Carbonyl reductase mutant based on cosubstrate catalytic capability optimization and application thereof

Abstract

The invention relates to a carbonyl reductase mutant based on cosubstrate catalytic ability optimization and application thereof, the mutant is constructed on the basis of a carbonyl reductase BaSDRX amino acid sequence as shown in SEQ ID No.2, the nucleotide sequence of the carbonyl reductase is as shown in SEQ ID No.1 in a sequence table, and the nucleotide sequence of the carbonyl reductase is as shown in SEQ ID No.2 in the sequence table. The mutant is single-point mutation or multi-point combined mutation on the basis of an amino acid sequence of carbonyl reductase BaSDRX as shown in SEQ ID No.2: threonine Thr133 at the 133 site, arginine Arg137 at the 137 site and glutamic acid Glu142 at the 142 site; when the mutant enzyme or a recombinant cell containing the mutant enzyme is applied to synthesis of (S)-1-(2, 6-difluorophenyl) ethanol through asymmetric reduction of 2, 6-difluoroacetophenone, high catalytic activity and high stereoselectivity are achieved, and high-optical-purity (S)-1-(2, 6-difluorophenyl) ethanol (eet; therefore, the method has a good industrial application and development prospect.

Description

Technical Field

[0001] This invention belongs to the fields of biocatalysis and protein engineering technology, and specifically relates to carbonyl reductase mutants with optimized co-substrate catalytic ability and their applications. The invention mainly involves carbonyl reductase mutants with optimized co-substrate catalytic ability and their encoding genes, recombinant expression vectors containing the carbonyl reductase mutant gene, genetically engineered bacteria, and the application of the aforementioned mutant enzyme or recombinant cells containing the mutant enzyme in the asymmetric reduction of 2,6-difluoroacetophenone to synthesize (S)-1-(2,6-difluorophenyl)ethanol. Background Technology

[0002] Chiral alcohols are important chiral skeleton compounds for the synthesis of high-value-added chemicals. The asymmetric reduction of pre-chiral ketones catalyzed by carbonyl reductases has become a preferred method for preparing chiral alcohols due to its significant advantages such as environmental friendliness, mild reaction conditions, and high stereoselectivity. It is generally believed that carbonyl reductases mainly rely on cofactors NADH or NADPH to provide hydride anions to achieve the reduction of ketones. However, nicotinamide cofactors are expensive and difficult to use stoichiometrically in large-scale production. Furthermore, biocatalytic asymmetric reduction reactions are often limited by the low catalytic efficiency of the enzyme itself. Therefore, the key to developing efficient bioreduction systems lies in achieving efficient regeneration of cofactors and improving the catalytic activity of the enzyme.

[0003] Substrate-coupled cofactor regeneration systems provide a simple and direct pathway for cofactor regeneration. In this system, a single enzyme simultaneously catalyzes the conversion of the host substrate into the target product, and utilizes an additional cosubstrate to drive the cofactor regeneration reaction. Many carbonyl reductases possess the ability to oxidize both glucose and / or isopropanol. Therefore, glucose and isopropanol are often used as cosubstrates for regenerating NAD(P)H in substrate-coupled systems. Glucose, as a natural substance in cellular metabolism, has good biocompatibility and cost advantages; while when isopropanol is used as a cosubstrate, its oxidation byproduct acetone has a low boiling point and is easily removed in situ, which is beneficial for the subsequent separation and purification of the target product. Furthermore, isopropanol can also act as a solubilizer, improving the diffusion properties of water-poor host substrates. However, the physicochemical properties of the host substrate (prochiral ketone) and the cosubstrate (glucose / isopropanol) often differ significantly, making it difficult for the enzyme's substrate-binding pocket to simultaneously possess the ability to efficiently convert both the host and cosubstrate, thus affecting the overall efficiency of the enzymatically catalyzed asymmetric reduction reaction.

[0004] Protein engineering is an effective means of customizing enzyme catalytic performance and has been successfully applied to improve the catalytic efficiency of carbonyl reductases. However, current research on the engineering modification of carbonyl reductases in substrate-coupled cofactor regeneration systems generally focuses on optimizing their catalytic performance on the main substrate, while the conversion ability of co-substrate is seriously neglected. Clearly, the dual catalytic efficiency of the enzyme on both the main and co-substrate is a key factor determining the overall performance of the substrate-coupled system. Unfortunately, the impact of mutations on the ability of carbonyl reductases to catalyze co-substrate has rarely been explored in depth, and research on the engineering modification of carbonyl reductases specifically from the perspective of optimizing the enzyme's catalytic performance on co-substrate (such as glucose and isopropanol) conversion is even more scarce.

