Figure 10The stereoselectivity of mutants Y51F / T62F / Y265F for 2a in Example 3; Figure 11 This refers to the stereoselectivity of the wild-type mutant PTGR2 for 2a in Example 3.

[0022] Figure 12 This is the standard sample 2a in Example 3. Detailed Implementation

[0023] The present invention will now be described in detail with reference to specific embodiments.

[0024] The amino acid and DNA sequences of the wild-type PTGR2 described in this invention are shown in SEQ ID NO. 1 and SEQ ID NO. 2.

[0025] The amino acid and DNA sequences of the PTGR2 mutants Y51F / T62F / Y265F are shown in SEQ ID NO. 3 and SEQ ID NO. 4.

[0026] The amino acid and DNA sequences of the GDH described in this invention are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0027] Table 1 Sequence information of PTGR2 wild type

[0028] Table 2. Sequence information of GDH

[0029] Example 1 Methods for designing and constructing PTGR2 mutants: Step 1: Referring to the reported crystal structure of the wild-type PTGR2 protein (PDB ID: 2ZB3), this study performed molecular docking simulations using AutoDock (AutoDock Vina 1.2.7) software. In the docking pretreatment, an active pocket was defined targeting the NADPH binding site. Subsequently, substrate 1a (( E )-3-(4-methoxybenzyl)hepta-4-one sequentially attaches to this active site, with a binding free energy ( ΔG _bind (This is used as an indicator of affinity. Based on empirical thresholds,) ΔG _bind <-6.5 kcal·mol -1 This is considered the standard for effective binding. By extracting the lowest-energy dominant docking conformation, the optimal binding mode between non-natural small molecules and the PTGR2 protein was determined, and the key amino acid residues in stable ligand binding were identified. Figure 1 ).

[0030] Step 2, conformational analysis showed that nine key amino acid residues in the active pocket were closely associated with substrate binding: Tyr51, Cys54, Thr62, Tyr64, Leu65, Tyr259, Tyr265, Arg286, and Leu290.

[0031] Step 3, plasmid amplification: The ultra-clean platform will be provided by Sangon Biotech (Shanghai) Co., Ltd. E. coli 5 μL of the top 10 pET-28a(+) PTGR2 glycerol bacteria was added to 5 mL of LB liquid medium containing kanamycin (Kana) resistance at pH 7.2. After overnight incubation at 37°C and 200 rpm, 1 mL of the overnight culture was added to 50 mL of LB liquid medium containing Kana resistance at a concentration of 50 μg / mL and incubated overnight.

[0032] Step 4, Plasmid extraction: Plasmid extraction was performed using the Rapid Plasmid Mini-Prep Kit DP105 (Tiangen Biotech (Beijing) Co., Ltd.). RNase A and TIANRed solution were added to P1 solution and mixed thoroughly, then stored at 4°C for later use. 4 mL of overnight culture containing the WT-PTGR2 gene was then taken. E. coli The top 10 bacterial cultures were centrifuged at 12,000 rpm for 1 min, and the supernatant was removed to collect the bacterial precipitate. 150 μL of pre-prepared P1 solution was added, and the precipitate was mixed by pipetting. Then, 150 μL of P2 solution was added, and the mixture was gently inverted 8 times to fully lyse the bacteria. Subsequently, 350 μL of P5 solution was added, and the mixture was rapidly inverted 20 times to renature the genetic material. After centrifuging at 12,000 rpm for 2 min, the supernatant was transferred to a CP3 adsorption column. After centrifuging again at 12,000 rpm for 30 sec to remove the liquid from the collection tube, 300 μL of PWT wash buffer was added to the CP3 adsorption column, and the column was centrifuged at 12,000 rpm for 30 sec. The wash buffer was discarded, and then the column was centrifuged at 12,000 rpm for 1 min to thoroughly remove any remaining wash buffer. Finally, the CP3 adsorption column was placed in an autoclaved Eppendorf tube, 100 μL of TB elution buffer was added, and the plasmid was collected by centrifugation at 12,000 rpm for 30 seconds. After determining the plasmid concentration using a microplate reader, the plasmid was rapidly frozen in liquid nitrogen and stored at -80°C for later use.

[0033] Step 5, Mutant library construction: 1. Primer design Primers with mutation sites (such as Y51F, which mutates tyrosine at position 51 to phenylalanine) were designed using the Novizan website (https: / / bio.vazyme.com) and Snap Gene software tools (Table 3). This ensured that both upstream and downstream primers contained mutation sites and spanned introns, with GC content below 60%, thus controlling the PCR reaction at a suitable annealing temperature. The designed primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and purified by HPLC. The lyophilized primer powder was dissolved in ultrapure water to prepare a 10 μM solution for later use.

