Hybridization principle is used to obtain high-activity amino peptidase mutants and their application
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGXI UNIV
- Filing Date
- 2026-02-04
- Publication Date
- 2026-06-09
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Figure CN122168568A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of enzyme engineering technology, specifically involving the use of the principle of hybridity to modify and obtain aminopeptidase mutants with high esterase activity. Background Technology
[0002] Enzyme heterogeneity refers to the ability of an enzyme to exhibit additional catalytic activity on substrates with different structures while maintaining its intrinsic catalytic function. This characteristic provides a theoretical basis for expanding the functional range of enzymes. Studies have shown that enzyme heterogeneity is usually related to the structural plasticity, conformational flexibility, and stability of different transition states of the active site, and is an important starting point for the functional evolution of new enzymes.
[0003] Aminopeptidases (APs) are a class of metal-dependent hydrolases widely found in bacteria, fungi, and higher organisms, capable of specifically cleaving amino acid residues from the N-terminus of polypeptides or proteins. These enzymes have significant applications in protein degradation, amino acid production, bioactive peptide preparation, and food processing. The catalytic performance of APs mainly depends on the spatial arrangement of key amino acid residues in their active site and the structural characteristics of the substrate-binding pocket, typically exhibiting high substrate specificity. With the rapid development of enzyme engineering and computer-aided protein design technologies, molecular modification of the active site of APs through site-directed mutagenesis, site-directed saturation mutagenesis, and protein structure optimization has become an important research direction for improving their catalytic performance and expanding their application potential.
[0004] However, natural aminopeptidases primarily exert their catalytic activity on peptide substrates, exhibiting generally weak catalytic activity against non-natural substrates, especially esters. This limits their application in complex industrial wastewater treatment, organic pollutant degradation, and green biocatalysis. Existing research largely focuses on enhancing the hydrolytic activity of aminopeptidases on peptide substrates, while studies on improving their hybrid esterase activity and expanding their substrate adaptability remain relatively scarce. Consequently, the functional diversity and catalytic efficiency of aminopeptidases in multi-substrate systems fail to meet the demands of practical industrial applications.
[0005] Therefore, rational molecular modification of aminopeptidases based on the principle of heterogeneity holds promise for enhancing their catalytic ability towards ester substrates without significantly affecting their intrinsic proteolytic activity and overall structural stability. However, effectively coordinating the relationship between substrate diversity, catalytic efficiency, and structural stability of aminopeptidases remains a pressing technical challenge in the field of enzyme engineering. Summary of the Invention
[0006] To overcome the problems of low catalytic efficiency and limited substrate adaptability of existing aminopeptidases in the degradation of ester compounds, this invention utilizes computer-aided design combined with enzyme engineering techniques such as site-directed mutagenesis and site-directed saturation mutagenesis to obtain an aminopeptidasease mutant with high esterase hybrid activity. Furthermore, this invention also provides the coding gene of the mutant, recombinant expression vector, recombinant strain, genetically engineered strain, and related applications, offering a feasible solution for the efficient biodegradation of ester pollutants.
[0007] One of the objectives of this invention is to provide a mutant aminopeptidase with high esterase activity obtained by modifying it using the principle of heterogeneity. The aminopeptidase mutant is obtained by the following mutation based on the wild-type aminopeptidase shown in SEQ ID NO.1: the amino acid at position 112 is mutated from serine to aspartic acid (S112D). Furthermore, the high-esterase-activity aminopeptidase mutant obtained by modifying it using the principle of heterogeneity can be obtained by generating the S112D-F352P mutant by causing a mutation at the F352P site based on S112D, and its amino acid sequence is shown in SEQ ID NO.3; Furthermore, the high-esterase-activity aminopeptidase mutant obtained by utilizing the principle of heterogeneity is obtained by further truncating the C-terminal propeptide of the S112D-F352P mutant shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.4.
[0008] A second objective of this invention is to provide applications of the aforementioned aminopeptidase mutant, particularly in the catalytic degradation of ester compounds.
[0009] A third objective of this invention is to provide a recombinant vector, a recombinant bacterial strain, and genetically engineered cells containing the aminopeptidase mutant encoding gene. The recombinant vector can be a conventional protein expression vector in the art, constructed through gene recombination technology. The expression vector includes pET22b(+), pET28a, pET32a, pGEX-4T, pMAL-p2x, and pMAL-c2X. The host cells of the recombinant bacterial strain include... E. coli BL21(DE3).
[0010] A fourth objective of this invention is to provide applications of the aforementioned aminopeptidase mutant encoding gene, recombinant vector, recombinant strain, and genetically engineered cells, particularly in the production of the aminopeptidase mutant. The genetically engineered cells contain and produce the aforementioned aminopeptidase S112D-F352P mutant and S112D-F352P-JD3 mutant; or contain the aforementioned recombinant vector and recombinant strain.