[0005] Previous studies have found that in substrate-coupled cofactor regeneration systems, the carbonyl reductase BaSDRX, derived from *Bacillus aryabhattai*, can catalyze the asymmetric reduction of 2,6-difluoroacetophenone with high stereoselectivity to generate (S)-1-(2,6-difluorophenyl)ethanol, a valuable product, with an enantiomeric excess (ee) value as high as 99.9%. This chiral alcohol is a key synthon in the preparation of sodium channel modulators and has potential applications in the treatment of channel-related diseases. However, the overall efficiency of BaSDRX in catalyzing the reduction of 2,6-difluoroacetophenone in substrate-coupled systems still falls short of practical application requirements. Therefore, it is necessary to improve the catalytic efficiency of this enzyme through relevant technologies to fully realize its application value. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, the present invention aims to provide a carbonyl reductase mutant based on optimized co-substrate catalytic ability and its application, providing a powerful biocatalyst for the synthesis of (S)-1-(2,6-difluorophenyl)ethanol. By enhancing the catalytic ability of BaSDRX for co-substrates (glucose and isopropanol), the activity of the biocatalytic system for the asymmetric reduction of 2,6-difluoroacetophenone to synthesize (S)-1-(2,6-difluorophenyl)ethanol is significantly improved.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A carbonyl reductase mutant with optimized co-substrate catalytic activity is constructed based on the carbonyl reductase BaSDRX amino acid sequence shown in SEQ ID No. 2, the nucleotide sequence of which is shown in SEQ ID No. 1 in the sequence listing. The mutant is a single-point mutation or multi-point combination mutation containing the following sites on the carbonyl reductase BaSDRX amino acid sequence shown in SEQ ID No. 2: threonine at position 133 (Thr133), arginine at position 137 (Arg137), and glutamate at position 142 (Glu142).

[0009] The mutant is formed by simultaneous substitution at two or three sites of the carbonyl reductase BaSDRX: Thr133, Arg137, and Glu142.

[0010] The carbonyl reductase mutants are as follows:

[0011] The threonine at position 133, Thr133, is mutated to histidine, Thr133His, i.e., T133H, which consists of the amino acid sequence shown in SEQ ID No. 4; the nucleotide sequence is shown in SEQ ID No. 3 in the sequence listing.

[0012] Arginine at position 137 was mutated to glutamine Arg137Gln, or R137Q, which consists of the amino acid sequence shown in SEQ ID No. 6; the nucleotide sequence is shown in SEQ ID No. 5 in the sequence listing.

[0013] The glutamic acid at position 142 was mutated to phenylalanine Glu142Phe, or E142F, which consists of the amino acid sequence shown in SEQ ID No. 8; the nucleotide sequence is shown in SEQ ID No. 7 of the sequence listing.

[0014] The glutamic acid at position 142 was mutated to alanine Glu142Ala, or E142A, which consists of the amino acid sequence shown in SEQ ID No. 10; the nucleotide sequence is shown in SEQ ID No. 9 in the sequence listing.

[0015] Furthermore, the mutants of the carbonyl reductase BaSDRX were combined to construct a combined mutant Thr133His / Glu142Phe, i.e., T133H / E142F, which mutates threonine at position 133 (Thr133) to histidine and glutamic acid at position 142 (Phenylalanine), and is composed of the amino acid sequence shown in SEQ ID No. 12; the nucleotide sequence is shown in SEQ ID No. 11 of the sequence listing.

[0016] Furthermore, mutants of the carbonyl reductase BaSDRX were combined to construct a combined mutant Thr133His / Arg137Gln / Glu142Ala, namely T133H / R137Q / E142A, which is composed of the amino acid sequence shown in SEQ ID No. 14 and the nucleotide sequence shown in SEQ ID No. 13 in the sequence listing.

[0017] The application of carbonyl reductase mutants optimized for co-substrate catalytic ability is applied to recombinant expression vectors for constructing nucleotide sequences of carbonyl reductase mutant genes. The recombinant expression vectors are constructed by linking the carbonyl reductase mutant nucleotide sequences to various vectors, including various plasmids, bacteriophages, or viral vectors, with pET-30a being preferred.

