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Article

Development and Biochemical Characterization of Quorum Quenching Enzyme from Deep-Sea Bacillus velezensis DH82

by
Xiaohui Sun
1,*,
Jia Liu
1,
Ying Yan
1,
Suping Yang
1,
Guangya Zhang
1 and
Hala F. Mohamed
2,*
1
College of Chemical Engineering, Huaqiao University, Xiamen 361021, China
2
Department of Botany and Microbiology, Faculty of Science, Al-Azhar University (Girls Branch), Cairo 11651, Egypt
*
Authors to whom correspondence should be addressed.
Microorganisms 2025, 13(8), 1717; https://doi.org/10.3390/microorganisms13081717
Submission received: 31 May 2025 / Revised: 5 July 2025 / Accepted: 14 July 2025 / Published: 22 July 2025
(This article belongs to the Special Issue Microbes in Aquaculture)
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Abstract

Quorum quenching (QQ) is of interest for potential application as a sustainable strategy for bacterial disease control via communication interruption. The QQ enzyme can be used as a good alternative antagonist to combat antibiotic abuse and bacterial resistance. Here, genomic DNA sequencing was performed on N-acyl homoserine lactonase from the deep-sea strain Bacillus velezensis DH82 with Cluster of Orthologous Groups of proteins (COGs) annotation. The homologous sequences with β-lactamase domain-containing protein were predicted to be potential QQ enzymes and were cloned and expressed to study their quorum quenching properties by comparing them with the reported enzyme AiiA3DHB. The experimental results of enzyme activity analysis and steady-state kinetics, as well as enzyme structure and substrate docking simulations and predictions, all consistently demonstrated that YtnPDH82 presented superior enzyme structural stability and higher degradation efficiency of N-acyl homoserine lactones than AiiADH82 under the effects of pH, and temperature, and performed better on short -chain and 3-O-substituted AHSLs. The findings revealed the structural and biochemical characterization of YtnPDH82 from the deep sea, which provide the capacity for further application in sustainable aquaculture as an alternative to antibiotics.

1. Introduction

Intensive and high-stocking-density aquaculture generates a high output of aquatic animals but also leads to a high risk of pathogen infection and frequent bacterial disease in aquatic animals. The traditional technique for solving this problem involves using antibiotics or chemical compounds to inhibit pathogens and prevent bacterial infection. However, the abuse of antibiotics and drug residues can cause bacterial drug resistance, which has increasingly raised concerns about food safety and environmental protection; thus, an alternative strategy for biological disease control is required to support the development of sustainable aquaculture.
Aquatic pathogens are mainly Gram-negative bacteria, such as Vibrio sp., Aeromonas sp., and Pseudomonas sp., that communicate with a common auto-inducer, N-acyl-L-homoserine lactone (AHL), for quorum sensing (QS) regulation [1]. The QS inhibitor [2,3], for example, the quorum quenching (QQ) enzymes, which degrade the AHL signaling molecules, including AHL-lactonase, -acylase, and -oxidoreductases, has been applied as an alternative antibacterial reagent with the advantages of precise targeting control and mitigation of bacterial antibiotic resistance. However, the application of QQ enzymes is still limited by their low catalytic efficiency and enzyme stability in comprehensive aquacultural environments, which are normally affected by high salt, multiple iron, pH, and temperature ranges.
Compared to land resources, marine microorganisms, especially those from the deep surface, normally contain homologous genes with low similarity to currently known sequences and exhibit specific functions [4,5]. In previous work, Bacillus velezensis DH82 was isolated from underlying sea water of the Western Pacific Yap trench at a depth of 6000 m, and was identified, using the quorum quenching function, as affecting the biofilm-forming ability and pathogenicity of P. aeruginosain [6] and V. parahemolyticus [7]. For further utilization of this strain, in this study, the potential QQ enzymes were predicted by performing whole-genome sequencing and COG annotation according to the homological sequence of the serine active site of N-acyl homoserine lactonase AiiA (EC:3.1.1.81) [8,9,10]. The predicted QQ enzymes were engineered for heterologous expression in E. coli to study their enzyme activity on AHL degradation and their stability against environmental factors.