[0011] The fifth objective of this invention is to provide a high-throughput screening method for screening aminopeptidase mutants with esterase activity: (1) using a sterilized 96-well deep-well plate as a culture container, adding 300 μL of sterilized LB medium containing 100 μg / mL ampicillin to each well; (2) randomly picking clones of the transformed recombinant strain using sterile tools and inoculating them into the 96-well deep-well plate, wherein well A1 is set as an empty vector negative control ( E. coli pET-22b(+)), well A2 was set as a positive control, and cultured overnight at 37 ℃ and 220 rpm with shaking; (3) After the culture was completed, 200 μL of the overnight culture was transferred to a sterile 96-well glycerol preservation plate, 200 μL of 40% glycerol solution was added to each well, mixed well and sealed, and stored at −80 ℃ for a long time; at the same time, 600 μL of TB medium containing 100 μg / mL ampicillin was added to each well of the original 96-well deep plate, and the culture was passaged at 37 ℃ and 220 rpm for 6 h; (4) After the bacteria grew to a suitable state, isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 1 mM was added to each well as an inducer, and the culture conditions were adjusted to 20 ℃ and 220 rpm, and expression was induced for 12 h; (5) After the induction was completed, the 96-well deep plate was placed at 4 ℃ with 4000 Centrifuge at rpm for 10 min, collect the bacterial pellet, and wash the bacterial cells once with 50 mM phosphate buffer at pH 7.4. Then, resuspend the bacterial cells in 200 μL of the same buffer. (6) Add 100 μL of NP-40 lysis buffer and 200 μL of lysozyme at a concentration of 1 mg / mL to the resuspended bacterial cells. Incubate at 37 °C with shaking for 1 h to achieve cell lysis. After lysis, centrifuge at 4000 rpm for 30 min at 4 °C and collect the supernatant as crude enzyme solution for enzyme activity determination. (7) Add 160 μL of 1 mM p-nitrophenyl palmitate substrate solution and 40 μL of crude enzyme solution to each well of the microplate and shake to mix. Use the same volume of phosphate buffer instead of enzyme solution for the blank control. (8) React at 60 °C for 10 min and measure the absorbance at 410 nm to evaluate enzyme activity. Beneficial effects
[0012] This invention combines rational design with site-directed mutagenesis, supplemented by high-throughput screening technology, to target bacteria derived from *Pseudomonas aeruginosa* (…). Pseudomonas aeruginosa Directed evolution of the aminopeptidase (PaAps) of GF31 was successfully carried out, resulting in aminopeptidase mutants S112D, S112D-F352P and S112D-F352P-JD3 with significantly enhanced esterase activity.
[0013] Compared with the wild type, the above mutants showed 5.39-fold, 18.14-fold, and 12.64-fold increased activity toward p-nitrophenyl palmitate (pNPP), respectively. In the application of catalytic degradation of ester compounds, they can hydrolyze the ester bonds in ester compounds to generate the corresponding acids and alcohols. Moreover, their catalytic activity is significantly higher than that of the corresponding wild-type aminopeptidase, which significantly broadens the application prospects of aminopeptidase in the biodegradation of ester pollutants and related industrial fields.
[0014] Based on the S112D mutant, fine-tuning of the local environment of F352P significantly improved the enzyme's catalytic efficiency and thermal stability; the truncation mutation did not achieve the corresponding co-evolution effect, so the catalytic efficiency and thermal stability of S112D-F352P-JD3 were lower than those of S112D-F352P, but still higher than those of the wild type.
[0015] Based on the S112D mutant, the binding kinetics between the enzyme and the hydrophobic substrate can be effectively regulated by the synergistic regulation of the F352P mutation and the removal of the propeptide, providing a feasible technical approach for optimizing the enzyme's substrate recognition ability and catalytic performance.
[0016] A high-throughput screening method is provided for screening aminopeptidase mutants with esterase activity. The method can rapidly and effectively screen enzyme molecules that exhibit significant catalytic activity in the degradation of ester compounds from a variety of aminopeptidase mutants, achieving rapid and sensitive detection of ester substrate hydrolysis reactions. It can simultaneously evaluate the substrate specificity, catalytic activity, and stability of aminopeptidase mutants, providing technical support for the development of multifunctional enzymes with mixed aminopeptidase and esterase activities. Attached Figure Description
[0017] Figure 1 The image shows the pET-22b(+)-APS recombinant plasmid.
[0018] Figure 2 The results show the esterase activity assays of 36 PaAps mutants obtained through multiple computational design.
[0019] Figure 3 Structural analysis of the substrate-binding pocket of the S112D mutant. (A) Amino acid composition of the substrate-binding pocket of the S112D mutant; (B) Spatial configuration of the substrate-binding pocket of the S112D mutant; (C) Charge distribution characteristics inside the substrate-binding pocket of the S112D mutant, in which the pocket exhibits obvious negative charge.
[0020] Figure 4 The enzyme activity of the S112D 5Å active pocket alanine scanning mutant was determined.
