Cyclohexene carboxylate hydrolase mutants, methods of making and using the same

Abstract

This invention relates to a cyclohexenecarbamate hydrolase mutant, its preparation method, and its applications. The cyclohexenecarbamate hydrolase mutant provided by this invention, when used as a catalyst in the asymmetric resolution of chiral cyclohexenecarbamate to prepare chiral cyclohexenecarboxylic acid, exhibits suitable conversion rates, high optical purity (chiral purity exceeding 99%), mild reaction conditions, environmental friendliness, simple operation, high yield, low cost, and ease of industrial scale-up. Therefore, the cyclohexenecarbamate hydrolase mutant and its gene of this invention have promising prospects for industrial application development.

Description

Technical Field

[0001] This invention relates to a cyclohexene carbamate hydrolase mutant, its preparation method and application, belonging to the field of biomedical genetic engineering technology. Background Technology

[0002] (S)-3-cyclohexene-1-carboxylic acid is a key chiral intermediate for the novel oral anticoagulant edoxaban. Currently, the Diels-Alder reaction is the main method for synthesizing this intermediate. However, this method has harsh reaction conditions, cumbersome steps, and low yield, resulting in high production costs.

[0003] In recent years, environmentally friendly enzymatic hydrolysis and resolution methods have attracted considerable attention from both academia and industry. Compared with chemical methods, enzymatic methods offer milder reaction conditions, better selectivity, and higher yields. Currently, the most studied enzymatic resolution methods fall into two main categories. One approach utilizes animal-derived esterases (primarily porcine liver esterase) for effective resolution, directly yielding (S)-3-cyclohexene-1-carboxylic acid (Tetrahedron Asymmetry, 2004, 15, 2057-2060). However, this enzyme is primarily extracted from animals, and in vitro expression methods can only achieve high efficiency in Origami strains, which cannot be cultured at high densities. Furthermore, porcine liver esterase expression requires molecular chaperones, further complicating in vitro expression. Therefore, animal-derived porcine liver esterases cannot be widely applied. The second type of enzyme is a microbial hydrolase. These enzymes exhibit higher activity than animal-derived esterases and can be efficiently expressed in BL21(DE3) series *E. coli* chassis strains, and are easily scaled up for high-density cultivation. Using microbial hydrolases, (R)-3-cyclohexene-1-carboxylate in cyclohexene carboxylate can be hydrolyzed, retaining (S)-3-cyclohexene-1-carboxylate. Further hydrolysis with NaOH yields (S)-3-cyclohexene-1-carboxylic acid. Therefore, the microbial hydrolase method for resolution has greater potential for industrial applications.

[0004] Currently, for the enzymatic resolution of (S)-3-cyclohexene-1-carboxylate, the enzymes derived from microorganisms have a significant advantage in research. Acinetobacter sp. WCHAc010052 (Chinese Patent CN112813131B) and Acinetobacter sp. The two enzymes in JNU9335 (Chinese patent CN111778229B). Compared to the latter, it is derived from... Acinetobacter sp.The former, WCHAc010052, exhibits higher activity. Furthermore, according to patent CN112813131B, the inventors mutated the enzyme to obtain a highly active mutant without significantly affecting its selectivity. The applicant's research revealed that both enzymes require a hydrolysis conversion rate of over 62% to obtain a product meeting the chiral purity requirements (>97%). Therefore, although the enzymes reported in the literature have high activity, their poor selectivity affects the final product yield and thus the final production cost. Therefore, the continued development of new highly selective cyclohexenecarbamate hydrolase mutants remains of practical significance for industrial production. Summary of the Invention

[0005] The technical problem to be solved by the present invention is the poor enantioselectivity of the enzymatic resolution preparation of (S)-3-cyclohexene-1-carboxylate. The invention provides a hydrolytic enzyme with higher selectivity derived from natural enzyme mutations and applies it to the resolution of 3-cyclohexene-1-carboxylate.

