Enzymatic synthesis of a dossamine chiral intermediate

Abstract

This invention discloses an enzymatically catalytic synthesis method for a chiral intermediate of dzoxadamine. Using methyl acetoacetate as a substrate, buffer, substrate, alcohol dehydrogenase, formate dehydrogenase, ammonium formate, and coenzyme are added to a reaction vessel. The reaction is carried out at 20–40°C for 2–24 hours. After extraction, separation, and rotary evaporation, the chiral intermediate (R)-3-hydroxybutyrate methyl ester is obtained. The alcohol dehydrogenase is a mutant of ADH1 derived from Rollstonella, and the amino acid sequence of ADH1 is shown in SEQ ID No. 1. This invention offers mild reaction conditions, high stereoselectivity, and environmental friendliness. It also boasts high conversion rate, high chiral purity, low enzyme dosage, tolerance to high substrate concentrations, and low preparation cost, making it suitable for industrial production. Furthermore, it avoids chiral resolution and heavy metal residues in the product, overcoming the shortcomings of chemical methods.

Description

Technical Field

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

[0002] Dozole (trade name Trusopt) is a topical ocular carbonic anhydrase inhibitor, a non-bacterial sulfonamide drug that inhibits the activity of carbonic anhydrase. It is an anti-glaucoma topical eye drop primarily used to lower intraocular pressure in patients with open-angle glaucoma and ocular hypertension. Developed by Merck, it was approved for marketing by the US FDA in 1995.

[0003] The pharmacological activity of dorzodamine is highly dependent on its S-configuration enantiomer, and one of the key aspects of its synthesis lies in obtaining high-optical-purity (R)-3-hydroxybutyrate methyl ester. As an indispensable chiral building block in the synthesis of dorzodamine, the construction of its chiral center must possess an extremely high enantiomeric excess, typically requiring an ee value > 99% to meet stringent pharmaceutical regulatory requirements. Early methods for the synthesis of (R)-3-hydroxybutyrate methyl ester primarily relied on chemical catalysis. While this method may be academically mature, it suffers from inherent and insurmountable drawbacks. Chemical methods usually require expensive chiral ligands or reagents, involve harsh reaction conditions, and commonly use organic solvents, generating significant amounts of waste, which contradicts the principles of green pharmaceuticals. In contrast, biocatalysis (especially ketone reductase-mediated asymmetric reduction) has gradually become a research hotspot, offering advantages such as mild conditions, high stereoselectivity, and environmental friendliness. However, early biocatalytic processes still suffer from low conversion rates, unstable optical selectivity, and poor process compatibility.

[0004] The representative synthetic route for (R)-3-hydroxybutyrate methyl ester is as follows:

[0005] (R)-3-hydroxybutyrate methyl ester (CN113416130A) was prepared using polyhydroxy fatty acid esters as raw materials and a mixed solution of methanesulfonic acid / methyl methanesulfonate and methanol as solvent. This method involves a highly corrosive reagent (methanesulfonic acid), requires high-quality materials for the reaction equipment, raises concerns about safe operation and waste disposal, and results in high raw material costs, which does not conform to the principles of green chemistry.

[0006] Methyl 3-hydroxybutyrate (CN1181041C) was prepared using octacarbonyldicobalt-pyridine compound-sodium salt as a catalyst and propylene oxide, carbon monoxide, and methanol as raw materials. This method produces propylene glycol and dimethyl ether as byproducts, and lacks chiral control, yielding a racemic mixture. An additional chiral resolution step is required to obtain the optically pure (R)-configuration. The theoretical maximum conversion rate for resolution is only 50%, resulting in a low yield.

[0007] Methyl (R)-3-hydroxybutyrate (CN101864458A) was prepared by microbial fermentation using starch as a carbon source and ethanol as an auxiliary substrate. This method involves a complex and lengthy process route, with a relatively low yield compared to chemical methods, and the product separation and purification are also quite complicated, leaving considerable room for process optimization.

[0008] Existing methods for preparing (R)-3-hydroxy-butyrate methyl ester are inefficient, have low substrate concentrations, and involve long reaction times and high costs, making them unsuitable for industrial production. For example, CN113416130B reports a reaction time of approximately 72 hours, with conversion efficiency insufficient for industrial applications. Other problems include insufficient enzyme stability, high coenzyme regeneration costs, and difficulties in product separation, further limiting its large-scale application. Summary of the Invention

[0009] The purpose of this invention is to provide an enzymatically catalytic synthesis method for dzozoleamine chiral intermediates, which features mild reaction conditions, high stereoselectivity, and is more environmentally friendly. It also boasts high conversion rate, high chiral purity, low enzyme dosage, tolerance to high substrate concentrations, low preparation cost, and suitability for industrial production. Furthermore, it avoids chiral resolution and heavy metal residues in the product, thus overcoming the shortcomings of chemical methods.

