Enzymatic synthesis of a roscovitine chiral intermediate

Abstract

The application discloses an enzyme catalytic synthesis method of a lusoitin chiral intermediate, and the method comprises the following steps: adding recombinant escherichia coli wet bacteria expressing alcohol dehydrogenase, recombinant escherichia coli wet bacteria expressing formate dehydrogenase, a buffer, a substrate 3-cyclopentyl-3-oxopropionitrile, a cosolvent, ammonium formate and coenzyme into a reaction container, and reacting at 25-35 DEG C for 2-24 hours; and after extraction, separation and rotary evaporation, the lusoitin chiral intermediate is obtained. The method has the advantages of mild reaction condition, environmental friendliness, high regional selectivity and stereoselectivity, high conversion rate, high chiral purity, low enzyme consumption, low preparation cost, suitability for industrial production, avoidance of chiral resolution and heavy metal residue in a product, and compensation for the shortcomings of a chemical method.

Description

Technical Field

[0001] This invention relates to the field of biosynthesis technology, and in particular to an enzyme-catalyzed synthesis method for a chiral intermediate of ruxolitinib. Background Technology

[0002] Ruxolitinib (trade name Jakafi) is a potent small molecule inhibitor of JAK1 / JAK2, widely used in the treatment of myelofibrosis, polycythemia vera, and graft-versus-host disease. The drug's molecule contains a key chiral center, the stereochemical purity of which directly affects the drug's binding efficiency to the target, efficacy, and safety. Therefore, developing efficient and highly selective chiral intermediate construction processes is crucial for improving the synthetic efficiency of ruxolitinib, reducing production costs, and achieving green manufacturing.

[0003] Currently, the synthesis of (3S)-3-cyclopentyl-3-hydroxypropionitrile intermediates mainly relies on chemical catalytic pathways, such as chiral borane reduction and transition metal-catalyzed asymmetric hydrogenation. However, these methods have significant drawbacks: 1) They require expensive chiral ligands (such as (S)-BINAP, chiral phosphine ligands, etc.) and metal catalysts (such as ruthenium and rhodium complexes), resulting in high production costs; 2) The reaction conditions are usually harsh, requiring high temperature, high pressure, or anhydrous and oxygen-free environments, making operation complex and posing safety risks; 3) The enantiomeric excess value (ee value) of the product is often difficult to consistently meet pharmaceutical-grade standards (usually >99%); 4) Metal residues may be introduced during the reaction, affecting drug safety and contradicting the development trend of green chemistry and sustainable production. To overcome the limitations of chemical methods, biocatalytic methods, due to their mild reaction conditions, high stereoselectivity, and environmental friendliness, have gradually become a research hotspot in the synthesis of chiral alcohols. In particular, asymmetric reduction reactions catalyzed by aldehyde-ketone reductases (AKRs) can directly convert precursor ketone substrates into corresponding chiral alcohols without chiral resolution, resulting in high atom economy. However, existing reports of natural AKR enzymes suitable for the synthesis of this intermediate often suffer from problems such as low catalytic efficiency, poor substrate tolerance, insufficient optical selectivity, and strong coenzyme dependence, making it difficult to meet the efficiency and economy requirements of industrial production.

[0004] Therefore, there is an urgent need in this field to develop a novel biocatalytic scheme with high catalytic activity, high stereoselectivity, and stable operation at high substrate concentrations, in order to achieve a green, economical, and scalable synthesis process for (3S)-3-cyclopentyl-3-hydroxypropionitrile. Summary of the Invention

[0005] The purpose of this invention is to provide an enzymatically catalytic synthesis method for chiral intermediates of ruxolitinib. The reaction conditions are mild and environmentally friendly, with high regioselectivity and stereoselectivity, high conversion rate, high chiral purity, low enzyme dosage, and low preparation cost. It is suitable for industrial production, avoids chiral resolution and heavy metal residues in the product, and makes up for the shortcomings of chemical methods.

[0006] The technical solution adopted by this invention to solve its technical problem is: An enzymatic synthesis method for a chiral intermediate of ruxolitinib involves adding recombinant Escherichia coli wet cells expressing alcohol dehydrogenase, recombinant Escherichia coli wet cells expressing formate dehydrogenase, buffer solution, substrate 3-cyclopentyl-3-oxopropionitrile, solubilizer, ammonium formate, and coenzyme to a reaction vessel, reacting at 25–35°C for 2–24 hours, and obtaining the chiral intermediate of ruxolitinib: (3S)-3-cyclopentyl-3-hydroxypropionitrile after extraction, separation, and rotary evaporation.

[0007] The core of this invention lies in the development of an enzymatic synthesis process for chiral intermediates of ruxolitinib, and the preparation of a mutant AKR3 alcohol dehydrogenase for the enzymatic synthesis of chiral intermediates of ruxolitinib. This mutant has high catalytic efficiency, low dosage, and high substrate concentration, achieving a dual improvement in conversion rate and chiral purity, and has significant advantages for industrial application.

