Artificially modified cephalosporin C acylase mutant and application thereof
By recombinantly and mutated CPC acylases, CPC acylase variants with novel sequence characteristics were constructed, solving the problem of low catalytic efficiency of natural enzymes and achieving efficient and stable 7-ACA production, supporting industrial applications.
Patent Information
- Application Number
- CN202511984699.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-26
- Publication Date
- 2026-02-24
AI Technical Summary
Existing natural CPC acylases have low specificity for cephalosporin C substrates, limited catalytic efficiency, and are scarce, which restricts the industrial application of one-step enzymatic production of 7-ACA.
By combining sequence analysis, recombination technology and mutation optimization, novel CPC acylase variants were constructed, and key amino acid mutations such as T140S, L161S, F294L, N297S, Y271S, T314I, I366M, V415I, A421V, and N481S were introduced to improve the enzyme's catalytic efficiency and substrate adaptability.
The novel CPC acylase mutant significantly improves the efficiency and stability of catalyzing CPC to 7-ACA, meeting industrial needs and providing an efficient, economical, and green production pathway.
Smart Images
Figure CN121555487A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology and relates to an artificially modified cephalosporin C acylase mutant and its application. Background Technology
[0002] 7-Aminocephalosporanic acid (7-ACA) is a key intermediate in the synthesis of cephalosporin antibiotics and has extremely high pharmaceutical value. Early industrial production mainly relied on the chemical lysis of cephalosporin C (CPC) to obtain 7-ACA. However, the chemical process requires harsh conditions, including low temperatures, strong acids or alkalis, and organic solvents, resulting in high energy consumption, significant pollution, and high safety risks. Therefore, green and mild enzymatic processes have gradually become the mainstream direction of research and development.
[0003] Currently, industrially applied enzymatic methods mainly include two-step and one-step methods. The two-step method involves D-amino acid oxidase (DAAO) oxidizing CPC to glutaryl-7-aminocephalosporanic acid (GL-7-ACA), followed by hydrolysis catalyzed by GL-7-ACA acylase to produce 7-ACA. Although the two-step method is widely used, it still has significant drawbacks: the byproduct hydrogen peroxide (H2O2) generated in the first step not only triggers CPC degradation and generates byproducts, increasing the difficulty of downstream separation and purification, but also reduces the stability of the relevant enzymes, leading to a decrease in overall yield.
[0004] In contrast, the one-step enzymatic method, which directly catalyzes the conversion of CPC to 7-ACA via CPC acylase, offers advantages such as a shorter process, fewer byproducts, and environmental friendliness, and is considered a more promising technological approach. However, the number of acylases in nature capable of efficiently recognizing and catalyzing CPC is very limited. Currently known active enzymes mainly originate from a few Pseudomonas strains, such as Pseudomonas sp. SE83, P. diminuta N176, and Pseudomonas sp. P130. However, these natural enzymes are essentially GL-7-ACA acylases, exhibiting extremely low catalytic efficiency for CPC, typically only 2-4% of their activity for GL-7-ACA. To date, no wild-type CPC acylases with high CPC substrate activity have been discovered, severely limiting the industrial application of the one-step enzymatic method.
[0005] To overcome the problem of limited enzyme resources, researchers have attempted to modify known CPC acylatases using protein engineering methods (such as site-directed mutagenesis, directed evolution, and DNA shuffling) to enhance their catalytic activity toward CPC. However, existing modification strategies are still limited by the diversity of natural sequences, and traditional modification methods are unable to effectively expand the functional space of enzymes. There is an urgent need to develop innovative technological pathways that can explore novel sequence combinations and significantly improve CPC acylation activity. Summary of the Invention
[0006] The purpose of this invention is to overcome the shortcomings of existing one-step enzymatic production technology of 7-aminocephalosporanic acid (7-ACA), and to solve the problems of low substrate specificity of natural cephalosporin C acylase (CPC acylase), limited catalytic efficiency, and scarce enzyme source. This invention provides a novel CPC acylase with higher enzyme activity and stronger substrate recognition ability, so as to meet the industrial demand for efficient, economical, and green one-step enzymatic production of 7-ACA.