[0034] 2. Polymerase chain reaction (PCR) Experimental procedure: Design a 20 μL PCR reaction system, using 20 ng of wild-type plasmid template. Specific material additions are shown in Table 4. The general PCR procedure is: 98℃ denaturation—60℃ annealing—72℃ extension for 35 rounds (Table 5).

[0035] 3. Digestion of plasmid templates The PCR products were digested using the DpnI restriction endonuclease.

[0036] The general procedure for digesting PCR products is as follows: 20 μL of reaction system, digested in an incubator at 37°C for 12 h (Table 6).

[0037] 4. Agarose gel electrophoresis Weigh 0.4g of agarose, add 40 mL of 1×TAE Buffer, and heat in a microwave oven until the agarose is completely dissolved. Add the nucleic acid dye Super Gel Blue, vortex thoroughly, and pour into the corresponding mold to cool and set. Take 4.5μL of the nucleic acid product after the PCR reaction, mix it with 0.5μL of 6×Loading Buffer, and load it into the loading well. Add 5μL of RealBand DNA molecular weight standard marker (250 bp~10000 bp) to the corresponding loading well, and perform electrophoresis at 150V for 40 min. Use a high-sensitivity chemiluminescence imager for imaging. After the corresponding 6k strips show obvious fluorescence, add digestive enzymes to digest the template. Figure 2 ).

[0038] 5. PCR products were transformed into DH5α competent cells. The PCR product, digested with DpnI restriction endonuclease, was introduced into DH5α competent cells and pre-cooled on ice for 30 min. The competent cells with the added PCR product were then heat-shocked in a 42°C water bath for 80 sec, followed immediately by cooling on ice for 5 min. Under aseptic conditions, 800 μL of antibiotic-free LB solution was added, and the cells were incubated at 37°C on a shaker at 200 rpm for 45 min. The culture was then centrifuged at 4000 rpm for 5 min. Under aseptic conditions, 100 μL of the bacterial culture (after removing the supernatant) was collected, gently mixed, and added to the corresponding Kana antibiotic plate for overnight incubation. Round, single colonies were picked and incubated overnight in LB liquid medium containing the corresponding antibiotic (Kana 50 μg / mL). The plasmid was dissolved in ultrapure water and sent to the sequencing center of Sangon Biotech (Shanghai) Co., Ltd. for sequencing. After confirming the correct sequencing results, the mutant plasmid was used for the next step of protein expression and purification. Figure 3 , Figure 4 ).

[0039] Table 3 Primer information with mutation sites

[0040] Table 4 PCR reaction system

[0041] Table 5 PCR reaction amplification program

[0042] Table 6. DpnI enzyme digestion reaction system

[0043] Example 2 Protein purification and concentration: Step 1: After thawing the collected bacterial cells on ice, resuspend the cells in 8 mL of Lysis Buffer (pH 8.0, containing 20 mM Tris, 150 mM NaCl, and 0.2% Triton X-100). If necessary, use cold water and sonication to aid dissolution. Use an ultrasonic cell disruptor to disrupt the cells (operating parameters: power 200W, total duration 5 min, working time 2 sec, interval 4 sec; cycle 3-5 times). After cell disruption, centrifuge at 4°C and 12000 rpm for 30 min. Filter the supernatant through a 0.45 μm aqueous filter membrane and transfer it to a sterile centrifuge tube for loading, separation, and purification.

[0044] The target protein was separated and purified using a Bio-Rad NGC chromatography system via a Ni-NTA metal affinity chromatography column. A 280 nm UV detector was used. The mobile phase consisted of equilibration buffer (pH 8.0, containing 20 mM Tris, 150 mM NaCl) and imidazole elution buffer (pH 8.0, 20 mM Trizma, 150 mM NaCl, 500 mM Imidazol). The column was washed with imidazole elution buffer at different concentration gradients (1 mM, 5 mM, 20 mM, 100 mM, 200 mM) for 5-8 column volumes to displace the His-Tag-containing target protein from the nickel column. The corresponding imidazole gradient elution buffers were then collected.