[0006] This invention is derived from Acinetobacter sp. Using the hydrolase VcHLA002 of WCHAc010052 as the initial enzyme, and based on the Alphafold2 protein structure modeling and structural analysis method, target sites were selected for site-directed saturation mutagenesis, ultimately obtaining a highly selective enzyme mutant.

[0007] Includes the following steps:

[0008] (1) Evaluate the dissociation effect of naturally occurring enzymes.

[0009] Specifically, the synthesis originates from microbial strains. Acinetobacter sp. The hydrolase VcHLA002 of WCHAc010052 was synthesized, and its activity and selectivity were tested: the synthesized VcHLA002 glycerol bacteria were cultured at 37℃, expressed at 25℃, and then the enzyme activity was tested at 20℃. The results showed that the enzyme had high activity but poor selectivity. Therefore, mutation is needed to obtain an enzyme with better selectivity.

[0010] (2) The amino acid sequence of the enzyme was modeled and analyzed using Alphafold2;

[0011] (3) The model was docked with the target raw materials to obtain the binding mode of the enzyme and the raw materials, and primers were synthesized at selected sites (primer synthesis was completed by the biotechnology company). The selected target sites were V133, D253 and V257;

[0012] (4) After obtaining the primers, optimize the PCR conditions and use the following conditions to amplify the gene and obtain the PCR product:

[0013] Pre-denaturation: 98℃ for 3 min

[0014] Denaturation: 98℃ for 10 seconds

[0015] Annealing: 57℃ for 15 seconds

[0016] Extended cooking time: 72℃ for 3 minutes

[0017] Extended time: 72℃ for 5 minutes

[0018] Number of cycles: 30

[0019] (5) The PCR product was transferred into competent cells, plated on a plate containing kanamycin resistance, and incubated overnight at 37°C to obtain single colonies. The single colonies were then picked into 96-well plates of V133, D253 and V257 respectively, incubated overnight at 37°C to preserve the bacteria, induced at 25°C for 18 hours, and centrifuged to obtain the mutant enzyme library;

[0020] (6) After diluting the mutant library, add phosphate buffer, then add 5 μL of raw material, and react at 15–30 °C for 30–90 min. The pH of the buffer is 5.0–9.0. After the reaction, the dominant sites are detected, and after reculturing, the samples are sequenced by Qingke Biotechnology. The sequencing results unexpectedly showed that the D253V and D253M mutants had better selectivity for the substrate raw material compared with the wild-type enzyme;

[0021] (7) The activity and selectivity of the better mutant D253V and the wild-type enzyme were re-cultured and expressed simultaneously. 150 mg of enzyme and 3.0 g of product were weighed and reacted at 15-30 °C. During the reaction, the pH was adjusted by 2 M NaOH (pH 5.0-9.0). The results showed that the selectivity of mutant D253V was better than that of wild-type enzyme.

[0022] In one aspect, the present invention provides a cyclohexene carbamate hydrolase mutant, the amino acid sequence of which is shown in Serial Number (ID): 4.

[0023] The cyclohexene carbamate hydrolase mutant described in this invention is a protein with a new amino acid sequence formed by replacing the aspartic acid residue at position 253 of the amino acid sequence shown in sequence number (ID): 2 with a valine residue.

[0024] The present invention also provides a gene encoding a cyclohexenecarbamate hydrolase mutant, the nucleotide sequence of which is shown in Serial Number (ID): 3.

[0025] The present invention also provides a recombinant expression vector containing the coding gene as shown in the above nucleotide sequence, such as sequence number (ID): 3.

[0026] The present invention also provides a method for preparing a cyclohexenecarbamate hydrolase mutant, which is obtained by site-directed saturation mutagenesis. The upstream primer of the site-directed saturation mutagenesis is: CTGACCGGCGCTNNKGTTGATTACGTT, and the downstream primer is: AACGTAATCAACMNNAGCGCCGGTCAG.

[0027] In another aspect, the present invention also provides the application of a cyclohexenecarbamate hydrolase mutant in the catalytic preparation of optically active (S)-3-cyclohexene-1-carboxylic acid from 3-cyclohexene-1-carboxylic acid.