[0007] Currently, publicly available CPC acylases are mainly derived from a few microbial strains. They exhibit low enzyme activity, insufficient affinity for CPC substrates, and limited stability under commonly used industrial process conditions, making them unsuitable for large-scale biocatalysis applications. To overcome this bottleneck, this invention utilizes a combination of sequence analysis, recombination technology, and mutation optimization to systematically engineer existing acylases, successfully obtaining a class of CPC acylase variants with novel sequence characteristics and significantly improved catalytic performance.
[0008] To achieve the above technical objectives, the present invention adopts the following technical solution:
[0009] First, based on three representative CPC acylase source sequences—Bosea sp. OK40, Pseudomonas sp. GK16, and Pseudomonas sp. SE83—this invention utilizes the Schema algorithm to analyze the compatibility between structural modules, delineates homologous fragment regions, and employs DNA shuffling technology to perform in vitro fragment recombination, constructing a large-scale recombinant gene library. Subsequently, through high-throughput expression screening and functional evaluation, a CPC acylase with a novel amino acid sequence was obtained, the amino acid sequence of which is shown in SEQ ID NO:1.
[0010] Building upon this foundation, to further enhance the enzyme's catalytic efficiency, substrate adaptability, and stability, this invention identified multiple key functional sites through sequence alignment and structural analysis, and introduced or combined amino acid mutations such as T140S, L161S, F294L, N297S, Y271S, T314I, I366M, V415I, A421V, and N481S. These single-point mutations or any combination thereof further expanded the enzyme's structural plasticity, resulting in the obtained CPC acylate exhibiting significantly superior activity and adaptability compared to the natural enzyme in catalyzing the CPC deamidation reaction.
[0011] Through the above construction and screening strategies, the novel CPC acylase and its mutants were finally obtained. They not only catalyze CPC to 7-ACA efficiently, but also show significant advantages in catalytic efficiency, substrate specificity and tolerance. This fully demonstrates that the new sequences obtained through recombination and mutation design have achieved effective optimization of the catalytic mechanism.
[0012] Therefore, the present invention provides a cephalosporin C acylase, the amino acid sequence of which is selected from:
[0013] (a) SEQ ID NO:1; or
[0014] (b) Mutants with one or more of the following amino acid mutations relative to SEQ ID NO:1: T140S, L161S, F294L, N297S, Y271S, T314I, I366M, V415I, A421V, N481S.
[0015] The present invention also provides a nucleic acid molecule encoding the cephalosporin C acylase.
[0016] Specifically, the nucleotide sequence of the nucleic acid molecule is selected from:
[0017] (a) SEQ ID NO:2 or its degenerate sequence; or
[0018] (b) Compared with SEQ ID NO:2, there are one or more codon mutations to introduce the coding sequence of any one or a combination of T140S, L161S, F294L, N297S, Y271S, T314I, I366M, V415I, A421V, N481S; or their degenerate sequences.
[0019] The present invention further provides a recombinant vector comprising the aforementioned nucleic acid molecule.
[0020] The present invention further provides a recombinant host cell containing the aforementioned recombinant vector.
[0021] The present invention further provides a method for preparing the recombinant host cell, comprising:
[0022] (a) Construct a recombinant vector containing the nucleic acid molecule;
[0023] (b) Introducing the recombinant vector into host cells;
[0024] (c) Screening to obtain the recombinant host cells.
[0025] Specifically, the host cell is any one of Escherichia coli, yeast, plant cells, or non-human animal cells.
[0026] The present invention also provides the use of the cephalosporin C acylase, its encoding gene, the recombinant vector, or the recombinant host cell in the preparation of 7-aminocephalosporanic acid.
[0027] The present invention particularly provides a method for preparing 7-aminocephalosporanic acid, which uses cephalosporin C as a substrate and cephalosporin C acylase as a catalyst to catalyze the reaction to generate 7-aminocephalosporanic acid.
[0028] Specifically, the reaction system used CPC sodium salt as the substrate, and the composition of the reaction system was 50 mM Na2HPO4-KH2PO4 buffer (pH 8.0), final substrate concentration of 40 mM, and final concentration of purified cephalosporin C acylase of 0.2 mg / mL; the reaction was carried out at 25°C.