2. Materials and Methods

2.1. Bacteria, Plasmids, and Reagents

The B. velezensis DH82 strain (GenBank: MK203035) was isolated from sea water samples taken from the Western Pacific Yap trench at a depth of 6000 m, and was kindly provided by the Third Institute of Oceanography (Xiamen, China). The competent cells of the E. coli DH5α and E. coli BL21 strains, Isopropyl-β-D-thiogalactopyranoside (IPTG), and Kanamycin, were purchased from Transgen (Beijing, China). The AHL reporter operon of LuxR-PluxI-lacO-RFP was provided by Xiamen University (Xiamen, China) [11]. N-Acyl-L-homoserine lactone hydrolase (PDB: 3DHB) from B. thuringiensis (GenBank: AY943832), named AiiA3DHB, was used as the positive control to analyze the activity of the predicted QQ enzyme.
The plasmid pET28a expression vector was purchased from Novagen (Houston, TX, USA) (Cat. 69864-3). The plasmid miniprep kit (Cat. GMK5999) and gel extraction kit (Cat. D2500-02) were purchased from Promega (Beijing, China). The AHLs, C6-HSL (Cat. 56395), 3-oxo-C6-HSL (Cat. K3255), C4-HSL (Cat. 09945), 3-OH-C4-HSL (Cat. 74359), 3-oxo-C10-HSL (Cat. O9014), and 3-oxo-C12-HSL (Cat. O9139), were purchased from Sigma-Aldrich (Shanghai, China).

2.2. Genomic DNA Sequencing

The bacterial culture of DH82 was incubated at 37 °C with shaking at 180 rpm for 12 h and was centrifuged at 8000 rpm for 10 min to harvest the bacterial pellets. Then, 100 ng of the genomic DNA of the strain DH82 was extracted from the pellets by using the CTAB method, and randomly digested into fragments of less than 500 bp by ultrasonication using the Covaris S220 to establish the cDNA library. The quality of cDNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), while the quantity of cDNA was assessed using a Qubit 3.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing was performed using the Illumina Hiseq×10 and PacBio RSII by Majorbio (Shanghai, China).

2.3. Gene Cloning

The sequences of AiiADH82 and YtnPDH82 were amplified from the genomic DNA of DH82 using PCR and the primers listed in Table 1, which were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). The PCR products were digested with the restriction enzymes NdeI, XhoI, and EcoRI (Takara, Beijing, China) and subsequently ligated to the multiple cloning sites of the pET28a vector using T4 ligase (Takara, Beijing, China). The engineered expression clone, which was driven by the T7 promoter and framed with 6× Histidine (His) at both N- and C-terminal ends to ensure compatibility with the target enzyme, was transferred to E. coli BL21 competent cells for protein expression.
The AHL reporter operon of LuxR-PluxI-lacO-RFP [11] was digested using restriction enzymes NdeI and HpaI, and then ligated into the pET28a plasmid. The re-engineered reporter plasmid was then transferred to E. coli BL21 competent cells to construct the reporter strain (named LuxR-RFP for short) in this study for rapid detection of AHLs.

2.4. Bacterial Culture and Protein Expression

The bacteria were cultured using Luria–Bertani (LB) media (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl, pH 7.0) containing 50 µg/mL kanamycin with shaking at 180 rpm at 37 °C. Then, 0.2 mM IPTG was inoculated after 2 h of incubation with the bacterial culture to induce the protein expression. The overnight cultured bacteria were washed and concentrated at 5:1 with PBS (pH 7.0), ultrasonically broken (operate for 3 s at 300 W then break for 6 s, and repeat 60 times) on ice, centrifuged at 4 °C at 10,000 rpm for 15 min, and the supernatant was harvested. The extracted crude enzyme was resuspended using lysis buffer (300 mM NaCl and 50 mM NaH2PO4 (pH 7.4)), then washed with imidazole elution buffer (300 mM NaCl, 200 mM imidazole, and 50 mM NaH2PO4 (pH 7.4)). High-affinity NI-NTA chromatography was used to purify the His-tagged enzyme. The purified enzyme was filter-sterilized and analyzed using SDS-PAGE. The concentration of protein was quantified using the Bradford assay.

2.5. In Vitro Rapid Assessment of AHLs Level

Next, 100 µL of filter-sterilized AHL solution (working concentration, 400 mM) was mixed with 100 µL of purified enzyme in triplicate, using PBS as a negative control at the same volume. The enzyme-AHL mixtures were incubated at 28 °C for 45 min, followed by an immediate addition of 10% SDS to stop the reaction. The reporter strain was incubated overnight in LB media (containing 50 µg/mL Kanamycin) at 37 °C with shaking at 200 rpm, and then inoculated with the above enzyme-AHL mixture, incubated at 25 °C with shaking at 180 rpm for 12 h, centrifuged at 8000× g at 4 °C, and resuspended in an equal volume of PBS. A fluorescence intensity of 620 nm (excitation wavelength of 584 nm) and an optical density of 600 nm (OD600) were measured using a Tecan Infinite M200 Pro. microplate reader (Tecan Group Ltd., Grödig, Austria) to determine the residual AHL levels in the samples.