[0021] Figure 5This is a schematic diagram of a high-throughput screening technique for the esterase activity of aminopeptidase mutants.
[0022] Figure 6 High-throughput screening results of site-directed saturation mutagenesis at the F352 site using the S112D mutant as a template.
[0023] Figure 7 This diagram illustrates the C-terminal propeptide cleavage and enzyme activation mechanism of PaAps. The C-terminal propeptide sequence is located between the PA domain and the peptidase domain, forming a "lid"-like structure that induces the enzyme to be in a closed, inactive conformation, thus blocking the active pocket. Cleavage of this disordered propeptide structure by truncating primers allows larger ester substrates to smoothly enter the active pocket and undergo a catalytic reaction, generating the corresponding acid and alcohol.
[0024] Figure 8 Optimal reaction temperature and thermal stability analysis for PaAps and its mutants. (A) Optimal reaction temperature; (B) Temperature stability.
[0025] Figure 9 Molecular dynamics simulation analysis of the local flexibility of the active pocket and its interaction with the substrate in S112D and S112D-F352P mutants. (A) Molecular interaction between S112D mutant and pNPP; (B) Molecular interaction between S112D-F352P mutant and pNPP; (C) Root mean square fluctuation (RMSF) analysis of S112D and S112D-F352P mutants; (D) Dihedral distribution analysis of the side chain of the key residue F352 in PaAps, S112D, and S112D-F352P.
[0026] Figure 10 Substrate channel characterization analysis for S112D-F352P and its C-terminal propeptide truncated mutant S112D-F352P-JD3.
[0027] Figure 11 Analysis of the dynamic volume changes and conformational characteristics of the active pockets of PaAps and its mutants. Detailed Implementation
[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below. However, it should be understood that the description herein is merely illustrative and not intended to limit the scope of the invention.
[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention. All reagents and instruments used herein are commercially available, and the characterization methods involved are described in relevant prior art and will not be repeated herein.
[0030] The wild-type strain has the following biosecurity number: CGMCC 7173 Strains and vectors: Escherichia coli expression vector pET-22b(+), host strain E. coli BL21(DE3).
[0031] LB medium: tryptone 10 g / L; yeast extract 5 g / L; NaCl 10 g / L; pH=7.4.
[0032] The amino acid and nucleotide sequences involved in this application are listed in the sequence listing, which forms part of this specification.
[0033] The sequence list is as follows: SEQ ID NO.1: TPGKPNPSICKSPLLVSTPLGLPRCLQASNVVKRLQKLEDIASLNDGNRAAATPGYQASVDYVKQTLQKAGYKVSVQPFPFTAYYPKGPGSLSATVPQPVTYEWEKDFTYLSQTEAGDVTAKVVP VDLSLGAGNTSTSGCEAEDFANFPAGSIALIQRGTCNFEQKAENAAAAGAAGVIIFNQGNTDDRKGLENVTVGESYEGGIPVIFATYDNGVAWSQTPDLQLHLVVDVVRKKTETYNVVAETRRGN PNNVVMVGAHLDSVFEGPGINDNGSGSAAQLEMAVLLAKALPVNKVRFAWWGAEEAGLVGSTHYVQNLAPEEKKKIKAYLNFDMIGSPNFGNFIYDGDGSDFGLQGPPGSAAIERLFEAYFRLRG QQSEGTEIDFRSDYAEFFNSGIAFGGLFTGAEGLKTEEQAQKYGGTAGKAYDECYHSKCDGITNINQDALEIHSDAMAFVTSWLSLSTKVVDDEIAAAGQKAQSRSLQMQKSASQIERWGHDFIK SEQ ID NO.2: SEQ ID NO.3: TPGKPNPSICKSPLLVSTPLGLPRCLQASNVVKRLQKLEDIASLNDGNRAAATPGYQASVDYVKQTLQKAGYKVSVQPFPFTAYYPKGPGSLSATVPQPVTYEWEKDFTYLDQTEAGDVTAKVVPVDLSLGAGNTSTSGCEAEDFANFPAGSIALIQRGTCNFEQKAENAAAAGAAGVIIFNQGNTDDRKGLENVTVGESYEGGIPVIFATYDNGVAWSQTPDLQLHLVVDVVRKKTETYNVVAETRRGNPNNVVMVGAHLDSVFEGPGINDNGSGSAAQLEMAVLLAKALPVNKVRFAWWGAEEAGLVGSTHYVQNLAPEEKKKIKAYLNFDMIGSPNFGNFIYDGDGSDPGLQGPPGSAAIERLFEAYFRLRGQQSEGTEIDFRSDYAEFFNSGIAFGGLFTGAEGLKTEEQAQKYGGTAGKAYDECYHSKCDGITNINQDALEIHSDAMAFVTSWLSLSTKVVDDEIAAAGQKAQSRSLQMQKSASQIERWGHDFIK