Hybrid acyltransferase mutants and their amide synthesis applications for ai-assisted design

Abstract

The application discloses a kind of AI auxiliary design hybrid acyltransferase mutant and its amide synthesis application.The mutant is to come with VIII carboxylic esterase hybrid acyltransferase EstCE1 as wild type, and is obtained by introducing combination mutation M1A, L155T and F245S, and its amino acid sequence is shown in SEQ ID NO:2.Compared with wild type, the amide activity of mutant in the application is increased to 17.5 times, catalytic efficiency (k cat / K m ) is increased to 16.7 times, while hydrolysis activity is significantly inhibited.The mutant can be catalyzed to synthesize p-acetylaminophenol in aqueous system with ethyl acetate as acyl donor and p-aminophenol as acyl acceptor, and the conversion rate is 4.07% in 10 hours, which is increased by 30.5 times compared with wild type.The mutant provided by the application has the advantages of high catalytic efficiency, low hydrolysis side reaction and mild reaction conditions, and has wide application prospect in the field of green synthesis of amide drugs.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and biocatalysis, specifically to a hybrid acyltransferase combination mutant obtained through artificial intelligence-assisted screening, and the application of this mutant in the catalytic acyl transfer reaction to prepare amide compounds, particularly in the catalytic synthesis of acetaminophen in an aqueous system. Background Technology

[0002] Acetaminophen (APAP) is one of the most widely used drugs for treating pain and fever. Its chemical synthesis process typically involves large amounts of industrial organic solvents and highly toxic Lewis acid catalysts, easily generating non-environmentally friendly byproducts. Enzymatic synthesis of amide bonds, due to its advantages such as mild reaction conditions, high selectivity, and environmental friendliness, has become an important development direction to replace traditional chemical synthesis.

[0003] Hybrid acyltransferases can catalyze the transfer of acyl groups from esters to amines in aqueous systems without the need for coenzymes or cofactors, achieving irreversible amidation and offering possibilities for green biomanufacturing. EstCE1, a hybrid acyltransferase from family VIII carboxylesterases, has the ability to catalyze the synthesis of esters, amides, carbonates, and carbamates in aqueous environments. However, natural hybrid acyltransferases generally suffer from drawbacks such as low catalytic efficiency, narrow substrate spectrum, and insufficient specificity for non-natural substrates. In particular, EstCE1 exhibits extremely low efficiency in catalyzing the synthesis of amide drugs with practical application value, severely restricting its industrial application.

[0004] In recent years, with the rapid development of artificial intelligence and machine learning technologies, AI-aided design strategies based on enzyme kinetic parameter prediction have opened up new avenues for the rational modification of enzyme molecules. The UniKP framework for enzyme kinetic parameter prediction, proposed by Luo Xiaozhou's team at the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, integrates pre-trained protein language models with substrate chemical features, achieving the prediction of enzyme transformation numbers k for both natural enzymes and enzyme variants. cat Michaelis constant K m and catalytic efficiency k cat / K m The comprehensive prediction of enzyme kinetic parameters is achieved through a combination of pre-trained models and molecular fingerprinting. The CataPro enzyme kinetic parameter prediction model, jointly proposed by Professor Li Weifeng's research group at Shandong University and Dr. Zheng Liangzhen's team at Shanghai Zhiyu Biotechnology, significantly improves the accuracy and generalization ability of kinetic parameter prediction by integrating pre-trained models with molecular fingerprinting. Therefore, developing a highly efficient hybrid acyltransferase mutant based on AI-aided design to achieve efficient catalytic synthesis of pharmaceutical amide compounds in aqueous phase is a key problem urgently needing to be solved in the field of enzyme engineering. Summary of the Invention

[0005] The purpose of this invention is to overcome the technical defects of existing mixed acyltransferases, such as low catalytic efficiency and poor amidation selectivity for non-natural aromatic amine substrates, and to provide a high-performance mixed acyltransferase combination mutant obtained by AI-assisted virtual screening, as well as the application of this mutant in the preparation of amide compounds.

[0006] The present invention adopts the following technical solution:

[0007] A mutant of a hybrid acyltransferase, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] A mutant of a hybrid acyltransferase, the mutant of which is based on the wild-type enzyme EstCE1 with mutation sites M1A, L155T and F245S; the amino acid sequence of EstCE1 is as shown in SEQ ID NO.1.

[0009] The application of the mutant of the hybrid acyltransferase of the present invention in the preparation of amide compounds.

[0010] The application of the mutant in the preparation of amide compounds involves using the mutant of the present invention as a catalyst in an aqueous reaction system to catalyze the reaction between an acyl donor and an acyl acceptor to generate amide compounds.

[0011] Preferably, the acyl donor is ethyl acetate, the acyl acceptor is p-aminophenol, and the amide compound is p-acetaminophen.