2.6. Quantitative Analysis of Enzyme Activity on AHL Degradation

We analyzed 200 µL of filter-sterilized 3-oxo-C6-HSL at final concentrations of 0.05, 0.5, 1.0, 1.5, and 2.0 µM using a C18 chromatographic column (Agilent, 1.8 μm, 2.1 × 50 mm) on ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-QTOF MS, Agilent, 1290-6545) to generate a standard curve, with AHL concentrations determined by the peak area at the mass-to-charge ratio of 214.

2.6.1. Characterization of Enzyme Activity

Filter-sterilized 3-oxo-C6-HSL (final concentration of 300 µM) was added with 100 µL of 0.5 mg/mL purified lactonase in 1 mL reaction volumes and incubated at pH 7.5 at 18, 25, 37, 45, 55, and 65 °C for 45 min. Sterilized water and non-treated AHL served as negative and positive controls, respectively. Reactions were terminated by heating at 100 °C for 10 min, and the residual AHLs were quantified using a C18 column on UPLC-QTOF MS, with the standard curve used to analyze the temperature effects on the enzyme activity.
Similarly, enzyme-AHL mixtures were incubated at 28 °C under pH 2.0, 3.0, 4.0, 5.0, and 6.0 for 45 min, with residual AHLs quantified to evaluate the pH effects.

2.6.2. Kinetic Analysis of Enzyme Activity

We mixed 100 µL of filter-sterilized AHLs at 200, 250, 300, and 350 µM with 100 µL of 0.5 mg/mL purified enzyme before incubating them at pH 6.5 and 28 °C for 45 min. Reactions were terminated at 100 °C for 10 min, and residual 3-oxo-C6-HSL was analyzed using a C18 column on UPLC-QTOF MS. Residual AHLs were determined by the peak area at m/z 214. Lineweaver–Burk plots were established to calculate the Km value and reaction rate (Vmax).

2.7. Three-Dimensional Structure Simulation and Functional Prediction

Three-dimensional (3D) enzyme structures were simulated and predicted using the I-TASSER server [12]; the simulated models with a high confidence score were analyzed using VMD 1.8.3 software to identify ligand-binding regions and active sites involved in AHL degradation for functional prediction.

2.8. Analysis of Enzyme-Substrate Docking

Molecular docking was simulated using the CHARMm-based molecular dynamics (MD) scheme in Discovery Studio 2019 [13]. Random ligand conformations were generated using high-temperature MD, translated into the enzyme binding site with rigid-body rotations, followed by simulated annealing. Docked ligand poses were recorded with the CDOCKER score, including internal ligand strain energy and receptor–ligand interaction energy (higher values indicate more favorable binding).

2.9. Statistical Analysis

T-tests were applied to compare enzyme activities, which were analyzed using GraphPad Prism 6. The p-value indicated differences between groups.

3. Results

3.1. Prediction and Phylogenetic Tree Analysis of Quorum Quenching Enzyme

According to the COG annotation from the sequencing results, as shown in Figure 1, a panel of proteins containing a conserved β-lactamase domain—which were consistent with the reported QQ lactonase families—were predicted to be QQ-related proteins due to their homologous serine active sites with potential AHL-binding ability. Metal-binding sites were categorized as metal-dependent hydrolases. Protein 4079 showed 91.6% consensus with the quorum-quenching lactonase AiiA from B. thuringiensis (PDB ID: 3DHB) [9,10,11], and was 98.40% homologous to AiiA from B. cereus Y2 (Swiss-Prot: Q08GP4.1) [14].
Protein 0540 was 79% homologous to YtnP from B. subtilis 168 (Swiss-Prot: O34760.2) [15], 26.14% to Y2-AiiA from B. cereus Y2 (Swiss-Prot: Q08GP4.1) [14], and 27.84% to AiiB from Agrobacterium fabrum C58 (Swiss-Prot: A9CKY2.1) [16].
Other predicted proteins were not reported as QQ enzymes: protein 0114 was 99.53% homologous to YqgX from B. velezensis G341 [17]; protein 1459 was identified as putative Bacillaene biosynthesis zinc-dependent hydrolase and was a 100% match to BaeB from B. velezensis UCMB5033 (GenBank: AIU81842.1; UniPro: S6FKG4) [18]; protein 1509 was a 59.1% match to probable metallo-hydrolase YqjP from B. subtilis 168 (UniPro: P54553) [15]; protein 1800 was 28.64% homologous to MBL fold metallo-hydrolase YflN [19] from Paenibacillus flagellates (UniProt: A0A2V5K1S6).
Phylogenetic analysis using an NCBI-BLAST (ElasticBLAST 1.4.0) with maximum likelihood clustering is shown in Figure 2. The results revealed that proteins 0114, 1459, and 1800 were 100% homologous to metallo-β-lactamases, while protein 1509 showed more than 70% similarity to Bacillus-derived sequences. Proteins 4079 and 0540 shared 93% and 88% similarity with AiiA from B. cereus Y2 and YtnP from B. mojavensis, respectively, and were named AiiADH82 and YtnPDH82 for further biological characterization.