[0018] The application of carbonyl reductase mutants with optimized co-substrate catalytic ability is carried out in genetically engineered bacteria expressing recombinant carbonyl reductase mutants. The mutants are obtained by transforming the recombinant expression vector into the host microorganism, preferably Escherichia coli, and more preferably E. coli BL21(DE3).

[0019] A method for preparing a carbonyl reductase mutant based on co-substrate catalytic activity optimization includes the following steps: culturing a recombinant expression transformant, inducing the production of a recombinant carbonyl reductase mutant protein, wherein the culture medium used for culturing the recombinant expression transformant is LB medium, and recombinant *E. coli* is inoculated into LB medium containing 50 μg / mL kanamycin and cultured until the optical density OD of the culture medium reaches a certain level. 600 When the concentration reaches 0.5–0.7, the recombinant carbonyl reductase mutant protein can be efficiently expressed under the induction of isopropyl-β-D-thiopyranoside IPTG at a final concentration of 0.1–1.0 mM. The LB medium is a solution of 10 g / L peptone, 5 g / L yeast extract and 10 g / L sodium chloride at pH 7.2.

[0020] An application of a carbonyl reductase mutant optimized for co-substrate catalytic activity is disclosed, specifically the application of the carbonyl reductase mutant or its genetically engineered strain in the catalytic asymmetric reduction of 2,6-difluoroacetophenone to prepare (S)-1-(2,6-difluorophenyl)ethanol. The reaction uses 2,6-difluoroacetophenone of formula (I) as the substrate, the purified carbonyl reductase mutant enzyme or its recombinant cells as the catalyst, and glucose and / or isopropanol as co-substrates in a conversion reaction system composed of a buffer solution at pH 5.5–10. After the reaction is complete, the reaction solution is separated and purified to obtain the carbonyl reductase mutant. When using the purified carbonyl reductase mutant enzyme as the catalyst, 0.2–5 mM of the coenzyme NADPH is also added.

[0021]

[0022] The initial concentration of 2,6-difluoroacetophenone in the conversion reaction system is 5–300 mmol / L; the concentration of carbonyl reductase is 0.1–2.0 mg / mL, or the amount of engineered bacteria containing carbonyl reductase mutant is 10–400 g / L based on the wet weight of the bacteria.

[0023] The conversion reaction was carried out in a buffer solution at pH 7.5.

[0024] The conversion reaction system also contains glucose or isopropanol at a mass concentration of 1-50% as a co-substrate.

[0025] The concentrations of glucose and isopropanol are 5% (w / w).

[0026] The method for separating and purifying the conversion reaction solution is as follows: After the reaction is completed, the conversion reaction solution is centrifuged, and the supernatant is extracted with an equal volume of ethyl acetate. The organic layer is the crude product containing the corresponding (S)-1-(2,6-difluorophenyl)ethanol. The crude product is purified to obtain the corresponding (S)-1-(2,6-difluorophenyl)ethanol.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] 1. This invention significantly improves the catalytic activity of carbonyl reductase in the asymmetric reduction of the main substrate 2,6-difluoroacetophenone to (S)-1-(2,6-difluorophenyl)ethanol in a substrate-coupled cofactor regeneration system by enhancing the catalytic ability of carbonyl reductase for co-substrate (glucose and isopropanol).

[0029] 2. The asymmetric reduction of (S)-1-(2,6-difluorophenyl)ethanol by the carbonyl reductase mutant or recombinant cells containing mutant protein of the present invention has high stereoselectivity and can synthesize (S)-1-(2,6-difluorophenyl)ethanol with high optical purity (ee>99%).

[0030] 3. The recombinant cells containing mutant proteins of this invention can efficiently catalyze the asymmetric reduction of 2,6-difluoroacetophenone in a glucose and / or isopropanol reaction system without the addition of any expensive coenzymes.

[0031] 4. The recombinant cells containing mutant proteins of this invention can be directly used as catalysts for the asymmetric reduction of 2,6-difluoroacetophenone to synthesize (S)-1-(2,6-difluorophenyl)ethanol without the need for enzyme purification, and the catalyst is easy to prepare.

[0032] 5. The carbonyl reductase mutant or recombinant cells containing mutant proteins purified by this invention can be processed in an aqueous system without the need for organic solvents or chiral auxiliary reagents, making it environmentally friendly.

[0033] 6. The carbonyl reductase mutant purified by this invention or recombinant cells containing mutant proteins can catalyze reactions under isothermal conditions of room temperature and pressure, which has the advantages of low energy consumption and high safety factor.