[0010] The technical solution adopted by this invention to solve its technical problem is:

[0011] An enzymatically catalytic synthesis method for a chiral intermediate of dzodamine involves using methyl acetoacetate as a substrate, adding buffer, substrate, alcohol dehydrogenase, formate dehydrogenase, ammonium formate, and coenzyme to a reaction vessel, reacting at 20–40°C for 2–24 hours, and obtaining the chiral intermediate (R)-3-hydroxybutyrate methyl ester after extraction, separation, and rotary evaporation.

[0012] The alcohol dehydrogenase is a mutant of ADH1 derived from Rollstonella, and the amino acid sequence of ADH1 is shown in SEQ ID No. 1.

[0013] This invention utilizes a novel mutant of ADH1 derived from Ralstonia sp., which is particularly suitable for the enzymatic synthesis of dzoxamine chiral intermediates. It exhibits high catalytic efficiency, low dosage, high substrate concentration, and good stability, achieving a dual improvement in conversion rate and chiral purity, and possesses significant advantages for industrial application.

[0014] Both alcohol dehydrogenase and formate dehydrogenase were added in the form of wet cells of recombinant genetically engineered bacteria.

[0015] The proportions of each raw material in the 1 L reaction system are as follows: 5–100 g of recombinant Escherichia coli wet cells expressing alcohol dehydrogenase, 10–100 g of recombinant Escherichia coli wet cells expressing formate dehydrogenase, 20–100 g of substrate, 0.1–1.0 M of ammonium formate, 0.1–2.0 mM of coenzyme, and the balance of buffer.

[0016] As a preferred embodiment, the proportions of each raw material in the 1 L reaction system are as follows: 5–100 g of recombinant Escherichia coli wet cells expressing alcohol dehydrogenase, 50–75 g of recombinant Escherichia coli wet cells expressing formate dehydrogenase, 20–100 g of substrate, 0.1–1.0 M of ammonium formate, 0.1–2.0 mM of coenzyme, and the balance of buffer.

[0017] Preferably, the mutant of ADH1 is selected from one of ADH1-T55F / L96A / V110F / D82F, ADH1-T55F / L96A / V110F / G129P, ADH1-T55F / L96A / V110F / N176E, ADH1-T55F / L96A / V110F / Q191R, ADH1-T55F / L96A / V110F / V214P, and ADH1-T55F / L96A / V110F / G129P / Q191R;

[0018] ADH1-T55F / L96A / V110F / D82F is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and D at position 82 is changed to F.

[0019] ADH1-T55F / L96A / V110F / G129P is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and G at position 129 is changed to P.

[0020] ADH1-T55F / L96A / V110F / N176E is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and N at position 176 is changed to E.

[0021] ADH1-T55F / L96A / V110F / Q191R is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and Q at position 191 is changed to R.

[0022] ADH1-T55F / L96A / V110F / V214P is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and V at position 214 is changed to P.

[0023] In the ADH1-T55F / L96A / V110F / G129P / Q191R mutant, the amino acid sequence is modified by changing T at position 55 to F, L at position 96 to A, V at position 110 to F, G at position 129 to P, and Q at position 191 to R. The L20P / T55F / L96F / V110F mutant exhibits the best reaction conversion rate (99%) and optical purity (greater than 99%).

[0024] Preferably, the buffer solution is selected from one of TEA buffer, PB buffer, Tris-HCl buffer, and HEPES buffer.

[0025] Preferably, the concentration of the TEA buffer is 0.05–0.20 M, and the pH value is 6.0–8.0; the concentration of the PB buffer is 0.05–0.10 M, and the pH value is 6.0–7.0; the concentration of the Tris-HCl buffer is 0.05–0.10 M, and the pH value is 7.0–8.0; and the concentration of the HEPES buffer is 0.01–0.10 M, and the pH value is 6.0–8.0.

[0026] Preferably, the coenzyme is NADP. + or NAD + .

[0027] An alcohol dehydrogenase, wherein the alcohol dehydrogenase is a mutant of ADH1 derived from Rollstonella, and the amino acid sequence of ADH1 is shown in SEQ ID No. 1;

[0028] The ADH1 mutant is selected from one of the following: ADH1-T55F / L96A / V110F / D82F, ADH1-T55F / L96A / V110F / G129P, ADH1-T55F / L96A / V110F / N176E, ADH1-T55F / L96A / V110F / Q191R, ADH1-T55F / L96A / V110F / V214P, and ADH1-T55F / L96A / V110F / G129P / Q191R.