SEQ ID NO.4: TPGKPNPSICKSPLLVSTPLGLPRCLQASNVVKRLQKLEDIASLNDGNRAAATPGYQASVDYVKQTLQKAGYKVSVQPFPFTAYYPKGPGSLSATVPQPVTYEWEKDFTYLDQTEAGDVTAKV VPVDLSLGAGNTSTSGCEAEDFANFPAGSIALIQRGTCNFEQKAENAAAAGAAGVIIFNQGNTDDRKGLENVTVGESYEGGIPVIFATYDNGVAWSQTPDLQLHLVVDVVRKKTETYNVVAET RRGNPNNVVMVGAHLDSVFEGPGINDNGSGSAAQLEMAVLLAKALPVNKVRFAWWGAEEAGLVGSTHYVQNLAPEEKKKIKAYLNFDMIGSPNFGNFIYDGDGSDPGLQGPPGSAAIERLFEA YFRLRGQQSEGTEIDFRSDYAEFFNSGIAFGGLFTGAEGLKTEEQAQKYGGTAGKAYDECYHSKCDGITNINQDALEIHSDAMAFVTSWLSLSTKVVDDEIAAAGQKAQSRSLQMQKSASQIE Example 1 Construction of single-point and multi-point mutants of aminopeptidase From Pseudomonas aeruginosa Using the wild-type aminopeptidase PaAps gene of GF31 as the target for modification (its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence encoding the enzyme is shown in SEQ ID NO.2), site-directed mutagenesis primers were designed. Using rapid PCR technology, with plasmid pET-22b(+)-APS as a template, a single-point mutation was introduced at position 112, and a site-directed saturation mutation was introduced at position 352 of the S112D mutant. The pET-22b(+)-APS recombinant plasmid map is shown below. Figure 1 As shown.
[0034] The primer design is as follows: S112D-F: 5'-TTCACCTACCTGgacCAGACCGAAGCTGGCGATG-3' (SEQ ID NO. 5).
[0035] S112D-R: 5'-TGgtcCAGGTAGGTGAAATCTTTTTCCCATTCG-3' (SEQ ID NO. 6).
[0036] F352N-F: 5'-TCTGATNKGGCCTGCAGGGTCCGCCGGGC-3' (SEQ ID NO. 7).
[0037] F352N-R: 5'-CAGGCCMNATCAGAGCCGTCGCCATCGTAG-3' (SEQ ID NO. 8).
[0038] PCR reaction system: PrimeSTAR® Max DNA Polymerase Ver.2 10μL, upstream primer 0.5μL (5pmol / μL), downstream primer 0.5μL (5pmol / μL), template DNA 0.5μL (50ng / μL), ddH2O 8.5μL.
[0039] PCR reaction procedure: Step 1, pre-denaturation at 98℃ for 1 min; Step 2, denaturation at 98℃ for 10 s; Step 3, annealing at 55℃ for 15 s; Step 4, extension at 72℃ for 4.5 min; repeat steps 2 to 4 35 times; Step 5, final extension at 72℃ for 5 min; Step 6, incubate at 12℃ for ∞.
[0040] Template removal and recombination: The PCR product was digested with DpnI enzyme at 37°C for 1 h to remove the methylated original template plasmid; then the linearized plasmid fragment was circularized and recombinated using homologous recombination enzyme at 37°C for 30 min.
[0041] Transformation and validation: Add 10 μL of the recombinant product to 100 μL of ice bath. E. coli In BL21(DE3) competent cells, the cells were incubated on ice for 30 min. The transformation product was then heat-shocked at 42°C for 45 s, rapidly cooled on ice for 2 min, and 900 μL of antibiotic-free LB liquid medium was added to the tubes. The cells were incubated at 37°C, 180 rpm / min for 1 h, centrifuged at 5000 rpm / min for 10 min, and 700 μL of supernatant was discarded, with the cells resuspended. 200 μL of the resuspended cells were spread onto LB agar plates containing 50 mg / mL kanamycin resistance and incubated upside down at 37°C for 12 h. Single colonies were picked and inoculated into 5 mL of LB liquid medium containing 50 mg / mL kanamycin resistance and incubated at 37°C for 12 h. The resulting culture was then sent to BGI Genomics for sequencing verification.
[0042] Example 2 Construction of aminopeptidase truncated mutant Based on the amino acid sequence of PaAps, three pairs of truncated primers were designed using Snapgene software. The amplification procedure and system for the target fragment were the same as above.
[0043] Primer-JD-F: 5'-AGCCGGCGATGGCCATGGGCACACC-3' (SEQ ID NO. 7).
[0044] Primer-JD-R: 5'-GTGGTGGTGCTCGAGGATCTGGCTTGCGCTTT-3' (SEQ ID NO. 8).
[0045] Primer-JD2-F: 5'-AGCCGGCGATGGCCATGGGCACACC-3' (SEQ ID NO. 9).