[0028] The structural formula of the 3-cyclohexene-1-carboxylate is shown below:

[0029] ,

[0030] R is selected from methyl, ethyl, isopropyl, or butyl.

[0031] The application of this invention involves using a cyclohexenecarbamate hydrolase mutant to hydrolyze (R)-3-cyclohexene-1-carbamate in a buffer solution to obtain (S)-3-cyclohexene-1-carbamate, followed by hydrolysis under alkaline conditions to obtain (S)-3-cyclohexene-1-carboxylic acid. The buffer solution is selected from citrate buffer and phosphate buffer; the pH of the buffer solution is 5–9. The reaction temperature is 15–30°C.

[0032] In this embodiment of the invention, during the hydrolysis of (R)-3-cyclohexene-1-carboxylate in the racemic mixture of 3-cyclohexene-1-carboxylate by the cyclohexenecarboxylate hydrolase mutant in a buffer solution, the pH needs to be adjusted to 5-9 with an alkali. The alkali used to adjust the pH is 0.5-5M NaOH, Na2CO3 or NaHCO3.

[0033] The beneficial effects of this invention are:

[0034] This invention provides a more selective hydrolase derived from natural enzyme mutations. The hydrolase mutant of this invention is applied to the resolution of 3-cyclohexene-1-carboxylate, with suitable conversion rate, high optical purity (chiral purity of over 99%), mild reaction conditions, environmental friendliness, simple operation, high yield, low cost, and easy industrial scale-up. Attached Figure Description

[0035] Figure 1 VcHLA002 protein structure model.

[0036] Figure 2 VcHLA002 active center and docking results.

[0037] Figure 3Electrophoresis images of nucleic acid from PCR products of V133, D253, and V257.

[0038] Figure 4 VcHLA002 mutation library. Detailed Implementation

[0039] Example 1: Synthesis derived from microbial strains Acinetobacter sp. WCHAc010052 hydrolase VcHLA002

[0040] The amino acid sequence at sequence number (ID): 2 was submitted to Sangon Biotech (Shanghai) Co., Ltd. for codon optimization in *E. coli*. After confirming the optimization results, the gene sequence was synthesized and spliced ​​into the pET28a vector, which was then transformed into competent *E. coli* cells. After activation culture, the glycerol bacteria were preserved. In the laboratory, 10 μL of glycerol bacteria were transferred into 4 mL of LB medium (containing kanamycin resistance) and cultured overnight at 37°C. Then, the culture was transferred into 200 mL of LB medium (containing kanamycin resistance) and cultured at 37°C until OD600nm = 0.6–0.8. Then, 200 μL of 50 mM IPTG (isopropyl-β-D-thiogalactoside) was added, and the culture was induced overnight at 25°C. After centrifugation at 8000 rpm, approximately 1.0 g of bacterial cells (after draining off excess water) was collected, which is the prepared crude enzyme.

[0041] 200 mg of bacterial culture was weighed and suspended in 80 mL of 200 mM dipotassium hydrogen phosphate / potassium dihydrogen phosphate buffer (pH 8.0). After stirring in a 20°C water bath for 5 min, 4.0 g of racemic methyl 3-cyclohexene-1-carboxylate was added. During the reaction, the pH was maintained at 7.5–8.0 using 2 M NaOH aqueous solution. 200 μL samples were taken at 70 min, 100 min, 130 min, and 180 min of reaction, quenched in 1 mL of methanol, filtered, and the conversion rate was determined by LC / MS. Additionally, at each time point, 1 mL of the reaction solution was extracted with 1 mL of dichloromethane, dried under nitrogen, and sent for analysis of the chiral purity of (S)-3-cyclohexene-1-carboxylate. The results are shown in Table 1.

[0042] Table 1

[0043]

[0044] As shown in Table 1, the enzyme synthesized in this experiment has high activity, but exhibits significant over-hydrolysis. Ideally, under a conversion rate of 50%, the chiral purity of (S)-3-cyclohexene-1-carboxylate methyl ester should be close to 100%. Therefore, this invention modifies the enzyme.