[0029] ADH1-T55F / L96A / V110F / D82F is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and D at position 82 is changed to F.

[0030] ADH1-T55F / L96A / V110F / G129P is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and G at position 129 is changed to P.

[0031] ADH1-T55F / L96A / V110F / N176E is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and N at position 176 is changed to E.

[0032] ADH1-T55F / L96A / V110F / Q191R is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and Q at position 191 is changed to R.

[0033] ADH1-T55F / L96A / V110F / V214P is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and V at position 214 is changed to P.

[0034] ADH1-T55F / L96A / V110F / G129P / Q191R is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, G at position 129 is changed to P, and Q at position 191 is changed to R.

[0035] The beneficial effects of this invention are:

[0036] Biocatalysis not only offers mild reaction conditions and is environmentally friendly, but also exhibits high regioselectivity and stereoselectivity. Furthermore, it avoids chiral resolution and heavy metal residues in the products, thus effectively compensating for the shortcomings of chemical methods.

[0037] The mutant of ADH1 of the present invention can catalyze the formation of (R)-3-hydroxybutyrate methyl acetoacetate from methyl acetoacetate. This method requires only one step to obtain the target product, uses fewer reagents, has mild reaction conditions, high catalytic activity, reduces production costs, and has broad application prospects and considerable market value. Attached Figure Description

[0038] Figure 1 This is a process flow diagram of the present invention. Detailed Implementation

[0039] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0040] 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.

[0041] 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 cells), or in other forms, such as the supernatant of cell lysis expressing the alcohol dehydrogenase or whole cell immobilization, or immobilization of free enzyme powder.

[0042] In the preparation method of the alcohol dehydrogenase of the present invention, the host cell for enzyme protein expression can be Escherichia coli BL21(DE3), and the recombinant plasmid can be pET28 plasmid. The molecular biological operations involved in the steps are all well-known 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 host cell (i.e., transformation), culture of bacterial cells in culture medium and enzyme production (i.e., fermentation).

[0043] In this invention, the LB liquid culture medium formula is: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl; the LB solid culture medium formula is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 15 g / L agar.

[0044] Example 1:

[0045] Obtaining recombinant bacterial cells expressing alcohol dehydrogenase:

[0046] Expression vectors for ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, AKR1, AKR2, AKR3, and AKR4 were synthesized externally (Shanghai Sangon Biotech). The target gene fragment was inserted into the commercial plasmid pET28 to complete the expression vector construction. The gene sequences are as follows: ADH1 (Gene Bank accession number: MG563348.1), ADH2 (Gene Bank accession number: NP_388505.1), ADH3 (Gene Bank accession number: WP_041589967.1), ADH4 (Gene Bank accession number: WP_011251899.1), ADH5 (Gene Bank accession number: BAA24528.1), ADH6 (Gene Bank accession number: AAP94029), ADH7 (Gene Bank accession number: ACB78183.1), AKR1 (Gene Bank accession number: XP_002550841.1), AKR2 (Gene Bank accession number: XP_004637721.1), AKR3 (Gene Bank accession number: CDO95597.1), AKR4 (Gene Bank accession number: CDO95597.1), AKR4 (Gene Bank accession number: CDO95597.1), AKR4 (Gene Bank accession number: CDO95597.1), AKR4 (Gene Bank accession number: CDO95597.1), AKR5 (Gene Bank accession number: CDO95597.1), AKR4 ... Login ID: XP_036665498.1.

[0047] (2) Enzyme protein expression was performed using Escherichia coli BL21(DE3). The constructed plasmids were sequentially transformed into the same strain of Escherichia coli and plated on LB agar.

[0048] The specific implementation is as follows:

[0049] First, the bacterial cells transformed with plasmid pET28 were spread on LB agar plates containing kanamycin resistance (50 mg / L kanamycin) and incubated at 37°C for 12 hours. Then, single colonies were picked and transferred to LB liquid medium (with additional kanamycin added to a final concentration of 50 mg / L) and incubated at 37°C in a shaker for 12 hours to prepare competent cells. Then, the plasmid pET28 containing the target gene was transformed into the competent cells and spread on LB agar plates containing kanamycin (50 mg / L) resistance and incubated at 37°C for 12 hours to obtain recombinant bacterial cells.

[0050] Obtaining recombinant bacterial cells expressing formate dehydrogenase:

[0051] (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.