3.2. Construction of Engineered Enzyme and Protein Expression

The expression clones of His-tagged AiiADH82 and YtnPDH82 were constructed on the pET28a plasmid and expressed in E. coli BL21. The sequence of the expression clones was confirmed via sequencing by Sangon Biotech (Shanghai) Co., Ltd.
The heterologously expressed proteins were analyzed using SDS-PAGE as shown in Figure 3. The His-tagged AiiADH82 at 31.99 kDa and YtnPDH82 at 35.82 kDa were observed in Lanes 3 and 4, respectively, whereas the crude enzyme extraction of AiiADH82 was observed in Lane 1 and YtnPDH82 in Lane 2. The concentrations of harvested purified AiiADH82 and YtnPDH82 were 0.594 mg/mL and 0.513 mg/mL, respectively, according to a Bradford assay.

3.3. Identification of Quorum Quenching Capacity on AHL Degradation

The activity of recombinant enzymes was assessed according to AHL degradation against a series of AHLs. Fluorescence intensity generated from LuxR-RFP was used to determine the residual AHLs in the reaction. As shown in Figure 4, both AiiADH82 and YtnPDH82 were observed with function on 3-oxo-C6-HSL, C6-HSL, 3-OH-C4-HSL, C4-HSL, 3-oxo-C12-HSL and 3-oxo-C10-HSL.

3.4. 3D Structural Simulation and Analysis

3.4.1. 3D Structure of Lactonase

The 3D structures of AiiADH82 and YtnPDH82 were simulated using I-TASSER, which employed structural alignment to match the structures in the PDB library.
YtnPDH82 was predicted with an average distance of all residue pairs in two structures (RMSD) at 4.1 ± 2.7 Å (highest C-score at 0.97 and TM-score at 0.85 ± 0.08). The model of the most typical structure (Figure 5A) is presented with a substrate-binding pocket formed by the α-helix (labeled in purple), the Zn2+-binding site (H111, H113, H191, and D212), the FEO (µ-oxo-diiron) binding site (H111, H113, D115, H116, H191, D212, and H257), and the active site at residue D115. As shown in the magnified view presented in Figure 5B, the first Zn2+-binding site is composed of nitrogen atoms on H111 and H191, and an oxygen atom on D212; the second Zn2+-binding site is composed of a nitrogen atom on H116 and H257, and an oxygen atom on D115. Both Zn2+ ions bind an oxygen atom after the opening of the ester ring on AHL.
The structure of AiiADH82 is depicted in Figure 5C and magnified in Figure 5D, with an average distance of all residue pairs in two structures (RMSD) at 2.6 ± 1.9 Å (highest TM-score at 0.998), containing a flexible substrate pocket, a Zn2+-binding site (residues H104, H106, H169, and D191), a substrate pocket (C14, F64, T67, F68, I73, H106, F107, D108, H109, E136, H169, D191, Y194, and H235), and an active site at residue D108.
The structural simulation indicates that YtnPDH82 is more stable with a fixed substrate pocket with an α-helix, and that AiiADH82 exhibits higher activity with a more flexible substrate pocket, which was verified in further experiments under the challenges of different temperatures and pH.

3.4.2. Capacity of Lactonase-AHL Docking

The capacity of enzyme-substrate docking was analyzed for the top 10 docked structures of the enzyme with a ligand pose (Table 2). A higher CDOCKER score means a greater tendency and a higher possibility for substrate binding. The results demonstrated that YtnPDH82 showed higher affinity for short-chain AHLs (notably C6-HSLs), while AiiADH82 preferred long-chain substrates such as C10-HSL and C12-HSL. YtnPDH82 showed enhanced activity toward 3-O-substituted C4- and C6-HSLs, while such a tendency was not observed in AiiADH82.