[0012] Preferably, the reaction conditions are: temperature 25-40℃, pH 6.0-8.0, and reaction time 0.5-10 hours.

[0013] Preferably, the concentration of p-aminophenol is 1-30 mM; the concentration of ethyl acetate is 50-500 mM; and the final concentration of the mutant is 0.5-10 μM.

[0014] This invention discloses an AI-assisted designed hybrid acyltransferase mutant and its application in amide synthesis. The mutant is obtained by introducing combined mutations M1A, L155T, and F245S into the wild-type hybrid acyltransferase EstCE1, which is a VIII carboxylesterase. Its amino acid sequence is shown in SEQ ID NO:2. Compared with the wild-type, the amidation activity of the mutant of this invention is increased by 17.5 times, and the catalytic efficiency (kJ / kJ / kJ) is significantly improved. cat / K mThe efficiency was increased by 16.7 times, while hydrolysis activity was significantly inhibited. This mutant can synthesize acetaminophen (APAP) in a one-step process in an aqueous system using ethyl acetate as the acyl donor and p-aminophenol as the acyl acceptor, achieving a conversion rate of 4.07% in 10 hours, which is 30.5 times higher than the wild type. The mutant provided by this invention has the advantages of high catalytic efficiency, low hydrolysis side reactions, and mild and green reaction conditions, and has broad application prospects in the field of green synthesis of amide drugs.

[0015] The specific explanation is as follows:

[0016] The UniKP and CataPro deep learning models were used to perform high-throughput virtual screening of the full-sequence single-point saturation mutant library of EstCE1, a hybrid acyltransferase from the VIII family of carboxylesterases, to predict the catalytic efficiency of each mutant for the acyl acceptor p-aminophenol (PAP). Preferred single-point mutation sites were screened and verified experimentally. Combination mutants were constructed by stacking these sites to obtain an engineered enzyme with both enhanced amidation activity and hydrolysis inhibition. This mutant was then applied to an aqueous reaction system, using ethyl acetate (EtOAc) as the acyl donor and p-aminophenol as the acyl acceptor, to catalyze the synthesis of acetaminophen (APAP).

[0017] The wild type of the hybrid acyltransferase mutant described in this invention is hybrid acyltransferase EstCE1, whose amino acid sequence is shown in SEQ ID NO.1.

[0018] The mutant contains a combination of mutation sites M1A, L155T and F245S, and is named ATS. The amino acid sequence of ATS is shown in SEQ ID NO.2.

[0019] The mutant ATS exhibited 17.5-fold increased PAP amidation activity compared to the wild-type, and its kJ / k ... cat / K m Increased to 16.7 times that of the wild type, while hydrolyzed k cat / K m Only 25.5% of the wild type, successfully realizing the shift from catalytic heterogeneity preference to amidation synthesis.

[0020] The mutant ATS maintained extremely high amidation activity and AT / H ratio within the range of 25-40°C and pH 6.0-8.0; after being stored at 4°C for 7 days, the residual amidation activity was still 11.4 times that of the wild type, demonstrating excellent operational stability.

[0021] The method for preparing the above mutant includes the following steps:

[0022] 1) Based on the deep learning models UniKP and CataPro, the dynamic parameters of the EstCE1 full-sequence single-point saturation mutant library were virtually screened, and the top 5 dominant single-point mutants predicted by each model were obtained.

[0023] 2) The single-point mutants screened in step 1) were subjected to gene expression, purification and amidation activity experiments to obtain the dominant single-point mutants;

[0024] 3) Combine and stack the advantageous single-point mutation sites verified in step 2) to construct combined mutation libraries of double mutants, triple mutants, and quadruple mutants;

[0025] 4) Gene expression, purification and amidation activity determination of the combined mutant constructed in step 3) were performed to screen and obtain the dominant three mutant ATS (M1A / L155T / F245S).

[0026] The evaluation method for virtual screening in step 1) is: by predicting the kinetic parameter k of a specific substrate PAP. cat K m The amidation reaction rate v under specific catalytic conditions was calculated using the Michaelis-Menten equation, and the ratio of the reaction rate to that of wild-type EstCE1 was used as a normalized score for ranking and pre-screening.

[0027] The gene expression methods in steps 2) and 4) are as follows: EstCE1 and its mutant genes are subcloned into the pET-28a plasmid vector, transformed into E. coli BL21(DE3), and after IPTG induction, the purified enzyme is obtained by NiSepharose 6 Fast Flow affinity chromatography and Superdex200 Increase 10 / 300 GL size exclusion chromatography.

[0028] The method for determining the amidation activity in step 2) is as follows: using ethyl acetate as the acyl donor and p-aminophenol as the acyl acceptor, the reaction is carried out in sodium phosphate buffer, and the amidation activity is calculated by detecting the amount of APAP generated by HPLC.