[0052] Step (2) is the same as step (2) for obtaining recombinant cells expressing alcohol dehydrogenase.

[0053] Example 2:

[0054] S1: Preparation of wet bacterial cells: Single colonies of the recombinant engineered bacteria ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, AKR1, AKR2, AKR3, AKR4, and formate dehydrogenase recombinant engineered bacteria prepared in Example 1 were placed into 10 mL of LB liquid medium and cultured at 37°C and 180 rpm for 8–10 h. A 1% inoculum was then 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 0.6–0.8, and then 0.1 mM IPTG was added. The culture was induced at 24°C and 180 rpm for 16 h. The cultured bacterial solution was poured into a 50 mL centrifuge tube and centrifuged at 10,000 rpm and 4°C for 10 min. The supernatant was discarded, and the bacterial cells were collected and weighed for subsequent use.

[0055] S2: Take 0.05 g of wet bacterial cells of ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, AKR1, AKR2, AKR3, and AKR4 respectively and add them to 2 mL EP tubes, and set up a blank control group.

[0056] S3: Add 0.05 g of wet formate dehydrogenase cells to each corresponding EP tube, and add 700 µL of PB buffer (0.1 M, pH 7.0), 100 µL of 10 M ammonium formate solution, and 25 mM NADP to each EP tube. + 100 µL of methyl acetoacetate solution and 100 µL of methyl acetoacetate (commercially available, Leyan Reagent) solution (final concentration of methyl acetoacetate system is 20 g / L) were placed in an EP tube and reacted at 30℃ and 1000 rpm for 4 h.

[0057] S4: After the reaction is complete, take 200 µL of the reaction solution and extract it twice with 200 µL of ethyl acetate. Centrifuge at 12000 rpm for 1 min, take the supernatant and send it for HPLC analysis.

[0058] The conversion rates and ee values ​​of each alcohol dehydrogenase were determined by HPLC and are shown in Table 1.

[0059] Table 1

[0060]

[0061] The results showed that ADH1 and ADH3 had the highest conversion rates, with ADH1 exhibiting better chirality and an R-type ee value of 86.24%.

[0062] Example 3: Construction and screening of alcohol dehydrogenase mutant libraries

[0063] 1. Starting strain:

[0064] Using ADH1, which was screened in Example 2, as the original strain, the plasmid pET28b(+)-ADH1 was activated and extracted, wherein the amino acid sequence of ADH1 is shown in SEQ ID No. 1.

[0065] SEQ ID No. 1:

[0066] MYRLLNKTAVITGGNSGIGLATAKRFVAEGAYVFIVGRRRKELEQAAAEIGRNVTAVKADVTKLEDLDRLYAIVREQRGSIDVLFANSGAIEQKTLEEITPEHYDRTFDVNVRGLIFTVQKALPL LRDGGSVILTSSVAGVLGLQAHDTYSAAKAAVRSLARTWTTELKGRSIRVNAVSPGAIDTPIIENQVSTQEEADELRAKFAAATPLGRVGRPEELAAAVLFLASDDSSYVAGIELFVDGGLTQV.

[0067] 2. Single-point mutation:

[0068] (1) Construction of mutant libraries

[0069] The ADH1 mutant library was prepared by site-directed mutagenesis. Using the vector pET28b(+)-ADH1 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 E. coli BL21(DE3) competent cells and plated on LB solid medium. The cells were incubated upside down at 37°C for 12–16 h.

[0070] Table 2 Primer List:

[0071] .

[0072] (2) HPLC screening

[0073] Randomly select positive single colonies and the original strain from the plate and inoculate them into 10 mL of LB liquid medium (with kanamycin added to the test tube to a final concentration of 50 mg / L) and incubate at 37°C and 180 rpm on a temperature-controlled shaker for 8–10 h. Inoculate the culture solution at a 1% inoculation rate into 100 mL of LB liquid medium (with kanamycin added to the shake flask to a final concentration of 50 mg / L) and incubate at 37°C and 180 rpm on a temperature-controlled shaker for 2–2.5 h (OD). 600 When the pH value is 0.6–0.8, add IPTG inducer to a final concentration of 0.1 mM and incubate for 16 hours in a temperature-controlled shaker at 24°C and 180 rpm. Pour the cultured bacterial solution into 50 mL centrifuge tubes and centrifuge at 10,000 rpm and 4°C for 10 min in a low-temperature high-speed centrifuge. Discard the supernatant, collect the bacterial cells, weigh them, and use them for subsequent applications.