[0034] In summary, this invention differs from existing strategies that generally focus on optimizing the main substrate. It innovatively addresses the issue by systematically engineering the protein of BaSDRX to enhance its catalytic activity towards co-substrate (glucose and isopropanol), providing a carbonyl reductase mutant with significantly enhanced catalytic activity and its nucleotide sequence, as well as a recombinant expression vector and recombinant engineered bacteria containing the corresponding mutant gene. High-optical-purity (S)-1-(2,6-difluorophenyl)ethanol can be prepared through asymmetric reduction using these carbonyl reductase mutants or recombinant cells containing the corresponding mutant protein. The carbonyl reductase mutant or recombinant cell asymmetric reduction method described in this invention for preparing (S)-1-(2,6-difluorophenyl)ethanol exhibits high catalytic activity and high stereoselectivity, enabling the synthesis of high-optical-purity (S)-1-(2,6-difluorophenyl)ethanol (ee>99%). The catalyst is easy to prepare, the reaction conditions are mild and environmentally friendly, and its recombinant cells can efficiently catalyze the asymmetric reduction of 2,6-difluoroacetophenone in a glucose and / or isopropanol reaction system without the addition of any coenzymes, showing great promise for industrial application development. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the specific activity of wild-type BaSDRX and its variants in the reduction of 2,6-difluoroacetophenone using glucose and isopropanol as co-substrates.

[0036] Figure 2 This is a schematic diagram of the conversion of carbonyl reductase BaSDRX and its mutants into 2,6-difluoroacetophenone.

[0037] Figure 3 This is a schematic diagram of the conversion of high concentrations of 2,6-difluoroacetophenone by the carbonyl reductase BaSDRX mutant T133H / R137Q / E142A. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0039] Example 1: Construction of mutants

[0040] A carbonyl reductase mutant with optimized co-substrate catalytic activity is constructed based on the carbonyl reductase BaSDRX amino acid sequence shown in SEQ ID No. 2, the nucleotide sequence of which is shown in SEQ ID No. 1 in the sequence listing. The mutant is a single-point mutation or multi-point combination mutation containing the following sites on the carbonyl reductase BaSDRX amino acid sequence shown in SEQ ID No. 2: threonine at position 133 (Thr133), arginine at position 137 (Arg137), and glutamate at position 142 (Glu142).

[0041] The mutant is formed by simultaneous substitution at two or three sites of the carbonyl reductase BaSDRX: Thr133, Arg137, and Glu142.

[0042] The mutants of the carbonyl reductase BaSDRX are as follows:

[0043] The threonine at position 133, Thr133, is mutated to histidine, Thr133His, or T133H, which consists of the amino acid sequence shown in SEQ ID No. 4.

[0044] Arginine at position 137 was mutated to glutamine Arg137Gln, or R137Q, which is composed of the amino acid sequence shown in SEQ ID No. 6;

[0045] The glutamic acid at position 142 was mutated to phenylalanine Glu142Phe, or E142F, which is composed of the amino acid sequence shown in SEQ ID No. 8;

[0046] The glutamic acid at position 142 was mutated to alanine (Glu142Ala, or E142A), which consists of the amino acid sequence shown in SEQ ID No. 10.

[0047] Furthermore, the mutants of the carbonyl reductase BaSDRX were combined to construct a combined mutant Thr133His / Glu142Phe, namely T133H / E142F, which mutates threonine at position 133 to histidine and glutamic acid at position 142 to phenylalanine. It consists of the amino acid sequence shown in SEQ ID No. 12.

[0048] Escherichia coli BL21(DE3).

[0049] The specific construction process of the mutant is as follows:

[0050] Using oligonucleotide fragments containing mutation points as primers (Table 1), the pET-30a recombinant plasmid containing the carbonyl reductase BaSDRX gene was amplified using the QuickChange™ method (Stratagene, La Jolla, CA).

[0051] Table 1 Primers for mutant construction

[0052]

[0053]

[0054] The underlined 'a' indicates a mutation site.

[0055] PCR reaction system: upstream primer (10 μM), 1.0 μL; downstream primer (10 μM), 1.0 μL; recombinant plasmid template, 10 ng; PrimerSTAR Max DNA Polymerase (2X), 12.5 μL; add ddH2O to a total volume of 25 μL.

[0056] PCR program: (1) 98℃, 1min; (2) 98℃, 10s; (3) 55℃, 10s; (4) 72℃, 6min. After cycling (2)-(4) 15 times, cool to 4℃.