[0029] This invention also provides the application of the above-mentioned mutant in the aqueous-phase catalytic synthesis of acetaminophen. The synthesis reaction is carried out in a sodium phosphate reaction buffer, the reaction system containing PAP and EtOAc, and the reaction is carried out at 25-40°C and 100-200 rpm, achieving a conversion rate of 4.07%, which is 30.5 times higher than that of the wild type.

[0030] In summary, addressing the issues of low catalytic efficiency and poor amidation selectivity of the wild-type mixed acyltransferase EstCE1 for the non-natural substrate PAP, this invention utilizes two deep learning models, UniKP and CataPro, to perform virtual screening of enzyme kinetic parameters using a single-point saturation mutant library of EstCE1. Experimental verification and site combination yielded the optimal mutant combination ATS (M1A / L155T / F245S). This mutant exhibits an amidation specificity 17.5 times higher than the wild-type and a hydrolysis catalytic efficiency reduced to 25.5% of the wild-type, successfully shifting the catalytic preference towards amide synthesis. In aqueous synthesis of acetaminophen, the conversion rate reached 4.07%, a 30.5-fold improvement over the wild-type, while also demonstrating excellent thermal and storage stability. This invention provides a highly efficient catalyst and a simple process for the green biosynthesis of amide drugs, showing significant promise for industrial applications. Attached Figure Description

[0031] Figure 1 Results of amidation activity assay for single-point mutants.

[0032] Figure 2 Results of amidation activity assay for the combined mutants.

[0033] Figure 3 Enzyme reaction kinetics curves of EstCE1-WT and its mutant ATS.

[0034] Figure 4 Thermal stability test results of EstCE1-WT and its mutant ATS.

[0035] Figure 5 pH stability test results of EstCE1-WT and its mutant ATS.

[0036] Figure 6 Storage stability test results of EstCE1-WT and its mutant ATS.

[0037] Figure 7 The yield of APAP synthesized by the amidation of PAP catalyzed by EstCE1-WT and its optimal combination mutant ATS as a function of time. Detailed Implementation

[0038] The following examples will further illustrate the method provided by the present invention.

[0039] Example 1: Saturation Mutation Screening Based on AI Prediction of Enzyme Kinetic Parameters

[0040] 1) EstCE1, a hybrid acyltransferase from family VIII carboxylesterases, is wild-type and its amino acid sequence is shown in SEQ ID NO.1. Based on this, a single-point saturation mutant library was constructed.

[0041] 2) The kinetic parameters (k) of the EstCE1 full-sequence single-point saturation mutant library were predicted using the UniKP framework based on unified pre-trained features. cat K m k cat / K m This technology enables high-throughput prediction. The proposed solution is publicly available in a paper published in Nature Communications by H. Yu et al. (DOI: 10.1038 / s41467-023-44113-1), and the related code has been publicly released by the research team in the academic community. In the protein feature extraction stage, the large-scale protein pre-trained language model ProtT5-XL-UniRef50 is used to perform high-dimensional semantic encoding of the amino acid sequence of the target enzyme. Average pooling is then used to extract a 1024-dimensional global sequence feature vector containing physicochemical properties and evolutionary information. In the substrate molecule characterization stage, the open-source cheminformatics tool RDKit is used to convert the SMILES string of the substrate PAP into a traditional molecular descriptor. After concatenating and fusing the multi-source features into a comprehensive input matrix, a pre-trained extreme random tree algorithm is used for efficient regression prediction. The model outputs the logarithmically transformed predicted values, which are then subjected to an inverse exponential transform to obtain the final kinetic parameters.

[0042] 3) The CataPro enzyme kinetic parameter prediction model, which integrates deep neural networks, is used for high-throughput prediction of the kinetic parameters of the EstCE1 full-sequence single-point saturation mutation library. This technical solution is disclosed in the paper published by Z. Wang et al. in Nature Communications (DOI: 10.1038 / s41467-025-58038-4), and the code is publicly available on GitHub under the MIT license. In the protein feature extraction stage, the large-scale protein pre-trained language model ProtT5-XL-UniRef50 is used to perform high-dimensional semantic encoding of the amino acid sequence of the target enzyme, and a 1024-dimensional global sequence feature vector containing physicochemical properties and evolutionary information is extracted by average pooling. In the substrate molecule characterization stage, a dual-channel characterization strategy is adopted. Based on traditional molecular fingerprinting, the small molecule pre-trained model MolT5 is introduced to extract the deep contextual semantic features of the substrate PAP. After concatenating and fusing the multi-source features into a comprehensive input matrix, it is input into the core deep neural network. The model outputs the logarithmically transformed predicted values, which are then subjected to inverse exponential transformation to obtain the final kinetic parameters.

