Magnetic immobilization of a quorum sensing signal hydrolase, AiiA

Abstract Magnetic immobilization of quorum sensing (QS) signal hydrolases provides a convenient solution for quenching QS process that is essential for bacterial biofilm formation and antimicrobial resistance. In the present study, a QS signal hydrolase, AiiA, was fused with a magnetic protein, MagR, and expressed in Escherichia coli. Magnetic immobilization of AiiA was achieved on Fe3O4‐SiO2 iron beads and was confirmed via SDS‐PAGE, zeta potential measurement, FTIR spectrometry, and SEM analysis. The magnetic immobilized AiiA exhibited activity in degrading the quorum sensing signal, C6‐HSL. This study opens a new avenue to actively immobilize enzymes via magnetic interaction and quench quorum sensing.

multifunctional coupling agents to bridge enzymes with supports/carriers, or by genetically modifying enzymes with fusion tags to provide the enzymes with binding affinity to supports/ carriers (Tischer & Kasche, 1999). Various proteins or domains with affinity to matrix have been used as fusion tags for immobilizing enzymes. Representatives include polyhistidine tag (Chen, Brash, & Funk, 1993), Strep II tag (Smyth, Odenthal, Merkl, & Paulsson, 2000), MBP tag (Lichty, Malecki, Agnew, Michelson-Horowitz, & Tan, 2005), and GST tag (Scheich, Sievert, & Bussow, 2003), which facilitate the purification of many proteins. However, interactions between conventional fusion tags rely on pH, salinity, and ionic strength of the environment, which may not be suited to the activity of tagged enzymes. The use of magnetic protein MagR as fusion tag has been recently reported for actively immobilizing GFP and lipase (Jiang, Zhang, Wang, Zhang, & Liu, 2017). The magnetic interactions between MagR and supports imply that the MagR-based immobilization is less affected by chemical environments. Therefore, the enzyme can be immobilized over a broad range of environmental conditions suiting its activity.
In the present study, the magnetic protein MagR was used as a fusion tag for magnetic immobilization of the QS signal hydrolases,

| Molecular biology manipulation
The quorum sensing signal hydrolase AiiA was expressed as MagR fusions. The DNA segments encoding MagR (GenBank Accession NP_573062) and AiiA (GenBank Accession AGA83379) were optimized for prokaryotic expression and synthesized by Bio Translation Lab. The coding sequence of AiiA was cloned into the multiple cloning sites of plasmid pET28a containing MagR, resulting in the AiiA protein placing at N-terminus of MagR. The resultant vector was named as pET28a[AiiA-MagR] with the sequence was confirmed by DNA sequencing.
The cell suspension was inoculated into LB medium and followed by shaking at 37°C until the value of OD600 reached 0.5-0.6. Protein expression was initiated by supplying with 0.2 mM of IPTG and was continuously shaken at 20°C for 10-14 hr. Cells were then harvested by centrifugation at 6,000 g for 10 min and stored at −80°C.

| Characterization of IB@[AiiA-MagR]
The morphology of IB@[AiiA-MagR] was imaged by using a S4800 (Hitachi Company) scanning electron microscope (SEM), fitted with a field-emission source operating at 5 kV. The zeta potential was measured using a Zetasizer Nano-ZS90 (Malvern, UK). The FTIR spectra of pellets were collected from 4,000 to 400 cm −1 , at a resolution of 4 cm −1 with 64 scans by using a Nexus (Thermo Fisher Scientific) Fourier transform infrared (FTIR) spectrometer.

| Enzymatic assay of IB@[AiiA-MagR]
The AHL activity of IB@ [AiiA-MagR] was assessed with a C6-HSL diffusion assay (Jiang et al., 2017)  The AHL activity of IB@[AiiA-MagR] was also quantitatively assessed through a colorimetrical assay of the purple pigment violacein (Mukherji, Varshney, Panigrahi, Suresh, & Prabhune, 2014). Aliquots of 20 μl phosphate buffer (pH 7.0) containing 1 mmol AHL (C6-HSL) and 1 mg IB@[AiiA-MagR] were incubated at 35°C for enzymatic degradation of C6-HSL. The reaction aliquot was taken hourly and diluted in 5 ml LB broth containing 100 μl of overnight culture of CV026 to terminate reaction, followed by incubating at 30°C for 16-18 hr. The violacein produced was extracted from the culture broth by dissolving the pigment in 1 ml DMSO. After removing the cell mass by centrifugation, the purple pigment production was quantitatively estimated by measuring the absorbance at 570 nm.
The catheter pieces were removed from the plate and washed three times using sterilized PBS to remove cell medium and nonadhered bacteria. The plates were dried at ambient temperature for 20 min and then stained with 0.1% crystal violet for 10 min. Residual solution was then removed, and samples were washed four times using sterilized Plates were air-dried before adding 1 ml 30% acetic acid to each well and recording spectrophotometric absorbance at 590 nm.

