Acylase based on ancestor sequence reconstruction and application thereof
By using ancestral sequence reconstruction and protein engineering, a cephalosporin C acylase with high catalytic activity and thermal stability was constructed, which solved the problem of low catalytic activity in existing enzymatic methods. This enabled a highly efficient and green one-step enzymatic production of 7-ACA, simplifying the process and reducing costs.
Patent Information
- Application Number
- CN202512011697.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-29
- Publication Date
- 2026-05-01
AI Technical Summary
Existing natural CPC acylases have low catalytic activity, making it difficult to meet the industrial requirements for efficient one-step enzymatic production of 7-ACA, and they also suffer from insufficient thermal stability.
Cephalosporin C acylase was constructed using ancestral sequence reconstruction technology. Combined with codon optimization and protein engineering, various single-point and multi-point mutants were prepared to improve the enzyme's catalytic activity and thermal stability.
This has enabled a highly efficient and green one-step enzymatic production of 7-ACA, reducing production costs, simplifying the process, and improving production efficiency.
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Figure CN121950771A_ABST
Abstract
Description
An acylase based on ancestor sequence reconstruction and its application Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to novel variants of cephalosporin C acylase, their encoding genes, recombinant vectors, and the construction of recombinant host cells, particularly novel cephalosporin C acylases obtained through ancestral enzyme reconstruction technology, and their application in the one-step enzymatic production of 7-aminocephalosporanic acid (7-ACA). Background Technology
[0002] 7-Aminocephalosporanic acid (7-ACA) is a key intermediate in the synthesis of various cephalosporin antibiotics and has extremely high pharmaceutical industrial value. Early industrial production primarily employed a chemical cleavage method, using cephalosporin C (CPC) as a substrate. Side chains were removed through harsh low-temperature conditions and organic solvent treatment to generate 7-ACA. While feasible, this chemical method suffers from high energy consumption, severe environmental pollution, and complex process control, and has been gradually replaced by enzymatic methods.
[0003] Existing enzymatic methods mainly include two-step and one-step enzymatic methods. The two-step enzymatic method uses D-amino acid oxidase (DAAO) to catalyze the conversion of CPC to glutaryl-7-aminocephalosporanic acid (GL-7-ACA), followed by GL-7-ACA acylation catalyzing the removal of the side chain to generate 7-ACA. This method has been industrialized and has advantages such as being green, energy-saving, and having high yields. However, because the byproduct hydrogen peroxide (H2O2) in the DAAO catalysis process degrades CPC and leads to byproduct formation, and because it requires two consecutive steps, the process is complex and the production cost is high.
[0004] To simplify the process, researchers developed a "one-step enzymatic method," which utilizes CPC acylase to directly catalyze the removal of the side chain from CPC to generate 7-ACA, thus avoiding the formation of intermediate products and their subsequent transformations. However, CPC acylases isolated from nature (such as those derived from Pseudomonas sp. SE83, Pseudomonas diminuta N176, and Pseudomonas sp. P130) generally exhibit low catalytic activity towards CPC, only 2-4% of the activity of their GL-7-ACA acylases, which is insufficient to meet the requirements of high conversion rates and high production efficiency for industrial production. Therefore, the one-step enzymatic method has not yet completely replaced the large-scale application of the two-step enzymatic method.
[0005] To address the aforementioned issues, this invention constructs CPC acylases using ancestral sequence reconstruction (ASR) technology, successfully obtaining novel CPC acylases with significantly enhanced catalytic activity, providing a new enzyme resource and solution for the efficient one-step enzymatic production of 7-ACA. Summary of the Invention
[0006] This invention discloses a cephalosporin C (CPC) acylase obtained through ancestral sequence reconstruction (ASR) technology, addressing the limitations of existing natural CPC acylases such as limited source, insufficient thermostability, and restricted substrate activity. The target ancestral node sequence was obtained through multiple sequence alignment, phylogenetic tree construction, and PAML software deduction. Codon optimization was performed on the obtained ancestral enzyme sequence to adapt it for efficient expression in prokaryotic hosts. This invention provides the amino acid sequence (SEQ ID NO:1) and its corresponding nucleotide sequence (SEQ ID NO:2) of this ancestral enzyme. This enzyme can be used for one-step enzymatic catalysis of CPC cleavage to generate 7-aminocephalosporanic acid (7-ACA), providing a new enzymatic solution for achieving green, efficient, and sustainable industrial production.