[0008] Preferably, the alcohol dehydrogenase is a mutant of AKR3; the amino acid sequence of AKR3 is shown in SEQ ID No. 1.

[0009] Preferably, the AKR3 mutant is selected from one of AKR3-G54W / D127A / L167R, AKR3-G54W / D127A / L231A, AKR3-G54W / D127A, AKR3-G54W / L167R, and AKR3-D127A / L167R. AKR3-G54W / D127A / L167R: The amino acid sequence shown in SEQ ID No. 1 has a G mutation at position 54 that is changed to W, a D mutation at position 127 that is changed to A, and an L mutation at position 167 that is changed to R. AKR3-G54W / D127A / L231A: The amino acid sequence shown in SEQ ID No.1 has a G mutation at position 54 that is changed to W, a D mutation at position 127 that is changed to A, and an L mutation at position 231 that is changed to A. AKR3-G54W / D127A: The amino acid sequence shown in SEQ ID No. 1 has a G mutation at position 54 that is changed to W, and a D mutation at position 127 that is changed to A; AKR3-G54W / L167R: The amino acid sequence shown in SEQ ID No.1 has a G mutation at position 54 that is changed to W, and an L mutation at position 167 that is changed to R; AKR3-D127A / L167R: The amino acid sequence shown in SEQ ID No.1 has a D mutation at position 127 to A and an L mutation at position 167 to R.

[0010] Preferably, the proportions of each raw material in the 1L reaction system are as follows: 10–200g of recombinant *E. coli* wet cells expressing alcohol dehydrogenase, 10–200g of recombinant *E. coli* wet cells expressing formate dehydrogenase, 20–200g of substrate, 50–500mL of solubilizer, 0.5–2.5 mol of ammonium formate, 0.1–2.5 mmol of coenzyme, and the balance being buffer. The recombinant *E. coli* wet cells expressing formate dehydrogenase, ammonium formate, and coenzyme constitute a coenzyme cycling system. More preferably, the recombinant *E. coli* wet cells expressing formate dehydrogenase are 50–100g.

[0011] Preferably, the co-solvent is at least one selected from ethyl acetate, butyl acetate, dibutyl phthalate, DMSO, methyl tert-butyl ether, dimethyl phthalate, and methanol.

[0012] Preferably, the buffer solution is selected from one of TEA buffer, PB buffer, and Tris-HCl buffer.

[0013] Preferably, the concentration of the TEA buffer is 0.05–0.20 M and the pH value is 6–8; the concentration of the PB buffer is 0.05–0.10 M and the pH value is 6–7; and the concentration of the Tris-HCl buffer is 0.05–0.10 M and the pH value is 7–8.

[0014] Preferably, the coenzyme is NADP. + or NAD + .

[0015] The beneficial effects of this invention are: 1. Biocatalysis not only has mild reaction conditions and is environmentally friendly, but also has high regioselectivity and stereoselectivity. It avoids chiral resolution and heavy metal residues in the products, thus making up for the shortcomings of chemical methods.

[0016] 2. The mutant of AKR3 of the present invention can catalyze the formation of (3S)-3-cyclopentyl-3-hydroxypropionitrile from 3-cyclopentyl-3-oxopropionitrile, wherein the mutant of AKR3, AKR3-G54W / D127A / L167R, has a reaction conversion rate of up to 99% and an optical purity of more than 99%.

[0017] 3. The method requires only one reaction step to obtain the target product, uses fewer reagents, has mild reaction conditions, and high catalytic activity, which reduces production costs and has broad application prospects and considerable market value. Attached Figure Description

[0018] Figure 1 This is a process flow diagram of the present invention; Figure 2 The gas chromatogram confirms the product of (3S)-3-cyclopentyl-3-hydroxypropionitrile. Detailed Implementation

[0019] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0020] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. Unless otherwise specified, the methods in the following embodiments are conventional methods in the field.

[0021] In the method of the present invention, the alcohol dehydrogenase can be a free enzyme (e.g., used in the form of enzyme powder), or in the form of cells expressing the alcohol dehydrogenase (e.g., wet bacterial cells). Or other forms, such as cell lysate supernatant or whole-cell immobilization expressing the alcohol dehydrogenase, or immobilization of free enzyme powder.

[0022] In the preparation method of the alcohol dehydrogenase of the present invention, the host microorganism for enzyme protein expression can be Escherichia coli. Escherichia coli BL21(DE3) can be used as the recombinant plasmid, and pET28 plasmid can be selected. The molecular biology operations involved in the procedure are all well-known and routine experimental procedures in the biological field, including gene acquisition (PCR), splicing of plasmid and target gene (i.e., vector construction), introduction of plasmid containing target gene fragment into bacterial cells (i.e., transformation), bacterial culture in culture medium and enzyme production (i.e., fermentation).