[0043] 4) Based on the enzyme catalytic conversion number k cat Calculation formula for maximum reaction rate V maxThe enzyme concentration [E0] was set to 0.0005 mM. Substituting V into the equations using the Michaelis-Menten and Lineweaver-Burk equations... max and the predicted K for PAP m Calculate the amide reaction rate v (mM / min), where the substrate concentration [S] is set to 15 mM.

[0044] 5) Using the amidation reaction rate v calculated from the predicted kinetic parameters of EstCE1-WT as a benchmark, the predicted calculation results of each mutant were normalized and used as the normalized score of the virtual screening evaluation system.

[0045] The top five dominant single-point mutants predicted by the UniKP model were Q18V, P240A, D350K, L155T, and Q276W; the top five dominant single-point mutants predicted by the CataPro model were M1A, R377D, F245S, R11L, and R282E. Based on the combined scores of the two models, 10 representative single-point mutations were selected for further experimental validation.

[0046] Example 2: Expression, purification, and amidation activity verification of single-point mutants

[0047] 1) The EstCE1 gene and its single-point mutant gene, which are fused with a histidine tag (His-tag) at the N-terminus, were subcloned into the Bam HI and Hind III restriction sites of the pET-28a plasmid vector by overlapping extension polymerase chain reaction (PCR), and then transformed into competent E.coli BL21(DE3) to obtain the genetically engineered expression strains of EstCE1 and its single-point mutant.

[0048] 2) Take 10 μL of the EstCE1 and its single-point mutant genetically engineered expression strain obtained in step 1) into 5 mL of LB liquid medium containing 5 μL of kanamycin solution (50 mg / mL) and incubate in a shaker at 37°C and 220 rpm for 12 h.

[0049] 3) Transfer 2.5 mL of bacterial culture to 300 mL of TB liquid medium containing 250 μL kanamycin solution (50 mg / mL), and incubate at 37°C and 220 rpm for 3-3.5 h. When the OD600 reaches between 0.6 and 0.8, add 120 μL of 1 M IPTG to make the final IPTG concentration 0.4 mM, and incubate at 16°C and 160 rpm for 20 h to induce expression.

[0050] 4) Centrifuge at 4°C and 4000 rpm for 30 min, discard the supernatant and collect the bacterial cells deposited at the bottom.

[0051] 5) The collected bacterial cells were resuspended in 30 mL of lysis buffer (50 mM sodium phosphate, 300 mM NaCl, pH 8.0), and then the bacterial cells were disrupted by an ultrasonic cell disruptor. After centrifuging the lysis buffer at 4°C and 10,000 rpm for 30 min, the precipitate was discarded and the supernatant bacterial lysis supernatant was collected.

[0052] 6) The supernatant was purified by Ni Sepharose 6 Fast Flow affinity chromatography. First, the nickel affinity chromatography column was equilibrated with lysis buffer. Then, non-specifically adsorbed proteins were eluted sequentially with wash buffers of 30 mM, 40 mM, and 50 mM imidazole (50 mM sodium phosphate, 300 mM NaCl, 30-50 mM imidazole, pH 8.0). Finally, the target protein was obtained and collected by washing with elution buffer (50 mM sodium phosphate, 300 mM NaCl, 300 mM imidazole, pH 8.0).

[0053] 7) Further purification of the target protein in the eluent was performed by size exclusion chromatography using a Superdex 200 Increase 10 / 300 GL column.

[0054] 8) Finally, the enzyme solution was desalted by ultrafiltration through a 10 kDa ultrafiltration tube and then stored at 4°C.

[0055] 9) The amidation activity was determined by high performance liquid chromatography (HPLC). 200 μL of 2.5 μM enzyme solution was added to 800 μL of reaction buffer (200 mM sodium phosphate, pH 8.0) containing 18.75 mM PAP and 625 mM EtOAc, making the reaction volume 1 mL. The final enzyme concentration was 0.5 μM, the final PAP concentration was 15 mM, and the final EtOAc concentration was 500 mM.

[0056] 10) After incubating the reaction system in an air shaker at 25°C and 150 rpm for 30 min, terminate the reaction by heat treatment in a water bath at 95°C for 5 min.

[0057] 11) After terminating the reaction, centrifuge the sample at 10,000 rpm for 5 min and discard the precipitate.