[0029] Furthermore, after the reaction is complete, the reaction is terminated by adding ammonium acetate buffer and methanol. Optionally, the reaction also includes a step of isolating or purifying the resulting 7-aminocephalosporanic acid.
[0030] The cephalosporin C acylase obtained through the technical solution of this invention possesses novel characteristic domains in its sequence structure. Furthermore, by introducing key mutations, synergistic optimization of the catalytic and substrate-binding regions was achieved, significantly improving its efficiency and process stability in catalyzing the formation of 7-ACA. This enzyme not only provides a new biocatalytic tool for one-step enzymatic production of 7-ACA but also offers a stable and efficient template for further engineering modification of CPC acylases. Attached Figure Description
[0031] Figure 1 The liquid phase spectrum and structural formula of CPC are shown.
[0032] Figure 2 Liquid phase diagram and structural formula of enzyme-catalyzed CPC to 7-ACA. Detailed Implementation
[0033] Example 1: Construction and acquisition of CPC acylase sequence using DNA shuffling technology
[0034] The amino acid sequences of CPC acylases derived from Bosea sp. OK40, Pseudomonas sp. GK16, and Pseudomonas sp. SE83 were used as initial templates (Table 1). First, the sequences were quality-screened, removing entries with deletions, premature termination, or obvious abnormalities. Then, multiple sequence alignment was performed to analyze the distribution characteristics of conserved and variable regions among the sequences. Based on this, and combined with previously reported homologous acylase structural information, the catalytic center, substrate-binding pocket, and structural fragments crucial to overall folding stability were identified. These regions were designated as protected areas that do not participate in cleavage during recombination, thereby avoiding adverse effects on the core structure and function of the enzyme.
[0035] After identifying the protected area, the processed template sequence was input into the Schema program for module division, and the structural incompatibility energy of different module recombination combinations was calculated to screen candidate recombination schemes with lower Schema energy. Simultaneously, sequence conservation analysis and protein physicochemical property prediction were combined to comprehensively evaluate the candidate sequences, thereby increasing the probability of obtaining recombinant CPC acylase sequences with good folding potential and expression stability. The final recombinase amino acid sequence is denoted as SEQ ID NO:1.
[0036] Table 1. Information on CPC acylases involved in this embodiment.
[0037]
[0038] Example 2: Expression of CPC acylase or its mutant gene
[0039] To obtain novel CPC acylase mutants with improved catalytic performance, a systematic structural and functional evaluation was conducted on the amino acid sequence and three-dimensional structure of the novel CPC acylase (SEQ ID NO:1) obtained in Example 1. First, substrate channel simulation analysis based on the CAVER program was performed on the enzyme structure to identify potential channel bottlenecks, channel radius variations, and key structural regions that may affect substrate entry into the active site. Simultaneously, protein residue interaction network analysis tools were used to quantify hydrogen bonding, hydrophobic interactions, and co-evolutionary characteristics within the structure to screen key node residues that play a central role in catalytic stability or conformational regulation. Combining the channel analysis and interaction network results with modeling of local structural fluctuation regions, candidate mutation sites including T140, L161, F294, N297, Y271, T314, I366, V415, A421, and N481 were identified.
[0040] Table 2. PCR reaction system
[0041]
[0042] Subsequently, site-directed mutagenesis primers were designed for the aforementioned sites, and single-point mutants such as T140S, L161S, F294L, N297S, Y271S, T314I, I366M, V415I, A421V, and N481S were constructed through site-directed mutagenesis. The PCR system composition is shown in Table 2, with a total system volume of 50 μL. The reaction conditions were: 98℃ pre-denaturation for 2 min; 30 cycles (98℃ for 10 s, 58℃ for 15 s, 72℃ for 5-15 s); and a final extension at 72℃ for 2 min. The amplified products were digested with DpnI and transformed into E. coli DH5α, and the mutations were confirmed to be correct by sequencing. After obtaining the single-point mutant plasmid, it was used as a template to successively stack mutation sites, and the same PCR-DpnI-screening process was used to construct two-point and multi-point combined mutants, ultimately obtaining a combined mutant gene containing multiple engineered mutations.