[0074] The concentrations and chirality of methyl acetoacetate and (R)-3-hydroxybutyrate methyl ester were determined by HPLC, and the conversion rates were calculated. The dominant strains were screened based on the conversion rates and chirality.

[0075] The dominant strains were sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing and stored at -80℃. The final dominant mutants were identified as ADH1-L20P, ADH1-E29K, ADH1-T55F, ADH1-T55W, ADH1-T55S, ADH1-L96F, ADH1-L96W, ADH1-L96A, ADH1-V110A, ADH1-V110F, ADH1-V110W, and ADH1-T149P.

[0076] 3. Iterative mutation

[0077] Using vectors pET28b(+)-ADH1-L20P, ADH1-E29K, ADH1-T55F, ADH1-T55W, ADH1-T55S, ADH1-L96F, ADH1-L96W, ADH1-L96A, ADH1-V110A, ADH1-V110F, ADH1-V110W, and ADH1-T149P as templates, primers were designed (primer design is the same as in Table 2), and polymerase chain reaction (PCR) was performed. Based on the HPLC screening procedure, dominant mutations were further screened from single-point mutants.

[0078] PCR reaction system (25µL): 1 µL upstream primer (10µM), 1 µL downstream primer (10µM), 12.5µL 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.

[0079] 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, 58℃ annealing for 15 s, 72℃ extension for 4 min), 72℃ final extension for 10 min, and incubation at 16℃.

[0080] Iterative mutants (multi-site mutants) were obtained:

[0081] ADH1-L20P / E29K, ADH1-L20P / T55F, ADH1-L20P / L96A, ADH1-L20P / V110F,

[0082] ADH1-L20P / T149P, ADH1-E29K / T55F, ADH1-E29K / L96A, ADH1-E269K / V110F, ADH1-E29K / T 149P, ADH1-T55F / L96A, ADH1-T55F / V110A, ADH1-T55F / T149P, ADH1-L96A / V110F, ADH1-L9 6A / T149P, ADH1-V110F / T149P, ADH1-L20P / E29K / T55F, ADH1-L20P / E29K / L96A, ADH1-L20 P / E29K / V110F, ADH1-L20P / E29K / T149P, ADH1-L20P / T55F / L96A, ADH1-L20P / T55F / V110F, ADH1-L20P / T55F / T149P, ADH1-L20P / L96A / V110F, ADH1-L20P / L96A / T149P, ADH1-L20P / V 110F / T149P, ADH1-E29K / T55F / L96A, ADH1-E29K / T55F / V110F, ADH1-E29K / T55F / T149P, AD H1-E29K / L96A / V110F, ADH1-E29K / L96A / T149P, ADH1-E29K / V110F / T149P, ADH1-T55F / L9 96F / V110F, ADH1-T55F / L96A / T149P, ADH1-T55F / V110F / T149P, ADH1-L96A / V110F / T149P.

[0083] 4. Catalytic activity detection

[0084] Single mutant strains, iterative mutant strains, and control strains (WT, ADH1 as control) were used as catalysts, with methyl acetoacetate as the substrate, to compare the catalytic activity of each mutant. The reaction system consisted of 1 mL of 1 mL of 1 mL of 1 mL of 50 g / L wet cell catalyst, 50 g / L substrate, and a pH 7.0, 0.1 M PB buffer solution. The reaction medium included 1 M ammonium formate, 100 g / L wet cell formate dehydrogenase, and 2.0 mM NADPH. The reaction was carried out at 30℃ and 1000 rpm for 2 h under vortexing conditions. 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. The concentrations and chirality of methyl acetoacetate and (R)-3-butyrate methyl ester in the ethyl acetate sample were determined by HPLC, and the conversion rates were calculated.

[0085] The screening results are shown in Table 3:

[0086] Table 3

[0087]

[0088] Finally, further screening yielded the dominant strain T55F / L96A / V110F, which is an ADH1 mutant.

[0089] 5. Room temperature stability test

[0090] Wet cells of WT and T55F / L96A / V110F were stored at 25°C in the dark. Samples were taken at 12 h and 24 h, and enzyme activity was measured under standard reaction conditions. The percentage of residual activity at different time points was calculated, with the initial activity defined as 100%.

[0091] Table 4

[0092]

[0093] The stability of the T55F / L96A / V110F mutant is slightly lower than that of the WT mutant.

[0094] Example 4: Construction of stable mutants

[0095] Given that the superior mutant ADH1-T55F / L96A / V110F (hereinafter referred to as M3) has decreased storage stability at room temperature compared to the wild type, in order to obtain an enzyme mutant with both high catalytic activity and excellent stability, this invention uses M3 as the starting strain and performs iterative saturation mutations targeting sites that may affect structural stability.