3.4.3. Prediction of Ester-Hydrolysis Activity on AHLs

The molecular dynamics were further analyzed using ligand poses and the average CDOCKER score (Figure 6) to predict their ester-hydrolysis activity against AHLs, according to the distance between the AHLs’ ester group and the active sites. The ligand poses showed that YtnPDH82 was closer to 3-oxo-C6-HSL (5.85 Å) than AiiA (11.09 Å), which indicated that YtnPDH82 had higher hydrolysis activity. However, AiiADH82 was closer to C6-HSL and 3-oxo-C12-HSL than YtnPDH82, suggesting that AiiADH82 had superior activity toward these substrates.

3.5. Qualification of AHL Degradation Activity

The classical AHL, 3-oxo-C6-HSL, was further used to quantitatively assess the enzyme activity of lactonases. A lower residual amount of AHLs indicates that the lactonase has a stronger degradation activity.
As shown in Figure 7A, after a 45-min catalytic reaction, AiiADH82 exhibited greater adaptability (with degradation activity all above 89.95%) across a broad pH range (2.0–6.0). Its activity peaked at pH 6.0 (95.61% degradation) with 1318.37 nM of residual AHL. In contrast, YtnPDH82 was sensitive to strong acidic conditions but demonstrated enhanced stability as the pH increased to 6.0, at which point its AHL-degradation activity reached a maximum of 99.99% with only 4.378 nM of residual AHL remaining.
Figure 7B illustrates that both YtnPDH82 and AiiADH82 reduced the AHL concentration to below 5 nM. Furthermore, YtnPDH82 showed higher stability and activity within the temperature range of 18–65 °C, with degradation rates against 3-oxo-C6-HSL all exceeding 99.99% and a peak value of 99.9987% at 18 °C. In comparison, AiiADH82 showed lower activity and a declining trend, with its highest activity of 99.9955% degradation achieved at 18 °C.

3.6. Steady-State Kinetics Characterization of Enzyme

The kinetic characterization of AiiADH82 and YtnPDH82 was further performed using the substrate of 3-oxo-C6-HSL, as shown in Table 3, according to the Lineweaver–Burk equation obtained from the standard curve. The Km value of YtnPDH82 was 15.33 mmol/L, and the Vmax was 0.33 mmol/min, while the Km of AiiADH82 was 7.8 mmol/L and 0.18 mmol/min.

4. Discussion

Bacteria produce various molecular signals to communicate through the QS system [20]. However, it remains unknown whether bacteria have evolved to produce signals via QS for communication or to degrade signals via QQ for competition [21,22]. Researchers have attempted to reveal the novel QS signals and the regulatory mechanisms of QS systems [1], as well as to discover novel QQ enzymes [23] to interfere with bacterial QS by degrading the relevant QS signals [24].
AiiA was initially classified as belonging to the metallo-β-lactamase superfamily due to its conserved HXHXDH region. Another conserved sequence, GHXXGX, was later identified in AiiA (PDB: 3DHB) from B. thuringiensis [10]. Both sequences constitute Zn2+-binding sites: the HXHXDH region binds Zn2+ and Tyr194 stabilizes the charge of the tetrahedral AHL substrate, thereby promoting nucleophilic attack; in contrast, the GHXXGX sequence binds Zn2+, stabilizing the charge of the alkoxide formed after the AHL ring opens to break the C-O ester bond [25,26].
YtnPDH82 was also classified as belonging to the metallo-β-lactamase superfamily, containing both the conserved HXHXDH and GHXXGX regions. The enzyme structure reveals a conserved active site with Asp115 substitution, similar to AiiA-like lactonase but with a distinctive feature—a “kinked” α-helix that forms part of a closed binding pocket that provides affinity and enforces selectivity for AHL substrates, similar to previously characterized AHL lactonase AidC [27].
The structural features of both lactonases were reflected in the enzyme activities on AHL degradation. For 3-oxo-C6-HSL, a signal molecule reported to be produced by numerous Gram-negative bacteria, and especially the primary signal for Vibrio sp. Pathogens [28], AiiADH82 has a Km value of 7.8 mM, showing no significant difference from other reported wild-type AiiAs [10]. In contrast, YtnPDH82, despite sharing the same active site, has a more extensive α-helix structure, resulting in lower substrate affinity with a Km of 15.33 mM. Additionally, the α-helical feature demonstrated its advantage for enzyme stability, as evidenced by a broader tolerance to changes in temperature. Additionally, YtnPDH82 exhibits higher kcat and kcat/Km values than AiiADH82, which is consistent with the ligand pose of the target C-O bond within the substrate pocket relative to the active site of Asp substitution.
For AHL compounds, differences in acyl chain length (4 to 20 carbons) or substituents at the 3-position of the N-acyl side chain (no substituent, keto, or hydroxy) all influence enzyme activity in substrate hydrolysis. The activities of both lactonases correlate positively with the substrate’s acyl chain length. Furthermore, YtnPDH82 showed higher activity toward short-chain substrates such as C4 and C6, whereas AiiADH82 performs better with longer-chain substrates such as C10-HSL and C12-HSL. Substrate effects on enzyme activity also differ slightly, as YtnPDH82 has higher activity toward substrates with oxygen-containing substituents (for example, it performs better on 3-oxo-C6-HSL than on C6-HSL), while AiiADH82 showed no significant activity toward short-chain substrates.
The QQ efficiency of lactonase was also evaluated using a quantitative analysis of the residual AHL concentrations. Considering the alkaline hydrolysis character of AHLs at pH values above 7 [29], the activity of lactonases was only accessed in the range of acidic conditions used in this study. Bacterial QS systems are threshold-dependent, relying on signal accumulation and removal [30]; for instance, the receptor protein LuxR requires a threshold concentration of 5 nM for the canonical signal molecule 3-oxo-C6-HSL [30,31]. Therefore, YtnPDH82 demonstrates an advantage over AiiADH82 in terms of QS regulation via 3-oxo-C6-HSL degradation, as evidenced by its higher degradation rates across different pH and temperature conditions, which indicates the greater potential and broader applicability of YtnPDH82 in regulating the AHL-mediated QS pathway in Gram-negative bacteria.