[0043] The correctly mutated recombinant plasmid was then transformed into E. coli BL21(DE3) competent cells. The transformed strains were inoculated onto LB solid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C. Single colonies were selected and inoculated into LB liquid medium containing kanamycin, and cultured at 37°C with shaking at 200 rpm until OD (out of control) was reached. 600 After reaching a concentration of 0.6–1.0, IPTG (final concentration 0.1 mmol / L) was added to induce protein expression, and the mixture was cultured at 25°C for another 12 hours.
[0044] After induction culture, the bacterial culture was centrifuged at 12000 rpm for 20 min (4℃) to collect the bacterial cells, and then treated with 50 mM Na2HPO4. 4- Cells were resuspended in KH2PO4 buffer (pH 6.0). Cell lysis was then performed using an ultrasonic disruptor (200W power, 5s operation, 5s interval, 20 min cycle, ice bath). The disrupted mixture was centrifuged at 12000 rpm for 30 min (4℃), and the supernatant was collected as the crude enzyme solution for subsequent enzyme activity assays and catalytic performance analysis.
[0045] Example 3: Isolation and purification of cephalosporin C acylase
[0046] Because the novel enzyme has a 6×His tag fused to its C-terminus, it was purified using Ni-NTA affinity chromatography. The purification steps are as follows:
[0047] a) The fermentation-induced bacterial broth was centrifuged and the supernatant was collected. The broth was then filtered through a 0.22 μm filter membrane to obtain a clear crude enzyme solution.
[0048] b) Ni-NTA column pre-equilibration, the equilibration buffer is 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0;
[0049] c) Load the sample at a flow rate of 0.5 mL / min to bind the His-tagged target protein to the packing material;
[0050] d) Wash the column bed with equilibration buffer containing 20 mM imidazole to remove non-specifically bound proteins;
[0051] e) Elute the target protein with an elution buffer containing 250 mM imidazole (50 mM NaH2PO4, 300 mM NaCl, pH 8.0);
[0052] f) Collect the elution fraction and dialyze it (molecular weight cutoff 10 kDa) in 50 mM Na2HPO4-KH2PO4 buffer (pH 6.0) to remove residual imidazole.
[0053] After purity was determined by SDS-PAGE, the enzyme solution after removing imidazole was temporarily stored at 4 degrees Celsius for subsequent enzyme activity analysis.
[0054] Example 4: CPC conversion activity assay
[0055] The catalytic activity of the purified enzyme for CPC was determined by high performance liquid chromatography (HPLC).
[0056] The analysis conditions are as follows:
[0057] The instrument was an Agilent 1260 Infinity liquid chromatography system; the column was a C18 reversed-phase column (4.6 mm × 250 mm, 5 μm); the mobile phase was a mixture of 20 mM ammonium acetate buffer (pH 7.2) and acetonitrile (95:5 v / v); the flow rate was 0.8 mL / min; the column temperature was 40 °C; the detection wavelength was 254 nm; and the injection volume was 10 μL.
[0058] The reaction system (1 mL) consisted of 50 mM Na₂HPO₄-KH₂PO₄ buffer (pH 8.0), 40 mM CPC substrate, and an appropriate amount of purified enzyme. The reaction was carried out at the corresponding temperature for 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 and centrifuged at 12000 rpm for 10 min (4℃). The supernatant was filtered through a 0.22 μm filter before analysis.
[0059] A standard curve was plotted using CPC and 7-ACA standards, and the product yield was calculated from the peak area to determine the enzyme catalytic activity. Enzyme activity was defined as the amount of enzyme required to catalyze the production of 1 μmol of 7-ACA per minute at pH 8.0, defined as 1 unit (U).
[0060] Example 5: Performance verification of CPC acylase and its mutants catalyzing the production of 7-ACA from CPC
[0061] The catalytic performance of the CPC acylase obtained in this invention was determined using the method described in Example 4. The reaction system used CPC sodium salt as the substrate, and the composition was 50 mM Na₂HPO₄-KH₂PO₄ buffer (pH 8.0), a final substrate concentration of 40 mM, a final purified enzyme concentration of 0.2 mg / mL, and a total volume of 1 mL. The reaction was carried out at 25°C for 1 h, and then terminated by adding 3 volumes of ammonium acetate buffer and 1 volume of methanol. After mixing, the mixture was placed on ice for 10 min, centrifuged at 12000 rpm for 10 min (4°C), and the supernatant was filtered through a 0.22 μm filter membrane for HPLC analysis.