[0058] 12) The APAP content in the sample was determined by HPLC to characterize the amidation activity of the enzyme. HPLC detection conditions: AQ-C18 column (250 mm × 4.6 mm, 5 µm, Yuexu Technology), UV detection of peak area at 245 nm wavelength, column oven temperature 37°C, mobile phase A (ultrapure water containing 0.1% phosphoric acid) and organic phase B (HPLC-grade methanol), flow rate 1 mL / min. Gradient elution was performed as shown in Table 1 below:

[0059] Table 1: Gradient elution time program for HPLC detection of p-aminophenol and p-acetaminophen

[0060]

[0061] The amidation activity unit (U) of an enzyme is defined as the amount of enzyme required to produce 1 μM APAP per minute under specific conditions, while the amidation specific activity (U / mg) of an enzyme is defined as the amount of APAP (μM) produced per 1 μg of enzyme per minute.

[0062] like Figure 1 Activity assays showed that the actual amidation activities of all nine mutants were higher than those of the wild type, demonstrating a positive evolutionary effect. M1A showed the best performance, with a specific activity of 45.7 U / mg, 7.1 times that of EstCE1-WT. F245S (41.6 U / mg), Q18V (39.4 U / mg), and L155T (39.2 U / mg) all exhibited excellent amidation performance, with improvements of more than 5 times compared to the wild type. The experimental results validated the effectiveness of the AI ​​model predictions, especially since M1A, which ranked first in CataPro predictions, was precisely the single-point mutation with the most significant activity improvement.

[0063] Example 3: Construction of combinatorial mutants and screening of amidation activity

[0064] 1) Four single-point mutation sites, M1A, Q18V, L155T and F245S, which showed the most significant enhancement of amidation activity in experimental verification, were selected and combined to construct a combined mutant library containing six double mutants (AV, AT, AS, VT, TS, VS), four triple mutants (AVT, ATS, AVS, VTS) and one quadruple mutant (AVTS), as shown in Table 2.

[0065] Table 2: List of combined mutations constructed based on single-point mutations with amidation performance advantages

[0066]

[0067] 2) By overlapping extension PCR, the genes of each combined mutant with the N-terminus fused with His-tag were subcloned into the Bam HI and Hind III restriction sites of the pET-28a plasmid vector, and then transformed into competent E.coli BL21(DE3) to obtain the genetically engineered expression strain of the combined mutant.

[0068] 3) Take 10 μL of the combined mutant gene-engineered expression strain obtained in step 2) into 5 mL of LB liquid medium containing 5 μL of kanamycin solution (50 mg / mL) and incubate in a shaker at 37°C and 220 rpm for 12 h.

[0069] 4) Transfer 2.5 mL of bacterial culture to 300 mL of TB liquid medium containing 250 μL kanamycin solution (50 mg / mL), and incubate at 37°C and 220 rpm for 3-3.5 h. When the OD600 reaches between 0.6 and 0.8, add 120 μL of 1 M IPTG to make the final IPTG concentration 0.4 mM, and incubate at 16°C and 160 rpm for 20 h to induce expression.

[0070] 5) Centrifuge at 4°C and 4000 rpm for 30 min, discard the supernatant and collect the bacterial cells deposited at the bottom.

[0071] 6) The collected bacterial cells were resuspended in 30 mL of lysis buffer (50 mM sodium phosphate, 300 mM NaCl, pH 8.0), and then the bacterial cells were disrupted by an ultrasonic cell disruptor. After centrifuging the lysis buffer at 4°C and 10,000 rpm for 30 min, the precipitate was discarded and the supernatant bacterial lysis supernatant was collected.

[0072] 7) The supernatant was purified by Ni Sepharose 6 Fast Flow affinity chromatography. First, the nickel affinity chromatography column was equilibrated with lysis buffer. Then, non-specifically adsorbed proteins were eluted sequentially with wash buffers of 30 mM, 40 mM, and 50 mM imidazole (50 mM sodium phosphate, 300 mM NaCl, 30-50 mM imidazole, pH 8.0). Finally, the target protein was obtained and collected by washing with elution buffer (50 mM sodium phosphate, 300 mM NaCl, 300 mM imidazole, pH 8.0).

[0073] 8) The target protein in the eluent was further purified by size exclusion chromatography using a Superdex 200 Increase 10 / 300 GL column, and finally desalted by ultrafiltration using a 10 kDa ultrafiltration tube. The purified and desalted enzyme solution was stored at 4°C.

[0074] 9) The amidation activity was determined by high performance liquid chromatography (HPLC). 200 μL of 2.5 μM enzyme solution was added to 800 μL of reaction buffer (200 mM sodium phosphate, pH 8.0) containing 18.75 mM PAP and 625 mM EtOAc, making the reaction volume 1 mL. The final enzyme concentration was 0.5 μM, the final PAP concentration was 15 mM, and the final EtOAc concentration was 500 mM.

[0075] 10) After incubating the reaction system in an air shaker at 25°C and 150 rpm for 30 min, terminate the reaction by heat treatment in a water bath at 95°C for 5 min.