[0045] Example 2: Modeling, Active Site Analysis, and Molecular Docking of VcHLA002 Protein Based on Alphafold2

[0046] Amino acid sequence comparison of the VcHLA002 protein revealed no clear protein structure. Alphafold2, another successful machine learning model following AlphaGo, boasts a structure prediction success rate exceeding 98% for human proteins. Therefore, this invention submits the amino acid sequence to the Alphafold2 platform for structural modeling, yielding the results shown in the attached figure. Figure 1 The protein structure is shown. For hydrolases, most contain a GXSXG catalytic structure (where S is the catalytic amino acid serine, G is glycine, and X is a variable amino acid) and an XGGG structure that forms an oxon center. In VcHLA002, GXSXG is located in the 199-203 region, and XGGG is located in the 127-129 region.

[0047] Then, the present invention docks the protein structure with methyl 3-cyclohexene-1-carboxylate (as shown in the attached diagram). Figure 2 (As shown). Analysis revealed that the structure of the methyl ester in the raw material is located near the catalytic group of the active site, while the cyclohexene structure interacts with sites V133, D253, V257, N329, and L249. Changing the size and polarity of these sites may lead to changes in the binding mode of the substrate in the binding pocket, thereby causing changes in enzyme activity and selectivity. Therefore, in the first round of evolution, this invention selects some amino acids that are tightly bound to the substrate for site-directed saturation mutagenesis, specifically amino acids V133, D253, and V257.

[0048] Example 3: Design of VcHLA002 site-directed saturation primers and construction of a site-directed mutagenesis library

[0049] After determining the target point, the present invention designs the following primers:

[0050] V257-F:GCTGATGTTGATTACNNKACCGATTACTACGCT

[0051] V257-R:AGCGTAGTAATCGGTMNNGTAATCAACATCAGC

[0052] D253-F: CTGACCGGCGCTNNNKGTTGATTACGTT

[0053] D253-R:AACGTAATCAACMNNAGCGCCGGTCAG

[0054] V133-F:GGCTTCGTTNNKGGCGGTATGGATAGC

[0055] V133-R:GCTATCCATACCGCCMNNAACGAAGCC,

[0056] Gene amplification was performed using the plasmid synthesized in Example 1 as a template. The PCR conditions were as follows:

[0057] Pre-denaturation: 98℃ for 3 min

[0058] Denaturation: 98℃ for 10 seconds

[0059] Annealing: 57℃ for 15 seconds

[0060] Extended cooking time: 72℃ for 3 minutes

[0061] Extended time: 72℃ for 5 minutes

[0062] Number of cycles: 30

[0063] After PCR, the products were detected by nucleic acid electrophoresis (see attached image). Figure 3 (As shown in the attached image). Nucleic acid electrophoresis results showed that the PCR product molecular size was between 4000-6000, consistent with the target product, indicating successful PCR. After digestion with nucleases, the product was chemically transformed into *E. coli* BL21(DE3) competent cells. The cells were plated on kanamycin-resistant plates and incubated overnight at 37°C to obtain single colonies (as shown in the attached image). Figure 4 (As shown).

[0064] Single clones were picked up using sterile pipette tips and transferred to 96-well plates (one 96-well plate per plate, for a total of 3 plates). 300 μL of LB medium containing kanamycin resistance was added, and the plates were incubated at 37°C (250 rpm) for 18 h. 100 μL of the bacterial culture was then transferred to a 350 μL preservation plate, along with 100 μL of 30% sterile glycerol, and stored at -80°C. The remaining sample was added to 400 μL of LB medium containing kanamycin resistance and incubated for 3 h. Then, 100 μL of LB medium containing kanamycin resistance and IPTG was added, and the plates were induced at 25°C (250 rpm) for 18 h. The plates were then centrifuged at 4000 rpm for 20 min, drained, and the site-directed saturation mutant library was obtained and frozen for screening.