5. Conclusions

In summary, both AiiADH82 and YtnPDH82 from the B. velezensis DH82 strain belong to the metallo-β-lactamase superfamily. They share the same conserved domains and active sites for AHL hydrolysis, but differ in the structural features of substrate pockets. Herein, AiiADH82 demonstrated similar biochemical properties to other reported lactonases and showed greater adaptability to pH fluctuations and higher activity toward long-chain substrates due to its more flexible substrate pocket in the enzyme structure. In contrast, the α-helix in YtnPDH82 confers substrate affinity and enforces selectivity, enabling it to perform better in degrading short-chain AHLs such as 3-oxo-C6-HSL. Additionally, YtnPDH82 displayed higher catalytic activity and greater stability under varying environmental conditions, such as temperature. These findings clarify the structural features and biochemical characterization of QQ enzymes in the probiotic B. velezensis DH82 strain and provide an experimental basis for the further application of YtnPDH82 in regulating AHL-mediated QS pathways.

Author Contributions

X.S. contributed to the conception of the study and drafted the manuscript; J.L. and Y.Y. performed the experiments and analyzed the data; G.Z. performed the simulation of enzyme-substrate docking; X.S. and J.L. contributed significantly to the analysis of the results; H.F.M. contributed to the proofreading of the manuscript; S.Y. helped analysis with constructive discussions; X.S. offered the funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

The project was sponsored by the National Key Research and Development Program of China (2022-20), the Key Research and Development Program of Xiamen (3502Z20234041), Xiamen Marine Youth Innovation Project (23YYZP017QCB25), the Fundamental Research Funds for the Central Universities (ZQN-1018) and Campus-Local cooperative Research Project of Nanping (N2023Y013).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author due to privacy or ethical restrictions.