[0076] 11) After terminating the reaction, centrifuge the sample at 10,000 rpm for 5 min and discard the precipitate.

[0077] 12) The APAP content in the sample was detected by the HPLC method described in Example 2 to characterize the amidation activity of the enzyme.

[0078] like Figure 2 All combinatorial mutants exhibited significantly enhanced catalytic performance compared to the wild type, with most combinatorial variants showing significantly superior activity compared to their corresponding single-site mutant parents. In the double mutants, the specific activities of AS (M1A / F245S) and VS (Q18V / F245S) reached 85.7 U / mg and 88.1 U / mg, respectively, representing an increase of more than 12-fold compared to EstCE1-WT. The triple mutant ATS (M1A / L155T / F245S) demonstrated the strongest amide synthesis capacity, with a specific activity as high as 113.8 U / mg, 17.5 times that of EstCE1-WT, and far exceeding its three constituent single-site mutants M1A (46.1 U / mg), L155T (39.5 U / mg), and F245S (41.8 U / mg), indicating a good synergistic effect among the three sites. The complete amino acid sequence of this mutant ATS is shown in SEQ ID NO.2. The specific activity of the tetra mutant AVTS (59.2 U / mg) was significantly lower than that of the tri-mutant ATS, indicating that excessive amino acid substitution can easily cause spatial conflicts in local structures and has a negative exotropic effect.

[0079] Example 4: Enzyme kinetics determination of the combined mutant ATS

[0080] The initial rates of the enzyme-catalyzed reactions of EstCE1-WT and the mutant ATS at different substrate concentrations were determined by HPLC. The enzyme-catalyzed reaction kinetic curves were obtained by fitting the Michaelis-Menten equation, and the corresponding kinetic parameters were calculated.

[0081] For the hydrolysis reaction kinetics, without the addition of PAP, the reaction system was incubated for 30 min at 25°C and 150 rpm in an air shaker at different EtOAc concentrations (10.0 mM, 50.0 mM, 100.0 mM, 150.0 mM, 200.0 mM, 300.0 mM, 400.0 mM, and 500.0 mM). The reaction was then terminated by heat treatment in a 95°C water bath for 5 min. The initial reaction rate was determined by HPLC. HPLC detection conditions: Sepax HP-C18 column (150 mm × 4.6 mm, 5 µm, Sepax Technology), UV detection of peak area at 210 nm wavelength, column oven temperature 26°C, mobile phase 100 mM sodium phosphate buffer (pH 3.1), flow rate 1.0 mL / min, isocratic elution.

[0082] For the amidation reaction kinetics, the initial reaction rate was determined under the following conditions: when the EtOAc concentration was 500 mM, the PAP concentrations were 1.0 mM, 2.0 mM, 3.0 mM, 5.0 mM, 10.0 mM, 15.0 mM, 20.0 mM, 25.0 mM, and 30.0 mM; when the EtOAc concentrations were 10.0 mM, 50.0 mM, 100.0 mM, 150.0 mM, 200.0 mM, 300.0 mM, 400.0 mM, and 500.0 mM, the PAP concentration was 15.0 mM. The reaction system was incubated in an air shaker at 25°C and 150 rpm for 30 min, and the reaction was terminated by heat treatment in a 95°C water bath for 5 min. The initial reaction rate was determined by the HPLC method described in Example 2.

[0083] The results are as follows Figure 3 As shown in Table 3-5, the maximum rate V of the hydrolysis reaction of ATS. max Compared to EstCE1-WT, its k-value for EtOAc hydrolysis is significantly reduced. cat / K m Only 26% of the wild type. In the amidation reaction, the catalytic efficiency k of ATS for the substrates PAP and EtOAc was... cat / K m Both achieved an order of magnitude improvement over the wild type: k for PAP cat / K m It is 1.088 min -1 / mM, which is 16.7 times that of EstCE1-WT; for EtOAc k cat / K mThe kcat of ATS for PAP was increased by 17.9-fold compared to wild-type. This indicates that multi-site combined mutations effectively promoted the transformation reaction following the binding of the acyl-enzyme intermediate to the nucleophilic reagent PAP. In a dual-substrate coexisting amidation reaction system, the kcat of ATS for PAP was significantly increased. m The value is slightly lower than that of the wild type, and the K value of EtOAc is also lower. m This reduces the percentage to 91.3% of the wild type, allowing the enzyme to form acyl-enzyme intermediates more efficiently.

[0084] Table 3: Hydrolysis kinetic parameters of EstCE1-WT and its mutant ATS a

[0085]

[0086] a Numerical rounding follows these rules: uncertainty is rounded to 1-2 significant digits, and the last digit of the parameter value is aligned with the last digit of the uncertainty.

[0087] b Er (Efficiency Ratio) represents the ratio of the catalytic efficiency of ATS to that of EstCE1-WT, and its expression is: .