[0057] After washing, the PCR product was digested with the restriction endonuclease DpnI, which specifically recognizes methylation sites, to degrade the template plasmid. The enzyme digestion reaction system and conditions were as follows: 17 μL of washed PCR product, 2.0 μL of 10× buffer, 1.0 μL of restriction endonuclease DpnI, and incubated at 37°C for 1 h.

[0058] The PCR product obtained after enzyme digestion was transformed into *E. coli* BL21(DE3) to obtain the corresponding recombinant *E. coli*. The recombinant *E. coli* was plated on kanamycin-containing plates and incubated overnight at 37°C. Randomly selected clones were then subjected to colony PCR identification and sequencing verification. The results showed that the recombinant expression vector containing the carbonyl reductase mutant gene was successfully transformed into the expression host *E. coli* BL21(DE3). The nucleotide sequences of the mutants T133H, R137Q, E142F, E142A, T133H / E142F, and T133H / R137Q / E142A are shown in SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, and SEQ ID No. 13 in the sequence listing, respectively. The corresponding amino acid sequences encoding the proteins are shown in SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, and SEQ ID No. 14 in the sequence listing.

[0059] Example 2: Induced expression of carbonyl reductase mutant

[0060] The preparation method of the carbonyl reductase mutant in Example 1 includes the following steps: culturing the recombinant expression transformant and inducing the recombinant carbonyl reductase mutant protein. The culture medium used for culturing the recombinant expression transformant can be any culture medium in the art that can enable the transformant to grow and produce the carbonyl reductase mutant protein of the present invention, preferably LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2. There are no special limitations on the culture method and conditions, as long as the transformant can grow and produce the carbonyl reductase mutant protein. A preferred method is as follows: the constructed engineered bacteria are inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C. Then, at a 1% inoculation rate (v / v), they are inoculated into 50 mL of LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 200 rpm until the bacterial concentration reaches OD0.05. 600 Add IPTG to a final concentration of 0.1 mM to 0.6, induce culture at 30℃ for 6 h, collect the cells by centrifugation at 4℃ and 4000 rpm for 10 min, and store at -80℃ for later use.

[0061] Example 3: Isolation and purification of carbonyl reductase mutants

[0062] In Example 2, the collected bacterial cells were suspended in 10 mL of Na₂HPO₄-NaH₂PO₄ buffer (100 mM, pH 8.0), shaken well, and then sonicated (effective time 10 min). The lysate was centrifuged at 12,000 rpm for 15 min to remove cell debris, and the supernatant (crude enzyme solution) was collected for subsequent enzyme separation and purification. The purification column was a Ni-NTA column with a packing volume of 5 mL. The Ni-NTA column was first equilibrated with loading equilibration buffer (20 mM sodium phosphate, 500 mM NaCl, and 20 mM imidazole, pH 7.4). The crude enzyme solution was loaded at a rate of 5 mL / min, and eluted with loading equilibration buffer to remove unadsorbed protein. Finally, the target protein was collected by elution with elution buffer (20 mM sodium phosphate, 500 mM NaCl, and 500 mM imidazole, pH 7.4). The enzyme solution was desalted using a HiTrap desalting column with a Na2HPO4-NaH2PO4 (100mM, pH 7.5) buffer solution. The resulting pure enzyme solution was stored at 4°C for later use.

[0063] Example 4: Catalytic performance of carbonyl reductase BaSDRX and its mutant in reducing 2,6-difluoroacetophenone

[0064] Specifically, this involves the application of the carbonyl reductase mutant or its genetically engineered bacteria in the catalytic asymmetric reduction of 2,6-difluoroacetophenone to prepare (S)-1-(2,6-difluorophenyl)ethanol. Using 2,6-difluoroacetophenone of formula (I) as the substrate, the carbonyl reductase mutant pure enzyme or its recombinant cells as the catalyst, and glucose and / or isopropanol as co-substrate, the reaction is carried out in a conversion reaction system composed of a buffer solution at pH 5.5–10. After the reaction is complete, the reaction solution is separated and purified to obtain the carbonyl reductase mutant. When using the carbonyl reductase mutant pure enzyme as the catalyst, the coenzyme NADPH also needs to be added.

[0065]

[0066] The reaction conditions can be selected according to the conventional conditions used in the art.