Acknowledgments

Thanks to Changan Xu for providing B. velezensis DH82 strain, and Baishan Fang for providing the AHL reporter operon of LuxR-PluxI-lacO-RFP.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Multiple alignment of β-lactamase domain-containing protein superfamily from DH82 strain. The residues that were completely conserved are represented by black shading. The residues that were not completely homologous were labelled in pink and light blue. The conserved regions HXHXDHA and GHXXGX were labelled with a red *.
Figure 1. Multiple alignment of β-lactamase domain-containing protein superfamily from DH82 strain. The residues that were completely conserved are represented by black shading. The residues that were not completely homologous were labelled in pink and light blue. The conserved regions HXHXDHA and GHXXGX were labelled with a red *.
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Microorganisms 13 01717 g002
Figure 2. Phylogenetic analysis of predicted enzymes. The nucleotide sequence of each predicted QQ enzyme was analyzed using NCBI-BLAST. Proteins 0114, 1459, 1509, 1800, 4079, and 0540 were respectively present in panels (A–F) labelled with a ◆.
Figure 2. Phylogenetic analysis of predicted enzymes. The nucleotide sequence of each predicted QQ enzyme was analyzed using NCBI-BLAST. Proteins 0114, 1459, 1509, 1800, 4079, and 0540 were respectively present in panels (A–F) labelled with a ◆.
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Microorganisms 13 01717 g003
Figure 3. SDS-PAGE analysis of engineered AiiADH82 and YtnPDH82. The engineered lactonases were expressed overnight at 37 °C in E. coli under induction with 0.4 mM IPTG; the expressed proteins were harvested and analyzed using SDS-PAGE. Lane M, marker; Lane 1, crude extraction from E. coli::pET28a/AiiADH82; Lane 2, crude extraction from E. coli::pET28a/YtnPDH82; Lane 3, purified AiiADH82; Lane 4, purified YtnPDH82.
Figure 3. SDS-PAGE analysis of engineered AiiADH82 and YtnPDH82. The engineered lactonases were expressed overnight at 37 °C in E. coli under induction with 0.4 mM IPTG; the expressed proteins were harvested and analyzed using SDS-PAGE. Lane M, marker; Lane 1, crude extraction from E. coli::pET28a/AiiADH82; Lane 2, crude extraction from E. coli::pET28a/YtnPDH82; Lane 3, purified AiiADH82; Lane 4, purified YtnPDH82.
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Microorganisms 13 01717 g004
Figure 4. QQ activity on AHL degradation. Activity of the predicted proteins was determined by the relative fluorescence intensity of LuxR-RFP. Degradation of 3-oxo-C6-HSL (cyan), C6-HSL (yellow), 3-OH-C4-HSL (green), C4-HSL (blue), 3-oxo-C12-HSL (purple), and 3-oxo-C10-HSL (orange) by AiiADH82 and YtnPDH82, compared with untreated AHLs as experimental control groups (ECGs). Controls include reporter without AHL (CG1 in red) and LB only (CG2 in black). Statistical analysis results are presented, with significant differences indicated by *** where p < 0.01 and ** where p < 0.05.
Figure 4. QQ activity on AHL degradation. Activity of the predicted proteins was determined by the relative fluorescence intensity of LuxR-RFP. Degradation of 3-oxo-C6-HSL (cyan), C6-HSL (yellow), 3-OH-C4-HSL (green), C4-HSL (blue), 3-oxo-C12-HSL (purple), and 3-oxo-C10-HSL (orange) by AiiADH82 and YtnPDH82, compared with untreated AHLs as experimental control groups (ECGs). Controls include reporter without AHL (CG1 in red) and LB only (CG2 in black). Statistical analysis results are presented, with significant differences indicated by *** where p < 0.01 and ** where p < 0.05.
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Microorganisms 13 01717 g005
Figure 5. Three-dimensional structural simulation and functional prediction. (A) YtnP 3D structure; (B) magnified view of YtnP substrate-binding site; (C) AiiA 3D structure; (D) magnified view of AiiA substrate-binding site. α-helix (purple band), β-sheet (yellow arrow), random coil (blue band), and other structures (cyan and white tubes) are shown. Red, blue, cyan, and white represent Oxygen (red), nitrogen (blue), carbon (cyan), and hydrogen atoms (white), respectively. The residues and atoms of zinc-binding sites are labeled in the magnified view.
Figure 5. Three-dimensional structural simulation and functional prediction. (A) YtnP 3D structure; (B) magnified view of YtnP substrate-binding site; (C) AiiA 3D structure; (D) magnified view of AiiA substrate-binding site. α-helix (purple band), β-sheet (yellow arrow), random coil (blue band), and other structures (cyan and white tubes) are shown. Red, blue, cyan, and white represent Oxygen (red), nitrogen (blue), carbon (cyan), and hydrogen atoms (white), respectively. The residues and atoms of zinc-binding sites are labeled in the magnified view.