[0045] Step 2: SDS-PAGE protein electrophoresis and concentration Using an SDS-PAGE gel rapid preparation kit, a 12% separating gel and a corresponding stacking gel were prepared based on the molecular weights of PTGR2 (38 kDa) and GDH (28 kDa). The protein samples were premixed with loading buffer, heated in a 95°C metal bath for 5 min, and then loaded into the corresponding protein loading tanks. Electrophoresis was performed using a protein electrophoresis apparatus at 80V for 90 min. After the samples entered the separating gel, the voltage was increased to 120V and electrophoresis continued until the bands were completely separated. After electrophoresis, the gel was stained with Coomassie Brilliant Blue for 10 min. After rinsing the gel with ultrapure water, detection was performed using a high-sensitivity chemiluminescence imaging system. Clear fluorescent bands were observed at 38 kDa for the PTGR2 mutant and at 28 kDa for GDH. Figure 5 , Figure 6 ).

[0046] The purified protein was concentrated to approximately 1 mL by centrifugation at 4000 rpm in a 4°C refrigerated centrifuge using a 10 kDa ultrafiltration tube. Pre-prepared protein stock solution (0.1 mol·L⁻¹) was then added. -1 10 mL of Tris-HCl (pH 7.5) was centrifuged again to replace the imidazole eluent. This process was repeated 3-4 times until all the imidazole eluent was replaced. The mixture was then centrifuged again to concentrate the solution to approximately 1 mL. The protein concentrations were determined using a multi-mode microplate reader. The concentrations of mutant PTGR2 protein T51F were 4 mg / mL, mutant Y51F / T62F protein were 6 mg / mL, mutant Y51F / T62F / Y265F protein were 6 mg / mL, and GDH protein was 10 mg / mL. The purified proteins were aliquoted and stored at -80°C for later use.

[0047] Example 3 Standard synthesis of substrate 1a and racemic standard 2a: Step 1: General Synthesis Method of Substrate 1a In a 250 mL round-bottom flask, p-methoxybenzaldehyde (50 mmol), the corresponding fatty ketone (50 mmol), and concentrated hydrochloric acid (15 mL) were added sequentially. The mixture was heated to reflux in an oil bath and stirred continuously for 4 h. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was complete, the system was cooled to room temperature and extracted with dichloromethane (3 × 15 mL). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 99:1-90:10) to obtain the target product 1a ( Figure 7 ).

[0048] Step 2: General Synthetic Method for Hydrogenation Products of α,β-Unsaturated Ketones In a round-bottom flask, substrate 1a (10 mmol) and 10% Pd / C (1.0 mmol) were suspended in anhydrous methanol (15 mL). The reaction system was subjected to three cycles of evacuation followed by hydrogen purging to completely replace the air, and then stirred continuously for 12 h under H2 atmosphere and at room temperature. After the reaction was completed, the mixture was purified by Celite. ® (Filter aid) filtration was used to remove the solid catalyst, and the filtrate was concentrated under reduced pressure to remove the solvent. The resulting crude product was further purified by preparative high-performance liquid chromatography (pre-HPLC) to obtain product 2a ( Figure 8 ).

[0049] Example 4 In this embodiment, a NADPH cofactor regeneration system was constructed using glucose dehydrogenase (GDH). The asymmetric reduction of substrate 1a was achieved using wild-type PTGR2, single-site mutant, and multi-site combination mutant as catalytic enzymes. The catalytic conversion rate and stereoselectivity of various mutants were systematically investigated.

[0050] 1. Enzyme cascade catalytic reaction at 0.1 mol·L⁻¹ -1 The reaction was carried out in Tris-HCl buffer (pH 7.0, containing acetonitrile as a co-solvent), with a total reaction volume of 0.5 mL per group. The reaction system components included: substrate 1a (final concentration 400 μmol·L⁻¹). -1 ), PTGR2 wild type or its mutant (0.5 mol%), GDH (1 mol%), NADP + (1.25 eq.) and glucose (50 eq.). The reaction mixture was placed in a constant temperature shaker and incubated at 37°C and 200 rpm for 16 h to complete the reduction reaction. After the reaction, the mixture was extracted with dichloromethane (3 × 1 mL), and the organic phases were combined and concentrated under reduced pressure. The residue was redissolved in isopropanol and brought to a final volume of 200 μL for subsequent chiral analysis.

[0051] 2. Yield analysis by gas chromatography-mass spectrometry (GC-MS): A quantitative standard curve was established using the external standard method on an Agilent 5977C GC-MS system. Chromatographic conditions were: injection port temperature 310℃; carrier gas: He; flow rate: 1.2 mL / min. -1 Split ratio 20:1; Column temperature program from 90℃ to 300℃, heating rate 15℃·min -1 The sample yield was calculated using the peak area response value of the target product.