[0096] 1. Selection of stable mutation sites and primer design

[0097] 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 mutation: K63L, D82F, G129P, N176E, Q191R, and V214P.

[0098] Using plasmid pET28b(+)-ADH1-M3 (T55F / L96A / V110F) as a template, specific primers were designed to perform site-directed mutagenesis at the aforementioned sites. The primer sequences are shown in the table below:

[0099] Table 5. Primer list for stable iterative mutations

[0100] .

[0101] 2. Construction and screening of mutant libraries

[0102] Following the PCR, DpnI digestion, transformation, and culture methods described in Example 3, single-point mutation libraries targeting the aforementioned six sites were constructed. Mutations at each site were performed using the M3 plasmid as a template.

[0103] 3. Initial stability screening and activity verification

[0104] Single colonies were randomly selected from each mutant library plate, and together with M3 and wild-type WT, they were subjected to small-scale expression and cell collection according to the method described in Example 3.

[0105] 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 min, followed immediately by cooling in an ice bath for 5 min. The bacterial suspension that was not heat-treated under the same conditions was used as the activity baseline (100%).

[0106] 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 min). 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%.

[0107] 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, G129P and Q191R, which showed the most significant improvement in thermal stability while maintaining the high level of catalytic activity of M3.

[0108] 4. Combination and acquisition of superior stable mutants

[0109] Using the positive single-point mutant plasmid pET28b(+)-ADH1-M3-G129P obtained through screening as a template, a two-point iterative mutant ADH1-T55F / L96A / V110F / G129P / Q191R was constructed through site-directed mutagenesis and named M4.

[0110] M4 was expressed, purified (specific methods can be found in Example 2), and subjected to comprehensive catalytic and stability tests. The results showed that while inheriting the ultra-high catalytic performance of M3 (conversion rate >99%, ee value >99.5%), the thermal stability, solvent tolerance, and storage stability of the M4 mutant were comprehensively and significantly improved, successfully solving the problem of decreased stability (Table 6).

[0111] Table 6 Initial screening data of stable iterative mutants

[0112] .

[0113] 5. Stability and Tolerance Verification

[0114] 1. Thermal stability test

[0115] 1.1 Melting Temperature (Tm) Determination: Differential scanning fluorescence (DSF) was used. Purified wild-type (WT) and mutants T55F / L96A / V110F / G129P / Q191R 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 (e.g., 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.

[0116] 1.2 Determination of the thermal inactivation half-life: Enzyme solutions of WT and T55F / L96A / V110F / G129P / Q191R were incubated in a 55℃ constant temperature water bath. Samples were taken at 0, 5, 15, 30, 60, 120, and 240 min, 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 (t1 / 2).

[0117] 1.3 Determination of residual activity after heat shock: The enzyme solution was incubated in a water bath at 50℃ and 60℃ for specified times (e.g., 30 min and 60 min), and immediately cooled in an ice bath. The enzyme activity was measured under standard conditions and compared with the activity of the same enzyme solution without heat treatment to calculate the percentage of residual activity.

[0118] 2. Organic solvent tolerance test

[0119] Enzyme solutions of WT and mutants T55F / L96A / V110F / G129P / Q191R were mixed with buffer solutions containing different volume fractions of organic solvent (20% DMSO and 30% methanol). The solutions were incubated with gentle shaking at 25°C for 2 h. After incubation, a suitable amount of the mixture was taken, and enzyme activity was measured under standard reaction conditions. The percentage of residual activity 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%.

[0120] 3. pH stability test

[0121] Prepare a series of buffer solutions with different pH values ​​(e.g., Britton-Robinson buffers from pH 5.0 to 10.0). Mix WT and T55F / L96A / V110F / G129P / Q191R enzyme solutions with equal volumes of buffer solutions at different pH values ​​to expose the enzymes to the target pH environment. Incubate at 4°C for 24 h. 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, using the enzyme activity incubated in the optimal pH buffer as 100%, to assess its pH stability range and tolerance limit.

[0122] Storage stability test

[0123] The WT and T55F / L96A / V110F / G129P / Q191R 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%.

[0124] The results of the comparison between stability and tolerance are shown in Table 7:

[0125] Table 7

[0126] .

[0127] The above data shows that the T55F / L96A / V110F / G129P / Q191R 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.