[0023] In this invention, the LB liquid culture medium formula is: 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L NaCl; the LB solid culture medium formula is: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, and 15 g / L agar.

[0024] Example 1: Obtaining recombinant bacterial cells expressing alcohol dehydrogenase: (1) Expression vectors for AKR3, AKR7, AKR20, AKR25, AKR38, AKR40, and AKR44 were synthesized externally (Shanghai Sangon Biotech). The target gene fragments were inserted into the commercial plasmid pET28 to complete the expression vector construction. The gene sequences are as follows: AKR3 (Gene Bank accession number: CDO95597.1), AKR7 (Gene Bank accession number: WP_168100556.1), AKR20 (Gene Bank accession number: WP_023468191.1), AKR25 (Gene Bank accession number: MCF7618489.1), AKR38 (Gene Bank accession number: WP_223637006.1), AKR40 (Gene Bank accession number: WP_134982026.1), and AKR44 (Gene Bank accession number: WP_013056240.1).

[0025] (2) Expression of enzyme proteins using Escherichia coli BL21(DE3). The plasmids that had been constructed were sequentially transformed into the same strain of E. coli cells and plated on LB agar plates. The specific implementation is as follows: The plasmid pET28 containing the target gene was transformed into competent cells, plated on LB agar plates containing kanamycin (50 mg / L) resistance, and cultured at 37°C for 12 hours to obtain recombinant cells.

[0026] Obtaining recombinant bacterial cells expressing formate dehydrogenase: (1) The formate dehydrogenase expression vector was synthesized externally (Shanghai Sangon Biotech). The commercial plasmid pET28 was used to insert the target gene fragment into pET28 to complete the construction of the expression vector. The formate dehydrogenase gene sequence is located in GenBank as AXT18256.1.

[0027] (2) Expression of enzyme proteins using Escherichia coli BL21(DE3). The plasmids that had been constructed were sequentially transformed into the same strain of E. coli cells and plated on LB agar plates. The specific implementation is as follows: The plasmid pET28 containing the target gene was transformed into competent cells, plated on LB agar plates containing kanamycin (50 mg / L) resistance, and cultured at 37°C for 12 hours to obtain recombinant cells.

[0028] Example 2: Preparation of wet bacterial cells of S1, AKR3, AKR7, AKR20, AKR25, AKR38, AKR40, and AKR44: The recombinant engineered bacteria AKR3, AKR7, AKR20, AKR25, AKR38, AKR40, and AKR44 prepared in Example 1 were inoculated (1% inoculum) into 10 mL of LB liquid medium and cultured at 37°C and 180 rpm for 8-10 h. Then, 1% of the bacterial culture was inoculated into 100 mL of LB liquid medium and cultured at 37°C and 180 rpm for 2-2.5 h (equivalent to OD). 600 The concentration was set at 0.6-0.8, and then 0.1 mM IPTG was added. The mixture was induced and cultured at 24°C and 180 rpm for 12 h. The cultured bacterial solution was poured into a 500 ml centrifuge cup and centrifuged at 8000 rpm and 4°C for 10 min. The supernatant was discarded, and the bacterial cells were collected and weighed for subsequent use.

[0029] Preparation of wet formate dehydrogenase cells: The recombinant formate dehydrogenase engineered bacteria prepared in Example 1 was inoculated (1% inoculum) into 10 mL LB liquid medium and cultured at 37°C and 180 rpm for 8-10 h. Then, 1% of the bacterial culture was inoculated into 100 mL LB medium and cultured at 37°C and 180 rpm for 2-2.5 h (equivalent to an OD600 value of 0.6-0.8). Then, 0.1 mM IPTG was added, and the culture was induced at 24°C and 180 rpm for 12 h. The cultured bacterial culture was poured into a 500 mL centrifuge cup and centrifuged at 8000 rpm and 4°C for 10 min. The supernatant was discarded, and the bacterial cells were collected and weighed for subsequent use.

[0030] S2. Take 0.05g of wet bacterial cells of AKR3, AKR7, AKR20, AKR25, AKR38, AKR40 and AKR44 respectively and add them to 2mL EP tubes, and set up a blank control group.

[0031] S3. Add 0.05 g of wet formate dehydrogenase cells to each EP tube. Add 700 µL of PB buffer (0.1 M, pH 7), 100 µL of 7 M ammonium formate solution, and 25 mM NADP to each EP tube. + 100 µL of solution, 100 µL of methyl ether solution of 3-cyclopentyl-3-oxopropionitrile (commercially available, Leyan Reagent) (final concentration of 3-cyclopentyl-3-oxopropionitrile system is 50 g / L), place the EP tube in a constant temperature mixer, and react at 30℃ and 1200 rpm for 2 h.

[0032] S4. After the reaction is complete, take 200 µL of the reaction solution, add 200 µL of ethyl acetate, shake to mix for 2 min, centrifuge at 12000 rpm for 1 min, take the supernatant and send it for HPLC analysis.