[0052] Stereoselectivity analysis by chiral high-performance liquid chromatography (HPLC): using a system equipped with ChiralCEL ® Enantiomeric excess values ​​were determined using an HPLC system with a chiral column (250 mm × 4.6 mm, 5 μm). Ee The determination was performed using the following methods: 220 nm detection wavelength, 20 °C column temperature, and 0.8 mL / min flow rate. -1 .

[0053] Fine catalytic characterization was performed using the three-key combination mutant Y51F / T62F / Y265F and wild-type PTGR2 as representatives, and the corresponding detection spectra are shown below: Figure 9 The external standard curve for product 2a; Figure 10 Stereoselectivity of mutants Y51F / T62F / Y265F for 2a Ee =80% ( S Configuration), yield = 87%; Figure 11 Stereoselectivity of wild-type PTGR2 for 2a Ee =3% ( S Configuration), yield = 30%; Figure 12 Standard sample 2a.

[0054] Using the same buffer system, substrate concentration, reaction temperature, reaction time, and detection and analysis methods as described above, parallel catalytic screening was conducted on single-site mutants and multi-site combined mutants of PTGR2 protein. The substrate conversion rate and product stereoselectivity of each mutant catalyzing substrate 1a are shown in Table 7.

[0055] Table 7. Yields and stereoselectivity of product 2a catalyzed by different PTGR2 mutants

[0056] Table 7 shows the yields and stereoselectivity of product 2a catalyzed by different PTGR2 mutants.

[0057] Note: Stereoselectivity values ​​are enantiomeric excess values ​​of the product. Ee (%) Ee A positive value represents the dominant product. S Configuration, a value of 0 represents no stereo selectivity.

[0058] The above embodiments are only used to illustrate the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, but should not be used to limit the scope of protection of the present invention. All equivalent transformations or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A prostaglandin reductase mutant, characterized in that, The mutant is based on the mouse wild-type PTGR2 protein and undergoes a site-directed mutation at at least one amino acid site selected from the following: tyrosine Y51 at position 51, cysteine ​​C54 at position 54, threonine T62 at position 62, tyrosine Y64 at position 64, leucine L65 at position 65, methionine M135 at position 135, tyrosine Y259 at position 259, tyrosine Y265 at position 265, and valine V289 at position 289. Each mutation site is independently mutated to any one of alanine A, methionine M, arginine R, phenylalanine F, tryptophan W, leucine L, valine V, and isoleucine I. The amino acid sequence of the wild-type PTGR2 is shown in SEQ ID NO.

1.