[0088] Table 4: Kinetic parameters of the amidation reaction of substrate PAP by EstCE1-WT and its mutant ATS a

[0089]

[0090] a Numerical rounding follows these rules: uncertainty is rounded to 1-2 significant digits, and the last digit of the parameter value is aligned with the last digit of the uncertainty.

[0091] b Er (Efficiency Ratio) represents the ratio of the catalytic efficiency of ATS to that of EstCE1-WT, and its expression is: .

[0092] Table 5: Kinetic parameters of the amidation reaction of EstCE1-WT and its mutant ATS with ethyl acetate substrate a

[0093]

[0094] a Numerical rounding follows these rules: uncertainty is rounded to 1-2 significant digits, and the last digit of the parameter value is aligned with the last digit of the uncertainty.

[0095] b Er (Efficiency Ratio) represents the ratio of the catalytic efficiency of ATS to that of EstCE1-WT, and its expression is: .

[0096] Example 5: Thermal stability test of mutants

[0097] The purified EstCE1-WT and mutant ATS were incubated at 25°C, 30°C, 35°C and 40°C in 200 mM sodium phosphate buffer at pH 8.0 for 30 min, and the amidation activity and hydrolysis activity were determined by HPLC.

[0098] Hydrolysis activity assay: 200 μL of 2.5 μM enzyme solution was added to 800 μL of reaction buffer (200 mM sodium phosphate, pH 8.0) containing 625 mM EtOAc, making the reaction volume 1 mL. The final concentration of EstCE1-WT or mutant ATS enzyme was 0.5 μM, and the final concentration of EtOAc was 500 mM. After incubation at 25°C and 150 rpm in an air shaker for 30 min, an equal volume of methanol was added to terminate the reaction. After centrifugation at 10000 rpm for 5 min, the precipitate was discarded, and the HAc content in the sample was detected using the HPLC method described in Example 4. HPLC detection conditions: Sepax HP-C18 column (150 mm × 4.6 mm, 5 µm, Sepax Technology), UV detection of peak area at 210 nm wavelength, column oven temperature 26°C, mobile phase 100 mM sodium phosphate buffer (pH 3.1), flow rate 1.0 mL / min, isocratic elution. The hydrolytic activity unit (U) of an enzyme is defined as the amount of enzyme required to produce 1 mM HAc per minute under specific conditions, while the amidation specific activity (U / mg) of an enzyme is defined as the amount of APAP produced per minute by 1 μg of enzyme (mM).

[0099] Amidation activity assay: 200 μL of 2.5 μM enzyme solution was added to 800 μL of reaction buffer (200 mM sodium phosphate, pH 8.0) containing 18.75 mM PAP and 625 mM EtOAc, resulting in a final reaction volume of 1 mL. The final enzyme concentration was 0.5 μM, the final PAP concentration was 15 mM, and the final EtOAc concentration was 500 mM. The mixture was incubated at 25°C and 150 rpm in an air shaker for 30 min, and then the reaction was terminated by heat treatment in a 95°C water bath for 5 min. After centrifugation at 10000 rpm for 5 min, the precipitate was discarded, and the APAP content in the sample was determined using the HPLC method described in Example 2.

[0100] like Figure 4As shown, in terms of hydrolysis activity ( Figure 4 a) Wild-type EstCE1-WT maintained a high hydrolysis rate at all test temperatures, with a slight increase with increasing temperature. In contrast, the hydrolysis activity of the mutant ATS was controlled at a low level throughout the 25-40°C range, indicating that this combined mutation could stably suppress competition from hydrolysis side reactions under different temperature conditions. Regarding amidation activity (b), ATS showed significantly higher activity than the wild-type throughout the entire test temperature range. The amidation activity of the wild-type was generally low, showing a slow upward trend with increasing temperature; while ATS reached its highest value (>110 U / mg) at 25°C, subsequently showing a certain downward trend with increasing temperature. Further comparison of AT / H values ​​( Figure 4 c) The AT / H ratio of ATS significantly exceeded that of the wild type at all temperatures, reaching its maximum at 25°C. Based on the dual consideration of the highest amidation activity and the optimal AT / H ratio, 25°C was determined to be the optimal reaction temperature for amidation synthesis of the mutant ATS.

[0101] Example 6: pH stability test of mutants

[0102] The purified EstCE1-WT and mutant ATS were incubated in 200 mM sodium phosphate buffer (pH 6.0, 7.0 and 8.0) at different pH conditions for 30 min, and the amidation activity and hydrolysis activity were determined by HPLC method in Example 5.