[0062] like Figure 2 As shown, the results indicate that the peak area of the CPC substrate decreased significantly, while the peak area of the product 7-ACA increased significantly, with a calculated yield of 2.6 g / L. This demonstrates that the CPC acylase (SEQ ID NO:1) of this invention possesses significant catalytic activity and can efficiently convert CPC to 7-ACA. For reference, Figure 1 The liquid phase spectrum and structural formula of the substrate CPC are presented.
[0063] Furthermore, according to the method described in Example 2, single-site, double-site, and multi-site mutants were constructed by introducing mutation sites such as T140S, L161S, F294L, N297S, Y271S, T314I, I366M, V415I, A421V, and N481S using SEQ ID NO:1 as a template. These mutants were also validated under the same conditions. HPLC results showed that all mutants could catalyze the conversion of CPC to 7-ACA, and the characteristic peak of 7-ACA was observed in the product region, indicating that these mutants maintained the basic catalytic function of CPC acylase (Table 3).
[0064] Table 3. CPC acylases and their mutants catalyze the formation of 7-ACA from CPC.
[0065]
[0066] In summary, the CPC acylase and its allelic mutants containing the aforementioned target mutation sites provided by this invention can both catalyze the production of 7-ACA from CPC, demonstrating stable biocatalytic performance. These enzyme mutants not only provide new candidate molecules for the sequence and functional expansion of CPC acylases, but also offer a broader enzymatic basis and engineering application potential for one-step enzymatic preparation of 7-ACA.
Claims
1. A cephalosporin C acylase, characterized in that, Its amino acid sequence is selected from: (a) SEQ ID NO:1; or (b) Mutants with one or more of the following amino acid mutations relative to SEQ ID NO:1: T140S, L161S, F294L, N297S, Y271S, T314I, I366M, V415I, A421V, N481S.
2. A nucleic acid molecule encoding the cephalosporin C acylase of claim 1.
3. The nucleic acid molecule as described in claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is selected from: (a) SEQ ID NO:2 or its degenerate sequence; or (b) Compared with SEQ ID NO:2, there are one or more codon mutations to introduce the coding sequence of any one or a combination of T140S, L161S, F294L, N297S, Y271S, T314I, I366M, V415I, A421V, N481S; or their degenerate sequences.
4. A recombinant vector, characterized in that, It includes the nucleic acid molecule as described in claim 2 or 3.
5. A recombinant host cell, characterized in that, The host cell contains the recombinant vector as described in claim 4.
6. A method for preparing the recombinant host cell of claim 5, characterized in that, include: (a) Constructing a recombinant vector containing the nucleic acid molecule of claim 3; (b) Introducing the recombinant vector into host cells; (c) Screening to obtain the recombinant host cells.
7. The method according to claim 6, characterized in that, The host cell is any one of Escherichia coli, yeast, plant cells, or non-human animal cells.
8. The use of the cephalosporin C acylase as described in claim 1, its encoding gene, the recombinant vector as described in claim 4, or the recombinant host cell as described in claim 5 in the preparation of 7-aminocephalosporanic acid.
9. A method for preparing 7-aminocephalosporanic acid, characterized in that, Using cephalosporin C as a substrate and the cephalosporin C acylase as described in claim 1 as a catalyst, a catalytic reaction is carried out to generate 7-aminocephalosporanic acid.
10. The method as described in claim 9, characterized in that, The reaction system used CPC sodium salt as the substrate and consisted of 50 mM Na2HPO4-KH2PO4 buffer (pH 8.0), a final substrate concentration of 40 mM, and a final concentration of purified cephalosporin C acylase of 0.2 mg / mL. The reaction was carried out at 25°C. Furthermore, after the reaction was completed, the reaction was terminated by adding ammonium acetate buffer and methanol.