[0067] Furthermore, the initial concentration of 2,6-difluoroacetophenone in the conversion reaction system is 5–300 mmol / L; the concentration of the carbonyl reductase is 0.1–2.0 mg / mL, or the amount of engineered bacteria containing the carbonyl reductase mutant is 10–400 g / L based on the wet weight of the bacteria.

[0068] Furthermore, the reaction was carried out in a buffer solution at pH 7.5.

[0069] Furthermore, glucose or isopropanol at a mass concentration of 1% to 50% is added as a co-substrate to the conversion reaction system.

[0070] Furthermore, the concentrations of glucose and isopropanol were 5% (w / w).

[0071] Further, the separation and purification method of the conversion reaction solution is as follows: after the reaction is completed, the conversion reaction solution is centrifuged, and the supernatant is extracted with an equal volume of ethyl acetate. The organic layer is the crude product containing the corresponding (S)-1-(2,6-difluorophenyl)ethanol. The crude product is purified to obtain the corresponding (S)-1-(2,6-difluorophenyl)ethanol. The method for purifying the crude product is a well-known technique in the art, and typically involves organic solvent extraction, chromatographic separation, and adsorption separation.

[0072] Under optimal conditions, the constructed mutants were used to catalyze the asymmetric reduction of 2,6-difluoroacetophenone using glucose or isopropanol as co-substrate, respectively. The conversion rates and stereoselectivity of wild-type BaSDRX and its mutants in catalyzing the reduction of 2,6-difluoroacetophenone are shown in Table 2. The single-point mutant T133H performed exceptionally well, achieving conversion rates 2.6 times and 1.7 times higher than the wild-type enzyme when using glucose and isopropanol as co-substrate, respectively. The mutant R137Q showed a 2.0-fold increase in conversion rate to the wild-type when using isopropanol as a co-substrate. The mutant E142A achieved a 3.2-fold increase in conversion rate to the wild-type when using isopropanol as a co-substrate; the mutant E142F achieved a 1.6-fold increase in conversion rate to the wild-type when using glucose as a co-substrate. The co-mutant T133H / E142F achieved a conversion rate of 83.9% with glucose as a co-substrate; while the co-mutant T133H / R137Q / E142A achieved a conversion rate as high as 98.3% with isopropanol as a co-substrate, a significant improvement of 6.9 times compared to the wild-type enzyme. All mutants maintained excellent stereoselectivity (ee value 99.9%).

[0073] Table 2. Asymmetric reduction of 2,6-difluoroacetophenone using glucose or isopropanol as co-substrate.

[0074]

[0075] Example 5: Specific activity of carbonyl reductase BaSDRX and its mutants in the asymmetric reduction of 2,6-difluoroacetophenone

[0076] Using glucose or isopropanol as co-substrate, the constructed combinatorial mutants were used to catalyze the asymmetric reduction of 2,6-difluoroacetophenone. The conversion and stereoselectivity of wild-type BaSDRX and its mutants in the reduction of 2,6-difluoroacetophenone are shown in the figures below. Figure 1 As shown, the specific activity of the combinatorial mutant T133H / E142F, with glucose as a cosubstrate, reached 34.1 mU / mg, which is 5.9 times that of the wild-type enzyme; the specific activity of the combinatorial mutant T133H / R137Q / E142A, with isopropanol as a cosubstrate, was significantly increased to 80.7 mU / mg, which is a significant increase of 27.9 times compared with the wild-type enzyme.

[0077] Example 6: Kinetic parameters of carbonyl reductase BaSDRX and its mutants

[0078] To elucidate the contribution mechanism of co-substrate catalytic activity to the overall performance improvement, the apparent kinetic parameters of wild-type BaSDRX and its mutants for catalyzing 2,6-difluoroacetophenone, glucose, isopropanol, and NADPH were determined and are shown in Table 3. The catalytic efficiencies (kJ / kJ) of the combined mutants T133H / E142F and T133H / R137Q / E142A for 2,6-difluoroacetophenone were also measured. cat / Km The enzyme efficiency was only increased by 1.3-fold and 1.6-fold, respectively. However, the catalytic efficiency for co-substrate was significantly improved. The co-mutant T133H / E142F showed a 12.8-fold increase in glucose catalytic efficiency compared to the wild type, while the co-mutant T133H / R137Q / E142A showed a 17.2-fold increase in isopropanol catalytic efficiency compared to the wild type, confirming that the enhanced co-substrate catalytic ability was the main driving force for the overall performance improvement. Furthermore, the enhanced glucose catalytic ability of the co-mutant T133H / E142F was attributed to the increased enzyme-substrate affinity (K0). m ) and improved effective binding level (k cat The enhanced catalytic activity of T133H / R137Q / E142A for isopropanol is mainly attributed to a significant increase in the effective binding level (k). cat All mutants showed reduced catalytic efficiency for NADPH, indicating that the mutations did not enhance the utilization of cofactors.