[0103] The results are as follows Figure 5 As shown, the hydrolysis activity of wild-type EstCE1-WT increased significantly with increasing pH, reaching its peak at pH 8.0. In contrast, the hydrolysis activity of the mutant ATS was significantly inhibited across the entire pH range and remained at a relatively low level with minimal pH variation. Regarding amidation activity, ATS exhibited excellent catalytic performance, with its amidation activity showing a significant increasing trend with increasing pH, reaching its peak at pH 8.0. Analysis of the AT / H ratio revealed a clear pH-dependent amidation preference for ATS, increasing with increasing pH from 6.0 to 8.0, reaching its highest level at pH 8.0. Combining the highest amidation activity with the optimal AT / H ratio, pH 8.0 was determined to be the optimal reaction pH for amidation synthesis of this mutant.

[0104] Example 7: Storage stability test of mutants

[0105] The purified EstCE1-WT or mutant ATS enzymes were stored in sodium phosphate buffer (200 mM, pH 7.5) at 4°C for 0, 1, 2, 3, 5 and 7 days. The amidation activity and hydrolysis activity were determined by HPLC as described in Example 5 on days 0, 1, 2, 3, 5 and 7 after enzyme purification.

[0106] The results are as follows Figure 6 As shown, the hydrolysis activities of both EstCE1-WT and ATS generally decreased during the 7-day storage period. By day 7, the residual hydrolysis activity of EstCE1-WT was 54.2%, while that of ATS was 48.9%. The absolute specific activity of ATS remained at a low level throughout, with only 10.7 U / mg remaining on day 7, equivalent to 30.2% of the hydrolysis activity of EstCE1-WT on day 7. This is beneficial for continuously suppressing the occurrence of hydrolysis side reactions in the catalytic amide synthesis reaction after long-term storage. On day 7, the residual amidation activity of ATS remained at 64.7% of that on day 0, and its activity decrease was 19.2% lower than that of EstCE1-WT, and 11.4 times that of EstCE1-WT on day 0, demonstrating its excellent amidation ability.

[0107] Example 8: Enzymatic Synthesis of Acetaminophen

[0108] 1) Using PAP as an acyl acceptor substrate, the acetylation synthesis of APAP was carried out in 200 mM sodium phosphate reaction buffer (pH 8.0). The reaction system contained 15 mM PAP and 5% (v / v) ethyl acetate. The final concentration of EstCE1-WT or mutant ATS enzyme was 10 μM.

[0109] 2) The reaction system was subjected to reaction at 25°C and 150 rpm for 0.5 h, 1 h, 2 h, 3 h, 5 h, 7 h and 10 h respectively.

[0110] 3) After the reaction was completed, the reaction was terminated by heat treatment in a 95°C water bath for 5 min, followed by centrifugation at 10000 rpm for 5 min, the precipitate was discarded, and the yield of APAP in the sample was detected by HPLC method as described in Example 2.

[0111] like Figure 7The results showed that EstCE1-WT exhibited almost no ability to catalyze the amidation of PAP, with an APAP yield of only 20.8 μM (conversion rate of 0.13%) after 10 hours of reaction, remaining at an extremely low baseline level. In contrast, the combinatorial mutant ATS showed a rapid increase in conversion rate during the first 3 hours of reaction, followed by a plateauing of the reaction rate, reaching a peak APAP yield of 609.5 μM at 10 hours, with a conversion rate of 4.1%, a 30.5-fold improvement over EstCE1-WT. This demonstrates that ATS successfully overcame the barrier of extremely low activity of the wild-type enzyme against the non-natural substrate PAP, significantly improving the efficiency of green biosynthesis of high-value amide drug molecules such as APAP.

[0112] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A mutant of a hybrid acyltransferase, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.

2.

2. A mutant of a hybrid acyltransferase, characterized in that, The mutant of the hybrid acyltransferase is based on the wild-type enzyme EstCE1 with mutation sites M1A, L155T and F245S; the amino acid sequence of EstCE1 is shown in SEQ ID NO.

1.

3. The use of the mutant of the hybrid acyltransferase of claim 1 or claim 2 in the preparation of amide compounds.

4. The application of the mutant as described in claim 3 in the preparation of amide compounds, wherein the mutant as described in claim 1 or 2 is used as a catalyst in an aqueous reaction system to catalyze the reaction of an acyl donor and an acyl acceptor to generate amide compounds.

5. The application according to claim 4, characterized in that, The acyl donor is ethyl acetate, the acyl acceptor is p-aminophenol, and the amide compound is p-acetaminophen.

6. The application according to claim 4, characterized in that, The reaction conditions are: temperature 25-40℃, pH 6.0-8.0, and reaction time 0.5-10 hours.

7. The method according to claim 5, characterized in that, The concentration of p-aminophenol was 1-30 mM; the concentration of ethyl acetate was 50-500 mM; and the final concentration of the mutant was 0.5-10 μM.

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