[0033] The conversion rates and ee values ​​of each alcohol dehydrogenase were determined by HPLC and are shown in Table 1. Table 1 .

[0034] The results showed that AKR3 and AKR7 had the highest conversion rates, with AKR3 exhibiting better chirality and an S-type ee value of 83.86%.

[0035] Example 3: Construction and screening of alcohol dehydrogenase mutant libraries 1. Starting strain: Using AKR3, which was screened in Example 2, as the original strain, the plasmid pET28b(+)-AKR3 was activated and extracted, wherein the amino acid sequence of AKR3 is shown in SEQ ID No. 1.

[0036] SEQ ID No. 1: MTTQQFFTLSNGNKIPAVAIVGTGTAWYKSEETDATFSQDLVNIVKETLDTVPGVVHLDAAEIYRTYPELGAALKDTKKPREEIFITDKYSTLLQLSENPTVALESSLKKLGVDYVDLYLIHSPIIDKNGTIDIETAWKHLEALYKSGKAKNIGVSNF TVEDLEKLLAVAEIKPQVNQIEFSPFLQNQTPGIVEFSQKNNILLEAYSPLGPLQKRPADSDKIGFYSYIAELTKKYSKSEAQVILLWVYKRGILPVTTSSKIERVQQAQDIFSFDLTDEEVKKITQLGLEQPALRLYWADVYTKYNSEAQKHHHHHH.

[0037] 2. Single mutation: (1) Construction of mutant libraries The AKR3 mutant library was prepared by site-directed mutagenesis. Using the vector pET28b(+)-AKR3 from the original strain as a template, primers were designed (see Table 2 for primer design) and polymerase chain reaction (PCR) was performed. The recombinant plasmid digested with DpnI was transferred into Escherichia coli BL21(DE3) competent cells, and the clones were inoculated into 10 mL LB agar plates and cultured at 37°C for 12–16 h.

[0038] Table 2 Primer List: .

[0039] (2) HPLC screening Randomly select positive clones and the original strain from the plate and inoculate them into 10 mL of LB liquid medium (with kanamycin, 50 mg / L added to the test tube), and incubate at 37°C and 200 rpm in a temperature-controlled shaker for 8-10 h. Inoculate 1% of the culture medium into 100 mL of LB liquid medium (with kanamycin, 50 mg / L added to the shake flask), and incubate at 37°C and 180 rpm in a temperature-controlled shaker for 2-2.5 h (equivalent to when the bacterial density OD... 600 When the pH value is 0.6~0.8, add IPTG inducer to a final concentration of 0.1mM and incubate for 12 hours in a temperature-controlled shaker at 24℃ and 180 rpm. Pour the cultured bacterial solution into an 800ml centrifuge cup and centrifuge at 8000rpm and 4℃ for 10 minutes in a low-temperature high-speed centrifuge. Discard the supernatant, collect the bacterial cells and weigh them for subsequent use.

[0040] The concentrations and chirality of 3-cyclopentyl-3-oxopropionitrile and (3S)-3-cyclopentyl-3-hydroxypropionitrile were determined by HPLC, and the conversion rates were calculated. The dominant strains were screened based on the conversion rates and chirality.

[0041] The dominant strains were sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing and stored at -80℃. The final dominant mutants were AKR3-G54W, AKR3-G54L, AKR3-G54A, AKR3-D127A, AKR3-D127N, AKR3-D127C, AKR3-L167R, AKR3-L167D, AKR3-L167F, AKR3-L231A, AKR3-L231G, AKR3-L231K, AKR3-Y225F, and AKR3-Y225W.

[0042] 3. Iterative mutation Using vectors pET28b(+)-AKR3-G54W, pET28b(+)-AKR3-G54L, pET28b(+)-AKR3-G54A, pET28b(+)-AKR3-D1 27A, pET28b(+)-AKR3-D127N, pET28b(+)-AKR3-D127C, pET28b(+)-AKR3-L167R, pET28b(+)-AKR Using 3-L167D, pET28b(+)-AKR3-L167F, pET28b(+)-AKR3-L231A, pET28b(+)-AKR3-L231G, pET28b(+)-AKR3-L231K, pET28b(+)-AKR3-Y225F, and pET28b(+)-AKR3-Y225W as templates, primers were designed (primer design is the same as in Table 2), and polymerase chain reaction (PCR) was performed. Following the HPLC screening procedure, dominant mutations were further screened based on single-point mutants.

[0043] PCR reaction system (25 µL): 1 µL forward primer (100 μM), 1 µL reverse primer (100 μM), 12.5 µL 2×Phanta buffer (Novizan, China), 0.5 µL dNTP mixture (10 mM each), 1 µL plasmid template, 0.5 µL DNA polymerase Phanta (Novizan, China) and 8.5 µL ultrapure water.