[0079] Table 3 Apparent kinetic parameters of wild-type BaSDRX and its variants

[0080]

[0081]

[0082] Example 7: Conversion of 2,6-difluoroacetophenone by carbonyl reductase BaSDRX and its mutants

[0083] Whole-cell catalytic systems, leveraging their endogenous NAD(P)H reserves to provide initial coenzymes for the reaction, represent an effective strategy for in-situ regeneration of cofactors. Simultaneously, the enzyme exhibits higher stability in the intracellular environment, eliminating the need for additional enzyme purification steps. Based on this, recombinant *E. coli* expressing the aforementioned mutant was used to catalyze the asymmetric reduction reaction of 2,6-difluoroacetophenone in whole cells. In the asymmetric reduction reaction of 10 mM 2,6-difluoroacetophenone (… Figure 2 Recombinant E. coli expressing the mutant T133H / R137Q / E142A achieved a conversion rate of >95% within 75 minutes using isopropanol as a co-substrate; recombinant E. coli expressing the mutant T133H / E142F achieved a conversion rate of >95% within 150 minutes using glucose as a co-substrate; while the wild-type enzyme achieved a conversion rate of only 20.0% after 240 minutes of whole-cell catalysis.

[0084] Example 8: Conversion of high concentrations of 2,6-difluoroacetophenone by the carbonyl reductase BaSDRX mutant T133H / R137Q / E142A

[0085] Based on the excellent performance of the mutant T133H / R137Q / E142A with isopropanol as a co-substrate, and the advantages of easy removal of isopropanol byproducts and solubilizing effect, this example conducts catalytic verification in the concentration range of 2,6-difluoroacetophenone within the range of 20-100 mM. Figure 3 The results showed that the mutant recombinant Escherichia coli whole-cell catalyst achieved a conversion rate of 95.6% after 900 minutes of reaction at a high substrate concentration of 100 mM, and the enantiomeric excess (ee) of the product (S)-1-(2,6-difluorophenyl)ethanol remained at 99.9%, demonstrating its potential application in substrate-coupled cofactor regeneration systems.

Claims

1. A carbonyl reductase mutant optimized based on co-substrate catalytic ability, characterized in that, The mutant was constructed based on the carbonyl reductase BaSDRX amino acid sequence shown in SEQ ID No. 2, and the carbonyl reductase nucleotide sequence is shown in SEQ ID No. 1 in the sequence listing. The mutant is a single-point mutation or multi-point combination mutation containing the following sites based on the carbonyl reductase BaSDRX amino acid sequence shown in SEQ ID No. 2: threonine at position 133 (Thr133), arginine at position 137 (Arg137), and glutamate at position 142 (Glu142).

2. The carbonyl reductase mutant based on co-substrate catalytic ability optimization according to claim 1, characterized in that, The mutant is formed by simultaneous substitution at two or three sites of the carbonyl reductase BaSDRX: Thr133, Arg137, and Glu142.

3. The carbonyl reductase mutant based on co-substrate catalytic ability optimization according to claim 1 or 2, characterized in that, The carbonyl reductase mutants are as follows: The threonine at position 133, Thr133, is mutated to histidine, Thr133His, i.e., T133H, which consists of the amino acid sequence shown in SEQ ID No. 4; the nucleotide sequence is shown in SEQ ID No. 3 in the sequence listing. Arginine at position 137 was mutated to glutamine Arg137Gln, or R137Q, which consists of the amino acid sequence shown in SEQ ID No. 6; the nucleotide sequence is shown in SEQ ID No. 5 in the sequence listing. The glutamic acid at position 142 was mutated to phenylalanine Glu142Phe, or E142F, which consists of the amino acid sequence shown in SEQ ID No. 8; the nucleotide sequence is shown in SEQ ID No. 7 of the sequence listing. The glutamic acid at position 142 was mutated to alanine Glu142Ala, or E142A, which consists of the amino acid sequence shown in SEQ ID No. 10; the nucleotide sequence is shown in SEQ ID No. 9 in the sequence listing.