[0128] Example 5: Buffer solution and pH optimization

[0129] Buffer optimization:

[0130] S1. Add 0.5 g each of ADH1-T55F / L96A / V110F / G129P / Q191R wet bacterial cells and formate dehydrogenase wet bacterial cells, 7 mL of buffer (0.1 M), 1 mL of 10 M ammonium formate solution, and 10 mM NADP to the reaction flask. + 1 mL of solution and 1 mL of 500 g / L methyl acetoacetate solution were used. The buffer solutions were prepared as pH 7.0 phosphate buffer, Tris-HCl buffer, HEPES buffer, and TEA buffer, and the reaction was carried out at 30 °C and 1000 rpm.

[0131] S2. After 2 hours 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:

[0132] Table 8

[0133]

[0134] The results showed that the highest conversion rate was achieved when the buffer solution was PB buffer at pH 7.0.

[0135] pH optimization:

[0136] S1. Add 0.5 g each of ADH1-T55F / L96A / V110F / G129P / Q191R wet bacterial cells and formate dehydrogenase wet bacterial cells, 7 mL of buffer (0.1 M), 1 mL of 10 M ammonium formate solution, and 10 mM NADP to the reaction flask. + 1 mL of solution and 1 mL of 500 g / L methyl acetoacetate solution were used. The buffer solution was set as PB buffer with pH 6.0, 6.5, 7.0, 7.5, and 8.0. The reaction was carried out at 30℃ and 1000 rpm.

[0137] S2. After 2 hours 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:

[0138] Table 9

[0139]

[0140] The results showed that the highest conversion rate (>99%) was achieved with PB buffer at pH 7.0, making it the optimal choice.

[0141] Example 6: Temperature Optimization

[0142] S1. Add 0.5 g each of ADH1-T55F / L96A / V110F / G129P / Q191R wet bacterial cells and formate dehydrogenase wet bacterial cells, 7 mL of PB buffer (0.1 M, pH 7.0), 1 mL of 10 M ammonium formate solution, and 10 mM NADP to the reaction flask. + 1 mL of solution and 1 mL of 500 g / L methyl acetoacetate solution were added. The reaction was carried out at 1000 rpm, with the temperature set at 20℃, 25℃, 30℃, 35℃, and 40℃.

[0143] S2. After 2 hours of reaction, 1 mL of the reaction solution was taken and extracted with an equal volume of ethyl acetate. The supernatant organic phase was collected by centrifugation and analyzed by HPLC. The results are shown in Table 10.

[0144] Table 10

[0145]

[0146] The results showed that the conversion rate was highest at 30℃ (>99%), making it the optimal temperature.

[0147] Example 7:

[0148] Add 0.5 g of wet ADH1-T55F / L96A / V110F / G129P / Q191R bacterial cells and 0.5 g of wet formate dehydrogenase bacterial cells, 6 mL of PB buffer (0.1 M, pH 7.0), 1 mL of 10 M ammonium formate solution, and 10 mM NADP to the reaction flask. + 1 mL of solution, 2 mL of 500 g / L methyl acetoacetate solution, and a total system volume of 10 mL were added and reacted at 30 °C and 1000 rpm.

[0149] After 6 h 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.

[0150] Single-factor substitutions are performed according to the conditions in Table 11 below:

[0151] Table 11

[0152] .

[0153] Example 8: 1L scale-up reaction

[0154] Take a 2 L three-necked flask and add 50 g each of ADH1-T55F / L96A / V110F / G129P / Q191R wet bacterial cells and formate dehydrogenase wet bacterial cells. Add 500 mL of PB buffer (0.1 M, pH 6.0), 100 mL of 10 M ammonium formate solution, and 5 mM NADP. + 100 mL of a 1000 g / L methyl acetoacetate (containing methyl tert-butyl ether) solution was added dropwise using a constant pressure funnel, with the substrate added completely over 2 hours. The mixture was placed in a 30°C water bath and stirred at 300 rpm.

[0155] At 3 h, 6 h, and 80 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.

[0156] After reacting for 80 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.

[0157] The crude product was dissolved in ethyl acetate using a vacuum pump followed by an oil pump. After the rotary evaporation, the crude product was sonicated until completely dissolved. 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 using an oil pump to obtain the final product.

[0158] The results of the above reaction are shown in Table 12. ADH1-T55F / L96A / V110F / G129P / Q191R can catalyze the conversion of 100 g / L methyl acetoacetate to (R)-3-hydroxy-butyrate methyl ester, with a conversion rate >99% and an ee value greater than 99%.

[0159] Table 12 Results of the scaled-up experiment:

[0160] .