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Microorganisms 13 01717 g006
Figure 6. Ligand binding to AHL substrates. (A–C) Ligand pose of AiiADH82 with 3-oxo-C6-HSL, C6-HSL and 3-oxo-C12-HSL; (D–F) ligand pose of YtnPDH82 with 3-oxo-C6-HSL, C6-HSLand 3-oxo-C12-HSL. Oxygen (red), nitrogen (blue), carbon (cyan), and hydrogen (white) atoms are shown. The distance between the active site (Dotted Asp residue) and the hydrolysis site of the ester oxygen is indicated by the white line.
Figure 6. Ligand binding to AHL substrates. (A–C) Ligand pose of AiiADH82 with 3-oxo-C6-HSL, C6-HSL and 3-oxo-C12-HSL; (D–F) ligand pose of YtnPDH82 with 3-oxo-C6-HSL, C6-HSLand 3-oxo-C12-HSL. Oxygen (red), nitrogen (blue), carbon (cyan), and hydrogen (white) atoms are shown. The distance between the active site (Dotted Asp residue) and the hydrolysis site of the ester oxygen is indicated by the white line.
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Microorganisms 13 01717 g007
Figure 7. The enzyme’s activity in degrading AHL. The enzyme activity’s effect on AHL degradation was determined by the percentage of residual AHL(initial 300 µM in 1 mL reaction). Residual AHL was quantified via LC-MS (m/z 214). (A) indicates pH effects; (B) indicates temperature effects. Samples with residual AHL lower than 5 nM are marked with *.
Figure 7. The enzyme’s activity in degrading AHL. The enzyme activity’s effect on AHL degradation was determined by the percentage of residual AHL(initial 300 µM in 1 mL reaction). Residual AHL was quantified via LC-MS (m/z 214). (A) indicates pH effects; (B) indicates temperature effects. Samples with residual AHL lower than 5 nM are marked with *.
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Table 1. Sequences of PCR primers in this study.
Table 1. Sequences of PCR primers in this study.
PrimerSequence
aiiA-F5′-GGAATTCCATATGACAGTAAAGAAGCTTTATTTC-3′
aiiA-R5′-CCGCTCGAGCGGTATATATATTCGAACACTTTACATCCCC-3′
ytnP-F5′-GGAATTCATGGAGACATTGAATATTGGGAATTTTC-3′
ytnP-R5′-CCGCTCGAGCGGTTTTTTCTCCCGTTGACAGATG-3′
Note: the sequences underline were respectively the restriction enzyme recognition sites of NdeI, XhoI and EcoRI.
Table 2. CDOCKER score of enzyme-substrate docking.
Table 2. CDOCKER score of enzyme-substrate docking.
SubstrateAiiADH82YtnPDH82p Value (t-Test)
3-OH-C4-HSL21.898 ± 1.64523.146 ± 1.7050.113
C4-HSL22.618 ± 1.84823.050 ± 0.9370.519
3-oxo-C6-HSL27.820 ± 1.49931.130 ± 0.8041.389 × 10−10
C6-HSL27.020 ± 1.56128.396 ± 0.8050.023
3-oxo-C10-HSL36.196 ± 1.76635.080 ± 1.7020.049
3-oxo-C12-HSL40.803 ± 1.17538.229 ± 1.4052.319 × 10−7
Table 3. Steady-state kinetic constants for the hydrolysis of 3-oxo-C6-HSL by lactonases.
Table 3. Steady-state kinetic constants for the hydrolysis of 3-oxo-C6-HSL by lactonases.
Km (mM)kcat (s−1)kcat/Km (M−1s−1)
AiiADH827.81.616 × 1032.071 × 105
YtnPDH8215.333.840 × 1032.505 × 105
Note: 100 µL of filter-sterilized 3-oxo-C6-HSL at concentrations of 200 µM, 250 µM, 300 µM, and 350 µM were mixed with 100 µL of 0.5 mg/mL purified enzyme at pH 6.5 and 28 °C for 45 min.
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Sun, X.; Liu, J.; Yan, Y.; Yang, S.; Zhang, G.; Mohamed, H.F. Development and Biochemical Characterization of Quorum Quenching Enzyme from Deep-Sea Bacillus velezensis DH82. Microorganisms 2025, 13, 1717. https://doi.org/10.3390/microorganisms13081717

AMA Style

Sun X, Liu J, Yan Y, Yang S, Zhang G, Mohamed HF. Development and Biochemical Characterization of Quorum Quenching Enzyme from Deep-Sea Bacillus velezensis DH82. Microorganisms. 2025; 13(8):1717. https://doi.org/10.3390/microorganisms13081717

Chicago/Turabian Style

Sun, Xiaohui, Jia Liu, Ying Yan, Suping Yang, Guangya Zhang, and Hala F. Mohamed. 2025. "Development and Biochemical Characterization of Quorum Quenching Enzyme from Deep-Sea Bacillus velezensis DH82" Microorganisms 13, no. 8: 1717. https://doi.org/10.3390/microorganisms13081717

APA Style

Sun, X., Liu, J., Yan, Y., Yang, S., Zhang, G., & Mohamed, H. F. (2025). Development and Biochemical Characterization of Quorum Quenching Enzyme from Deep-Sea Bacillus velezensis DH82. Microorganisms, 13(8), 1717. https://doi.org/10.3390/microorganisms13081717

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