[0065] Example 4: Screening of site-directed saturation mutation libraries

[0066] Take the 96-well plates (V133, D253, V257) from Example 3, add 200 μL of 200 mM pH 7.0 dipotassium hydrogen phosphate / potassium dihydrogen phosphate buffer, resuspend, and then take 20 μL of the resuspended bacterial cells into a new 96-well plate, add 180 μL of 200 mM pH 8.0 dipotassium hydrogen phosphate / potassium dihydrogen phosphate buffer. Add 5 μL of racemic methyl 3-cyclohexene-1-carboxylate, shake, and react at 20°C and 250 rpm for 50–55 min. Immediately afterwards, add 800 μL of methanol to quench the reaction. After centrifugation, send for analysis and detection. Furthermore, the applicant selected the enzyme at the corresponding position used in samples with a conversion rate greater than 50%, recultured, and sequenced. The screening results and sequencing results of mutant D253 are shown in Table 2.

[0067] Table 2

[0068]

[0069] Note: Due to the micro-expression in the well plate, there will be differences in the cell volume or protein expression level of the same genotype, resulting in differences in enzyme conversion rates for the same genotype on the same plate. However, for enzymes of the same genotype, the conversion rate corresponds to the ee value.

[0070] The results in Table 2 show a mismatch between the high conversion rate and the ee value of the D253 plate samples. For the unmutated wild-type enzymes (samples D11 and D6), a higher conversion rate should correspond to a higher ee value. However, for the mutant D253C (sample B12), the conversion rate reached 73.82%, but the ee(s)% was only 69.36%, indicating that this mutant reduces selectivity and is considered an unfavorable mutation.

[0071] The conversion rates of wild-type enzyme (sample D11) and mutant D253V (samples A12, A11, A2) were similar, but the ee value was significantly lower than that of the latter three samples, indicating that the selective separation effect of mutant D253V was significantly improved.

[0072] The conversion rates of the wild-type enzyme (sample D6) and the mutant D253V (sample A4) were similar, but the ee value of the mutant D253V was much lower than that of the wild-type enzyme, further indicating that the mutant D253V was superior to the wild-type enzyme.

[0073] In addition, other mutants at this site, such as D253M, D253G, D253T, and D253I, also have significant advantages. However, since many advantageous mutations are D253V, this mutant will be the preferred choice for subsequent experimental verification.

[0074] Example 5: Repetitive expression and activity verification of mutant VcHLA002_D253V and wild-type enzyme VcHLA002_WT

[0075] A2 and wild-type glycerol bacteria from D253 plates stored at -80℃ were transferred to 4 ml of LB medium (containing kanamycin resistance) and incubated overnight at 37℃. Then, they were transferred to 200 ml of LB medium (containing kanamycin resistance) and incubated at 37℃ until OD600nm = 0.6-0.8. Then, 200 μL of 50 mM IPTG (isopropyl-β-D-thiogalactoside) was added and incubated overnight at 25℃. After centrifugation at 8000 rpm, about 1.0 g of bacterial cells were collected (after draining off the water), which is the crude enzyme.

[0076] 150 mg of wet mutant VcHLA002_D253V cells and wild-type enzyme VcHLA002 cells were weighed and resuspended in 60 mL of 200 mM dipotassium hydrogen phosphate / potassium dihydrogen phosphate buffer (pH 8.0). The mixture was stirred in a 20°C water bath for 5 min, and then 3.0 g of racemic methyl 3-cyclohexene-1-carboxylate was added. During the reaction, the pH was maintained at 7.5–8.0 using 2 M NaOH aqueous solution. 200 μL samples were taken every 30 min, quenched in 2 mL of methanol, filtered, and the conversion rate was detected by LC / MS (sampling was stopped when the conversion rate approached 60%). Additionally, at each time point, 1 mL of the reaction solution was extracted with 1 mL of dichloromethane, dried under nitrogen, and sent for analysis of the chiral purity of (S)-3-cyclohexene-1-carboxylate. The results are shown in Table 3.

[0077] Table 3

[0078]

[0079] Experimental results show that, with minimal enzyme usage, the mutant VcHLA002_D253V product can achieve the required chiral purity within a short time. Furthermore, compared to the wild-type VcHLA002_WT, the mutant achieves 98.6% chiral purity at approximately 55.5% concentration, while the wild-type enzyme only achieves 96.2% chiral purity at 63.6%. Therefore, the mutant VcHLA002_D253V exhibits superior selectivity.