2. The prostaglandin reductase mutant according to claim 1, characterized in that, The mutants include: mutating tyrosine at position 64 to phenylalanine, leucine, valine, and isoleucine, respectively; mutating methionine at position 135 to alanine and phenylalanine, respectively; mutating tyrosine at position 259 to alanine; mutating tyrosine at position 265 to tryptophan, leucine, valine, and isoleucine, respectively; and mutating valine at position 289 to alanine; Simultaneously mutate tyrosine at positions 51 and 265 to alanine; simultaneously mutate tyrosine at positions 259 and 265 to alanine; mutate tyrosine at position 51 to methionine and simultaneously mutate tyrosine at position 265 to arginine; simultaneously mutate tyrosine at positions 51, 259, and 265 to alanine; mutate tyrosine at position 51 to phenylalanine and simultaneously mutate threonine at position 62 to phenylalanine; mutate tyrosine at positions 51, 62, and 265 to phenylalanine, phenylalanine, and alanine, respectively; simultaneously mutate tyrosine at position 51 and 265 to phenylalanine and alanine; mutate threonine at position 62 and tyrosine at position 265 to phenylalanine and alanine, respectively. Mutate to phenylalanine and alanine; mutate tyrosine at positions 51, 259, and 265 to phenylalanine, alanine, and alanine, respectively; mutate tyrosine at position 51 to alanine, and simultaneously mutate threonine at position 62 to phenylalanine; mutate threonine at position 62 and tyrosine at position 259 to phenylalanine and alanine, respectively; mutate tyrosine at positions 64 and 265 simultaneously to phenylalanine; mutate threonine at positions 62, 259, and 265 to phenylalanine, alanine, and alanine, respectively; mutate tyrosine at positions 51 and 259 to phenylalanine and alanine, respectively; mutate threonine at positions 62 and 259 simultaneously to phenylalanine; mutate threonine at positions 51 and 265 to phenylalanine, respectively; mutate threonine at positions 64 and 265 simultaneously to phenylalanine; mutate threonine at positions 62 ... Simultaneously mutate tyrosine at position 65 to phenylalanine; simultaneously mutate tyrosine at positions 51 and 64 to phenylalanine; mutate tyrosine at position 64 to phenylalanine and simultaneously mutate tyrosine at position 265 to alanine; mutate tyrosine at positions 64 and 259 to phenylalanine respectively; simultaneously mutate threonine at positions 62 and 64 to phenylalanine; simultaneously mutate tyrosine at positions 62 and 265 to phenylalanine respectively; simultaneously mutate tyrosine at positions 51, threonine at positions 62, and 64 to phenylalanine; simultaneously mutate tyrosine at positions 51, 62, and 265 to phenylalanine; simultaneously mutate tyrosine at positions 51, 64, and 265 to phenylalanine; simultaneously mutate tyrosine at positions 62, 64, and 265 to phenylalanine; mutate tyrosine at positions 62, 64, and 265 to phenylalanine; simultaneously ...5, 64, and 265 to phenylalanine; simultaneously mutate tyrosine at positions 65, 64, and 265 to phenylalanine; simultaneously mutate tyrosine at positions 65, Simultaneously mutate tyrosine at positions 4 and 265 to phenylalanine; mutate threonine at position 62, tyrosine at position 64, and tyrosine at position 265 to phenylalanine, alanine, and alanine, respectively; mutate tyrosine at position 51 to alanine and tyrosine at position 265 to phenylalanine; mutate tyrosine at positions 51 and 64 to phenylalanine and tyrosine at position 265 to alanine; mutate tyrosine at position 51 to alanine and tyrosine at position 64 to phenylalanine; simultaneously mutate tyrosine at positions 259 and 265 to phenylalanine; mutate tyrosine at position 51 to phenylalanine, threonine at position 62 to tryptophan, tyrosine at position 64 to tryptophan, and tyrosine at position 265 to phenylalanine;The tyrosine at position 51 is mutated to phenylalanine, and the cysteine ​​at position 54 is mutated to tryptophan; the threonine at position 62 is mutated to tryptophan, and the tyrosine at position 64 is mutated to phenylalanine; the tyrosine at position 51 is mutated to phenylalanine, the threonine at position 62 is mutated to tryptophan, and the tyrosine at position 265 is mutated to phenylalanine.

3. A method for constructing the prostaglandin reductase mutant according to claim 1 or 2, characterized in that, The process includes the following steps: performing molecular docking on the wild-type crystal structure of PTGR2 to identify key amino acid residue sites; designing specific mutant primers for the target sites and performing whole-plasmid PCR amplification using the wild-type plasmid as a template; digesting the PCR product with DpnI restriction endonuclease to remove the wild-type template plasmid; transforming the digested product into competent E. coli cells and sequencing to verify the correct mutant plasmid.

4. The method for constructing a prostaglandin reductase mutant according to claim 3, characterized in that, Molecular docking sets the binding free energy ΔG _bind <-6.5 kcal·mol -1 As a threshold for effective substrate binding, the dominant binding conformation with the lowest binding free energy is screened to determine the mutation site.

5. A method for expressing and purifying the prostaglandin reductase mutant according to claim 1 or 2, characterized in that, Import the correctly sequenced mutant plasmid. E. coli BL21(DE3) competent cells were induced to express mutants heterologously by adding IPTG at low temperature. The cells were collected by centrifugation and the intracellular proteins were released by sonication. The proteins were then purified by affinity chromatography, concentrated by ultrafiltration, and identified by SDS-PAGE electrophoresis to determine the molecular weight and purity of the proteins.

6. A whole-cell co-expression strain based on the prostaglandin reductase mutant of claim 1 or 2, characterized in that, Constructed by co-expressing the PTGR2 mutant gene and the glucose dehydrogenase gene in the E. coli host, it possesses the ability to regenerate NADPH cofactor in situ.

7. The use of the prostaglandin reductase mutant according to claim 1 or 2 in catalyzing asymmetric reduction reactions of α,β-unsaturated ketone compounds.

8. The application of an enzyme cascade reduction system in catalyzing asymmetric reduction reactions of α,β-unsaturated ketones, characterized in that, The enzyme cascade reduction system comprises the prostaglandin reductase mutant of claim 1 or 2 and glucose dehydrogenase.

9. The application according to claim 7 or 8, characterized in that, The α,β-unsaturated ketone is (E)-3-(4-methoxybenzyl)hepta-4-one.

10. The application according to claim 8, characterized in that, In the reaction system, glucose is used as a cofactor and regeneration substrate to achieve NADPH cycle regeneration.

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