[0161] 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 dextrin intermediate, characterized in that, Using methyl acetoacetate as a substrate, buffer, substrate, alcohol dehydrogenase, formate dehydrogenase, ammonium formate and coenzyme were added to the reaction vessel and reacted at 20-40°C for 2-24 hours. After extraction, separation and rotary evaporation, the chiral intermediate (R)-3-hydroxybutyrate methyl ester of polyazolamine was obtained. The alcohol dehydrogenase is a mutant of ADH1 derived from Rollstonella, and the amino acid sequence of ADH1 is shown in SEQ ID No. 1; The ADH1 mutant is selected from one of the following: ADH1-T55F / L96A / V110F / D82F, ADH1-T55F / L96A / V110F / G129P, ADH1-T55F / L96A / V110F / N176E, ADH1-T55F / L96A / V110F / Q191R, ADH1-T55F / L96A / V110F / V214P, and ADH1-T55F / L96A / V110F / G129P / Q191R. ADH1-T55F / L96A / V110F / D82F is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and D at position 82 is changed to F. ADH1-T55F / L96A / V110F / G129P is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and G at position 129 is changed to P. ADH1-T55F / L96A / V110F / N176E is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and N at position 176 is changed to E. ADH1-T55F / L96A / V110F / Q191R is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and Q at position 191 is changed to R. ADH1-T55F / L96A / V110F / V214P is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and V at position 214 is changed to P. ADH1-T55F / L96A / V110F / G129P / Q191R is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, G at position 129 is changed to P, and Q at position 191 is changed to R.

2. The enzyme-catalyzed synthesis method according to claim 1, characterized in that, Both alcohol dehydrogenase and formate dehydrogenase were added in the form of wet cells of recombinant genetically engineered bacteria.

3. The enzyme-catalyzed synthesis method according to claim 2, characterized in that, The proportions of each raw material in the 1 L reaction system are as follows: 5–100 g of recombinant Escherichia coli wet cells expressing alcohol dehydrogenase, 10–100 g of recombinant Escherichia coli wet cells expressing formate dehydrogenase, 20–100 g of substrate, 0.1–1.0 M of ammonium formate, 0.1–2.0 mM of coenzyme, and the balance of buffer.

4. The enzyme-catalyzed synthesis method according to claim 3, characterized in that, The proportions of each raw material in the 1 L reaction system are as follows: 5–100 g of recombinant Escherichia coli wet cells expressing alcohol dehydrogenase, 50–75 g of recombinant Escherichia coli wet cells expressing formate dehydrogenase, 20–100 g of substrate, 0.1–1.0 M of ammonium formate, 0.1–2.0 mM of coenzyme, and the balance of buffer.

5. The enzyme-catalyzed synthesis method according to any one of claims 1-4, characterized in that, The buffer solution is selected from one of TEA buffer, PB buffer, Tris-HCl buffer, and HEPES buffer.

6. The enzyme-catalyzed synthesis method according to claim 5, characterized in that, The concentration of the TEA buffer is 0.05–0.20 M, and the pH value is 6.0–8.0; the concentration of the PB buffer is 0.05–0.10 M, and the pH value is 6.0–7.0; the concentration of the Tris-HCl buffer is 0.05–0.10 M, and the pH value is 7.0–8.0; the concentration of the HEPES buffer is 0.01–0.10 M, and the pH value is 6.0–8.

0.

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

8. An alcohol dehydrogenase, characterized in that, The alcohol dehydrogenase is a mutant of ADH1 derived from Rollstonella, and the amino acid sequence of ADH1 is shown in SEQ ID No. 1; The ADH1 mutant is selected from one of the following: ADH1-T55F / L96A / V110F / D82F, ADH1-T55F / L96A / V110F / G129P, ADH1-T55F / L96A / V110F / N176E, ADH1-T55F / L96A / V110F / Q191R, ADH1-T55F / L96A / V110F / V214P, and ADH1-T55F / L96A / V110F / G129P / Q191R. ADH1-T55F / L96A / V110F / D82F is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and D at position 82 is changed to F. ADH1-T55F / L96A / V110F / G129P is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and G at position 129 is changed to P. ADH1-T55F / L96A / V110F / N176E is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and N at position 176 is changed to E. ADH1-T55F / L96A / V110F / Q191R is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and Q at position 191 is changed to R. ADH1-T55F / L96A / V110F / V214P is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, and V at position 214 is changed to P. ADH1-T55F / L96A / V110F / G129P / Q191R is an ADH1 amino acid sequence where T at position 55 is changed to F, L at position 96 is changed to A, V at position 110 is changed to F, G at position 129 is changed to P, and Q at position 191 is changed to R.

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