[0044] The PCR program set according to the Phanta Super-Fidelity DNA Polymerase Manual (Novizan, China) is as follows: 95℃ pre-denaturation for 5 min, then 30 cycles (95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 4 min), 72℃ final extension for 10 min, and incubation at 16℃.

[0045] Iterative mutants (multi-site mutants) were obtained: AKR3-G54W / D127A, AKR3-G54W / L167R, AKR3-G54W / L231A, AKR3-G54W / Y225F, AKR3-D127A / L167R、AKR3-D127A / L231A、AKR3-D127A / Y225F、AKR3-L167R / L231A、AKR3-L167R / Y225F、 AKR3-L231A / Y225F, AKR3-G54W / D127A / L167R, AKR3-G54W / D127A / L231A, AKR3-G54W / D127 A / Y225F, AKR3-G54W / L167R / L231A, AKR3-G54W / L167R / Y225F, AKR3-D127A / L167R / L231A.

[0046] 4. Catalytic activity detection Single mutant strains, iterative mutant strains, and control strains (WT, AKR3 as control) were used as catalysts, with 3-cyclopentyl-3-oxopropionitrile as substrate, to compare the catalytic activity of each mutant. The reaction system consisted of 1 mL of 1 mL of 100 mM PB buffer (pH 7.0), with catalyst at 50 g / L wet cell weight, substrate at 100 g / L, and ammonium formate 1.2 M, formate dehydrogenase at 50 g / L wet cell weight, NADPH 2.5 mM, and 0.1 mL of methyl ether as a co-solvent. The reaction was carried out at 30 °C and 1200 rpm for 1 h. 200 μL of the reaction solution was then added to 50 μL of 6 M hydrochloric acid (to terminate the reaction), followed by extraction twice with 200 μL of ethyl acetate. The ethyl acetate phases were combined. For ethyl acetate samples, the concentrations and chirality of 3-cyclopentyl-3-oxopropionitrile and (3S)-3-cyclopentyl-3-hydroxypropionitrile were determined by HPLC according to Example 1, and the conversion rate was calculated.

[0047] The screening results are shown in Table 3: Table 3 .

[0048] Finally, further screening yielded the dominant strain G54W / D127A / L167R, which is an AKR3 mutant.

[0049] 5. Room temperature stability test Wet cells of WT and G54W / D127A / L167R were stored at 25°C in the dark. Samples were taken at 12h and 24h, and enzyme activity was measured under standard reaction conditions. The percentage of residual activity at different times was calculated with the initial activity as 100%, and the results are shown in Table 4. Table 4 .

[0050] The stability of the G54W / D127A / L167R mutant is slightly lower than that of the WT mutant.

[0051] Example 4-1: Construction of stable mutants Given that the results measured in step 5 of Example 3 show that the storage stability of the dominant mutant AKR3-G54W / D127A / L167R (hereinafter referred to as M3) at room temperature is lower than that of the wild type, in order to obtain an enzyme mutant with both high catalytic activity and excellent stability, this invention takes M3 as the starting point and performs iterative saturation mutations on sites that may affect structural stability.

[0052] 1. Selection of stable mutation sites and primer design Based on the analysis of the homologous three-dimensional structures of the M3 mutant, we selected the following sites that may enhance structural rigidity, hydrophobic stacking, or surface salt bridges for iterative mutagenesis: T33V, A217P, S219P, K233E, and L295D. Using plasmid pET28b(+)-AKR3-M3 (G54W / D127A / L167R) as a template, we designed specific primers for site-directed mutagenesis targeting the above sites. The primer sequences are shown in Table 5 below. Table 5. Primer list for stable iterative mutations .

[0053] 2. Construction and screening of mutant libraries Following the PCR, DpnI digestion, transformation, and culture methods described in Example 3, single-point mutation libraries targeting the aforementioned five sites were constructed. Mutations at each site were performed using the M3 plasmid as a template.

[0054] 3. Initial stability screening and activity verification Single clones were randomly selected from each mutant library plate, and together with the M3 and WT controls, were subjected to small-scale expression and cell collection according to the method described in Example 3.

[0055] Initial stability screening: The obtained wet bacterial cells were resuspended in an equal volume of pH 7.0 phosphate buffer and vortexed to mix. 100 μL of the bacterial suspension was transferred to a PCR tube and heat-treated in a 55°C metal bath for 30 minutes, followed immediately by cooling on ice for 5 minutes. The bacterial suspension without heat treatment under the same conditions was used as the activity baseline (100%).

[0056] Activity determination: Both heat-treated and untreated samples were tested for activity according to the catalytic reaction system described in step 4 of Example 3 (reaction time shortened to 30 minutes). The conversion rate was determined and the residual activity of thermal stability (%) was calculated as (conversion rate of heat-treated sample / conversion rate of untreated sample) × 100%.

[0057] Initial screening results showed that most mutants exhibited improved thermal stability compared to M3, but some mutants showed a decrease in initial catalytic activity. Considering both thermal stability, residual activity, and initial conversion rate, we screened out two single-point mutants, T33V and A217P, which showed the most significant improvement in thermal stability while maintaining the high level of catalytic activity of M3.