[0007] This invention further provides a method for protein engineering based on the ancestral enzyme sequence. Using mutant primers, the ancestral enzyme is constructed site-by-site, successfully producing various single-site mutants, including W188I, H256T, Y263T, F291L, N301S, R335V, Y391S, F415V, N416T, and N482S. These single-site mutants can serve as templates for subsequent two-site or multi-site combined mutations. By introducing new mutation sites into a single-site mutant vector, multi-site mutants can be gradually constructed. This systematic mutational construction approach provides a comprehensive and scalable molecular basis for subsequent directed evolution, sequence optimization, and performance enhancement.
[0008] To achieve the above objectives, the present invention adopts the following technical solution:
[0009] This invention provides a cephalosporin C acylase, the amino acid sequence of which is shown in SEQ ID NO:1, or based on the amino acid sequence shown in SEQ ID NO:1, any one or more of the following amino acid mutations are present: G140T, L161S, F294L, F297N, Y271S, A421V, N481S, W188I, H256T, Y263T, F291L, N301S, R335V, Y391S, F415V, N416T, N482S.
[0010] The present invention further provides the encoding polynucleotide of the cephalosporin C acylase.
[0011] Specifically, the encoding polynucleotide sequence is selected from: the sequence shown in SEQ ID NO:2, or its codon-optimized equivalent sequence; a coding sequence containing one or more codon mutations compared to SEQ ID NO:2 to introduce any one or a combination of G140T, L161S, F294L, F297N, Y271S, A421V, N481S, W188I, H256T, Y263T, F291L, N301S, R335V, Y391S, F415V, N416T, N482S; or their degenerate sequences.
[0012] The present invention provides a recombinant vector comprising the aforementioned encoding polynucleotide.
[0013] The present invention further provides a recombinant host cell containing the aforementioned recombinant vector.
[0014] The present invention further provides a method for preparing the recombinant host cells, comprising the following steps:
[0015] The recombinant vector as described in claim 4 is introduced into Escherichia coli host cells; host cells containing the recombinant vector are obtained by screening.
[0016] The present invention also provides the cephalosporin C acylase, the encoding polynucleotide, the recombinant vector, and the application of the recombinant host cell in the catalytic production of 7-aminocephalosporanic acid.
[0017] This invention also provides a method for the catalytic production of 7-aminocephalosporanic acid, which uses cephalosporin C as a substrate and the cephalosporin C acylase as a catalytic enzyme to catalyze the reaction to produce 7-aminocephalosporanic acid.
[0018] Specifically, the catalytic reaction is carried out at 15-40℃ and pH 6.0-9.0 for 30-120 min, and then ammonium acetate buffer and methanol are added to terminate the reaction.
[0019] More specifically, the reaction system, with a total volume of 1 mL, consisted of pH 8.050 mM Na2HPO4-KH2PO4 buffer, 40 mM cephalosporin C substrate, and 200 μL of enzyme. Attached Figure Description
[0020] Figure 1 shows the liquid phase spectrum and structural formula of enzyme-catalyzed CPC to 7-ACA at 45℃. Detailed Implementation
[0021] The specific steps of the present invention are illustrated below through examples, but are not limited to these examples.
[0022] Unless otherwise stated, the terms used in this invention generally have the meanings commonly understood by those skilled in the art.
[0023] The present invention will now be described in further detail with reference to specific embodiments and data. It should be understood that these embodiments are merely illustrative of the invention and are not intended to limit the scope of the invention in any way.
[0024] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art.
[0025] Example 1: Reconstruction of the ancestral sequence of acylase
[0026] Using the CPC acylase amino acid sequences from Bosea sp. OK40, Pseudomonas sp. GK16, Pseudomonas sp. SE83, Pseudomonas diminuta N176, and Pseudomonas sp. P130 strains as templates (Table 1), homologous sequences were retrieved from the NCBI database, totaling 2027. The retrieved sequences were deredundant using CD-HIT software, and sequences with less than 40% similarity to the three template sequences were selected as candidate sequences for ancestral sequence reconstruction.
[0027] Table 1. Information on CPC acylases involved in this embodiment.
[0028]
[0029] Clustal Omega multiple sequence alignment was performed using the candidate sequences mentioned above, and a phylogenetic tree was constructed based on the alignment results using maximum likelihood (ML) and Bayesian inference. Subsequently, the ancestral sequence was inferred from the phylogenetic tree using the codeml module in PAML 4.9 software, selecting later ancestral nodes and predicting their amino acid sequences. The obtained sequences were further validated using MEGA-X software and used as the reconstructed ancestral sequences for subsequent experiments. The resulting cephalosporin acylase ancestral sequence AS8 (its amino acid sequence is shown in SEQ ID NO: 1) was obtained.