[0046] Primer-JD2-R: 5'-GTGGTGGTGCTCGATCTGGCTTGCGCTTTC-3' (SEQ ID NO. 10).
[0047] Primer-JD3-F: 5'-AGCCGGCGATGGCCATGGGCACACC-3' (SEQ ID NO. 11).
[0048] Primer-JD3-R: 5'-GTGGTGGTGCTCGAGTTCGATCTGGCTTGCGCT-3' (SEQ ID NO. 12).
[0049] The plasmid pET-22b(+)-APS was double-digested with NcoI and XhoI. The specific digestion system is shown in Table 1. After gently mixing with a pipette, the mixture was placed at 37℃ for 2 hours to complete the digestion reaction.
[0050]
[0051] Agarose gel electrophoresis and gel recovery: Weigh 0.5 g of agarose, add 50 mL of 1×TAE buffer, heat until completely dissolved, then add 1 μL of Gold View dye and mix thoroughly. Pour the mixture into a gel well with the comb inserted and let it stand for about 30 min. Take 10 μL of PCR product and mix it with 3 μL of 10×Loading Buffer, then add it to the wells of the solidified agarose gel. Perform electrophoresis at 120 V for 30 min. After electrophoresis, observe the bands under UV light to verify the site-directed mutagenesis PCR products. The verified PCR products are treated with DpnI restriction endonuclease to digest the methylated template DNA. For plasmid DNA fragments with band lengths consistent with expectations, recovery is performed using the Novizan gel recovery kit, following the kit instructions.
[0052] The amplified target gene and the gel-recovered vector were added according to the system shown in Table 2, and then incubated at 50°C for 15 min. After the reaction, the reaction product was placed on ice for 1-2 min to terminate the reaction and complete the recombination reaction. The reaction product was directly transformed into the target gene according to the method in Example 1. E. coli BL21(DE3) competent cells.
[0053]
[0054] Example 3 Expression and purification of aminopeptidase mutant The mutant strains that were correctly sequenced in Examples 1 and 2 were transferred to glass vials containing 3 ml of LB liquid medium containing 100 μg / mL ampicillin and cultured at 37°C and 180 rpm / min for 12 h. Subsequently, 2% (v / v) of the seed culture was inoculated into fresh LB medium containing the corresponding antibiotic and cultured at 37°C and 220 rpm until the OD600 reached 0.6-0.8. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mM and the culture was transferred to 20°C for 12 h to induce the expression of the target enzyme.
[0055] The fermentation broth was centrifuged at 10,000 rpm for 10 min at 4°C to collect the bacterial cells. The cells were then washed once with 50 mM, pH 8.0 PBS buffer and resuspended. The bacterial suspension was then sonicated in an ice bath under the following conditions: 3 seconds on, 5 seconds off, for a total of 20 min. The resulting lysate was centrifuged at 10,000 rpm for 10 min at 4°C to collect the supernatant, which is the crude enzyme solution.
[0056] Protein purification was performed at 4°C. First, the Ni-NTA affinity chromatography column was equilibrated with buffer A (50 mM phosphate buffer, 0.3 M NaCl, pH 7.4), and the crude enzyme solution was loaded at a flow rate of 1 mL / min. After loading, unbound contaminating proteins were eluted with buffer B (50 mM phosphate buffer, 0.3 M NaCl, 20 mM imidazole, pH 7.4); then, the target protein was eluted with buffer C (50 mM phosphate buffer, 0.3 M NaCl, 250 mM imidazole, pH 7.4). The collected target protein was purified by SDS-PAGE, desalted using a BeyoDesalt™ G-25 desalting column, and concentrated using Amicon® Ultra ultrafiltration tubes for subsequent enzymatic characterization. Protein concentration was determined using the Beyotime Bradford Protein Analyzer.
[0057] Example 4 Enzymatic Properties Determination of Aminopeptidase PaAps and its Mutants Using L-leucine-p-nitroaniline and p-nitrophenyl palmitate as substrates, the enzyme activity and thermostability of wild-type and mutant enzymes were determined. The specific methods are as follows: Assay for aminopeptidase activity: 3.8 mL of 50 mM Tris-HCl buffer (pH 8.0) containing 500 mg / L Leu-pNA was added, and 0.2 mL of enzyme solution diluted to a suitable concentration was added to make the total reaction volume 4.0 mL. The reaction was carried out in a parallel synthesizer at 80 °C and 150 rpm for 10 min, followed by the addition of 0.2 mL of glacial acetic acid to terminate the reaction. During the reaction, the release of p-nitroaniline (pNA) caused an increase in the absorbance of the system at 405 nm. The absorbance was detected using an Epoch 2 microplate reader (Agilent, USA), with a system without enzyme solution serving as a blank control. One unit (U) of aminopeptidase activity was defined as the amount of enzyme that hydrolyzes Leu-pNA to produce 1 μmol of pNA per minute at pH 8.0 and 80 °C.