4. The carbonyl reductase mutant based on co-substrate catalytic ability optimization according to claim 1 or 2, characterized in that, The mutants of the carbonyl reductase BaSDRX were combined to construct a combined mutant Thr133His / Glu142Phe, i.e., T133H / E142F, which mutates threonine at position 133 (Thr133) to histidine and glutamic acid at position 142 (glutamic acid) to phenylalanine. It consists of the amino acid sequence shown in SEQ ID No. 12; the nucleotide sequence is shown in SEQ ID No. 11 in the sequence listing.

5. The carbonyl reductase mutant based on co-substrate catalytic ability optimization according to claim 1 or 2, characterized in that, The mutants of the carbonyl reductase BaSDRX were combined to construct a combined mutant Thr133His / Arg137Gln / Glu142Ala, namely T133H / R137Q / E142A, which is composed of the amino acid sequence shown in SEQ ID No. 14 and the nucleotide sequence shown in SEQ ID No. 13 in the sequence listing.

6. The application of the carbonyl reductase mutant based on co-substrate catalytic ability optimization according to any one of claims 1-5, characterized in that, It is used in recombinant expression vectors for constructing nucleotide sequences of carbonyl reductase mutant genes. The recombinant expression vectors are constructed by linking carbonyl reductase mutant nucleotide sequences to various vectors, including various plasmids, bacteriophages or viral vectors, preferably pET-30a.

7. The application of the carbonyl reductase mutant based on co-substrate catalytic ability optimization according to any one of claims 1-5, characterized in that, The invention is applied to genetically engineered bacteria that express recombinant carbonyl reductase mutants and is obtained by transforming the recombinant expression vector into a host microorganism, wherein the host microorganism is preferably Escherichia coli, and more preferably E. coli BL21(DE3).

8. The method for preparing carbonyl reductase mutants based on co-substrate catalytic ability optimization according to any one of claims 1-7, characterized in that, The process includes the following steps: culturing the recombinant expression transformant, inducing the production of the recombinant carbonyl reductase mutant protein, wherein the culture medium used for culturing the recombinant expression transformant is LB medium, and recombinant Escherichia coli is inoculated into LB medium containing 50 μg / mL kanamycin and cultured until the optical density OD of the culture medium reaches a certain level. 600 When the concentration reaches 0.5–0.7, the recombinant carbonyl reductase mutant protein can be efficiently expressed under the induction of isopropyl-β-D-thiopyranoside IPTG at a final concentration of 0.1–1.0 mM. The LB medium is a solution of 10 g / L peptone, 5 g / L yeast extract and 10 g / L sodium chloride at pH 7.

2.

9. The application of the carbonyl reductase mutant based on co-substrate catalytic ability optimization according to any one of claims 1-5, characterized in that, Specifically, this involves the application of the carbonyl reductase mutant or its genetically engineered bacteria in the catalytic asymmetric reduction of 2,6-difluoroacetophenone to prepare (S)-1-(2,6-difluorophenyl)ethanol. Using 2,6-difluoroacetophenone of formula (I) as the substrate, the carbonyl reductase mutant pure enzyme or its recombinant cells as the catalyst, and glucose and / or isopropanol as co-substrate, the reaction is carried out in a conversion reaction system composed of a buffer solution at pH 5.5–10. After the reaction is complete, the reaction solution is separated and purified to obtain the carbonyl reductase mutant. When using the carbonyl reductase mutant pure enzyme as the catalyst, 0.2–5 mM of the coenzyme NADPH also needs to be added.

10. The application of the carbonyl reductase mutant based on co-substrate catalytic ability optimization according to claim 9, characterized in that, The initial concentration of 2,6-difluoroacetophenone in the conversion reaction system is 5–300 mmol / L; the concentration of carbonyl reductase is 0.1–2.0 mg / mL, or the amount of engineered bacteria containing carbonyl reductase mutant is 10–400 g / L based on the wet weight of the bacteria. The conversion reaction was carried out in a buffer solution at pH 7.

5. The conversion reaction system also contains glucose or isopropanol at a mass concentration of 1-50% as a co-substrate. The concentrations of glucose and isopropanol are 5% (w / w); The method for separating and purifying the conversion reaction solution is as follows: After the reaction is completed, the conversion reaction solution is centrifuged, and the supernatant is extracted with an equal volume of ethyl acetate. The organic layer is the crude product containing the corresponding (S)-1-(2,6-difluorophenyl)ethanol. The crude product is purified to obtain the corresponding (S)-1-(2,6-difluorophenyl)ethanol.

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