[0030] Example 2: Expression of cephalosporin C acylase and progenitor enzyme
[0031] The predicted ancestral cephalosporin C acylase sequence was codon optimized and synthesized by a commissioned synthesis agency. The synthesized gene fragment was then cloned into the pET-28a(+) vector after double enzyme digestion, resulting in a 6×His tag at the C-terminus of the target protein. After the recombinant plasmid was verified to be correct by Sanger sequencing, it was transformed into *E. coli* BL21(DE3) competent cells.
[0032] After obtaining the expression plasmid of the ancestral enzyme, to screen for potential key functional residues and construct its derived mutants, the ancestral enzyme sequence and three-dimensional structure were first comprehensively evaluated based on multiple sequence alignment, structural prediction, and structural simulation analysis. Multiple sequence alignment identified variable regions in homologous enzyme sequences that exhibited conserved differences or were related to substrate recognition. Furthermore, AlphaFold2 or homology modeling methods were used to obtain a three-dimensional structural model of the ancestral enzyme, and molecular docking was combined with simulations of substrate-enzyme active site binding patterns. Amino acid positions that might affect substrate localization, catalytic efficiency, or stability were screened from key residues surrounding the substrate (including inside the pocket and adjacent distal regulatory sites). Based on the above structure-function analysis, candidate sites such as W188, H256, Y263, F291, N301, R335, Y391, F415, N416, and N482 were identified for subsequent site-directed mutagenesis construction.
[0033] Based on this, to obtain the corresponding derived mutants, mutation primers designed for the specific sites mentioned above were used to perform site-by-site PCR amplification of the ancestral enzyme expression plasmid, thereby constructing single-point mutations such as W188I, H256T, Y263T, F291L, N301S, R335V, Y391S, F415V, N416T, and N482S. The composition of the PCR reaction system is shown in Table 2, with a total system volume of 50 μL. After the amplification products were digested with DpnI to remove the unmutated template plasmid, they were transformed into E. coli DH5α for screening mutant-positive clones.
[0034] Table 2. PCR reaction system
[0035]
[0036] All ancestral enzyme plasmids and their mutant expression plasmids, confirmed by sequencing, were transformed into *E. coli* BL21(DE3) competent cells. The transformed strains were inoculated into LB solid or liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37 °C with shaking (200 rpm). Subsequently, 1% (v / v) seed culture was inoculated into fresh LB liquid medium (containing 50 μg / mL kanamycin) and cultured at 37 °C until the OD600 reached 0.6–1.0. IPTG was then added to a final concentration of 0.1 mmol / L for induction, and the cells were cultured at 25 °C for another 12 h to obtain the expressed ancestral enzymes and mutant proteins.
[0037] Example 3: Activity test of cephalosporin acylase and various mutant enzymes
[0038] 1. Preparation of cephalosporin acylase and various mutant enzymes
[0039] After induction, the bacterial culture was centrifuged at 12,000 rpm for 20 min (4℃) to collect the bacterial cells, and the supernatant was discarded. The bacterial cells were weighed, and for every 1 g of wet bacterial weight, the cells were resuspended in 10 mL of 50 mM Na2HPO4-KH2PO4 buffer (pH 8.0). The cells were then disrupted using an ultrasonic cell disruptor (200 W, 5 s intervals, 5 s intervals, 20 min total, ice bath). The disrupted solution was centrifuged at 12,000 rpm for 30 min (4℃), and the supernatant was collected as the enzyme solution for the reaction.
[0040] The reaction system consisted of 50 mM Na₂HPO₄-KH₂PO₄ buffer (pH 8.0), 40 mM CPC substrate, and 200 μL of enzyme, with a total volume of 1 mL. The reaction time was 30–120 min. To terminate the reaction, 300 μL of ammonium acetate buffer and 100 μL of methanol were added to every 100 μL of reaction solution. After mixing, the mixture was incubated on ice for 10 min, followed by centrifugation at 12000 rpm for 10 min (4 °C). The supernatant was filtered through a 0.22 μm filter and analyzed using liquid chromatography.
[0041] 2. High-performance liquid chromatography (HPLC) method for analyzing the catalytic activity of substrate CPC and product 7ACA.