[0058] Esterase activity assay: Solution A was prepared as a 10 mM palmitic acid p-nitrobenzene solution in isopropanol and stored in a brown bottle at 4°C. Solution B was prepared as 50 mM pH 8.0 phosphate buffer with 1% (v / v) Triton X-100 added and incubated in a water bath at 60°C for 5 min. Solutions A and B were thoroughly mixed at a ratio of 1:9 to obtain a working solution with a substrate concentration of 1 mM. 2.8 mL of each substrate solution was placed in three reaction flasks, and 200 μL of enzyme solution was added to each. A blank control group was set up, using an equal volume of phosphate buffer instead of enzyme solution. The reaction system was incubated at 60°C and 220 rpm for 10 min, and then 200 μL of anhydrous ethanol was added to terminate the reaction. The absorbance was then measured at 410 nm. Enzyme activity was defined as the amount of enzyme required to catalyze the hydrolysis of palmitic acid p-nitrobenzene to produce 1 μmol of p-nitrophenol per minute at 410 nm, defined as one enzyme activity unit (U).
[0059] High-throughput screening method for esterase activity: A sterilized 96-well deep-well plate was used as the culture container. 300 μL of sterilized LB medium containing 100 μg / mL ampicillin was added to each well. Subsequently, clones of the transformed recombinant strain were randomly picked using sterile tools and inoculated into the 96-well deep-well plate, with well A1 serving as a negative control for empty vector. E. colipET-22b(+)) was used, with well A2 set as a positive control, and cultured overnight with shaking at 37 ℃ and 220 rpm. After culture, 200 μL of the overnight culture was transferred to a sterile 96-well glycerol plate, and 200 μL of 40% glycerol solution was added to each well. The plate was mixed, sealed, and stored at −80 ℃ for long-term preservation. Simultaneously, 600 μL of TB medium containing 100 μg / mL ampicillin was added to each well of the original 96-well deep-well plate, and the plate was passaged at 37 ℃ and 220 rpm for 6 h. After the cells reached a suitable growth state, isopropyl-β-D-thiogalactoside (IPTG) was added to each well as an inducer at a final concentration of 1 mM, and the culture conditions were adjusted to 20 ℃ and 220 rpm for 12 h of induction expression. After induction, the 96-well deep-plate was centrifuged at 4000 rpm for 10 min at 4 ℃, and the bacterial pellet was collected. The pellet was washed once with 50 mM phosphate buffer (pH 7.4), and then resuspended in 200 μL of the same buffer. 100 μL of NP-40 lysis buffer and 200 μL of 1 mg / mL lysozyme were added to the resuspended pellet, and the mixture was incubated at 37 ℃ with shaking for 1 h to achieve cell lysis. After lysis, the pellet was centrifuged at 4000 rpm for 30 min at 4 ℃, and the supernatant was collected as the crude enzyme solution for enzyme activity assay. 160 μL of 1 mM p-nitrophenyl palmitate substrate solution and 40 μL of crude enzyme solution were added to each well of the microplate, and the mixture was shaken to mix. The blank control was replaced with the same volume of phosphate buffer instead of the enzyme solution. The reaction was carried out at 60 ℃ for 10 min, and the absorbance was measured at 410 nm to assess enzyme activity.
[0060] Thermal stability determination: The enzyme solution was incubated in a 60℃ constant temperature water bath. Residual enzyme activity was measured at different time points. Using the initial enzyme activity as 100%, a curve showing the change in enzyme activity over incubation time was plotted. The enzyme half-life (T1 / 2) was calculated by fitting the curve. The experimental results are shown below. Figure 8 As shown.
[0061] The test results are shown below. Figure 2 , Figure 4 and Figure 8 The results showed that among the 36 PaAps mutants obtained through multiple computational design, S59H, S112D, S112I, and W300F exhibited the most significant increases in esterase activity, especially the S112D mutant, whose esterase activity was approximately 5.39 times higher than the wild type. Alanine scanning of the amino acid residue sites within the 5 Å activity pocket of S112D revealed a significant increase in esterase activity at the F352 site. Site-directed saturation mutagenesis was performed at the F352 site, resulting in the S112D-F352P mutant. Figure 3 , Figure 6 Furthermore, we performed C-terminal truncation mutations on the wild-type PaAps and the S112D mutant, finding that the esterase activity of PaAps-JD3 increased to 228.52%, and the aminopeptidase activity was also partially enhanced. This indicates that moderate removal of the C-terminal propeptide can effectively reduce the spatial restriction at the substrate channel inlet, making it easier for mixed substrates to enter the active pocket, thereby accelerating the catalytic process. Therefore, we performed a superposition mutation in this truncation mode on the locally finely tuned S112D-F352P mutant, obtaining the S112D-F352P-JD3 mutant. Compared with the wild type, the activities of the mutants S112D-F352P and S112D-F352P-JD3 on p-nitrobenzene palmitate (pNPP) were increased by 18.14-fold and 12.64-fold, respectively.