[0058] 4. Combination and acquisition of superior stable mutants Using the positive single-point mutant plasmid pET28b(+)-AKR3-M3-T33V obtained from screening as a template, and with the corresponding primers in Table 5, the two-point iterative mutant G54W / D127A / L167R / T33V / A217P was constructed through site-directed mutagenesis and named M5.

[0059] M5 was expressed, purified (specific methods can be found in Example 2), and subjected to comprehensive catalytic and stability tests. Table 6 shows that the M5 mutant, while inheriting the ultra-high catalytic performance of M3 (conversion rate >99%, ee value >99.5%), exhibited comprehensive and significant improvements in thermal stability, solvent tolerance, and storage stability, successfully resolving the issue of decreased stability.

[0060] Table 6 Initial screening data of stable iterative mutants .

[0061] 5. Stability and Tolerance Verification 1. Thermal stability test 1.1 Melting Temperature (Tm) Determination: Differential scanning fluorescence (DSF) was used. Purified wild-type (WT) and G54W / D127A / L167R / T33V / A217P mutant enzymes were diluted separately in the same reaction buffer (50 mM sodium phosphate buffer, pH 7.0) to a final concentration of 0.1 mg / mL. A fluorescent dye (SYPRO Orange) was added. On a real-time quantitative PCR instrument, the temperature was increased from 25°C to 95°C at a rate of 1°C / min, and the fluorescence signal was monitored. The melting temperature (Tm) of the protein was determined by derivative curve analysis. Each sample was tested in triplicate.

[0062] 1.2 Determination of the heat inactivation half-life: WT and G54W / D127A / L167R / T33V / A217P enzyme solutions were incubated in a 55℃ constant temperature water bath. Samples were taken at 0, 5, 15, 30, 60, 120, and 240 minutes, and immediately cooled in an ice bath. Residual enzyme activity was then measured under standard reaction conditions (30℃). The relative activity at each time point was calculated with the initial activity as 100%. The time required for half of the enzyme activity to be lost was calculated by fitting a first-order inactivation kinetic equation; this is the half-life (t0). 1 / 2 ).

[0063] 1.3 Determination of residual activity after heat shock: Incubate the enzyme solution in a water bath at 50°C and 60°C for specified times (e.g., 1 hour and 30 minutes), and immediately cool it in an ice bath. Measure the enzyme activity under standard conditions and compare it with the activity of the same enzyme solution without heat treatment to calculate the percentage of residual activity.

[0064] 2. Organic solvent tolerance test WT and G54W / D127A / L167R / T33V / A217P enzyme solutions were mixed with buffer solutions containing different volume fractions of organic solvent (20% DMSO and 30% methanol), respectively. The solutions were incubated with gentle shaking at 25°C for 2 hours. After incubation, a suitable amount of the mixture was taken, and enzyme activity was measured under standard reaction conditions. The residual activity percentage after organic solvent treatment was calculated, with the activity of the enzyme solution incubated in the same buffer without organic solvent incubation defined as 100%.

[0065] 3. pH stability test Prepare a series of buffer solutions with different pH values ​​(e.g., Britton-Robinson buffers pH 5.0-10.0). Mix WT and G54W / D127A / L167R / T33V / A217P enzyme solutions with equal volumes of buffer solutions at different pH values ​​to place the enzymes in the target pH environment. Incubate at 4°C for 24 hours. After incubation, adjust the sample pH back to the standard reaction pH (e.g., 7.0) and immediately measure enzyme activity. Calculate the residual activity under each pH condition, taking the enzyme activity incubated in the optimal pH buffer as 100%, to assess its pH stability range and tolerance limit.

[0066] Storage stability test The WT and G54W / D127A / L167R / T33V / A217P enzyme solutions were placed in standard storage buffer and stored at 4°C and 25°C, respectively, protected from light. Samples were taken periodically (e.g., on days 0, 7, and 30), and enzyme activity was measured under standard reaction conditions. The percentage of residual activity at different storage times and temperatures was calculated, with the activity at the beginning of storage as 100%.

[0067] The results of the comparison between stability and tolerance are shown in Table 7: Table 7 .

[0068] The above data shows that the G54W / D127A / L167R / T33V / A217P mutation not only greatly improves catalytic efficiency and enantioselectivity, but also significantly strengthens the three-dimensional structure of the enzyme through synergistic effects, making it exhibit far greater resilience than the wild type when facing harsh industrial conditions such as high temperature, organic solvents, and extreme pH.