| Construction and expression of MagR-Tagged AiiA
In the present study, a plasmid harboring the DNA sequence encoding segments of AiiA and MagR was constructed to facilitate expression of MagR-tagged AiiA in E. coli host. The sequence of protein AiiA was based on AHL from Bacillus cereus isolated from striped catfish (Pangasianodon hypophthalmus) pond (Tinh, Dung, Trung, & Thuy, 2013). The sequence of protein MagR was based on magnetic receptor, isoform A from Drosophila melanogaster (Cyranoski, 2015). The resultant recombinant protein was named as AiiA-MagR with the MagR protein placing at C-terminus of AiiA.

| Magnetic immobilization of recombinant AiiA-MagR
The expressed AiiA-MagR was subjected to interact with Fe 3 O 4 iron beads (IBs). The binding of AiiA-MagR to IBs was evidenced with SDS-PAGE analysis (Figure 2, Panel a). It was observed that significant amount of impurity was removed by wash. The binding capacity  -MagR]. Bottom panel, the quantitative analysis of the relative and absolute degradation of C6-HSL based on pigment production that was indicated by area and density purple color IBs and IB@[AiiA-MagR] was observed in the range of 2,800-3,600 cm −1 and 1,500-1,700 cm −1 (Figure 3, Panels a-c). After interaction with AiiA-MagR, two characteristic peaks were detected at 3,566 cm −1 and 3,587 cm −1 that were assigned to stretching vibrations of primary amines as well as the band at 2,974 cm −1 was identified as secondary amine band. The characteristic bands in amide I region between 1,500 and 1,700 cm −1 correspond to the secondary structure of the polypeptide backbone. After binding with AiiA-MagR, there was a red shift of the absorbance peak from 1.630 cm −1 to 1,648 cm −1 as well as the appearance of the characteristic peak at 1,548 cm −1 that was assigned to the bending vibration of amine.
These data supported that IBs were coated with AiiA-MagR.
The morphological changes of IB after coating with AiiA-MagR protein were further investigated with SEM ( Figure 4). A coating layer was observed on IB@[AiiA-MagR], which made the surface of IB@[AiiA-MagR] rougher than that of IB. Therefore, it can be concluded that the magnetic immobilization of AiiA-MagR on IB has been successfully achieved.

| CON CLUS ION
Enzyme immobilization is important in modern biotechnology and with applications in broad areas such as industrial production of antibiotics and beverages, diagnosis and treatment of diseases, and treatment of sewage and industrial effluents. Enzyme immobilization can be achieved with various techniques such as affinity-tag binding, adsorption on supports, entrapment, cross-linkage, and covalent bond. With gene fusion technology, a segment of peptide as a fusion tag can be engineered into a target protein at the genetic level and provide it with biochemical properties of the imported fusion tag. In the present study, a magnetic protein, MagR, as a fusion tag was placed at C-terminus of a quorum sensing signal hydrolase, AiiA.
The resultant recombinant protein AiiA-MagR was successfully expressed in the E. coli host. The interaction between AiiA-MagR and Fe 3 O 4 -SiO 2 iron beads (IBs) was observed and confirmed by SDS-PAGE, zeta potential measurement, FTIR spectrometry, and SEM analysis. The binding capacity of IB to AiiA-MagR was 5.1 ± 0.3 mg protein per gram IBs. The AiiA-MagR coating on IBs remained activity in degrading the quorum sensing signal, C6-HSL. The activity of AiiA-MagR coating on IBs was detected in inhibiting biofilm formation of P. aeruginosa PA01 on sterile catheters. This study proved that the magnetic protein MagR could be used as a magnetic fusion tag to AiiA and facilitated the active magnetic immobilization of this QS signal hydrolase as well as reduced biofilm formation of bacteria. This magnetic immobilized AiiA-MagR can be further used for quenching QS and inhibiting biofilm formation in various environments.

ACK N OWLED G EM ENT
This work was financially supported by the National Natural Science Foundation of China (31771032, 51572206). We thank Professor Zhu Hu (China University of Petroleum, China) for assistance in assaying enzymatic degradation of C6-HSL.

CO N FLI C T O F I NTE R E S T
The authors declare no financial or commercial conflict of interest.