[0062] Its dynamic characteristics were measured, as shown in Table 3.
[0063] The kcat / Km value of the S112D-F352P is 355.57M. -1 S -1 The efficiency of S112D-F352P was significantly higher than that of the wild type, indicating that the fine-tuning of the local environment of F352P significantly improved the enzyme's catalytic efficiency. Corresponding thermostability half-life (t1 / 2) analysis showed that at 60℃, the t1 / 2 of S112D-F352P was 144.10 min, significantly higher than that of wild-type PaAps, further confirming that these mutations played a positive role in improving the enzyme's thermostability. However, the catalytic efficiency and thermostability of S112D-F352P-JD3 were lower than those of S112D-F352P, but still higher than those of the wild type, indicating that the truncated mutation did not achieve the corresponding co-evolutionary effect.
[0064] Example 5: Substrate channel identification and molecular dynamics simulation analysis of aminopeptidase PaAps and its mutants The substrate channels and molecular dynamics of the active pocket in wild-type and mutant strains were simulated using CAVER and GROMACS software to analyze the channel size, active pocket size, and structural stability. The mutant topology file was generated using the AMBER99SB force field, and the substrate topology parameters were generated using GAFF (General AMBER Force Field). The AM1-BCC charge was calculated using the Anttecamber tool and converted to a GROMACS-compatible file using an ACPYPE script. The complex system was placed in a trislanted box composed of TIP3P water molecules, ensuring a minimum distance of 1.0 nm between the protein surface and the box edge. Electroneutrality was achieved by adding appropriate amounts of Na+ and Cl−, and the salt concentration was adjusted to 150 mM to simulate physiological conditions. The system was first subjected to energy minimization to eliminate undesirable contacts and high-energy conformations, followed by a two-stage equilibration: a 100 ps NVT ensemble (isothermal and isovolume) equilibration, with the temperature maintained at 310 K using a V-rescale temperature coupler; and a subsequent 100 ps NPT ensemble (isothermal and isobaric) equilibration, with the pressure maintained at 1 bar. After equilibration, productive molecular dynamics (MD) simulations were performed on each complex system for 100 ns. The Leap-frog integration algorithm was used during the production phase, with an integration step size of 2 fs. All hydrogen bonds and bond lengths were constrained using the LINCS algorithm. Temperature and pressure were maintained using a V-rescale temperature coupler and a Parrinello-Rahman pressure coupler, respectively. The simulation trajectory was saved every 100 ps. The simulation results were analyzed and visualized using VMD and OriginPro software.
[0065] See the mechanism of flexible local conformation regulation. Figure 9Within the amino acid sequence 329–390, the root mean square fluctuation (RMSF) of the S112D-F352P mutant was generally higher than that of the S112D mutant, with particularly significant differences at F352 and its adjacent residues, indicating that this mutation significantly enhances the local conformational flexibility of the active pocket entrance region. Specifically, the RMSF value at the F352 site in S112D-F352P was 0.46 Å, higher than the 0.39 Å in S112D, indicating that the F352P mutation effectively improves the dynamic tunability of the substrate channel region. Further dihedral analysis of the side chains showed that the aromatic side chain of F352 in wild-type PaAps has a stable conformation, exhibiting typical "gated" characteristics, while the F352P mutation, due to the replacement of the aromatic side chain with a proline ring structure, eliminates the original rigid constraints, significantly enhancing the flexibility of the active pocket entrance region and exerting a synergistic regulatory effect on the conformation of adjacent residues. Substrate interaction analysis further revealed that the binding mode of substrate pNPP in the S112D-F352P mutant changed from being dominated by hydrophobic interactions to being stabilized by a combination of hydrophobic interactions and hydrogen bonds. The newly added stable hydrogen bond with GLU105 significantly optimized the substrate binding configuration and transition state environment, thereby facilitating substrate conversion and product release and improving the overall catalytic efficiency.
[0066] Substrate channel characterization of S112D-F352P and its S112D-F352P-JD3 mutants is described in [link to analysis]. Figure 10 The S112D-F352P mutant forms five structurally sound substrate channels. Channels 2, 3, and 4 are located inside the active pocket, with channel 2 at the pocket entrance playing a dominant role in substrate entry into the active site. Channels 1 and 5 are interconnected and located outside the active pocket, forming auxiliary substrate transport pathways. The bottleneck radii of these channels are within a reasonable range, enabling directional transport and appropriate retention of the substrate during entry into the active pocket, thus promoting efficient catalytic reactions. In contrast, the S112D-F352P-JD3 mutant forms only two substrate channels, both concentrated inside the active pocket. Although the bottleneck radii are larger, the reduced number of channels and their relatively dispersed conformational distribution decrease channel stability and substrate transport directionality, thus limiting further improvements in catalytic efficiency.