[0042] The analytical conditions were as follows: An Agilent 1260 Infinity liquid chromatograph was used, with a C18 reversed-phase column (4.6 mm × 250 mm, 5 μm). The mobile phase was a mixture of 20 mM ammonium acetate solution (pH 7.2) and acetonitrile, with a volume ratio of 95:5. The flow rate was 0.8 mL / min, the column temperature was 40℃, the detection wavelength was 254 nm, and the injection volume was 10 μL. The retention time of each sample was approximately 8–9 min, and the peak areas of the substrate CPC and the product 7-ACA were recorded. Standard curves were plotted using CPC and 7-ACA standard samples, and the product yield was calculated by converting the peak areas to determine the enzyme's catalytic activity.
[0043] 3. Activity test results under different temperature conditions
[0044] To further evaluate the catalytic activity of the ancestral CPC acylase AS8 and its single-point mutants (including W188I, H256T, Y263T, F291L, N301S, R335V, Y391S, F415V, N416T, and N482S) constructed based on mutant primers under different temperature conditions, the prepared enzyme solutions of each enzyme were added to the standard reaction system and reacted at 15 °C, 37 °C, and 45 °C for 60 min. The reaction termination procedure, centrifugation conditions, and HPLC analysis methods were all performed as described in Examples 3 and 4.
[0045] Test results show that, at 45℃, as shown in Figure 1, a clear peak of 7-ACA formation can be detected during the CPC reaction catalyzed by AS8, indicating that the enzyme maintains good structural stability and high catalytic efficiency even at higher temperatures (Table 3). At 37℃, a significant product peak can still be detected, indicating that its enzyme activity remains at a high level. However, at 15℃, only trace amounts of product are detected, further demonstrating that this reconstructed ancestral enzyme is more suitable for catalytic reactions at higher temperatures. The mutants constructed based on the ancestral enzyme all exhibit a wide temperature adaptation range, maintaining good catalytic ability at 37-45℃. The acquisition of these variants provides expanded space for further enzyme molecule modification and enhances the application potential of the ancestral enzyme system in the industrial enzymatic production of 7-ACA.
[0046] Table 3. Catalysis of CPC to 7-ACA by ancestral enzymes and their mutants under different temperature conditions
[0047] .
Claims
1. A cephalosporin C acylase, characterized in that, Its amino acid sequence is shown in SEQ ID NO:1, or it contains any one or more of the following amino acid mutations based on the amino acid sequence shown in SEQ ID NO:1: W188I, H256T, N301S, R335V, Y391S, F415V, N416T, N482S.
2. The polynucleotide encoded by the cephalosporin C acylase according to claim 1.
3. The polynucleotide encoding according to claim 2, characterized in that, The encoding polynucleotide sequence is selected from: the sequence shown in SEQ ID NO:2, or its codon-optimized equivalent sequence; a coding sequence containing one or more codon mutations compared to SEQ ID NO:2 to introduce any one of W188I, H256T, N301S, R335V, Y391S, F415V, N416T, N482S; or their degenerate sequences.
4. A recombinant vector, characterized in that, It contains the encoded polynucleotide as described in claim 2 or 3.
5. A recombinant host cell, characterized in that, It contains the recombinant vector as described in claim 4.
6. A method for preparing the recombinant host cell as described in claim 5, characterized in that, Includes the following steps: The recombinant vector as described in claim 4 is introduced into Escherichia coli host cells; host cells containing the recombinant vector are obtained by screening.
7. The cephalosporin C acylase of claim 1, the encoding polynucleotide of claim 2 or 3, the recombinant vector of claim 4, and the recombinant host cell of claim 5 in the catalytic production of 7-aminocephalosporanic acid.
8. A method for catalytic production of 7-aminocephalosporanic acid, characterized in that: Using cephalosporin C as a substrate and the cephalosporin C acylase as described in claim 1 as a catalytic enzyme, a reaction is catalyzed to generate 7-aminocephalosporanic acid.
9. The method as described in claim 8, characterized in that, The catalytic reaction is carried out at 15-40℃ and pH 6.0-9.0 for 30-120 min, and then ammonium acetate buffer and methanol are added to terminate the reaction.
10. The method as described in claim 8, characterized in that, The reaction system, with a total volume of 1 mL, consisted of pH 8.0 50 mM Na2HPO4-KH2PO4 buffer, 40 mM cephalosporin C substrate, and 200 μL of enzyme.