[0069] Example 4-2: Screening of Co-solvents The steps are as follows: S1. Add 0.5g each of AKR3-G54W / D127A / L167R / T33V / A217P wet bacterial cells and formate dehydrogenase wet bacterial cells, 7 mL of PB buffer (0.1 M, pH 7), 1 mL of 12 M ammonium formate solution, and 10 mM NADP to the reaction flask. + 1 mL of solution and 1 mL of a 1000 g / L co-solvent solution of 3-cyclopentyl-3-oxopropionitrile. Ethyl acetate, butyl acetate, isobutyl acetate, Tween 20, Span 80, DMSO (dimethyl sulfoxide), methyl tert-butyl ether, and methanol were used as co-solvents, and the reaction was carried out at 30 °C and 800 rpm.

[0070] S2. After 1 h of reaction, take 1 mL of the reaction solution, add an equal volume of ethyl acetate for extraction, centrifuge, and send the upper organic phase for HPLC analysis. The results are shown in Table 8: Table 8 .

[0071] The results showed that the highest conversion rate (>99%) was achieved when methyl tert-butyl ether was used as the co-solvent, and its chirality was S-type with an ee value of 99.568%.

[0072] Example 5: Buffer solution and pH optimization Buffer optimization: S1. Add 0.5 g each of AKR3-G54W / D127A / L167R / T33V / A217P wet bacterial cells and formate dehydrogenase wet bacterial cells, 7 mL of buffer (0.1 M), 1 mL of 12 M ammonium formate solution, and 10 mM NADP to the reaction flask. + 1 mL of a 1000 g / L solution of 3-cyclopentyl-3-oxopropionitrile in methyl tert-butyl ether was prepared. The buffer solution was prepared as a pH 7.0 solution of PB, Tris-HCl, and TEA. The reaction was carried out at 30 °C and 800 rpm.

[0073] S2. After 0.5 h of reaction, take 1 mL of the reaction solution, add an equal volume of ethyl acetate for extraction, centrifuge, and send the upper organic phase for HPLC analysis. The results are shown in Table 9: Table 9 .

[0074] The results showed that the highest conversion rate was achieved when the buffer solution was a TEA buffer at pH 7.0.

[0075] pH optimization: S1. Add 0.5 g each of AKR3-G54W / D127A / L167R / T33V / A217P wet bacterial cells and formate dehydrogenase wet bacterial cells, 7 mL of buffer (0.1 M), 1 mL of 12 M ammonium formate solution, and 10 mM NADP to the reaction flask. + 1 mL of solution and 1 mL of 1000 g / L methyl tert-butyl ether solution of 3-cyclopentyl-3-oxopropionitrile were used. The buffer solutions were prepared as TEA buffers with pH values ​​of 6.0, 6.5, 7.0, 7.5, and 8.0, and the reaction was carried out at 30 °C and 800 rpm.

[0076] S2. After 1 h of reaction, take 1 mL of the reaction solution, add an equal volume of ethyl acetate for extraction, centrifuge, and send the upper organic phase for HPLC analysis. The results are shown in Table 10: Table 10 .

[0077] The results showed that the highest conversion rate (>99%) was achieved with a TEA buffer at pH 6.5, making it the optimal choice.

[0078] Example 6: Add 0.5 g of AKR3-G54W / D127A / L167R / T33V / A217P wet bacterial cells and 0.5 g of formate dehydrogenase wet bacterial cells, 7 mL of PB buffer (0.1 M, pH 7), 1 mL of 25 M ammonium formate solution, and 10 mM NADP to the reaction flask. + The total system volume was 10 mL, consisting of 1 mL of a 2000 g / L methyl tert-butyl ether solution containing the substrate. The substrate was 3-cyclopentyl-3-oxopropionitrile. The reaction was carried out at 30 °C and 800 rpm. The process flow is shown below. Figure 1 .

[0079] After 6 hours of reaction, 1 mL of the reaction solution was taken, and an equal volume of ethyl acetate was added for extraction. The upper organic phase was then centrifuged and sent for HPLC analysis.

[0080] Single-factor substitutions should be performed according to the following conditions: Table 11 .

[0081] Example 7: 1L scale-up reaction Take a 2000 mL three-necked flask and add 50 g each of AKR3-G54W / D127A / L167R / T33V / A217P wet bacterial cells and formate dehydrogenase wet bacterial cells. Add 500 mL of TEA buffer (0.1 M, pH 6.5), 100 mL of 25 M ammonium formate solution, 100 mL of 10 mM NADP+ solution, and 300 mL of 1000 g / L 3-cyclopentyl-3-oxopropionitrile (containing methyl tert-butyl ether) solution (add the substrate dropwise using a constant pressure funnel, allowing 2 hours for complete addition). Place the flask in a 30°C water bath and stir at 300 rpm.

[0082] At 3 h, 6 h, and 10 h of reaction time, 500 µL of the reaction solution was taken, and an equal volume of ethyl acetate was added for extraction. The extract was then analyzed by TLC.

[0083] After reacting for 10 h and confirming complete conversion, the reaction solution was extracted with ethyl acetate. For the first extraction, an equal volume of ethyl acetate was added, and for the second and third extractions, half a volume of ethyl acetate was added (extraction: after shaking and mixing, centrifuge at 8000 rpm for 5 min, collect the supernatant, and absorb water using anhydrous Na2SO4). After extraction, the extract was rotary evaporated.