[0067] The dynamic characteristics and structural physicochemical properties of the active pocket are analyzed in [reference needed]. Figure 11Molecular dynamics simulations based on the 10–100 ns timescale and SiteMap physicochemical parameter analysis results show that the active pocket of the S112D-F352P mutant achieves synergistic optimization in terms of volume dynamics, spatial stability, and microenvironment characteristics. Compared with the single S112D mutant, the S112D-F352P active pocket has a larger average volume and a significantly reduced volume fluctuation amplitude, and its spatial displacement trajectory is more concentrated, indicating that this mutation effectively enhances conformational stability while improving pocket flexibility. Further physicochemical property analysis shows that the F352P mutation significantly improves the hydrophobicity of the active pocket and improves the hydrophobic-hydrophilic balance, thereby forming a microenvironment more conducive to the binding and stable localization of hydrophobic ester substrates. In contrast, while S112D-F352P-JD3 further increased the active pocket volume and enhanced the overall exposure by truncating the propeptide, the complete removal of the propeptide weakened the local conformational constraints, resulting in significant volume and spatial fluctuations in the active pocket over time. Some conformations even experienced instantaneous collapse, leading to a decrease in pocket stability and substrate localization ability. This structurally limited further synergistic improvement in its catalytic efficiency.
[0068] According to the Gibbs binding free energy analysis of MM / PBSA in Table 4, the ΔG_bind of S112D-F352P was -20.85 kcal / mol, significantly lower than that of S112D (-17.35 kcal / mol), indicating that local conformational regulation enhanced the substrate binding capacity. However, the ΔG_bind of S112D-F352P-JD3 further decreased to -22.64 kcal / mol, but the enzyme activity was lower than that of the S112D-F352P mutant, indicating that excessive conformational freedom led to an increase in non-productive binding, offsetting part of the local optimization effect of F352P.
[0069]
[0070] Example 6: Determination of the affinity of aminopeptidase PaAps and its mutants for cypermethrin The interaction between S112D and its mutants and the hydrophobic small molecule substrate cypermethrin was quantitatively detected using bio-layer interferometry (BLI), as shown in Table 5.
[0071] The molecular recognition and binding abilities of different mutants to substrates were systematically evaluated by measuring the binding rate constant (Kon), dissociation rate constant (Koff), and equilibrium dissociation constant (KD). The results showed that the KD of S112D and cypermethrin was 1.77 × 10⁻⁻⁻⁶. 6M has low binding stability; after introducing the F352P mutation, KD decreased to 5.57 × 10⁻ 7 The mutation at site M significantly reduced the dissociation rate, indicating that the mutation at this site enhances the binding stability of the active pocket to hydrophobic substrates through a conformational constraint effect. Further analysis of the F352P mutant with propeptide cleavage yielded S112D-F352P-JD3, which had a KD of 7.24 × 10⁻⁻⁶. 7 The increased binding rate Kon (M) indicates that propeptide removal improves substrate accessibility to the active pocket, but the increased dissociation rate Koff reflects a decrease in pocket conformational stability. These results demonstrate that the synergistic regulation of site mutation and propeptide removal can effectively modulate the binding kinetics between the enzyme and hydrophobic substrates, providing a feasible technical approach for optimizing enzyme substrate recognition and catalytic performance.
Claims
1. An aminopeptidase mutant, characterized in that, The aminopeptidase mutant is obtained by any of the following mutations based on the wild-type aminopeptidase shown in SEQ ID NO.1: the 112th amino acid is mutated from serine to aspartic acid, i.e., the S112D mutant; based on S112D, a mutation is performed at the F352P site, i.e., the S112D-F352P mutant is obtained, the amino acid sequence of which is shown in SEQ ID NO.3; based on the S112D-F352P mutant shown in SEQ ID NO.3, its C-terminal propeptide is further truncated, i.e., the S112D-F352P-JD3 mutant is obtained, the amino acid sequence of which is shown in SEQ ID NO.
4.
2. The encoding gene of the aminopeptidase mutant according to claim 1.
3. A recombinant vector or recombinant strain containing the encoding gene of claim 2, wherein the recombinant expression vector comprises pET22b(+), pET28a, pET32a, pGEX-4T, pMAL-p2x, and pMAL-c2X; and the host cell of the recombinant strain comprises E. coli BL21(DE3).
4. Genetically engineered cells containing the recombinant vector or recombinant strain of claim 3, wherein the genetically engineered cells contain and express the aminopeptidase S112D-F352P mutant and S112D-F352P-JD3 mutant of claim 1.
5. The use of the recombinant vector, recombinant strain or genetically engineered cell as described in claim 3 or 4 in the production of the mutant as described in claim 1.
6. The application of the aminopeptidase mutant according to any one of claims 1 to 4 in the treatment of ester contaminants.
7. The application of the aminopeptidase mutant as described in claim 1 in the catalytic degradation of ester compounds.