[0080] Example 6: Asymmetric resolution reaction of different esters catalyzed by mutant VcHLA002_D253V

[0081] Different substrates were catalytically resolved using the mutant VcHLA002_D253V, and the experimental results are shown in Table 4.

[0082] Table 4

[0083]

[0084] Experimental results show that the mutant VcHLA002_D253V exhibits high efficiency and activity in resolving methyl 3-cyclohexene-1-carboxylate. Structurally, the modified enzyme has a smaller activity space, which is beneficial for controlling the chirality of small molecule substrates.

[0085] Example 7: Selective hydrolysis of mutant VcHLA002_D253V

[0086] Five g of the mutant VcHLA002_D253V wet bacterial body was weighed and suspended in 500 mL of 200 mM dipotassium hydrogen phosphate / potassium dihydrogen phosphate buffer (pH 8.0). The mixture was stirred in a 20°C water bath for 5 min, and then 100 g of racemic methyl 3-cyclohexene-1-carboxylate was added. During the reaction, the pH was maintained at 7.5–8.0 using 2 M NaOH aqueous solution. 200 μL of sample was taken every 30 min, quenched in 2 mL of methanol, filtered, and the conversion rate was determined by HPLC. The chiral purity of the sample was then tested. The results showed that when the conversion rate was 53.81%, the chiral purity of methyl (S)-3-cyclohexene-1-carboxylate was 97.59%. After the reaction, the sample was extracted twice with 100 mL of toluene, and the toluene phases were combined.

[0087] To the toluene solution of (S)-3-cyclohexene-1-carboxylic acid methyl ester, 400 mL of toluene, 200 mL of water, and 15.4 g of NaOH were added. The mixture was heated to 75 °C and reacted for 4 h. No residual raw material was detected, indicating the reaction was complete. Toluene was removed by separation. The pH of the aqueous phase was adjusted to 2–3 with hydrochloric acid, followed by extraction with dichloromethane. The solvent was removed to obtain a total of 41.5 g of (S)-3-cyclohexene-1-carboxylic acid. The overall yield of the two steps was 41.5%, and the chiral purity was 97.7%.

Claims

1. A cyclohexenecarbamate hydrolase mutant, characterized in that, The amino acid sequence is shown in sequence number (ID):

4.

2. The cyclohexenecarbamate hydrolase mutant according to claim 1, characterized in that, The cyclohexene carbamate hydrolase mutant is a protein with a new amino acid sequence formed by replacing the aspartic acid residue at position 253 of the amino acid sequence shown in sequence number (ID):2 with a valine residue.

3. A gene encoding a cyclohexenecarbamate hydrolase mutant as described in claim 1 or 2, characterized in that, Its nucleotide sequence is shown in sequence number (ID):

3.

4. A recombinant expression vector, characterized in that, It includes the coding gene as described in claim 3.

5. The use of the cyclohexenecarbamate hydrolase mutant according to claim 1 or 2 in the catalytic preparation of optically active (S)-3-cyclohexene-1-carboxylic acid from 3-cyclohexene-1-carboxylic acid.

6. The application according to claim 5, characterized in that, The structural formula of the 3-cyclohexene-1-carboxylate is shown below: R is selected from methyl, ethyl, isopropyl, or butyl.

7. The application according to claim 6, characterized in that, The application involves using a cyclohexenecarbamate hydrolase mutant to hydrolyze (R)-3-cyclohexene-1-carbamate in a buffer solution to obtain (S)-3-cyclohexene-1-carbamate, which is then hydrolyzed under alkaline conditions to obtain (S)-3-cyclohexene-1-carboxylic acid.

8. The application according to claim 7, characterized in that, The buffer solution is selected from citrate buffer and phosphate buffer; the pH of the buffer solution is 5-9.

9. The application according to claim 7, characterized in that, The reaction temperature is 15–30℃.

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