[0084] The crude product was dissolved in a small amount of ethyl acetate after rotary evaporation using a vacuum pump followed by an oil pump. The crude product was then solubilized with sonication until a small amount of particles remained. An appropriate volume of petroleum ether was placed in a beaker and placed on a magnetic stirrer. The crude product dissolved in ethyl acetate was slowly added dropwise to the petroleum ether (while the rotor was stirring in the petroleum ether) until a large amount of solid precipitated. Stirring was then stopped, and the mixture was allowed to stand for 10 minutes. The solid was then removed using a vacuum filter and dried by rotary evaporation with an oil pump to obtain the final product.

[0085] The reaction results are shown in Table 12. AKR3-G54W / D127A / L167R / T33V / A217P can catalyze the formation of (3S)-3-cyclopentyl-3-hydroxypropionitrile from 150 g / L of 3-cyclopentyl-3-oxopropionitrile. Product confirmation is shown in [Table 12]. Figure 2 The reaction conversion rate is >99%, and the ee value is greater than 99%.

[0086] Table 12 Results of the scaled-up experiment: .

[0087] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. An enzymatically catalyzed synthesis method for a chiral intermediate of ruxolitinib, characterized in that, Recombinant Escherichia coli wet cells expressing alcohol dehydrogenase, recombinant Escherichia coli wet cells expressing formate dehydrogenase, buffer, substrate 3-cyclopentyl-3-oxopropionitrile, solubilizer, ammonium formate and coenzyme were added to the reaction vessel. The reaction was carried out at 25-35℃ for 2-24 hours. After extraction, separation and rotary evaporation, the chiral intermediate of ruxolitinib: (3S)-3-cyclopentyl-3-hydroxypropionitrile was obtained. The alcohol dehydrogenase is a mutant of AKR3; the amino acid sequence of AKR3 is shown in SEQ ID No. 1; The AKR3 mutant is selected from one of AKR3-G54W / D127A / L167R, AKR3-G54W / D127A / L231A, AKR3-G54W / D127A, AKR3-G54W / L167R, AKR3-D127A / L167R, and AKR3-G54W / D127A / L167R / T33V / A217P. AKR3-G54W / D127A / L167R: The amino acid sequence shown in SEQ ID No. 1 has a G mutation at position 54 that is changed to W, a D mutation at position 127 that is changed to A, and an L mutation at position 167 that is changed to R; AKR3-G54W / D127A / L231A: The amino acid sequence shown in SEQ ID No. 1 has a G mutation at position 54 that is changed to W, a D mutation at position 127 that is changed to A, and an L mutation at position 231 that is changed to A. AKR3-G54W / D127A: The G at position 54 of the amino acid sequence shown in SEQ ID No. 1 is mutated to W, and the D at position 127 is mutated to A; AKR3-G54W / L167R: The amino acid sequence shown in SEQ ID No.1 has a G mutation at position 54 that is changed to W, and an L mutation at position 167 that is changed to R; AKR3-D127A / L167R: The amino acid sequence shown in SEQ ID No. 1 has a D mutation at position 127 that is changed to A, and an L mutation at position 167 that is changed to R; AKR3-G54W / D127A / L167R / T33V / A217P: The amino acid sequence shown in SEQ ID No. 1 has the following mutations: G at position 54 is mutated to W, D at position 127 is mutated to A, L at position 167 is mutated to R, T at position 33 is mutated to V, and A at position 217 is mutated to P. The proportions of each raw material in the 1L reaction system are as follows: 10-200g of recombinant Escherichia coli wet cells expressing alcohol dehydrogenase, 10-200g of recombinant Escherichia coli wet cells expressing formate dehydrogenase, 20-200g of substrate, 50-500mL of cosolvent, 0.5-2.5mol of ammonium formate, 0.1-2.5 mmol of coenzyme, and the balance of buffer.

2. The enzyme-catalyzed synthesis method according to claim 1, characterized in that, The co-solvent is at least one selected from ethyl acetate, butyl acetate, dibutyl phthalate, DMSO, methyl tert-butyl ether, dimethyl phthalate, and methanol.

3. The enzyme-catalyzed synthesis method according to claim 1, characterized in that, The buffer solution is selected from one of TEA buffer, PB buffer, and Tris-HCl buffer.

4. The enzyme-catalyzed synthesis method according to claim 3, characterized in that, The concentration of the TEA buffer is 0.05–0.20 M, and the pH value is 6–8; the concentration of the PB buffer is 0.05–0.10 M, and the pH value is 6–7; the concentration of the Tris-HCl buffer is 0.05–0.10 M, and the pH value is 7–8.

5. The enzyme-catalyzed synthesis method according to claim 1, characterized in that, The coenzyme is NADP. + or NAD + .

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